Β-thalassemia Patients Research Articles

Red blood cell alloimmunization in blood transfusion-dependent β thalassemia major patients in Sana’a City-Yemen

The development of erythrocyte alloantibodies complicates transfusion therapy in β thalassemia major patients. These antibodies increase the need for blood and intensify transfusion complications. Data on erythrocyte alloimmunization is scarce in Yemeni thalassemia patients. We studied the frequency of alloimmunization in multitransfused β-thalassemia major patients and investigated risk factors that affect antibody formation. Blood samples were taken from 100 β thalassemia major patients who received multitransfused leukodepleted packed red-blood cells. Antibody screening and identification were performed by indirect antiglobulin test using the gel column technique. All patients were tested for autoantibodies using autocontrol and direct antiglobulin test. No adsorption test was done as no autoantibodies were detected in any patient. In our study of 100 β-thalassemia patients, 50 were male and 50 were female with ages ranging from 1 to 30 years. Alloantibodies were present in 6% of patients, while no autoantibodies were detected. Of the 17 alloantibodies identified, the majority were directed against Kell (41.2%) and Rh (29.4%) blood groups. Alloimmunization was significantly associated with age group and sex (p = 0.013, p = 0.030), respectively in β thalassemia major patients. The development of alloantibodies was not significantly associated with duration, total number of transfusions and splenectomy (P = 0.445, P = 0.125, P = 0.647). No autoantibodies found in patients with β thalassemia major. The study found low rates of erythrocyte alloimmunization in multitransfused β-thalassemia major patients, but significant alloantibodies were produced primarily from Kell and Rh blood groups, suggesting the need for providing phenotypically matched cells for selective antigens to improve transfusion efficiency.

Role of feature tracking cardiac magnetic resonance imaging in early detection of cardiac dysfunction in β-thalassemia patients recovered from COVID-19: A cross-sectional study.

β-Thalassemia patients may have cardiac complications due to iron overload, which puts them at higher risk of cardiac complications induced by coronavirus disease 2019 (COVID-19) compared with the normal population. The present study aims to evaluate early cardiovascular complications following iron overload in β-thalassemia patients who had early recovery from COVID-19 by cardiac magnetic resonance imaging (MRI) and feature-tracking technique. Thirty-two confirmed COVID-19-recovered β-thalassemia cases were evaluated within 3 weeks to 3 months after a positive reverse-transcriptase polymerase chain reactionCOVID-19 test. Both the heart and liver of all patients were examined using cardiac MRI. We analyzed 32 patients with mean age of 32.84 ± 6.45 years at baseline. Left ventricular global strain values were significantly associated with myocardial T2*. A cut-off value of -15.08% for global longitudinal strain (GLS) with sensitivity and specificity of 90% and 61.1% (p = 0.017), 32.33% for global radial strain (GRS) with sensitivity and specificity of 80% and 94.4% (p = 0.001) and -16.21 for global circumferential strain(GCS), with sensitivity and specificity of 80% and 89.9% (p = 0.013) may indicate cardiac iron overload. GLS, GRS, and GCS were significantly decreased in patients with myocardial T2* <20 ms (iron overload), while no significant change was observed in the right and left ventricular ejection fraction (RV- and LVEF). Cardiac MRI feature-tracking may be helpful in the early detection of cardiac complications resulting from iron overload in β-thalassemia patients who had early recovery from COVID-19.

Diabetes mellitus progression in β-thalassaemia major patients: The impact of iron overload

ABSTRACT Regular blood transfusion therapy and enhanced iron absorption in patients with β-thalassemia major lead to progressive accumulation of iron in the patient’s tissues. Iron overload disrupts the function of pancreatic β-cell, deteriorates insulin resistance, increases blood glucose levels, and precipitates diabetes mellitus (DM). A family history of DM, obesity, sedentary lifestyle, and age increase the risk of DM in β-thalassemia patients. The prevalence of DM among individuals with β-thalassemia major is on the rise despite considerable screening and management efforts. Therefore, more research is still warranted to fully understand the molecular underpinnings linking β-thalassemia major and the onset and progression of DM to develop more robust screening and management approaches. In this context, no reliable biomarkers are currently available to predict or detect DM in such patients. In this review, we elaborate on key tentative links between thalassemia and DM and expand on established metabolic alterations that associate with DM in patients with β-thalassemia major. The review also addresses the prevalence rates and patient management strategies and underscores the need for identifying DM-related predictive and diagnostic biomarkers in β-thalassemia major patients.

