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2.
Haematologica ; 2021 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-33882639

RESUMO

Despite its high prevalence in children with sickle cell anemia (SCA), the pathophysiology of silent cerebral infarcts (SCIs) remains elusive. The main objective of this study was to explore the respective roles of major determinants of brain perfusion in SCA children with no past or current history of intracranial or extracranial vasculopathy. We used a multimodal approach based notably on perfusion imaging Arterial spin labelling (ASL) MRI and Near Infra-Red Spectroscopy (NIRS), as well as biomarkers reflecting blood rheology and endothelial activation. Out of 59 SCA patients (mean age 11.4} 3.9 yrs), 8 (13%) had a total of 12 SCIs. Children with SCIs had a distinctive profile characterized by decreased blood pressure, impaired blood rheology, increased P-selectin levels, and marked anemia. Although ASL perfusion and oximetry values did not differ between groups, comparison of biological and clinical parameters according to the level of perfusion categorized in terciles showed an independent association between high perfusion and increased sP-selectin, decreased RBC deformability, low HbF level, increased blood viscosity and no alpha-thalassemia deletion. NIRS measurements did not yield additional novel results. Altogether, these findings argue for early MRI detection of SCIs in children with no identified vasculopathy and suggest a potential role for ASL as an additional screening tool. Early treatment targeting hemolysis, anemia and endothelial dysfunction should reduce the risk of this under diagnosed and serious complication.

3.
Haematologica ; 106(5): 1303-1310, 2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32241839

RESUMO

Diamond Blackfan anemia (DBA) is predominantly an autosomal dominant inherited red cell aplasia primarily caused by pathogenic germline variants in ribosomal protein genes. DBA due to pathogenic RPL35A variants has been associated with large 3q29 deletions and phenotypes not common in DBA. We conducted a multi-institutional genotype-phenotype study of 45 patients with DBA associated with pathogenic RPL35A germline variants and curated the variant data on 21 additional cases from the literature. Genotype-phenotype analyses were conducted comparing patients with large deletions versus all other pathogenic variants in RPL35A. Twenty-two of the 45 cases had large deletions in RPL35A. After adjusting for multiple tests, a statistically significant association was observed between patients with a large deletion and steroid-resistant anemia, neutropenia, craniofacial abnormalities, chronic gastrointestinal problems, and intellectual disabilities (p<0.01) compared with all other pathogenic variants. Non-large deletion pathogenic variants were spread across RPL35A with no apparent hot spot and 56% of the individual family variants were observed more than once. In this, the largest known study of DBA patients with pathogenic RPL35A variants, we determined that patients with large deletions have a more severe phenotype that is clinically different from those with non-large deletion variants. Genes of interest also deleted in the 3q29 region that could be associated with some of these phenotypic features include LMLN and IQCG. Management of DBA due to large RPL35A deletions may be challenging due to complex problems and require comprehensive assessments by multiple specialists including immunologic, gastrointestinal, and developmental evaluations to provide optimal multidisciplinary care.


Assuntos
Anemia de Diamond-Blackfan , Anemia de Diamond-Blackfan/genética , Estudos de Associação Genética , Humanos , Mutação , Fenótipo , Proteínas Ribossômicas/genética
5.
Blood ; 136(11): 1262-1273, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32702755

RESUMO

Diamond-Blackfan anemia (DBA) was the first ribosomopathy described and is a constitutional inherited bone marrow failure syndrome. Erythroblastopenia is the major characteristic of the disease, which is a model for ribosomal diseases, related to a heterozygous allelic variation in 1 of the 20 ribosomal protein genes of either the small or large ribosomal subunit. The salient feature of classical DBA is a defect in ribosomal RNA maturation that generates nucleolar stress, leading to stabilization of p53 and activation of its targets, resulting in cell-cycle arrest and apoptosis. Although activation of p53 may not explain all aspects of DBA erythroid tropism, involvement of GATA1/HSP70 and globin/heme imbalance, with an excess of the toxic free heme leading to reactive oxygen species production, account for defective erythropoiesis in DBA. Despite significant progress in defining the molecular basis of DBA and increased understanding of the mechanistic basis for DBA pathophysiology, progress in developing new therapeutic options has been limited. However, recent advances in gene therapy, better outcomes with stem cell transplantation, and discoveries of putative new drugs through systematic drug screening using large chemical libraries provide hope for improvement.


