Brain perfusion changes in beta-thalassemia.

BACKGROUND: Brain injury in hereditary hemoglobinopathies is commonly attributed to anemia-related relative hypoperfusion in terms of impaired oxygen blood supply. Supratentorial and infratentorial vascular watershed regions seem to be especially vulnerable, but data are very scarce. AIMS: We investigated a large beta-thalassemia sample with arterial spin labeling in order to characterize regional perfusion changes and their correlation with phenotype and anemia severity. METHODS: We performed a multicenter single-scanner cross-sectional 3T-MRI study analyzing non-invasively the brain perfusion in 54 transfusion-dependent thalassemia (TDT), 23 non-transfusion-dependent thalassemia (NTDT) patients and 56 Healthy Controls (HC). Age, hemoglobin levels, and cognitive functioning were recorded. RESULTS: Both TDT and NTDT patients showed globally increased brain perfusion values compared to healthy controls, while no difference was found between patient subgroups. Using age and sex as covariates and scaling the perfusion maps for the global cerebral blood flow, beta-thalassemia patients showed relative hyperperfusion in supratentorial/infratentorial watershed regions. Perfusion changes correlated with hemoglobin levels (p = 0.013) and were not observed in the less severely anemic patients (hemoglobin level > 9.5 g/dL). In the hyperperfused regions, white matter density was significantly decreased (p = 0.0003) in both patient subgroups vs. HC. In NTDT, white matter density changes correlated inversely with full-scale Intelligence Quotient (p = 0.007) while in TDT no correlation was found. CONCLUSION: Relative hyperperfusion of watershed territories represents a hemodynamic hallmark of beta-thalassemia anemia challenging previous hypotheses of brain injury in hereditary anemias. A careful management of anemia severity might be crucial for preventing structural white matter changes and subsequent long-term cognitive impairment.

Cognitive impairment and hippocampal neuronal damage in ß-thalassaemia mice.

ß-Thalassaemia is one of the most common genetic diseases worldwide. During the past few decades, life expectancy of patients has increased significantly owing to advance in medical treatments. Cognitive impairment, once has been neglected, has gradually become more documented. Cognitive impairment in ß-thalassaemia patients is associated with natural history of the disease and socioeconomic factors. Herein, to determined effect of ß-thalassaemia intrinsic factors, 22-month-old ß-thalassaemia mouse was used as a model to assess cognitive impairment and to investigate any aberrant brain pathology in ß-thalassaemia. Open field test showed that ß-thalassaemia mice had decreased motor function. However, no difference of neuronal degeneration in primary motor cortex, layer 2/3 area was found. Interestingly, impaired learning and memory function accessed by a Morris water maze test was observed and correlated with a reduced number of living pyramidal neurons in hippocampus at the CA3 region in ß-thalassaemia mice. Cognitive impairment in ß-thalassaemia mice was significantly correlated with several intrinsic ß-thalassaemic factors including iron overload, anaemia, damaged red blood cells (RBCs), phosphatidylserine (PS)-exposed RBC large extracellular vesicles (EVs) and PS-exposed medium EVs. This highlights the importance of blood transfusion and iron chelation in ß-thalassaemia patients. In addition, to improve patients' quality of life, assessment of cognitive functions should become part of routine follow-up.

miR-214 aggravates oxidative stress in thalassemic erythroid cells by targeting ATF4.

Oxidative damage to erythroid cells plays a key role in the pathogenesis of thalassemia. The oxidative stress in thalassemia is potentiated by heme, nonheme iron, and free iron produced by the Fenton reaction, due to degradation of the unstable hemoglobin and iron overload. In addition, the levels of antioxidant enzymes and molecules are significantly decreased in erythrocytes in α- and ß-thalassemia. The control of oxidative stress in red blood cells (RBCs) is known to be mediated by microRNAs (miRNAs). In erythroid cells, microR-214 (miR-214) has been reported to respond to external oxidative stress. However, the molecular mechanisms underlying this phenomenon remain unclear, especially during thalassemic erythropoiesis. In the present study, to further understand how miR-214 aggravates oxidative stress in thalassemia erythroid cells, we investigated the molecular mechanism of miR-214 and its regulation of the oxidative status in thalassemia erythrocytes. We have reported a biphasic expression of miR-214 in ß- and α-thalassemia. In the present study the effect of miR-214 expression was investigated by using miR -inhibitor and -mimic transfection in erythroid cell lines induced by hemin. Our study showed a biphasic expression of miR-214 in ß- and α-thalassemia. Subsequently, we examined the effect of miR-214 on erythroid differentiation in thalassemia. Our study reveals the loss-of-function of miR-214 during translational activation of activating transcription factor 4 mRNA, leading to decreased reactive oxygen species levels and increased glutathione levels in thalassemia erythroid cell. Our results suggest that the expression of activating transcription factor 4 regulated by miR-214 is important for oxidative stress modulation in thalassemic erythroid cells. Our findings can help to better understand the molecular mechanism of miRNA and transcription factors in regulation of oxidative status in erythroid cells, particularly in thalassemia, and could be useful for managing and relieving severe anemia symptoms in patients in the future.