Identification and characterization of CHD4-associated eRNA as a novel modulator of fetal hemoglobin levels in β-thalassemia

Fetal-to-adult hemoglobin switching is controlled by programmed silencing of γ-globin while the re-activation of fetal hemoglobin (HbF) is an effective strategy for ameliorating the clinical severity of β-thalassemia and sickle cell disease. The identification of enhancer RNAs (eRNAs) related to the fetal (α2γ2) to adult hemoglobin (α2β2) switching remains incomplete. In this study, the transcriptomes of GYPA+ cells from six β-thalassemia patients with extreme HbF levels were sequenced to identify differences in patterns of noncoding RNA expression. It is interesting that an enhancer upstream of CHD4, an HbF-related core subunit of the NuRD complex, was differentially transcribed. We found a significantly positive correlation of eRNA-CHD4 enhancer-gene interaction using the public database of FANTOM5. Specifically, the eRNA-CHD4 expression was found to be significantly higher in both CD34+ HSPCs and HUDEP-2 than those in K562 cells which commonly expressed high level of HbF, suggesting a correlation between eRNA and HbF expression. Furthermore, prediction of transcription binding sites of cis-eQTLs and the CHD4 genomic region revealed a putative interaction site between rs73264846 and ZNF410, a known transcription factor regulating HbF expression. Moreover, in-vitro validation showed that the inhibition of eRNA could reduce the expression of HBG expression in HUDEP-2 cells. Taken together, the findings of this study demonstrate that a distal enhancer contributes to stage-specific silencing of γ-globin genes through direct modulation of CHD4 expression and provide insights into the epigenetic mechanisms of NuRD-mediated hemoglobin switching.

Kiosk 8R-FA-06 - Feature Tracking Cardiac Magnetic Resonance Imaging and Early Detection of Cardiac Dysfunction in β-thalassemia Patients Recovered FBom COVID-19

Kiosk 8R-FA-06 - Feature Tracking Cardiac Magnetic Resonance Imaging and Early Detection of Cardiac Dysfunction in β-thalassemia Patients Recovered FBom COVID-19

Beta-Thalassemia Major and Myocardial Iron Overload: A Longitudinal Study with Magnetic Resonance Imaging.

Patients with β-thalassemia major depend on lifelong transfusion, resulting in tissue iron overload. This longitudinal retrospective observational study aims to assess myocardial and liver iron overload using magnetic resonance imaging (MRI) and investigate the lag between myocardial and liver iron unloading in β-thalassemia patients undergoing chelation therapy. Beta-thalassemia major patients with at least two MRI studies between 2016 and 2020 were enrolled. Myocardial and liver iron overload were defined as T2 ∗ less than 20 and 2.1, respectively. Outcomes included mortality, myocardial and liver T2 ∗ changes, and systolic dysfunction assessed by cardiac MRI. Fifty-five patients with a mean age of 24.62 ± 7.94 years, a mean follow-up duration of 24.3 ± 12.9 months, and a mean ferritin level of 1475.75 ± 771.12 ng/mL were enrolled. All of the abovementioned patients only took deferoxamine as the iron-chelating medication. Mortality occurred in three patients (5.5%) during follow-up. Liver T2 ∗ significantly increased (p value <0.05), while myocardial T2 ∗ showed a nonsignificant increase. Iron unloading of the myocardium was not significantly different from that of the liver and did not result in a significant lag (56% vs. 44%; p value = 0.419). Baseline myocardial T2 ∗ correlated with extramedullary hematopoiesis, weekly number of deferoxamine injections (p value <0.01), timing between the transfusions, and serum ferritin (p value <0.05). Liver T2 ∗ reduced during deferoxamine chelation therapy, while myocardial T2 ∗ remained unchanged. No significant lag was observed between myocardial and liver iron unloading. Further studies are required to elucidate these findings.