Assuntos
Anemia de Diamond-Blackfan , Anormalidades Múltiplas/genética , Adenosina Desaminase/sangue , Adenosina Desaminase/genética , Anemia de Diamond-Blackfan/diagnóstico , Anemia de Diamond-Blackfan/genética , Anemia de Diamond-Blackfan/metabolismo , Anemia de Diamond-Blackfan/terapia , Pré-Escolar , Anormalidades Congênitas/genética , Diagnóstico Diferencial , Gerenciamento Clínico , Resistência a Medicamentos , Eritrócitos/enzimologia , Retardo do Crescimento Fetal/etiologia , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/fisiologia , Heterogeneidade Genética , Terapia Genética , Glucocorticoides/uso terapêutico , Proteínas de Choque Térmico HSP70/metabolismo , Transplante de Células-Tronco Hematopoéticas , Humanos , Lactente , Recém-Nascido , Peptídeos e Proteínas de Sinalização Intercelular/sangue , Peptídeos e Proteínas de Sinalização Intercelular/genética , Modelos Biológicos , Mutação , Síndromes Neoplásicas Hereditárias/genética , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/fisiologia , Proteína Supressora de Tumor p53/fisiologia
6.
Front Physiol ; 11: 576, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32595519

RESUMO

Despite the fact that Red Blood Cells (RBCs) have been intensively studied in the past 50 years to characterize mechanical phenotypes associated with both healthy and pathological states, only ektacytometry (i.e., laser diffractometry) is currently used by hematologists to screen for RBC membrane disorders. Therefore, the development of new diagnostic tools able to perform analysis at the scale of a single cell, over a statistically relevant population, would provide important complementary information. But these new diagnostic tools would have to be able to discriminate between different disorders causing a change in RBCs mechanical properties. We evaluated the mechanical response of artificially rigidified RBCs flowing through a microfluidic constriction. The geometry consists in a 50 µm wide channel with a succession of 14 tooth-like patterns, each composed of a 5 µm wide and 10 µm long constriction, associated with a 25 µm wide and 10 µm long enlargement. RBCs deformability was altered using two chemical treatments, known to affect RBCs membrane surface area and membrane deformability, lysolecithine (LPC) and diamide, respectively. Differences between samples were highlighted by the representation of the inverse of the shape recovery time (1/τ r ), versus the extension at the exit of the constriction, D out . The results demonstrate that our approach is able to provide a direct signature of RBCs membrane composition and architecture, as it allows discriminating the effect of changes in RBCs membrane surface area from changes in RBCs membrane deformability. Finally, in order to evaluate the potential of our microsystem to detect pathological cells, we have performed preliminary experiments on patients with Hereditary Spherocytosis (HS) or Sickle Cell Anemia (SCA).