Endocrinopathies in Hemoglobinopathies: What Is the Role of Iron?

Hemoglobinopathies, including ß-thalassemia and sickle cell disease (SCD), are common genetic blood disorders. Endocrine disorders are frequent manifestations of organ damage observed mainly in patients with ß-thalassemia and rarely in SCD. Iron overload, oxidative stress-induced cellular damage, chronic anemia, and HCV infection contribute to the development of endocrinopathies in ß-thalassemia. The above factors, combined with vaso-occlusive events and microcirculation defects, are crucial for endocrine dysfunction in SCD patients. These endocrinopathies include diabetes mellitus, hypothyroidism, parathyroid dysfunction, gonadal and growth failure, osteoporosis, and adrenal insufficiency, affecting the quality of life of these patients. Thus, we aim to provide current knowledge and data about the epidemiology, pathogenesis, diagnosis, and management of endocrine disorders in ß-thalassemia and SCD. We conducted a comprehensive review of the literature and examined the available data, mostly using the PubMed and Medline search engines for original articles. In the era of precision medicine, more studies investigating the potential role of genetic modifiers in the development of endocrinopathies in hemoglobinopathies are essential.

Oxidative Stress in Beta-thalassemia Patients: Role of Enzymatic and Non-enzymatic Modulators.

BACKGROUND: Oxidative stress is a pathophysiological state that arises due to an imbalance created between ROS generation and the antioxidant potential of the host cell. Transfusion- dependent beta-thalassemia major patients are at high risk of cellular and molecular damages induced by ROS mainly due to iron overload caused by repetitive blood transfusion. OBJECTIVES: To analyze oxidative stress status levels in ß-thalassemia patients. To analyze the expression profile of enzymatic (NOS2, OGG1, HuR, SOD2) and non-enzymatic (VDR) redox regulators in ß-thalassemia patients. To assess polymorphism in VDR (rs2228570) and NOS2 (rs944725) in ß-thalassemia patients. To analyze serum vitamin D levels of ß-TM patients compared to healthy individuals. METHODS: The present case-control study aimed to identify Vitamin D levels in the serum of ß-thalassemia patients and compared it with healthy subjects. The study further analyzed VDR FOKI (rs2228570) polymorphism through ARMS-PCR. Expression profiling of VDR, anti-oxidant enzyme (SOD2 and GPx), and their respective regulator (HuR and NrF2) transcripts was done by the 2-ΔΔCt method. RESULTS: The study reports that there is no a significant difference between the Vitamin D levels among healthy and patients. VDR polymorphism analysis (rs2228570) demonstrates that although the C allele is prevalent in the study cohort, the frequency of the T allele is comparatively higher in ß-thalassemia patients as compared to healthy subjects. Furthermore, patients express lower levels of anti-oxidant enzymes despite having increased oxidative stress. CONCLUSION: The study reports that ß-thalassemia patients are at higher risk of cellular and molecular damages induced by oxidative stress and their associated pathologies inefficient enzymatic and non-enzymatic anti-oxidant defense systems.

CHMMOTv1 - cardiac and hepatic multi-echo (T2*) MRI images and clinical dataset for Iron overload on thalassemia patients.

INTRODUCTION: Regarding deep learning networks in medical sciences for improving diagnosis and treatment purposes and the existence of minimal resources for them, we decided to provide a set of magnetic resonance images of the cardiac and hepatic organs. DATABASE DESCRIPTION: The dataset included 124 patients (67 women and 57 men) with thalassemia (THM), the age range of (5-52) years. Patients were divided into two groups: with follow-up (1-5 times) at time intervals of about (5-6) months and without follow-up. T2* and, R2* values, the results of the Cardiac and Hepatic overload report (normal, mild, moderate, severe), and laboratory tests including Ferritin, Bilirubin (D, and T), AST, ALT, and ALP levels were provided as an Excel file. Also, the details of the patients' Echocardiogram data have been made available. This dataset CHMMOTv1) has been published in Mendeley Dataverse and also is accessible through the web at: http://databiox.com .