Investigating the level of Hba1c and insulin level in β-thalassemia patients

Background: β-thalassemia is an inherited blood disorder characterized by reduced or no synthesis of β globin chain, resulting in chronic anemia, so blood transfusion is required as curative therapy. Repeated blood transfusions lead to iron overload that can lead to multiple organ damage, including pancreatic organs.Objective: This study aimed to describe HbA1c and insulin levels of β-thalassemia patients. The study also tested whether there was a significant difference in insulin and HbA1c levels among patients with different β-thalassemia categories. Method: This research was an analytic observational study. The samples were taken by total sampling and involved 30 patients, and the examination was carried out using the patient's blood plasma.Result: The study found that samples had low HbA1c levels. There was no significant mean difference (p&gt;0.05) between insulin and HbA1c in the mild, moderate, and severe clinical degree groups. There was no significant difference in average (p&gt;0.05) insulin and HbA1c in the thalassemia sufferers with allele β+ and β0 types.Conclusion: There was a decrease in insulin and HbA1c in patients with β-thalassemia, which may be caused by damage pancreatic organ damage.

Comparison of Capillary Zone Electrophoresis with High-pressure Liquid Chromatography in the Evaluation of Hemoglobinopathies

The capillary zone electrophoresis (CZE) and high-performance liquid chromatography (HPLC) methods were compared in terms of HbA2 measurement for the assessment of hemoglobinopathies. CZE was compared with HPLC for the evaluation of patients without hemoglobinopathy (n=321), with β-thalassemia trait (n=113), and with common (HbD-Punjab, E, C, S/A, and S/S) and rare (HbS/D, O-Arap, Lepore, G-Coushata, Setif, Hamadan, Q-Iran, and H) variants (n=21). The reference range for HbA2 was determined by CZE. Among patients without hemoglobinopathy, the median (2.5th-97.5th percentiles) values were 97.4% (97.0-98.0%) and 97.5% (96.6-98.4%) for HbA (p=0.060) and 2.4% (1.6-3.0%) and 2.5% (1.6-3.1%) for HbA2 (p<0.001) by HPLC and CZE, respectively. The reference range for HbA2 was 1.6-3.1% by CZE. In the comparison of methods for HbA2, there was a constant error of 0.255 (confidence interval: 0.062-0.448) and bias of 0.10% (limit of agreement: 0.33-0.53), and higher values were obtained with CZE. A strong correlation was observed between the methods (r=0.782). Interrater agreement was almost perfect for clinical diagnosis (ϰ=0.911). The two methods detected and identified the common variants similarly. All rare variants, except HbH by HPLC and HbS/D by CZE, were detected as separate peaks by both methods. The two methods were in agreement regarding the preliminary identification of β-thalassemia patients. Different Hb variants were detected by both methods but with possible methodological interference for HbA2 measurements. CZE is a reliable and simple alternative for the evaluation of hemoglobinopathies. The standardization of HbA2 measurements should be prioritized as more techniques become available in routine laboratory practice.

New Synthetic Isoxazole Derivatives Acting as Potent Inducers of Fetal Hemoglobin in Erythroid Precursor Cells Isolated from β-Thalassemic Patients.