7.
Blood Adv ; 4(8): 1760-1769, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32343795

RESUMO

Diamond-Blackfan anemia (DBA) is a congenital pure red cell aplasia associated with congenital abnormalities and cancer predisposition. Allogeneic hematopoietic stem cell transplantation (HSCT) can correct the hematological phenotype and is indicated in transfusion-dependent patients. In 70 children reported to the German DBA and French HSCT registries, HSCT was performed from 1985 to 2017. Median age at HSCT was 5.5 years (range, 0.9-17.3 years). Two-thirds of patients (64%) were transplanted from a matched sibling donor (MSD), and most procedures were performed after the year 1999 (73%). Primary engraftment was achieved in all patients. One patient developed secondary graft failure. Cumulative incidence of acute graft-versus-host disease (GVHD) was 24% for °II-IV (95% confidence interval [CI], 16% to 37%) and 7% for °III-IV (95% CI, 3% to 17%); cumulative incidence of chronic GVHD was 11% (95% CI, 5% to 22%). The probability of chronic GVHD-free survival (cGFS) was 87% (95% CI, 79% to 95%) and significantly improved over time (<2000: 68% [95% CI, 47% to 89%] vs ≥2000: 94% [95% CI, 87% to 100%], P < .01). cGFS was comparable following HSCT from a MSD and an unrelated donor (UD). Of note, no severe chronic GVHD or deaths were reported following MSD-HSCT after 1999. The difference of cGFS in children transplanted <10 years of age compared with older patients did not reach statistical significance (<10 years: 90% [95% CI, 81% to 99%] vs 10-18 years 78% [95% CI, 58% to 98%]). In summary, these data indicate that HSCT is efficient and safe in young DBA patients and should be considered if a MSD or matched UD is available. HSCT for transfusion dependency only must be critically discussed in older patients.


Assuntos
Anemia de Diamond-Blackfan , Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Adolescente , Idoso , Anemia de Diamond-Blackfan/terapia , Criança , Doença Enxerto-Hospedeiro/etiologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Estudos Retrospectivos , Doadores não Relacionados
8.
Kidney Int Rep ; 5(3): 348-357, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32154456

RESUMO

Introduction: Anion exchanger 1 (AE1) (SLC4A1 gene product) is a membrane protein expressed in both kidney and red blood cells (RBCs): it exchanges extracellular bicarbonate (HCO3 -) for intracellular chloride (Cl-) and participates in acid-base homeostasis. AE1 mutations in kidney α-intercalated cells can lead to distal renal tubular acidosis (dRTA). In RBC, AE1 (known as band 3) is also implicated in membrane stability: deletions can cause South Asian ovalocytosis (SAO). Methods: We retrospectively collected clinical and biological data from patients harboring dRTA due to a SLC4A1 mutation and analyzed HCO3 - and Cl- transports (by stopped-flow spectrophotometry) and expression (by flow cytometry, fluorescence activated cell sorting, and Coomassie blue staining) in RBCs, as well as RBC membrane stability (ektacytometry). Results: Fifteen patients were included. All experience nephrolithiasis and/or nephrocalcinosis, 2 had SAO and dRTA (dRTA SAO+), 13 dominant dRTA (dRTA SAO-). The latter did not exert specific RBC membrane anomalies. Both HCO3 - and Cl- transports were lower in patients with dRTA SAO+ than in those with dRTA SAO- or controls. Using 3 different extracellular probes, we report a decreased expression (by 52%, P < 0.05) in dRTA SAO+ patients by fluorescence activated cell sorting, whereas total amount of protein was not affected. Conclusion: Band 3 transport function and expression in RBCs from dRTA SAO- patients is normal. However, in SAO RBCs, impaired conformation of AE1/band 3 corresponds to an impaired function. Thus, the driver of acid-base defect during dominant dRTA is probably an impaired membrane expression.

9.
J Clin Med ; 9(1)2020 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-31947773

RESUMO

Perceived exertion is an important self-limiting factor influencing functional capacity in patients with sickle cell anemia (SCA). Exercise-related hemoglobin desaturation (EHD) may occur during a six-minute walking test (6MWT) and could influence the perceived rate of exertion. The aims of this study were (1) to compare the 6MWT responses (heart rate, perceived rate of exertion, and distance covered) between SCA children with and without EHD, and (2) to test the associations between EHD and several biological/physiological parameters. Nine of 51 SCA children (18%) at steady state (mean age 11.9 ± 3.8 years) exhibited EHD at the end of the 6MWT. The rate of perceived exertion increased with exercise in the two groups, but reached higher values in the EHD group. Heart rate and performance during the 6MWT did not differ between the two groups. The magnitude of change in SpO2 during the 6MWT was independently associated with the red blood cell (RBC) deformability and RBC aggregates strength. This study demonstrates that SCA children with EHD during a 6MWT have a higher rate of perceived exertion than non-EHD children despite a similar physiological demand, and that abnormal RBC rheology determinants appear to be significant contributors.