The clinical value of hsa-miR-190b-5p in peripheral blood of pediatric ß-thalassemia and its regulation on BCL11A expression.

BACKGROUND: The B cell CLL/lymphoma 11A (BCL11A) is a key regulator of hemoglobin switching in ß-thalassemia (ß-thal). Previous study has suggested that dysregulated microRNAs are involved in the regulation of BCL11A expression. The aim of this study was to investigate the clinical value of hsa-miR-190b-5p in ß-thal, and to confirm the regulatory effect of hsa-miR-190b-5p on BCL11A expression. METHODS: The peripheral blood of 25 pediatric ß-thal patients and 25 healthy controls were selected, and qRT-PCR was used to analyze the levels of hsa-miR-190b-5p and BCL11A mRNA. The relationship between hsa-miR-190b-5p expression and hematological parameters was assessed by Pearson's correlation test. The diagnostic power of hsa-miR-190b-5p was evaluated by ROC curves analysis. The direct integration between hsa-miR-190b-5p and BCL11A 3'-UTR was confirmed by luciferase reporter assay. RESULTS: Hsa-miR-190b-5p expression in pediatric ß-thal was upregulated, and negatively correlated with the MCH and HbA levels, but positively correlated with the HbF level. Hsa-miR-190b-5p showed a good diagnostic capability for pediatric ß-thal equivalent to that of HbA2 (AUC: 0.760 vs. 0.758). Moreover, the levels of BCL11A mRNA in pediatric ß-thal were decreased, and hsa-miR-190b-5p had a negative correlation with BCL11A mRNA expression (r = -0.403). BCL11A was a target gene of hsa-miR-190b-5p. The mRNA and protein levels of BCL11A were diminished by introduction of hsa-miR-190b-5p, whereas its expression was upregulated by knockdown of hsa-miR-190b-5p. CONCLUSIONS: Hsa-miR-190b-5p expression was upregulated in pediatric ß-thal and might be an effective diagnostic biomarker. BCL11A was negatively regulated by hsa-miR-190b-5p, which might provide new target for the treatment of pediatric ß-thal.

Pattern and clinical correlates of renal iron deposition in adult beta-thalassemia major patients.

We evaluated pattern and clinical correlates of renal T2* measurements in adult ß-thalassemia major (ß-TM) patients. Ninety ß-TM patients (48 females, 38.15 ± 7.94 years), consecutively enrolled in the Extension-Myocardial Iron Overload in Thalassemia network, underwent T2* magnetic resonance imaging (MRI) for quantification of iron overload (IO) in kidneys, liver, pancreas, and heart. Ten (11.1%) patients showed renal IO (T2* < 31 ms). Global kidney T2* values did not show a correlation with gender, age, splenectomy, regular transfusions or chelation starting age, pre-transfusion hemoglobin, and serum ferritin levels. Global kidney T2* values showed an inverse correlation with MRI liver iron concentration (LIC) values (R = - 0.349; p = 0.001) and a positive correlation with global pancreas T2* values (R = 0.212; p = 0.045). Frequency of renal IO was significantly higher in patients with cardiac IO than in patients without cardiac IO (50.0% vs. 6.3%; p = 0.001). A significant inverse association was detected between global kidneys T2* values and lactate dehydrogenase (LDH) (R = - 0.529; p < 0.0001). In multivariate regression analysis, MRI LIC and LDH were the strongest predictors of global kidney T2* values. A MRI LIC > 4.83 mg/g dw predicted the presence of renal IO (sensitivity = 90.0%; specificity = 61.2%). Global kidney T2* values were inversely correlated with uric acid (R = - 0.269; p = 0.025). In conclusion, in adult ß-TM patients, renal iron deposition is not common and is linked to both hemolysis and total body iron overload.

The Effects of Vitamin D on Myocardial Function Demonstrated by Speckle-Tracking Echocardiography in Children with Beta Thalassemia.