Induction of fetal hemoglobin (HbF) is highly beneficial for patients carrying β-thalassemia, and novel HbF inducers are highly needed. Here, we describe a new class of promising HbF inducers characterized by an isoxazole chemical skeleton and obtained through modification of two natural molecules, geldanamycin and radicicol. After preliminary biological assays based on benzidine staining and RT-qPCR conducted on human erythroleukemic K562 cells, we employed erythroid precursors cells (ErPCs) isolated from β-thalassemic patients. ErPCs weretreated with appropriate concentrations of isoxazole derivatives. The accumulation of globin mRNAs was studied by RT-qPCR, and hemoglobin production by HPLC. We demonstrated the high efficacy of isozaxoles in inducing HbF. Most of these derivatives displayed an activity similar to that observed using known HbF inducers, such as hydroxyurea (HU) or rapamycin; some of the analyzed compounds were able to induce HbF with more efficiency than HU. All the compounds were active in reducing the excess of free α-globin in treated ErPCs. All the compounds displayed a lack of genotoxicity. These novel isoxazoles deserve further pre-clinical study aimed at verifying whether they are suitable for the development of therapeutic protocols for β-thalassemia.

Amlodipine Therapy in β-Thalassemia Patients: A Systematic Review and Meta-Analysis on Ferritin Levels and Liver MRI T2*

Background and aim: We conducted a review to determine the efficacy of amlodipine alongside iron chelators on serum ferritin levels and liver T2-weighted magnetic resonance imaging (MRI T2*) in β-thalassemia patients. Methods: Systematic search was conducted in multiple databases, including Web of Science, PubMed, Scopus, Embase, Cochrane Library, ClinicalTrials.gov, the Iranian Registry of Clinical Trials (IRCT), ProQuest, OpenGrey, and Web of Science Conference Proceedings Citation Index. The search was closed in January 2023. Primary outcomes were comprised of liver MRI T2* (millisecond (msec)) and serum ferritin levels (ng/mL). Results: Seven studies (n = 227) were included in the study. The pooled Cohen’s d for serum ferritin was estimated at −0.46, 95% confidence interval (CI) −1.11 to 0.19 and p = 0.16 (I2 86.23%, p &lt; 0.0001). The pooled mean difference for serum ferritin was −366.44 ng/mL, 95% CI −844.94 to 112.05, and p = 0.13 (I2 81.63%, p &lt; 0.0001). After a meta-regression based on the length of using amlodipine, a coefficient for the mean difference was also −23.23 ng/mL and 95% CI −155.21 to 108.75. The coefficient obtained from a meta-regression as per the amlodipine dose at 5 mg/day than 2.5 to 5 mg/day anchored at −323.49 ng/mL and 95% CI −826.14 to 1473.12. A meta-regression according to the baseline values of serum ferritin discovered a coefficient of 1.25 ng/mL and 95% CI 0.15 to 2.35. Based on two included studies (n = 96), the overall Cohen’s d for liver MRI T2* was 2.069, 95% CI −0.896 to 5.035, and p = 0.17 (I2 96.31%, p&lt; 0.0001). The synthesized mean difference for liver MRI T2* was 8.76 msec, 95% CI −4.16 to 21.67, and p = 0.18 (I2 98.38%, p &lt; 0.000). Conclusion: At a very low level of evidence, probably using amlodipine at a dose of 2.5 to 5 mg a day, up to a year, alongside iron chelators slightly decreases serum ferritin levels in iron-overloaded thalassemia cases by nearly 366 ng/mL (23 ng/mL per month). The liver MRI T2* might also rise to 8.76 msec upon co-therapy with amlodipine.

MALAT1: A novel hypothesis on the pathology of vascular injury in patients with β-thalassemia

Vascular injury is a common consequence in β-thalassemia patients with severe anemia. Vascular cell damage can result in cardiomyopathy, thrombosis, and persistent ulceration. Although it has been suggested that injury is caused by iron accumulation and oxidative stress, the exact molecular mechanism remains unknown. Here, we highlight the potential role of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in this pathophysiology. This hypothesis is based on the literature, which demonstrates that lncRNA expression of MALAT1-lncRNA is linked to vascular damage, wound healing capability, and cardiomyopathy. Furthermore, various technologies can be employed to manipulate the expression of MALAT1-lncRNA. In light of these studies, assessment of MALAT1 expression may enhance our understanding of the molecular pathophysiology of vascular damage in β-thalassemia. If confirmed, elevated MALAT1 expression could serve as a promising target for expediting the healing process in pathogenic tissues. Additionally, MALAT1 may serve as a surrogate biomarker for therapeutic management of patients with β-thalassemia who have vascular complications.

