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1.
Eur Biophys J ; 48(7): 635-643, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31302726

RESUMO

In this work, wild-type and heterozygous ß-thalassaemic mice were enriched with 57Fe via gastrointestinal absorption to characterize in greater detail the iron complexes then identifiable via Mössbauer spectroscopy. The 57Fe enrichment method was validated and Mössbauer spectra were obtained at 80 K from blood samples from wild-type and ß-thalassaemic mice at 1, 3, 6, and 9 months of age. As expected, the haemoglobin levels of the thalassaemic mice were lower than from normal mice, indicating anaemia. Furthermore, significant amounts of ferritin-like iron were observed in the thalassaemic mice samples, which decreased with mouse age, reflecting the pattern of reticulocyte count reduction reported in the literature.


Assuntos
Isótopos de Ferro/metabolismo , Isótopos de Ferro/farmacologia , Espectroscopia de Mossbauer , Talassemia beta/sangue , Talassemia beta/metabolismo , Animais , Absorção Intestinal , Camundongos , Camundongos Endogâmicos C57BL
4.
Ann Hematol ; 98(9): 2045-2052, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31243572

RESUMO

Thalassemia has a high prevalence in Thailand. Oxidative damage to erythroid cells is known to be one of the major etiologies in thalassemia pathophysiology. Oxidative stress status of thalassemia is potentiated by the heme, nonheme iron, and free iron resulting from imbalanced globin synthesis. In addition, levels of antioxidant proteins are reduced in α-thalassemia and ß-thalassemia erythrocytes. However, the primary molecular mechanism for this phenotype remains unknown. Our study showed a high expression of miR-144 in ß- and α-thalassemia. An increased miR-144 expression leads to decreased expression of nuclear factor erythroid 2-related factor 2 (NRF2) target, especially in α-thalassemia. In α-thalassemia, miR-144 and NRF2 target are associated with glutathione level and anemia severity. To study the effect of miR-144 expression, the gain-loss of miR-144 expression was performed by miR inhibitor and mimic transfection in the erythroblastic cell line. This study reveals that miR-144 expression was upregulated, whereas NRF2 expression and glutathione levels were decreased in comparison with the untreated condition after miR mimic transfection, while the reduction of miR-144 expression contributed to the increased NRF2 expression and glutathione level compared with the untreated condition after miR inhibitor transfection. Moreover, miR-144 overexpression leads to significantly increased sensitivity to oxidative stress at indicated concentrations of hydrogen peroxide (H2O2) and rescued by miR-144 inhibitor. Taken together, our findings suggest that dysregulation of miR-144 may play a role in the reduced ability of erythrocyte to deal with oxidative stress and increased RBC hemolysis susceptibility especially in thalassemia.


Assuntos
Eritrócitos/metabolismo , MicroRNAs/biossíntese , Fator 2 Relacionado a NF-E2/biossíntese , Estresse Oxidativo , Regulação para Cima , Talassemia alfa/metabolismo , Talassemia beta/metabolismo , Eritrócitos/patologia , Feminino , Glutationa/biossíntese , Glutationa/genética , Hemólise , Humanos , Peróxido de Hidrogênio/metabolismo , Células K562 , Masculino , MicroRNAs/genética , Fator 2 Relacionado a NF-E2/genética , Talassemia alfa/genética , Talassemia alfa/patologia , Talassemia beta/genética , Talassemia beta/patologia
5.
Free Radic Res ; 53(7): 791-799, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31198069

RESUMO

Oxidative stress caused as a result of iron overload is implicated in clinical manifestation of beta-thalassemia/haemoglobin E (ß-Thal/HbE). In this study, we investigated the cellular adaptation against oxidative stress in ß-Thal/HbE patients. Twenty-four paediatric ß-Thal/HbE patients and 22 healthy controls were recruited in the study. Blood samples from patients exhibited iron overload, elevation of lipid peroxidation, and marked diminution in the reduced glutathione (GSH) level. However, expression of glutamate-cysteine ligase catalytic (GCLC) subunit, a key enzyme in GSH biosynthesis, was up-regulated when compared with that in controls. GCLC protein levels were correlated with serum iron. There was an enhanced binding activity of the oligonucleotide probe for Nrf2-driven antioxidant response element (ARE) to nuclear protein from blood mononuclear cells of thalassemia subjects. In conclusion, ß-Thal/HbE patients exhibit elevated plasma levels of GCLC expression and Nrf2-ARE binding activity, which may account for their adaptive survival response to oxidative stress.