10.
J Clin Invest ; 130(4): 2097-2110, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-31961825

RESUMO

Despite the effective clinical use of steroids for the treatment of Diamond Blackfan anemia (DBA), the mechanisms through which glucocorticoids regulate human erythropoiesis remain poorly understood. We report that the sensitivity of erythroid differentiation to dexamethasone is dependent on the developmental origin of human CD34+ progenitor cells, specifically increasing the expansion of CD34+ progenitors from peripheral blood (PB) but not cord blood (CB). Dexamethasone treatment of erythroid-differentiated PB, but not CB, CD34+ progenitors resulted in the expansion of a newly defined CD34+CD36+CD71hiCD105med immature colony-forming unit-erythroid (CFU-E) population. Furthermore, proteomics analyses revealed the induction of distinct proteins in dexamethasone-treated PB and CB erythroid progenitors. Dexamethasone treatment of PB progenitors resulted in the specific upregulation of p57Kip2, a Cip/Kip cyclin-dependent kinase inhibitor, and we identified this induction as critical; shRNA-mediated downregulation of p57Kip2, but not the related p27Kip1, significantly attenuated the impact of dexamethasone on erythroid differentiation and inhibited the expansion of the immature CFU-E subset. Notably, in the context of DBA, we found that steroid resistance was associated with dysregulated p57Kip2 expression. Altogether, these data identify a unique glucocorticoid-responsive human erythroid progenitor and provide new insights into glucocorticoid-based therapeutic strategies for the treatment of patients with DBA.


Assuntos
Anemia de Diamond-Blackfan/metabolismo , Inibidor de Quinase Dependente de Ciclina p57/biossíntese , Dexametasona/farmacologia , Resistência a Medicamentos/efeitos dos fármacos , Células Precursoras Eritroides/metabolismo , Regulação para Cima/efeitos dos fármacos , Adulto , Anemia de Diamond-Blackfan/tratamento farmacológico , Anemia de Diamond-Blackfan/patologia , Antígenos CD/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/biossíntese , Células Precursoras Eritroides/patologia , Feminino , Humanos , Masculino
11.
Fetal Diagn Ther ; 47(2): 156-164, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31505487

RESUMO

BACKGROUND: Rare causes of fetal anemia requiring intrauterine transfusion (IUT) are challenging for fetal medicine specialists. OBJECTIVES: The aim of this study was to describe the perinatal patterns and prognosis in a consecutive series of fetuses transfused for fetal anemia of rare or unknown etiology, and to propose a protocol of investigation for fetal anemia of undetermined cause and for the management of subsequent pregnancies. METHOD: We conducted a retrospective descriptive study on fetuses transfused for severe anemia of rare or unknown etiology managed in our national referral center (Centre National de Référence d'Hémobiologie Périnatale) and born between 2010 and 2017. RESULTS: During the study period, 584 IUT were performed in 253 fetuses. Among those IUT, 23 (3.9%) were performed for a rare or unknown cause of anemia in 13 fetuses (5.1% of transfused fetuses). The median gestational age at diagnosis was 26 weeks of gestation (WG; range 21-33). Hemoglobin levels ranged from 1.6 to 9.1 g/dL (0.18-0.83 multiples of median) before the first IUT. The fetuses received between 1 and 6 IUT (39% received at least 2 IUT). The definitive etiologies for central anemia were: congenital syphilis, neonatal poikilocytosis, type II congenital dyserythropoietic anemia (CDA), and neonatal hemochromatosis. There was 1 case with suspected type I CDA and 1 with suspected Diamond-Blackfan anemia. There was 1 case of peripheral anemia, secondary to cerebral hemorrhages of different ages, related to a variant of the COL4A1 gene. In 6 fetuses corresponding to 4 mothers, no precise diagnosis was found despite a complete workup. In our series, there were 8 live births, 4 terminations of pregnancy, and 1 intrauterine fetal death. CONCLUSIONS: Fetal anemia of rare or unknown diagnosis represents 5% of all transfused fetuses in our cohort. Fetal and neonatal anemias can be recurrent in further pregnancies, with variable expressivity.