When should gene therapy be considered for transfusion-dependent β-thalassemia patients?

When should gene therapy be considered for transfusion-dependent β-thalassemia patients?

A retrospective study of glucose homeostasis, insulin secretion, sensitivity/resistance in non- transfusion-dependent β-thalassemia patients (NTD- β Thal): reduced β-cell secretion rather than insulin resistance seems to be the dominant defect for glucose dysregulation (GD).

Non-transfusion - dependent β-thalassemias (NTD-βThal) can cause iron overload and serious iron-related organ complications as endocrine dysfunction, including glucose dysregulation (GD). We retrieved data of all NTD- β Thal patients referred consecutively to a single Outpatient Italian Clinic from October 2010 to April 2023. All patients underwent a standard 3-h oral glucose tolerance test (OGTT) for analysis of glucose homeostasis, insulin secretion and sensitivity/resistance (IR), using conventional surrogate indices derived from the OGTT. The collected data in NTD- β Thal patients were compared to 20 healthy subjects. Seventeen of 26 (65.3 %) NTD- β Thal patients (aged: 7.8 -35.1 years) had normal glucose tolerance, 1/26 (3.8 %) had impaired fasting glucose (IFG), 5/26 (19.2 %) impaired glucose tolerance (IGT), 1/26 (3.8%) IFG plus IGT and 2/26 (7.6%) plasma glucose (PG) level ≥155 mg/dL 1-h after glucose load. GD was observed exclusively in young adult patients; none of them had diabetes mellitus (DM). These findings were associated with a low insulinogenic index (IGI) and oral disposition index. HOMA-IR and QUICKI were not significantly different compared to controls. Interestingly, in young adult patients, ISI-Matsuda index was statistically higher compared to the control group, suggesting an increased insulin sensitivity. This study reported a high prevalence of GD in young adults with NTD- β Thal. The documented reduction of IGI rather than the presence of IR, indicates reduced insulin secretory capacity as the pathophysiological basis of dysglycemia that may represent a novel investigational path for future studies on the mechanism(s) responsible for GD in NTD- β Thal patients.

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

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. To analyze oxidative stress status levels in β-thalassemia patients. To analyze the expression profile of enzymatic (NOS2, OGG1, HuR, SOD2) and non-enzymatic (VDR) redox regulators in β-thalassemia patients. To assess polymorphism in VDR (rs2228570) and NOS2 (rs944725) in β-thalassemia patients. To analyze serum vitamin D levels of β-TM patients compared to healthy individuals. The present case-control study aimed to identify Vitamin D levels in the serum of β-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. 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 β-thalassemia patients as compared to healthy subjects. Furthermore, patients express lower levels of anti-oxidant enzymes despite having increased oxidative stress. The study reports that β-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.

Thalassaemia Patients with Polymorphism of COL1A1 Sp1 are at Greater Risk of Spine Degenerative Changes.

To explore the association of the COL1A1 Sp1 polymorphism with decreased bone density and spinal changes in β-thalassaemia patients. Cross-sectional, comparative study. Place and Duration of the Study: University of Health Sciences, Lahore, from May 2020 to June 2021. A total of 110 participants (55patients and 55 controls) of either gender, with ages ranging from 18-40 years were enrolled in the study. Bone density parameters including T-score, Z-score, bone-transmission time (BTT), and amplitude-dependent speed of sound (ADSOS) were assessed by ultrasound bone profiler. Lumbar spine radiographs were collected from patients and assessed for spine changes. Genotype analysis was done by HRM-PCR. Data were analysed using SPSS version 23. All bone density parameters were significantly lower in β-thalassaemai patients (p<0.001). Spine degenerative changes were more obvious in patients with age <25 years (p=0.04). Loss of lumbar lordosis was seen in 74.5% of the patients. The frequency of mutant allele (ss) was 7.3% while heterozygous (Ss) frequency was found to be 33.6%. The polymorphism showed significant association with T-scores (p=0.03) and ADSOS (p=0.02) in patients. Radiographic grades were higher in osteopenic and osteoporotic patients (p=0.04). The association of polymorphism with decreased bone density in β-thalassaemia patients revealed the potential role of genetics in bone changes and related disorders. Bone Density, COL1A1 Polymorphism, Osteoporosis, Thalassaemia.