Assuntos
Glutamato-Cisteína Ligase/metabolismo , Sobrecarga de Ferro/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/fisiologia , Talassemia beta/metabolismo , Adolescente , Criança , Feminino , Humanos , Sobrecarga de Ferro/sangue , Masculino , Fator 2 Relacionado a NF-E2/sangue , Regulação para Cima , Talassemia beta/sangue
6.
Ann Hematol ; 98(8): 1827-1834, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31190133

RESUMO

In this study, we aimed to investigate the pattern and association of genetic mutations occurring within the alpha hemoglobin-stabilizing protein (AHSP) gene among HbE beta thalassemia patients with varying phenotypic expressions. Fifty-four diagnosed cases of HbE beta thalassemia (transfusion dependent and independent) were included in the study. Among them, 38 patients with similar genotypes (IVS 1-5, alpha gene deletion and triplication, Xmn polymorphism) were selected for further analysis. AHSP gene sequencing was done for these 38 samples to study associated mutations in AHSP gene. HbE beta thalassemia patients with similar genotypes but different phenotypic expressions were found to have mutations in the AHSP gene. There were five mutations found most prevalent among the samples analyzed for AHSP gene sequencing. Among these, two mutations were from intron 1 region of AHSP and three mutations were found in exon 3. The most prevalent mutation was found at the Oct binding site at intron 1 of AHSP. The mutations in exon 3 were more prevalent among the TDT groups. A mutation in exon 3 changing the amino acid (33rd) from serine to phenylalanine was found to be associated with only TDT group. This study documents that among the HbE beta thalassemia patients with varying severity, an exon mutation in AHSP is significantly prevalent only among the TDT group. Further understanding of the mechanism will shed light upon the impact of AHSP in modifying the disease severity in thalassemia.


Assuntos
Proteínas Sanguíneas/genética , Deleção de Genes , Duplicação Gênica , Hemoglobina E/genética , Chaperonas Moleculares/genética , Talassemia beta/genética , Adolescente , Adulto , Sequência de Bases , Proteínas Sanguíneas/metabolismo , Transfusão de Sangue/estatística & dados numéricos , Criança , Pré-Escolar , Análise Mutacional de DNA , Eritrócitos/metabolismo , Eritrócitos/patologia , Éxons , Feminino , Expressão Gênica , Genótipo , Hemoglobina E/metabolismo , Humanos , Íntrons , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Fenótipo , Estrutura Secundária de Proteína , Índice de Gravidade de Doença , Talassemia beta/metabolismo , Talassemia beta/patologia , Talassemia beta/terapia
7.
Eur J Pharmacol ; 854: 398-405, 2019 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-31039344

RESUMO

Hemoglobinopathies, such as ß-thalassemia, and sickle cell disease (SCD) are caused by abnormal structure or reduced production of ß-chains and affect millions of people worldwide. Hereditary persistence of fetal hemoglobin (HPFH) is a condition which is naturally occurring and characterized by a considerable elevation of fetal hemoglobin (HbF) in adult red blood cells. Individuals with compound heterozygous ß-thalassemia or SCD and HPFH have milder clinical symptoms. So, HbF reactivation has long been sought as an approach to mitigate the clinical symptoms of ß-thalassemia and SCD. Using CRISPR-Cas9 genome-editing strategy, we deleted a 200bp genomic region within the human erythroid-specific BCL11A (B-cell lymphoma/leukemia 11A) enhancer in KU-812, KG-1, and K562 cell lines. In our study, deletion of 200bp of BCL11A erythroid enhancer including GATAA motif leads to strong induction of γ-hemoglobin expression in K562 cells, but not in KU-812 and KG-1 cells. Altogether, our findings highlight the therapeutic potential of CRISPR-Cas9 as a precision genome editing tool for treating ß-thalassemia. In addition, our data indicate that KU-812 and KG-1 cell lines are not good models for studying HbF reactivation through inactivation of BCL11A silencing pathway.