Assuntos
Anemia/terapia , Transfusão de Sangue Intrauterina , Doenças Fetais/terapia , Aborto Induzido , Anemia/sangue , Anemia/diagnóstico , Anemia/etiologia , Biomarcadores/sangue , Transfusão de Sangue Intrauterina/efeitos adversos , Feminino , Morte Fetal/etiologia , Doenças Fetais/sangue , Doenças Fetais/diagnóstico , Doenças Fetais/etiologia , Hemoglobina Fetal/metabolismo , Idade Gestacional , Humanos , Nascido Vivo , Gravidez , Estudos Retrospectivos , Fatores de Risco , Resultado do Tratamento
12.
Nucleic Acids Res ; 48(2): 770-787, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31799629

RESUMO

Variants in ribosomal protein (RP) genes drive Diamond-Blackfan anemia (DBA), a bone marrow failure syndrome that can also predispose individuals to cancer. Inherited and sporadic RP gene variants are also linked to a variety of phenotypes, including malignancy, in individuals with no anemia. Here we report an individual diagnosed with DBA carrying a variant in the 5'UTR of RPL9 (uL6). Additionally, we report two individuals from a family with multiple cancer incidences carrying a RPL9 missense variant. Analysis of cells from these individuals reveals that despite the variants both driving pre-rRNA processing defects and 80S monosome reduction, the downstream effects are remarkably different. Cells carrying the 5'UTR variant stabilize TP53 and impair the growth and differentiation of erythroid cells. In contrast, ribosomes incorporating the missense variant erroneously read through UAG and UGA stop codons of mRNAs. Metabolic profiles of cells carrying the 5'UTR variant reveal an increased metabolism of amino acids and a switch from glycolysis to gluconeogenesis while those of cells carrying the missense variant reveal a depletion of nucleotide pools. These findings indicate that variants in the same RP gene can drive similar ribosome biogenesis defects yet still have markedly different downstream consequences and clinical impacts.


Assuntos
Anemia de Diamond-Blackfan/genética , Processamento Pós-Transcricional do RNA/genética , Proteínas Ribossômicas/genética , Ribossomos/genética , Regiões 5' não Traduzidas/genética , Adolescente , Adulto , Anemia de Diamond-Blackfan/patologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Criança , Células Eritroides , Feminino , Humanos , Masculino , Mutação/genética , Precursores de RNA/genética , RNA Mensageiro/genética , Sequenciamento Completo do Exoma
13.
Am J Hum Genet ; 105(5): 1040-1047, 2019 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-31630789

RESUMO

Variants in genes encoding ribosomal proteins have thus far been associated with Diamond-Blackfan anemia, a rare inherited bone marrow failure, and isolated congenital asplenia. Here, we report one de novo missense variant and three de novo splice variants in RPL13, which encodes ribosomal protein RPL13 (also called eL13), in four unrelated individuals with a rare bone dysplasia causing severe short stature. The three splice variants (c.477+1G>T, c.477+1G>A, and c.477+2 T>C) result in partial intron retention, which leads to an 18-amino acid insertion. In contrast to observations from Diamond-Blackfan anemia, we detected no evidence of significant pre-rRNA processing disturbance in cells derived from two affected individuals. Consistently, we showed that the insertion-containing protein is stably expressed and incorporated into 60S subunits similar to the wild-type protein. Erythroid proliferation in culture and ribosome profile on sucrose gradient are modified, suggesting a change in translation dynamics. We also provide evidence that RPL13 is present at high levels in chondrocytes and osteoblasts in mouse growth plates. Taken together, we show that the identified RPL13 variants cause a human ribosomopathy defined by a rare skeletal dysplasia, and we highlight the role of this ribosomal protein in bone development.