Pyruvate Kinase Thermostability Is Associated with Red Blood Cell Adhesion, Deformability and Oxygen Affinity in Patients with Sickle Cell Disease

Introduction: Sickle cell disease(SCD) is caused by a mutation in the beta globin gene that causes formation of abnormal hemoglobin S (HbS). Red blood cells (RBCs) from patients with SCD are less deformable and take on the shape of sickle upon deoxygenation. This results in multiple downstream effects including RBC adhesion, microvascular occlusion and chronic hemolysis. RBCs rely solely on anaerobic glycolysis to produce adenosine triphosphate (ATP), which is needed to maintain the cell's structure and function. Pyruvate kinase (PK) is a key enzyme involved in the last step of glycolysis. We have previously demonstrated a decrease in PK activity and stability as a novel pathophysiological feature of SCD. Currently there are several clinical trials ongoing investigating the efficacy of PK activation by small molecules such as mitapivat (NL8517, NCT04000165, NCT05031780), AG-946 (NCT04536792) and etavopivat (NCT04624659, NCT04987489). In this study we aim to investigate how PK properties correlate to sickle RBC properties in order to explore if impaired PK function is associated with specific features of the complex SCD pathophysiology. Methods: Homozygous HbS (HbSS) and HbS/ β0 thalassemia patients were eligible to participate. Patients who received blood transfusion (&amp;lt;3 months) were excluded. Dense RBCs (MCHC &amp;gt;41 g/dL, Advia 120/2120 Siemens) and HbF and HbS levels (Tosoh G8) were measured. PK and hexokinase (HK) activity were measured on purified RBCs. PK thermostability (reflected by % residual PK activity) was measured on purified RBCs lysates after one hour of incubation (53°C). RBC adhesion to laminin (Biolamina) was measured using a microfluidic device (IBIDI µ-Slide I 0.4). RBC hydration (Ohyper), RBC deformability (EImax) and point of sickling (PoS) were assessed by osmotic and oxygen gradient ektacytometry using the Laser-Optical Rotational Red Cell Analyzer (Lorrca, RR Mechatronics). Oxygen affinity (P50) was measured with the Hemox-Analyzer (TCS). Statistical analysis was performed in Graphpad Prism (Pearson's correlation). Results: Fifty-seven patients with SCD (53 HbSS, 4 HbS/β 0 thalassemia) were included of which 17 were children (median age 14 years [range 6-17]) and 40 were adults (median age 36 years [range 18-58]). Baseline characteristics are depicted in Table 1. Figure 1 shows a heatmap with correlation coefficients of all laboratory parameters from Table 1 as measured in this cohort. When focusing on PK properties we found that patients with SCD and lower PK thermostability had significantly higher absolute reticulocyte counts (ARC, p&amp;lt;0.0001), lower hemoglobin (p=0.012) and HbF (p=0.020) levels and increased RBC adhesion to laminin (p=0.007). Furthermore, PK thermostability was significantly correlated with EImax (p=0.017) and inversely correlated with PoS (p=0.011) and P50 (p=0.003), indicating that patients with less stable RBC PK had less deformable RBCs which sickled at a higher oxygen tension. RBC adhesion to laminin was significantly correlated with both ARC (p=0.0003) and dense RBCs (p=0.019). To explore whether our findings were a reflection of reticulocytosis we fractionated whole blood samples of HbSS patients according to cell density. We found that more RBCs from the most dense fraction adhered to laminin than from the less dense, reticulocyte enriched, fraction, even though RBCs from this less dense fraction had reduced PK-thermostability. Additionally, the most dense RBCs had the lowest PK activity, were less deformable and had a higher PoS (data not shown). Altogether these findings indicate that the reduced PK thermostability we found in our SCD cohort is associated with an overall functionally impaired RBC population with increased RBC adhesion and ARC. We found no correlation of markers of RBC dehydration (Ohyper and % dense RBCs) with PK thermostability which suggests that RBCs with reduced PK thermostability are not more dehydrated. Discussion/conclusion: This study shows for the first time a significant correlation between PK thermostability in sickle RBCs and RBC functions, such as adhesion, deformability and oxygen affinity. Our results suggest that enhancing the activity and stability of PK, with PK activators, is an attractive target in SCD that might improve other pathophysiological targets outside RBC metabolism. Future studies are needed to explore how activation of PK will affect these altered sickle RBC properties in vivo.