Assuntos
Sistemas CRISPR-Cas/genética , Proteínas de Transporte/genética , Hemoglobina Fetal/metabolismo , Deleção de Genes , Terapia Genética/métodos , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Talassemia beta/terapia , Sequência de Bases , Edição de Genes , Humanos , Células K562 , Proteínas Repressoras , Talassemia beta/genética , Talassemia beta/metabolismo , gama-Globinas/genética
8.
Eur J Pharmacol ; 855: 285-293, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31100414

RESUMO

Fetal hemoglobin (HbF) induction is a cost-effective therapeutic approach for the treatment of ß-hemoglobinopathies like ß-thalassemia and sickle cell anemia. The present study discusses the potential of thiourea derivatives as new class of compounds that induce the fetal hemoglobin production. HbF inducing effect of thiourea derivatives was studied using experimental cell system, the human erythroleukemic K562 cell line. Erythroid induction of K562 cells was studied by the benzidine/H2O2 reaction, total hemoglobin production was estimated by plasma hemoglobin assay kit, and γ-globin gene expression by RT-qPCR, whereas fetal hemoglobin production was estimated by flow cytometry and immunofluorescence microscopy. The results indicated that newly synthesized thiourea derivative are potent inducers of erythroid differentiation of K562 cells with an increased γ-globin gene expression and fetal hemoglobin production. Moreover, these compounds showed no cytotoxic effect and inhibition on K562 cells at HbF inducing concentrations. It is important to note that hydroxyurea is a cytotoxic chemotherapeutic agent and have deleterious side effects, reflecting the need to identify new safe and effective HbF induces. These results signify thiourea derivatives as promising HbF inducers, with the potential to be studied against hematological disorders, including ß-thalassemia and sickle cell anemia.


Assuntos
Hemoglobina Fetal/biossíntese , Tioureia/análogos & derivados , Tioureia/farmacologia , Talassemia beta/tratamento farmacológico , Talassemia beta/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Eritroides/efeitos dos fármacos , Células Eritroides/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células K562 , Tioureia/uso terapêutico , Talassemia beta/patologia
9.
Ann Hematol ; 98(8): 1813-1826, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31098739

RESUMO

Pregnant patients with ß-thalassemia are more likely to have progressive anemia which expose them to risk of adverse pregnancy outcomes, blood transfusion, and iron overload. Results from our previous study indicated that Colla corii asini (CCA, E'jiao), a natural ingredient of traditional Chinese medicine, could significantly increase hemoglobin level of pregnant women with ß- thalassemia, but the underlying molecular mechanism was unclear. Thus, we applied high-throughput transcriptome sequencing to study the transcriptomic change before and after the CCA treatment. Twenty eligible pregnant women were recruited and randomized to either the CCA treatment group or the blank control group in a 3:1 ratio. Patients in the treatment group orally received daily 15 g CCA powder for 4 weeks. We analyzed the therapeutic effect indexes and the transcriptomic change in subjects' peripheral blood before and after treatment. We found that ß CD 41-42(-TTCT)/ßA was the main genotype of the subjects. The regulatory impact of CCA treatment became more evident among the subjects of genotype ß CD 41-42(-TTCT)/ßA. Gene ontogenesis analysis revealed that the top five molecular functions of differentially expressed genes were involved in membrane functionality and cellular structure. We further identified two consistent upregulated genes ZNF471 and THOC5 in the effective treatment group, which were engaged in Kruppel-associated box (KRAB) domain-containing zinc-finger protein pathway and THOC5 pathway, respectively. Based on our current findings, we hypothesize that the anti-anemia effect of CCA on pregnant women with ß-thalassemia might be related to translation regulation of spectrin synthesis, membrane stability, and eventually prolonged the life span of erythrocytes.