OBJECTIVE: Beta thalassemia major is an inherited hemoglobin disorder resulting in chronic hemolytic anemia. Cardiac involvement is the main cause of death in patients. Speckle-tracking echocardiography is a feasible method for the evaluation of cardiac function via an assessment of the longitudinal deformation of the myocardium through the cardiac cycle. The aim of our study is to evaluate the association between vitamin D deficiency and deformation of the left ventricular myocardium measured by speckle-tracking echocardiography in children with thalassemia major. METHODS: In this prospective study, 33 thalassemic patients with vitamin D deficiency were enrolled. Cardiac magnetic resonance T2* value, conventional echocardiography, and speckle tracking, and also left ventricular longitudinal and circumferential strain values were measured. Myocardial functions of the patients with vitamin D deficiency or insufficiency were evaluated by speckle-tracking echocardiography before and after vitamin D replacement. RESULTS: The mean age of the patients was 15.4 ± 3.09 years. Vitamin D level was deficient in 30 (90%) and insufficient in 3 (10%) of them. Speckle-tracking analysis showed a significantly decreased absolute value of the left ventricular global longitudinal strain before vitamin D replacement. A significant improvement in the global longitudinal strain was detected after vitamin D replacement (P < 0.05). A statistically significant increase was observed in parameters showing left ventricular systolic and diastolic functions after vitamin D replacement. CONCLUSION: Vitamin D deficiency is frequently observed and causes decreased contractility in thalassemic patients. In our study, we observed that our patients' cardiac functions had improved after vitamin D replacement therapy.

The association between steatosis and liver damage in transfusion-dependent beta thalassaemia patients.

Non-alcoholic fatty liver disease (NAFLD) is a global health problem. Iron is the leading cause of liver damage in patients with transfusion-dependent thalassaemia (TDT), and data on the contribution of NAFLD to liver damage in TDT is lacking. Forty-five heavily transfused TDT patients who did not have biochemical or ultrasonic evidence of liver cirrhosis were evaluated for effects of iron overload, including the presence of diabetes mellitus, hypogonadism, serum ferritin, R2-MRI-liver, and liver enzymes alanine aminotransferase and aspartate aminotransferase. Liver fibrosis and steatosis were estimated using transient elastography (TE). Nine (20%) patients had significant steatosis (S1), and their body mass index (BMI) and liver fibrosis scores were higher than in patients without significant steatosis (S0) (p = 0.03 and p = 0.004, respectively). On regression analysis, the controlled attenuation parameter (CAP) score (i.e., degree of liver steatosis) was associated only with increasing BMI. The TE score (i.e., degree of liver fibrosis) was associated with increasing age, CAP score, male gender, and presence of diabetes. Neither liver steatosis nor fibrosis showed significant association with the liver iron concentration or iron-related organ damage (hypogonadism). In this cohort of TDT patients, steatosis of the liver, which is associated with increasing BMI, appeared to increase the risk of liver fibrosis.

Stroke propensity in the Th3+/ mouse model of ß-thalassemia intermedia.

ß-thalassemia is associated with multiple hematological and cerebrovascular symptoms linked to a hypercoagulable state that has not been fully replicated in animal models for the development of stroke treatments. Herein we compared the physiological properties and responses to transient cerebral hypoxia-ischemia (tHI) between six-month-old wildtype and heterozygous Th3/+ mice, a model of non-transfusion-dependent ß-thalassemia intermedia (ß-TI). We found that Th3/+ mice developed microcytic anemia, splenomegaly, higher platelet counts, and increased platelet-erythrocyte plus erythrocyte-leukocyte aggregates. Furthermore, Th3/+ mice showed diminished cerebrovascular reactivity (CVR) and cortical oxygen saturation under repetitive hypercapnic challenges. When subjected to a sub-threshold tHI insult, platelets and leukocytes in Th3/+ mice adhered to the cerebrovascular wall or formed aggregates, while their counterparts flew through smoothly in wildtype mice. Subsequently, Th3/+ mice showed increased fibrin deposition around cerebral blood vessels and larger infarction than wildtype mice, especially in female Th3/+ mice. Collectively these results showed that Th3/+ mice mimic key clinical features and a propensity to thromboembolism in ß-TI patients. The hypercoagulable state in Th3/+ mice is likely caused by multiple hematological and CVR anomalies that are similar, but are not identical to those in the mouse model of sickle cell anemia. As such, we suggest that Th3/+ mice are a useful model to study the pathological mechanisms and prophylactic stroke treatments in thalassemia patients.