Assuntos
Doenças do Desenvolvimento Ósseo/genética , Nanismo/genética , Mutação de Sentido Incorreto/genética , Proteínas de Neoplasias/genética , Proteínas Ribossômicas/genética , Anemia de Diamond-Blackfan/genética , Animais , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL
14.
Sci Rep ; 9(1): 6771, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31043643

RESUMO

Osmotic gradient ektacytometry is the gold standard to assess red blood cell (RBC) deformability. It has been proposed that, when measured in isotonic condition, RBC deformability at low shear stress would depend on membrane elasticity while it would be influenced by internal viscosity when measured at high shear stress, but this hypothesis needs to be further addressed. Healthy RBCs were rigidified by treatment with lysolecithine (LPC), diamide or nystatine associated with hyperosmolar solutions (OSMO), which reduces membrane surface area, decreases membrane elasticity or promotes cell dehydration, respectively. Diamide treatment resulted in a decrease in isotonic RBC deformability at all shear stresses tested (i.e. from 0.3 to 30 Pa). LPC and OSMO treatments caused a decrease in isotonic RBC deformability above 3 Pa only. Isotonic RBC deformability from patients with hereditary spherocytosis or sickle cell disease was mainly decreased above 1.69 Pa. Our findings indicate that decreased isotonic RBC deformability at shear stresses above 3 Pa would be related to a reduction in the surface-area-to-volume ratio and/or to a loss of membrane elasticity and/or to an increase in internal viscosity while a decrease of RBC deformability below 3 Pa would reflect a loss of membrane elasticity.


Assuntos
Viscosidade Sanguínea , Deformação Eritrocítica , Membrana Eritrocítica/patologia , Eritrócitos Anormais/patologia , Eritrócitos/patologia , Esferocitose Hereditária/patologia , Estresse Mecânico , Elasticidade , Humanos , Soluções Isotônicas , Reologia
15.
Am J Hematol ; 94(6): 667-677, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30916803

RESUMO

MYH9-related disease (MYH9-RD) is a rare, autosomal dominant disorder caused by mutations in MYH9, the gene encoding the actin-activated motor protein non-muscle myosin IIA (NMIIA). MYH9-RD patients suffer from bleeding syndromes, progressive kidney disease, deafness, and/or cataracts, but the impact of MYH9 mutations on other NMIIA-expressing tissues remains unknown. In human red blood cells (RBCs), NMIIA assembles into bipolar filaments and binds to actin filaments (F-actin) in the spectrin-F-actin membrane skeleton to control RBC biconcave disk shape and deformability. Here, we tested the effects of MYH9 mutations in different NMIIA domains (motor, coiled-coil rod, or non-helical tail) on RBC NMIIA function. We found that MYH9-RD does not cause clinically significant anemia and that patient RBCs have normal osmotic deformability as well as normal membrane skeleton composition and micron-scale distribution. However, analysis of complete blood count data and peripheral blood smears revealed reduced hemoglobin content and elongated shapes, respectively, of MYH9-RD RBCs. Patients with mutations in the NMIIA motor domain had the highest numbers of elongated RBCs. Patients with mutations in the motor domain also had elevated association of NMIIA with F-actin at the RBC membrane. Our findings support a central role for motor domain activity in NMIIA regulation of RBC shape and define a new sub-clinical phenotype of MYH9-RD.