Assuntos
Gelatina/uso terapêutico , Regulação da Expressão Gênica/efeitos dos fármacos , Fármacos Hematológicos/uso terapêutico , Medicina Tradicional Chinesa/métodos , Proteínas Nucleares/genética , Proteínas Repressoras/genética , Talassemia beta/tratamento farmacológico , Administração Oral , Adulto , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Eritrócitos/patologia , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Proteínas Nucleares/agonistas , Proteínas Nucleares/metabolismo , Gravidez , Proteômica/métodos , Proteínas Repressoras/agonistas , Proteínas Repressoras/metabolismo , Transdução de Sinais , Espectrina/genética , Espectrina/metabolismo , Transcriptoma/efeitos dos fármacos , Talassemia beta/genética , Talassemia beta/metabolismo , Talassemia beta/patologia
10.
Biomed Res Int ; 2019: 6573497, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31119181

RESUMO

Background: Endocrinopathies are common in patients with ß-thalassemia major despite parenteral iron chelation therapy with deferoxamine. Prevalence of abnormal glucose metabolism in previous studies was controversial. The aim of this study was to discuss the prevalence of abnormal glucose metabolism in ß-thalassemia major based on a meta-analysis. Methods: PubMed, ScienceDirect, Springerlink, Ovid, Web of Science, MEDLINE, Wanfang database, and Chinese National Knowledge Internet were searched for relevant articles. Two authors selected the articles according to the inclusion criteria and then extracted the data. The prevalence of diabetes mellitus (DM) in ß-thalassemia major was defined as the primary outcome. The prevalence with the 95% confidence interval (95%CI) was used to evaluate the proportion of abnormal glucose metabolism and other endocrine disorders in patients with ß-thalassemia major. Subgroup analyses were applied to explore the prevalence in different regions. Sensitivity analysis and publication bias assessment were also conducted. Results: A total of 44 studies with 16605 cases were included in this analysis. Diabetes mellitus was present in 6.54% (95% CI: 5.30%-7.78%). The fixed subgroup study revealed that the region with the highest prevalence was the Middle East (prevalence= 7.90%, 95% CI: 5.75%-10.05%). The accumulated meta-analysis revealed that the prevalence of DM in ß-thalassemia major was relatively steady in each year. The prevalence of impaired fasting glucose (IFG), impaired glucose tolerance (IGT), and other endocrine disorders in ß-thalassemia major was 17.21% (95% CI: 8.43%-26.00%), 12.46% (95% CI: 5.98%-18.94%), and 43.92% (95% CI: 37.94%-49.89%), respectively. Sensitivity analysis showed that the pooled results were robust; publication bias assessment revealed that there was no significant evidence that the pooled results were influenced by publication bias. Conclusion: High prevalence of endocrine disorders involving abnormal glucose metabolism was detected in ß-thalassemia major. Treatment and prevention measurements may be necessary to prevent growth and endocrine problems.


Assuntos
Diabetes Mellitus/epidemiologia , Doenças do Sistema Endócrino/epidemiologia , Glucose/metabolismo , Talassemia beta/epidemiologia , Terapia por Quelação , Desferroxamina/uso terapêutico , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Doenças do Sistema Endócrino/complicações , Doenças do Sistema Endócrino/metabolismo , Doenças do Sistema Endócrino/patologia , Intolerância à Glucose , Humanos , Quelantes de Ferro/uso terapêutico , Oriente Médio/epidemiologia , Talassemia beta/complicações , Talassemia beta/metabolismo , Talassemia beta/patologia
11.
Hematology ; 24(1): 426-438, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30947625

RESUMO

OBJECTIVES: Beta-thalassemias are a group of recessively autosomal inherited disorders of hemoglobin synthesis, which, due to mutations of the beta-globin gene, lead to various degrees of defective beta-chain production, an imbalance in alpha/beta-globin chain synthesis, ineffective erythropoiesis, and anemia. Improved survival in thalassemic patients has led to the emergence of previously unrecognized complications, such as renal disease. METHODS: A comprehensive literature review through PubMed was undertaken to summarize the published evidence on the epidemiology and pathophysiology of renal disease in thalassemia. Literature sources published in English since 1990 were searched, using the terms beta-thalassemia, renal disease. RESULTS: Renal disease is considered to be the 4th cause of morbidity among patients with transfusion dependent thalassemia. Chronic anemia, hypoxia and iron overload are the main mechanisms implicated in development of renal injury, whereas several studies also suggested a contributive role of iron chelators. DISCUSSION AND CONCLUSION: Kidney disease may develop through progressive renal tubular and glomerular damage; thus, its early recognition is important in order to prevent and/or reverse deterioration. This review will provide an insight on the involved mechanisms implicated in kidney disease in thalassemic patients and will discuss the updates on diagnosis and prevention of renal complications in thalassemia.