Long-read sequencing on the SMRT platform enables efficient haplotype linkage analysis in preimplantation genetic testing for ß-thalassemia.

PURPOSE: This study aimed to evaluate the value of long-read sequencing for preimplantation haplotype linkage analysis. METHODS: The genetic material of the three ß-thalassemia mutation carrier couples was sequenced using single-molecule real-time sequencing in the 7.7-kb region of the HBB gene and a 7.4-kb region that partially overlapped with it to detect the presence of 17 common HBB gene mutations in the Chinese population and the haplotypes formed by the continuous array of single-nucleotide polymorphisms linked to these mutations. By using the same method to analyze multiple displacement amplification products of embryos from three families and comparing the results with those of the parents, it could be revealed whether the embryos carry disease-causing mutations without the need for a proband. RESULTS: The HBB gene mutations of the three couples were accurately detected, and the haplotype linked to the pathogenic site was successfully obtained without the need for a proband. A total of 68.75% (22/32) of embryos from the three families successfully underwent haplotype linkage analysis, and the results were consistent with the results of NGS-based mutation site detection. CONCLUSION: This study supports long-read sequencing as a potential tool for preimplantation haplotype linkage analysis.

Impaired neutrophil extracellular trap formation in ß-thalassaemia/HbE.

Neutrophil dysfunction contributes to a high susceptibility to severe bacterial infection which is a leading cause of morbidity and mortality in ß-thalassaemia/HbE, especially in splenectomised patients. This study demonstrated another abnormality of neutrophil function, namely neutrophil extracellular trap (NET) formation in splenectomised and non-splenectomised ß-thalassaemia/HbE patients who had iron overload. A classification system of morphological NET formation using confocal microscopy was developed, and samples were categorized into early and late phases which were subdivided into web-like and non-web structures. At baseline, neutrophils from non-splenectomised patients (58 ± 4%) and splenectomised patients (65 ± 3%) had higher early phase NETs than those from normal subjects (33 ± 1%). As a mimic of iron overload and infection, haemin/PMA/LPS treatment led to a significant reduction of early NETs and an increase of late NETs in neutrophils from normal and non-splenectomised patients. Interestingly, neutrophils from splenectomised patients had impaired development of late NETs. This suggests that during infection bacteria might not be trapped and may spread from the site of infection resulting in higher susceptibility to severe bacterial infection in splenectomised patients.

Interrelationship between liver T2*-weighted magnetic resonance imaging and acoustic radiation force impulse elastography measurement results and plasma ferritin levels in children with ß-thalassemia major.

PURPOSE: To evaluate correlation and agreement between T2*-weighted magnetic resonance imaging (T2*-wMRI), acoustic radiation force impulse elastography (ARFI-e) measurement results of liver and plasma ferritin levels (PFLs) in children with ß-thalassemia major (ß-TM). METHODS: The study included 40 pediatric patients (aged 64-216 months; 14 girls, 26 boys) receiving blood transfusion and chelation therapy. To detect the severity of liver iron overload (LIO) and concomitant parenchymal fibrosis, T2*-wMRI and ARFI-e measurements were performed from the right lobe segments. Student's t-test, Mann-Whitney U, ANOVA, Spearman's test and ICC were used for statistical analysis. RESULTS: After the measurements of T2*-wMRI, patients were grouped as normal in 4 (10%), mild in 11 (27.5%), moderate in 21 (52.5%), and severe in 4 (10%) cases in terms of LIO. Combined moderate and severe groups had significantly higher ARFI-e and PFL values than the combination of other groups (p = .001, p = .040). The ARFI-e measurements of boys were found to be significantly higher than those of girls (p = .023). A strong negative correlation between T2*-wMRI and ARFI-e and a moderate negative correlation between T2*-wMRI and PFL were detected (p;r = 0.001;-0.606, p;r = 0.009; -0.407). A strong positive correlation was found between ARFI-e values and PFL (p;r = 0.001; 0.659). The optimal cut-off value of ARFI-e to predict liver fibrosis because of moderate&severe LIO was determined to be 1.29 M/s (80% sensitivity and 88% specificity). A moderate agreement was observed between the T2*-wMRI and ARFI-e methods [ICC: 0.680, 95% CI: (0.470 to 0.817)]. CONCLUSION: Given the strong correlation and moderate agreement between ARFI-e and T2*-wMRI, ARFI -e could be used to monitor LIO in children with ß-TM.

Impaired bone marrow microenvironment and stem cells in transfusion-dependent beta-thalassemia.