Assuntos
Actinas , Membrana Eritrocítica , Eritrócitos Anormais , Perda Auditiva Neurossensorial , Mutação , Cadeias Pesadas de Miosina , Trombocitopenia/congênito , Actinas/genética , Actinas/metabolismo , Membrana Eritrocítica/genética , Membrana Eritrocítica/metabolismo , Membrana Eritrocítica/patologia , Eritrócitos Anormais/metabolismo , Eritrócitos Anormais/patologia , Feminino , Perda Auditiva Neurossensorial/genética , Perda Auditiva Neurossensorial/metabolismo , Perda Auditiva Neurossensorial/patologia , Humanos , Masculino , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Trombocitopenia/genética , Trombocitopenia/metabolismo , Trombocitopenia/patologia
17.
Blood ; 133(12): 1358-1370, 2019 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-30700418

RESUMO

Diamond-Blackfan anemia (DBA) is a congenital erythroblastopenia that is characterized by a blockade in erythroid differentiation related to impaired ribosome biogenesis. DBA phenotype and genotype are highly heterogeneous. We have previously identified 2 in vitro erythroid cell growth phenotypes for primary CD34+ cells from DBA patients and following short hairpin RNA knockdown of RPS19, RPL5, and RPL11 expression in normal human CD34+ cells. The haploinsufficient RPS19 in vitro phenotype is less severe than that of 2 other ribosomal protein (RP) mutant genes. We further documented that proteasomal degradation of HSP70, the chaperone of GATA1, is a major contributor to the defect in erythroid proliferation, delayed erythroid differentiation, increased apoptosis, and decreased globin expression, which are all features of the RPL5 or RPL11 DBA phenotype. In the present study, we explored the hypothesis that an imbalance between globin and heme synthesis may be involved in pure red cell aplasia of DBA. We identified disequilibrium between the globin chain and the heme synthesis in erythroid cells of DBA patients. This imbalance led to accumulation of excess free heme and increased reactive oxygen species production that was more pronounced in cells of the RPL5 or RPL11 phenotype. Strikingly, rescue experiments with wild-type HSP70 restored GATA1 expression levels, increased globin synthesis thereby reducing free heme excess and resulting in decreased apoptosis of DBA erythroid cells. These results demonstrate the involvement of heme in DBA pathophysiology and a major role of HSP70 in the control of balanced heme/globin synthesis.


Assuntos
Anemia de Diamond-Blackfan/patologia , Diferenciação Celular , Células Eritroides/patologia , Fator de Transcrição GATA1/metabolismo , Globinas/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Heme/metabolismo , Anemia de Diamond-Blackfan/metabolismo , Proliferação de Células , Células Cultivadas , Células Eritroides/metabolismo , Feminino , Seguimentos , Haploinsuficiência , Humanos , Lactente , Recém-Nascido , Masculino , Mutação , Fenótipo , Prognóstico , RNA Interferente Pequeno , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo
18.
Am J Hum Genet ; 103(6): 930-947, 2018 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-30503522

RESUMO

Diamond-Blackfan anemia (DBA) is a rare bone marrow failure disorder that affects 7 out of 1,000,000 live births and has been associated with mutations in components of the ribosome. In order to characterize the genetic landscape of this heterogeneous disorder, we recruited a cohort of 472 individuals with a clinical diagnosis of DBA and performed whole-exome sequencing (WES). We identified relevant rare and predicted damaging mutations for 78% of individuals. The majority of mutations were singletons, absent from population databases, predicted to cause loss of function, and located in 1 of 19 previously reported ribosomal protein (RP)-encoding genes. Using exon coverage estimates, we identified and validated 31 deletions in RP genes. We also observed an enrichment for extended splice site mutations and validated their diverse effects using RNA sequencing in cell lines obtained from individuals with DBA. Leveraging the size of our cohort, we observed robust genotype-phenotype associations with congenital abnormalities and treatment outcomes. We further identified rare mutations in seven previously unreported RP genes that may cause DBA, as well as several distinct disorders that appear to phenocopy DBA, including nine individuals with biallelic CECR1 mutations that result in deficiency of ADA2. However, no new genes were identified at exome-wide significance, suggesting that there are no unidentified genes containing mutations readily identified by WES that explain >5% of DBA-affected case subjects. Overall, this report should inform not only clinical practice for DBA-affected individuals, but also the design and analysis of rare variant studies for heterogeneous Mendelian disorders.