Assuntos
Hipóxia , Sobrecarga de Ferro , Nefropatias , Talassemia beta , Feminino , Humanos , Hipóxia/complicações , Hipóxia/metabolismo , Hipóxia/mortalidade , Hipóxia/terapia , Sobrecarga de Ferro/complicações , Sobrecarga de Ferro/metabolismo , Sobrecarga de Ferro/mortalidade , Sobrecarga de Ferro/terapia , Nefropatias/etiologia , Nefropatias/metabolismo , Nefropatias/mortalidade , Nefropatias/terapia , Masculino , Talassemia beta/complicações , Talassemia beta/metabolismo , Talassemia beta/mortalidade , Talassemia beta/terapia
12.
Blood ; 133(21): 2255-2262, 2019 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-30704988

RESUMO

The thalassemias are compelling targets for therapeutic genome editing in part because monoallelic correction of a subset of hematopoietic stem cells (HSCs) would be sufficient for enduring disease amelioration. A primary challenge is the development of efficient repair strategies that are effective in HSCs. Here, we demonstrate that allelic disruption of aberrant splice sites, one of the major classes of thalassemia mutations, is a robust approach to restore gene function. We target the IVS1-110G>A mutation using Cas9 ribonucleoprotein (RNP) and the IVS2-654C>T mutation by Cas12a/Cpf1 RNP in primary CD34+ hematopoietic stem and progenitor cells (HSPCs) from ß-thalassemia patients. Each of these nuclease complexes achieves high efficiency and penetrance of therapeutic edits. Erythroid progeny of edited patient HSPCs show reversal of aberrant splicing and restoration of ß-globin expression. This strategy could enable correction of a substantial fraction of transfusion-dependent ß-thalassemia genotypes with currently available gene-editing technology.


Assuntos
Edição de Genes , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas , Sítios de Splice de RNA , Processamento de RNA , Globinas beta , Talassemia beta , Sistemas CRISPR-Cas , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Mutação Puntual , Globinas beta/biossíntese , Globinas beta/genética , Talassemia beta/genética , Talassemia beta/metabolismo , Talassemia beta/terapia
13.
PLoS Comput Biol ; 15(1): e1006680, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30608934

RESUMO

It is well known that iron is an essential element for life but is toxic when in excess or in certain forms. Accordingly there are many diseases that result directly from either lack or excess of iron. Yet many molecular and physiological aspects of iron regulation have only been discovered recently and others are still elusive. There is still no good quantitative and dynamic description of iron absorption, distribution, storage and mobilization that agrees with the wide array of phenotypes presented in several iron-related diseases. The present work addresses this issue by developing a mathematical model of iron distribution in mice calibrated with ferrokinetic data and subsequently validated against data from mouse models of iron disorders, such as hemochromatosis, ß-thalassemia, atransferrinemia and anemia of inflammation. To adequately fit the ferrokinetic data required inclusion of the following mechanisms: a) transferrin-mediated iron delivery to tissues, b) induction of hepcidin by transferrin-bound iron, c) ferroportin-dependent iron export regulated by hepcidin, d) erythropoietin regulation of erythropoiesis, and e) liver uptake of NTBI. The utility of the model to simulate disease interventions was demonstrated by using it to investigate the outcome of different schedules of transferrin treatment in ß-thalassemia.


Assuntos
Distúrbios do Metabolismo do Ferro , Ferro , Modelos Biológicos , Talassemia beta , Animais , Biologia Computacional , Ferro/metabolismo , Ferro/fisiologia , Distúrbios do Metabolismo do Ferro/metabolismo , Distúrbios do Metabolismo do Ferro/fisiopatologia , Fígado/metabolismo , Camundongos , Transferrina , Talassemia beta/metabolismo , Talassemia beta/fisiopatologia
14.
Blood ; 133(8): 852-856, 2019 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-30617196