Beta-thalassemia (BT) is a hereditary disease caused by abnormal hemoglobin synthesis with consequent ineffective erythropoiesis. Patients with thalassemia major are dependent on long-term blood transfusions with associated long-term complications such as iron overload (IO). This excess iron can result in tissue damage, impaired organ function, and increased morbidity. Growing evidence has demonstrated that IO contributes to impairment of the bone marrow (BM) microenvironment that largely impacts the function of BM mesenchymal stem cells, hematopoietic stem cells, and endothelial cells. In this article, we review recent progress in the understanding of iron metabolism and the perniciousness induced by IO. We highlight the importance of understanding the cross-talk between BM stem cells and the BM microenvironment, particularly the pathological effect of IO on BM stem cells and BT-associated complications. We also provide an update on recent novel therapies to cure transfusion-dependent beta-thalassemia and iron overload-induced complications for their future clinical application.

Use of rapid cardiac magnetic resonance imaging to guide chelation therapy in patients with transfusion-dependent thalassaemia in India: UMIMI study.

AIMS: To explore the impact of incorporating a faster cardiac magnetic resonance (CMR) imaging protocol in a low-middle-income country (LMIC) and using the result to guide chelation in transfusion-dependent patients. METHODS AND RESULTS: A prospective UK-India collaborative cohort study was conducted in two cities in India. Two visits 13 months apart included clinical assessment and chelation therapy recommendations based on rapid CMR results. Participants were recruited by the local patient advocate charity, who organized the patient medical camps. The average scanning time was 11.3 ± 2.5 min at the baseline and 9.8 ± 2.4 min (P < 0.001) at follow-up. The baseline visit was attended by 103 patients (mean age 25 years) and 83% attended the second assessment. At baseline, 29% had a cardiac T2* < 20 ms, which represents significant iron loading, and 12% had left ventricular ejection fraction <60%, the accepted lower limit in this population. Only 3% were free of liver iron (T2* ≥ 17 ms). At 13 months, more patients were taking intensified dual chelation therapy (43% vs. 55%, P = 0.002). In those with cardiac siderosis (baseline T2* < 20 ms), there was an improvement in T2*-10.9 ± 5.9 to 13.5 ± 8.7 ms, P = 0.005-and fewer were classified as having clinically important cardiac iron loading (T2* < 20 ms, 24% vs. 16%, P < 0.001). This is the first illustration in an LMIC that incorporating CMR results into patient management plans can improve cardiac iron loading. CONCLUSION: For thalassaemia patients in an LMIC, a simplified CMR protocol linked to therapeutic recommendation via the patient camp model led to enhanced chelation therapy and a reduction in cardiac iron in 1 year.

Association of the Degree of Erythroid Expansion and Maturation Arrest with the Clinical Severity of ß0-Thalassemia/Hemoglobin E Patients.

INTRODUCTION: ß-Thalassemia/hemoglobin E represents one-half of all the clinically severe ß-thalassemias worldwide. Despite similar genetic backgrounds, patients show clinical heterogeneity ranging from nearly asymptomatic to transfusion-dependent thalassemia. The underlying disease modifying factors remain largely obscure. METHODS: To elucidate the correlation between ineffective erythropoiesis and ß0-thalassemia/hemoglobin E (HbE) disease severity, in vitro culture of erythroid cells derived from patients with different clinical symptoms was established. Cell proliferation, viability, and differentiation were investigated. To identify potential molecular mechanisms leading to the arrested erythroid maturation, the expression levels of erythropoiesis modifying factors were measured. RESULTS: The ß0-thalassemia/HbE cells exhibited enhanced proliferation, limited differentiation, and impaired erythroid terminal maturation but did not show accelerated erythroblast differentiation and increased cell death. Erythroblasts derived from mild patients showed the highest proliferation rate with a faster cell division time, while erythroblasts derived from severe patients displayed extremely delayed erythroid maturation. Downregulation of growth differentiation factor 11 and FOXO3a was observed in mild ß0-thalassemia/HbE erythroblasts, while upregulation of heat shock protein 70 and activin receptor 2A was revealed in severe erythroblasts. DISCUSSION/CONCLUSION: The degree of erythroid expansion and maturation arrest contributes to the severity of ß0-thalassemia/HbE patients, accounting for the disease heterogeneity. The findings suggest a restoration of erythroid maturation as a promising targeted therapy for severe patients.