Assuntos
Anemia de Diamond-Blackfan/genética , Adolescente , Criança , Pré-Escolar , Estudos de Coortes , Exoma/genética , Éxons/genética , Feminino , Deleção de Genes , Estudos de Associação Genética/métodos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Mutação/genética , Fenótipo , Proteínas Ribossômicas/genética , Ribossomos/genética , Análise de Sequência de RNA/métodos , Sequenciamento Completo do Exoma/métodos
19.
F1000Res ; 72018.
Artigo em Inglês | MEDLINE | ID: mdl-30228860

RESUMO

Diamond-Blackfan anemia (DBA) is a rare congenital hypoplastic anemia characterized by a block in erythropoiesis at the progenitor stage, although the exact stage at which this occurs remains to be fully defined. DBA presents primarily during infancy with macrocytic anemia and reticulocytopenia with 50% of cases associated with a variety of congenital malformations. DBA is most frequently due to a sporadic mutation (55%) in genes encoding several different ribosomal proteins, although there are many cases where there is a family history of the disease with varying phenotypes. The erythroid tropism of the disease is still a matter of debate for a disease related to a defect in global ribosome biogenesis. Assessment of biological features in conjunction with genetic testing has increased the accuracy of the diagnosis of DBA. However, in certain cases, it continues to be difficult to firmly establish a diagnosis. This review will focus on the diagnosis of DBA along with a description of new advances in our understanding of the pathophysiology and treatment recommendations for DBA.


Assuntos
Anormalidades Múltiplas , Anemia de Diamond-Blackfan/diagnóstico , Anemia de Diamond-Blackfan/terapia , Humanos
20.
Mol Ther ; 26(10): 2523-2532, 2018 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-30077612

RESUMO

Macrophage migration inhibitory factor (MIF) is elevated in patients with acute kidney injury (AKI) and is suggested as a potential predictor for renal replacement therapy in AKI. In this study, we found that MIF also plays a pathogenic role and is a therapeutic target for AKI. In a cisplatin-induced AKI mouse model, elevated plasma MIF correlated with increased serum creatinine and the severity of renal inflammation and tubular necrosis, whereas deletion of MIF protected the kidney from cisplatin-induced AKI by largely improving renal functional and histological injury, and suppressing renal inflammation including upregulation of cytokines such as interleukin (IL)-1ß, tumor necrosis factor-alpha (TNF-α), IL-6, inducible nitric oxide synthase (iNOS), MCP-1, IL-8, and infiltration of macrophages, neutrophils, and T cells. We next developed a novel therapeutic strategy for AKI by blocking the endogenous MIF with an MIF inhibitor, ribosomal protein S19 (RPS19). Similar to the MIF-knockout mice, treatment with RPS19, but not the mutant RPS19, suppressed cisplatin-induced AKI. Mechanistically, we found that both genetic knockout and pharmacological inhibition of MIF protected against AKI by inactivating the CD74-nuclear factor κB (NF-κB) signaling. In conclusion, MIF is pathogenic in cisplatin-induced AKI. Targeting MIF with an MIF inhibitor RPS19 could be a promising therapeutic potential for AKI.


Assuntos
Injúria Renal Aguda/terapia , Inflamação/terapia , Oxirredutases Intramoleculares/genética , Fatores Inibidores da Migração de Macrófagos/genética , Proteínas Ribossômicas/administração & dosagem , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/genética , Injúria Renal Aguda/patologia , Animais , Antígenos de Diferenciação de Linfócitos B/genética , Apoptose/efeitos dos fármacos , Cisplatino/efeitos adversos , Terapia Genética , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/patologia , Oxirredutases Intramoleculares/antagonistas & inibidores , Rim/efeitos dos fármacos , Rim/patologia , Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Camundongos , Camundongos Knockout , NF-kappa B/genética , Necrose , Proteínas Ribossômicas/genética , Transdução de Sinais/efeitos dos fármacos
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