RESUMO

ß-hemoglobinopathies, such as sickle cell disease and ß-thalassemia, result from mutations in the adult ß-globin gene. Reactivating the developmentally silenced fetal γ-globin gene elevates fetal hemoglobin levels and ameliorates symptoms of ß-hemoglobinopathies. The continued expression of fetal γ-globin into adulthood occurs naturally in a genetic condition termed hereditary persistence of fetal hemoglobin (HPFH). Point mutations in the fetal γ-globin proximal promoter can cause HPFH. The -113A>G HPFH mutation falls within the -115 cluster of HPFH mutations, a binding site for the fetal globin repressor BCL11A. We demonstrate that the -113A>G HPFH mutation, unlike other mutations in the cluster, does not disrupt BCL11A binding but rather creates a de novo binding site for the transcriptional activator GATA1. Introduction of the -113A>G HPFH mutation into erythroid cells using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system increases GATA1 binding and elevates fetal globin levels. These results reveal the mechanism by which the -113A>G HPFH mutation elevates fetal globin and demonstrate the sensitivity of the fetal globin promoter to point mutations that often disrupt repressor binding sites but here create a de novo site for an erythroid activator.


Assuntos
Anemia Falciforme , Hemoglobina Fetal , Fator de Transcrição GATA1/metabolismo , Regulação da Expressão Gênica , Mutação Puntual , Elementos de Resposta , Talassemia beta , Anemia Falciforme/genética , Anemia Falciforme/metabolismo , Linhagem Celular , Hemoglobina Fetal/biossíntese , Hemoglobina Fetal/genética , Fator de Transcrição GATA1/genética , Globinas beta/genética , Globinas beta/metabolismo , Talassemia beta/genética , Talassemia beta/metabolismo
15.
Clin Biochem ; 65: 24-28, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30503531

RESUMO

OBJECTIVE: The aims of this study were to establish the reference intervals for HbA2 and HbF in a Guizhou population of reproductive age, and to determine the cut-off value of HbA2 for ß-thalassemia carrier screening. METHODS: Hemoglobin analysis was performed on 832 individuals without hypochromic microcytic anemia to calculate the reference intervals for HbA2 and HbF. Three hundred and ninety one ß-thalassemia carriers and non ß-thalassemia individuals were analyzed for their HbA2 levels followed by detecting ß-globin gene mutations, then cut-off value of HbA2 for ß-thalassemia carrier screening was determined using ROC curve analysis. RESULTS: The reference interval for HbA2 in overall normal individuals was 2.3%-3.1%, and reference intervals for HbF in normal males and females (including normal females and pregnant women) were 0-0.5% and 0-1.0% respectively. The cut-off values of HbA2 for ß-thalassemia carrier screening in males, non-pregnant women, pregnant women and the overall set were 4.40%, 3.75%, 3.70% and 3.95% respectively. CONCLUSION: Gender and pregnancy status had no obvious influence on reference interval for HbA2. The HbF level was higher in females than in males, but pregnancy status had no obvious influence on HbF level. Cut-off value of HbA2 for ß-thalassemia carrier screening was obviously affected by gender but not by pregnancy status.


Assuntos
Hemoglobina Fetal/metabolismo , Hemoglobina A2/metabolismo , Talassemia beta/metabolismo , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Gravidez , Padrões de Referência , Reprodução/fisiologia , Adulto Jovem
16.
J Clin Invest ; 129(2): 598-615, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30422819

RESUMO

Current thalassemia gene therapy protocols require the collection of hematopoietic stem/progenitor cells (HSPCs), in vitro culture, lentivirus vector transduction, and retransplantation into myeloablated patients. Because of cost and technical complexity, it is unlikely that such protocols will be applicable in developing countries, where the greatest demand for a ß-thalassemia therapy lies. We have developed a simple in vivo HSPC gene therapy approach that involves HSPC mobilization and an intravenous injection of integrating HDAd5/35++ vectors. Transduced HSPCs homed back to the bone marrow, where they persisted long-term. HDAd5/35++ vectors for in vivo gene therapy of thalassemia had a unique capsid that targeted primitive HSPCs through human CD46, a relatively safe SB100X transposase-based integration machinery, a micro-LCR-driven γ-globin gene, and an MGMT(P140K) system that allowed for increasing the therapeutic effect by short-term treatment with low-dose O6-benzylguanine plus bis-chloroethylnitrosourea. We showed in "healthy" human CD46-transgenic mice and in a mouse model of thalassemia intermedia that our in vivo approach resulted in stable γ-globin expression in the majority of circulating red blood cells. The high marking frequency was maintained in secondary recipients. In the thalassemia model, a near-complete phenotypic correction was achieved. The treatment was well tolerated. This cost-efficient and "portable" approach could permit a broader clinical application of thalassemia gene therapy.