Base editing of the HBG promoter induces potent fetal hemoglobin expression with no detectable off-target mutations in human HSCs

Reactivating silenced γ-globin expression through the disruption of repressive regulatory domains offers a therapeutic strategy for treating β-hemoglobinopathies. Here, we used transformer base editor (tBE), a recently developed cytosine base editor with no detectable off-target mutations, to disrupt transcription-factor-binding motifs in hematopoietic stem cells. By performing functional screening of six motifs with tBE, we found that directly disrupting the BCL11A-binding motif in HBG1/2 promoters triggered the highest γ-globin expression. Via a side-by-side comparison with other clinical and preclinical strategies using Cas9 nuclease or conventional BEs (ABE8e and hA3A-BE3), we found that tBE-mediated disruption of the BCL11A-binding motif at the HBG1/2 promoters triggered the highest fetal hemoglobin in healthy and β-thalassemia patient hematopoietic stem/progenitor cells while exhibiting no detectable DNA or RNA off-target mutations. Durable therapeutic editing by tBE persisted in repopulating hematopoietic stem cells, demonstrating that tBE-mediated editing in HBG1/2 promoters is a safe and effective strategy for treating β-hemoglobinopathies.

Evaluation of some heart enzymes and Iron levels in β-thalassemia patients in Thi-Qar City, Iraq

Beta thalassemia is one of the most common hereditary diseases in the world caused by a deficiency of globin chains. Heart disease is one of the main complications of this disease as a result of excess iron deposition in the cardiac tissues. Total of 100 patients of 2-18 years diagnosed with ß-TM were employed in the current study and admitted to Thi-Qar Center of Hereditary Blood Diseases in Thi-Qar city, Iraq, and 80 healthy participants, matched by age, and geographical area were adopted as control group. The current study included evaluating of serum Troponin I (c.TnI), Creatine kinase-MB isoenzyme (CK-MB), Apelin, aspartate aminotransferase (AST), and Lactate Dehydrogenase enzyme (LDH) of studied groups. The finding revealed a significant increase (p&lt;0.01) of c.TnI, CK-MB, AST, and LDH levels as well as a significant decrease (p&lt;0.01) in apelin level in all patients with ß-TM compared to the control group. Pearson's correlation coefficient (r) was also found between the biochemical parameters studied for ß-TM patients with ferritin level, were found a significant correlation (p&lt;0.01) between ferritin level with CK-MB, LDH, and AST levels while there was no significant correlation (P&gt;0.01) through apelin and c.TnI levels. The finding showed a clinical predictor to damage cardiac tissues in the near term, which portends the use of more efficient treatment protocols to remove excess iron from ß-TM patients.