Assuntos
Eritrócitos , Regulação da Expressão Gênica , Terapia Genética , Células-Tronco Hematopoéticas/metabolismo , Talassemia beta , gama-Globinas , Adenoviridae , Animais , Linhagem Celular , Modelos Animais de Doenças , Eritrócitos/metabolismo , Eritrócitos/patologia , Vetores Genéticos , Células-Tronco Hematopoéticas/patologia , Humanos , Camundongos , Camundongos Transgênicos , Transdução Genética , Talassemia beta/genética , Talassemia beta/metabolismo , Talassemia beta/patologia , Talassemia beta/terapia , gama-Globinas/biossíntese , gama-Globinas/genética
17.
Mol Ther ; 27(1): 137-150, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30424953

RESUMO

Editing the ß-globin locus in hematopoietic stem cells is an alternative therapeutic approach for gene therapy of ß-thalassemia and sickle cell disease. Using the CRISPR/Cas9 system, we genetically modified human hematopoietic stem and progenitor cells (HSPCs) to mimic the large rearrangements in the ß-globin locus associated with hereditary persistence of fetal hemoglobin (HPFH), a condition that mitigates the clinical phenotype of patients with ß-hemoglobinopathies. We optimized and compared the efficiency of plasmid-, lentiviral vector (LV)-, RNA-, and ribonucleoprotein complex (RNP)-based methods to deliver the CRISPR/Cas9 system into HSPCs. Plasmid delivery of Cas9 and gRNA pairs targeting two HPFH-like regions led to high frequency of genomic rearrangements and HbF reactivation in erythroblasts derived from sorted, Cas9+ HSPCs but was associated with significant cell toxicity. RNA-mediated delivery of CRISPR/Cas9 was similarly toxic but much less efficient in editing the ß-globin locus. Transduction of HSPCs by LVs expressing Cas9 and gRNA pairs was robust and minimally toxic but resulted in poor genome-editing efficiency. Ribonucleoprotein (RNP)-based delivery of CRISPR/Cas9 exhibited a good balance between cytotoxicity and efficiency of genomic rearrangements as compared to the other delivery systems and resulted in HbF upregulation in erythroblasts derived from unselected edited HSPCs.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/fisiologia , Terapia Genética/métodos , Células-Tronco Hematopoéticas/metabolismo , Anemia Falciforme/genética , Anemia Falciforme/metabolismo , Anemia Falciforme/terapia , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Células-Tronco Hematopoéticas/citologia , Hemoglobinopatias/genética , Hemoglobinopatias/metabolismo , Hemoglobinopatias/terapia , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Plasmídeos/genética , RNA Guia/genética , RNA Guia/metabolismo , Talassemia beta/genética , Talassemia beta/metabolismo , Talassemia beta/terapia
18.
Blood Transfus ; 17(3): 165-170, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30036179

RESUMO

BACKGROUND: Transfusion dependency determines iron overload in thalassaemia major, with devastating complications. Significant liver iron overload has been observed from early childhood and we aimed to evaluate factors that could predict liver iron overload at the first magnetic resonance imaging (MRI). MATERIALS AND METHODS: All transfusion-dependent children who underwent MRI to assess iron overload were retrospectively studied. Age, weight, height, blood requirement, chelation drug and dosage, serum ferritin and liver enzymes were evaluated at three specific steps: start of transfusion regimen, start of chelation therapy, and first MRI. RESULTS: Among 198 patients, 25 children met inclusion criteria. No differences were detected in all the assessed parameters at start of transfusion regimen and chelation therapy (p>0.05) between patients with good iron balance (liver iron concentration [LIC] <7 mg Fe/g dry weight [dw]) and liver iron overload (LIC >7). At the first MRI, patients with iron overload had significantly higher serum ferritin (3,080.3±1,078.5 vs 1,672.0±705.3 ng/mL; p<0.01) while patients with good iron control maintained a stable ferritin value from the start of chelation therapy but showed significantly lower height Z-score (-1.48±1.02 vs -0.36±1.55; p=0.04). Serum ferritin >1,770 ng/mL was detected as the best threshold for predicting liver iron overload at the first MRI (p=0.0003). CONCLUSION: In order to prevent liver iron overload at the first MRI, children should maintain a stable level of serum ferritin below 1,770 from the start of chelation therapy. However, strict monitoring of growth is mandatory.


Assuntos
Sobrecarga de Ferro , Ferro/metabolismo , Fígado , Imagem por Ressonância Magnética , Talassemia beta , Terapia por Quelação , Pré-Escolar , Feminino , Humanos , Lactente , Sobrecarga de Ferro/diagnóstico por imagem , Sobrecarga de Ferro/tratamento farmacológico , Sobrecarga de Ferro/metabolismo , Sobrecarga de Ferro/patologia , Fígado/diagnóstico por imagem , Fígado/metabolismo , Masculino , Valor Preditivo dos Testes , Estudos Retrospectivos , Talassemia beta/diagnóstico por imagem , Talassemia beta/tratamento farmacológico , Talassemia beta/metabolismo
19.
Hematology ; 24(1): 20-25, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30095041

RESUMO

BACKGROUND: Cardiomyocytes are particularly susceptible to complications from iron loading. The blood transfusions in thalassaemia major create loading of iron that cannot be naturally excreted. Apolipoprotein E and Glutathione S-transferase act as the scavenger of free radicals, which are generated due to excess iron. The variants of Apolipoprotein E (ApoE) and Glutathione S-transferase (GST) may play a role in oxidative damage-induced cardiomyopathy, so we aimed to study the association of genetic variants of these genes on diastolic dysfunction in our patients. MATERIALS AND METHODS: One hundred and five ß-thalassaemia patients older than 10 years were enrolled for the study. Two-dimensional and M-mode echocardiography analysis was done in all patients. Genotyping of the genetic variants of aforementioned genes was done using the PCR-RFLP method. Serum Glutathione S-transferase levels were estimated by ELISA. RESULTS: Diastolic dysfunction was observed in 24 (22.8%) patients, whereas left ventricular hypertrophy was present in 37(35.2%) patients. There was a significant association of GSTM1 null allele with diastolic dysfunction only. Serum GST levels were also positively correlated with e/a and e/e' ratio. Positive association of ApoE E2 allele with the diastolic dysfunction was also seen. CONCLUSIONS: Patients having Glutathione S-transferase M1 allele and Apolipoprotein E E2 allele are predisposed to oxidative stress-induced cardiac injury.


Assuntos
Apolipoproteínas E/genética , Predisposição Genética para Doença , Glutationa Transferase/genética , Polimorfismo de Fragmento de Restrição , Disfunção Ventricular Esquerda/genética , Talassemia beta/genética , Adolescente , Adulto , Apolipoproteínas E/metabolismo , Criança , Feminino , Glutationa Transferase/metabolismo , Humanos , Masculino , Estresse Oxidativo/genética , Disfunção Ventricular Esquerda/metabolismo , Disfunção Ventricular Esquerda/patologia , Talassemia beta/metabolismo , Talassemia beta/patologia
20.
Genomics ; 111(1): 67-75, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-29309842

RESUMO

Diseases and pathological ailments are known to perplex clinicians and researchers with their varied clinical manifestations. Such variations are mostly attributed to the complex interplays between numerous molecular players and their modifiers. This complexity in turn baffles scientists further to tweak multiple players together when attempting to identify definitive therapeutic interventions. In this pursuit, researchers often tend to ignore one of the commonest known genetic variations - single nucleotide polymorphisms (SNPs) in non-coding genetic regions. In this study, we demonstrate how SNPs in critical genes and their miRNA regulators may play a crucial role in varied clinical manifestations using the beta-thalassemia clinical spectrum and fetal hemoglobin levels (HbF) as an illustration. A methodological approach using freely available bioinformatics tools was able to identify SNPs in pre-miRNA regions, pre-miRNA flanking regions and miRNA binding sites which in turn are expected to alter the translation process and thereby the expression of HbF.


Assuntos
Hemoglobina Fetal/genética , MicroRNAs/genética , Talassemia beta/genética , Ilhas de CpG , Bases de Dados Genéticas , Regulação da Expressão Gênica , Heterogeneidade Genética , Perfil Genético , Humanos , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Domínios e Motivos de Interação entre Proteínas , Talassemia beta/metabolismo
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