Effect of hydroxyurea on SP1, LIN28B, IGF2BP3, COL4A5, BCL2, gamma globin genes expression: an in vitro study

BackgroundIn some β-thalassemia intermedia patients, hydroxyurea (HU) increases hemoglobin and HbF levels. However, HUs’ effects molecular mechanism is still unclear.MethodsIn this study, a weighted gene co-expression network analysis was conducted on the GSE109186 dataset. The genes LIN28A, COL4A5, SP1, BCL2, and IGF2BP3 were identified as hub genes involved in the γ-gene switching process. The effect of HU treatment at doses of 50, 100, and 150 μM for 12, 24, and 48 h on the K562 cell line was examined by using qRT-PCR to measure the expression levels of these hub genes.ResultsSP1 gene expression decreased after treatment with 50, 100, and 150 μM HU for 12, 24, and 48 h. The expression of the LIN28A gene was tripled at three concentrations of 50, 100, and 150 μM for 12 h. The IGF2BP3 gene expression was doubled after 24 and 48 h at a concentration of 150 μM HU. Regarding COL4A5 gene expression, except at 12 h after treatment at a concentration of 50 μM, a significant increase was observed in other concentrations and times. The BCL2 gene expression pattern at all concentrations decreased significantly after 12 h. The γ gene showed a significant increase compared to the control group after 24 and 48 h at the different concentrations.ConclusionThe results showed that in HU-treated cells, changes in the expression of LIN28A, COL4A5, SP1, and IGF2BP3 genes were accompanied by an increase in γ-gene expression. By elucidating precisely the mechanism of γ-to-β gene switching, we can hope for less complicated drugs.

Gene Therapy of Transfusion-Dependent β-Thalassemia Patients with Quick Engraftment of Reinfused Hematopoietic Stem Cells: An Investigator-Initiated Trial of KL003

Introduction In recent years, autologous hematopoietic stem cell (HSC)-based gene therapy has emerged as one of the most promising curative therapies for the treatment of severe hereditary anemia, including β-thalassemia major. Although several clinical trials of gene therapy for β-thalassemia major patients have been completed in developed countries, clinical data of gene therapy remain scarce for patients in developing countries. These patients are often in poor clinical condition because of severe iron overload and insufficient blood transfusion due to limited blood supply. They account for the majority of the β-thalassemia major patients in developing countries. Unlike in developed countries, most β-thalassemia major patients in developing countries cannot survive to adulthood as a consequence of irregular treatments due to the limitation of medical facilities and/or personal financial conditions. Methods We conducted a non-randomized, single-dose, open-label investigator-initiated clinical trial (ChiCTR2200055565) in representative patients in southern China, including those with ferritin above 5000 µg/L. Among the 11 β-thalassemia major patients (aged 6-22) we enrolled, 6 of the patients were in poor clinical conditions (e.g., medium (3000-5000 µg/L ferritin) to severe (&amp;gt;5000 µg/L ferritin) iron overload) due to inconsistent blood transfusions and iron chelation therapy before enrollment (Table). We treated patients using their own HSCs after transduced ex vivo with KL003, a lentiviral vector expressing a functional β-globin gene. We then monitored patients closely for safety and evaluated efficacy of KL003, including stem cell engraftment, hematopoietic reconstitution/recovery, and blood transfusion requirement. Results Blood transfusion was no longer needed for all 11 patients from median day 14 (range: 10-30 days) after treatment. The longest duration of no blood transfusion has been 18 months. The median engraftment time was 14 days for neutrophils (range: 8-15 days) and 14 days (range: 11-33 days) for platelets. This is a significant improvement compared with much longer engraftment time from previous autologous HSC-based gene therapies in the literature, which were around 4 weeks for neutrophils and 6 weeks for platelets. It is worth noting that the platelet engraftment time (33 days) for patient No. 07, who had a severe iron overload, was much longer than others (Table). This may represent a common challenge for treating patients with severe iron overload. The safety profile was consistent with that typical of busulfan-based myeloablation conditioning. There were no severe adverse events associated with KL003. Conclusion Our clinical trial results demonstrate potentially curative results in β-thalassemia major patients, including those in poor clinical condition. In addition, the optimized transduction process of HSCs may have contributed to maintaining stemness of HSCs, which is essential for quick engraftment and often gets lost during ex vivo manipulation. This has led to significantly faster engraftment and better efficacy. Thus, KL003 could be a potential functional cure for β-thalassemia major patients.