Search Results (74)

Background/Objectives: Red blood cell (RBC) parameters are routinely used to screen for α- and β-thalassemia traits as part of prenatal diagnosis for severe fetal thalassemia in countries with a high prevalence of the disease. In clinical practice, the same cut-off values for these parameters are applied to both females and males. However, given that the normal reference ranges for some RBC parameters differ significantly between sexes, sex-specific cut-off values may be more appropriate, especially in combination. To date, the effectiveness of RBC indices in males for predicting α- and β-thalassemia traits has not been evaluated. The objectives of this study are to assess the diagnostic performance of individual and combined RBC parameters in detecting α⁰-thalassemia traits among non-anemic males. Methods: This diagnostic study is a secondary analysis of prospectively collected data from our project on prenatal control of severe thalassemia. The study population comprised male partners of pregnant women who underwent thalassemia screening during their first antenatal visit. RBC parameters, including hemoglobin (Hb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW), and RBC count, were measured for each participant. Carrier status for the α0-thalassemia Southeast Asian (SEA) genotype was confirmed by using a polymerase chain reaction (PCR)-based method. The diagnostic performance of each RBC parameter and their combinations, based on predictive models generated using logistic regression, was evaluated and compared using receiver operating characteristic (ROC) curves. Results: A total of 486 Thai males were recruited for the study, including 137 individuals with the α⁰-thalassemia trait and 349 with a normal α-thalassemia genotype (control group). All RBC parameters, except for Hct, differed significantly between the two groups. Among the individual indices, MCH exhibited the highest diagnostic accuracy, followed by MCV, with areas under the curve (AUCs) of 0.981 and 0.973, respectively. An MCH cut-off value of 26 pg and an MCV cut-off value of 80 fL provided a sensitivity of 100% for both indices, with specificities of 88.5% and 86.8%, respectively. The combination predictive model provided the best diagnostic performance, achieving an AUC of 0.987, which was slightly but significantly higher than that of any individual parameter. This model yielded a sensitivity of 100% and a significantly higher specificity of 90.8% at a cut-off probability of 7.0%. Conclusions: MCH and MCV demonstrated excellent screening performance for identifying α0-thalassemia carriers in males. However, the combination model exhibited even greater accuracy while reducing the false-positive rate. Implementing this model could minimize the need for unnecessary PCR testing, leading to substantial cost savings. Full article

Beta-hemoglobinopathies such as beta-thalassemia and sickle cell disease are severe genetic blood disorders affecting the beta globin chain of haemoglobin A (α2β2). Activation of delta globin, the non-alpha globin of HbA2 (α2δ2), could represent a possible approach to improve the clinical severity of these pathologies. Notably, the therapeutic potential of delta globin has been demonstrated in previous studies using a mouse model of beta-thalassemia and sickle cell disease. The present study evaluated delta globin gene activation by small molecules in erythroid cells isolated from transgenic murine foetal liver. A screening of 119 molecules, selected for their potential in drug repurposing, was performed without prior selection based on specific pathways of interest. Three candidates—Nexturastat, Stattic and Palbociclib—were found to have high efficacy on delta globin expression. Palbociclib also proved effective in increasing gamma globin expression. All of these compounds have pharmacokinetic profiles that are beneficial for clinical application, providing potential inducer agents of HbA2 that could have therapeutic effects in the treatment of beta-hemoglobinopathies. Full article

β-globin gene cluster regulation involves complex mechanisms to ensure proper expression and function in RBCs. During development, switching occurs as γ-globin is replaced by β-globin. Key regulators, like BCL11A and ZBTB7A, repress γ-globin expression to facilitate this transition with other factors, like KLF1, LSD1, and PGC-1α; these regulators ensure an orchestrated transition from γ- to β-globin during development. While these mechanisms have been extensively studied, circRNAs have recently emerged as key contributors to gene regulation, but their role in β-globin gene cluster regulation remains largely unexplored. Although discovered in the 1970s, circRNAs have only recently been recognized for their functional roles, particularly in interactions with RNA-binding proteins. Understanding how circRNAs contribute to switching from γ- to β-globin could lead to new therapeutic strategies for hemoglobinopathies, such as sickle cell disease and β-thalassemia. This review uses the circAtlas 3.0 database to explore circRNA expressions in genes related to switching from γ- to β-globin expression, focusing on blood, bone marrow, liver, and spleen. It emphasizes the exploration of the potential interactions between circRNAs and RNA-binding proteins involved in β-globin gene cluster regulatory mechanisms, further enhancing our understanding of β-globin gene cluster expression. Full article

β-thalassemia, a hereditary hemoglobinopathy, is caused by reduced or absent synthesis of the β-globin chains of hemoglobin. Three clinical conditions are recognized: β-thalassemia major, β-thalassemia intermedia, and β-thalassemia minor (β-Thal⁺). This latter condition occurs when an individual inherits a mutated β-globin gene from one parent. In erythrocytes from β-Thal⁺ subjects, the excess α-globin chains produce unstable α-tetramers, which can induce substantial oxidative stress leading to plasma membrane and cytoskeleton damage, as well as deranged cellular function. In the present study, we hypothesized that increased oxidative stress might lead to structural rearrangements in erythrocytes from β-Thal⁺ volunteers and functional alterations of ion transport proteins, including band 3 protein. The data obtained showed significant modifications of the cellular shape in erythrocytes from β-Thal⁺ subjects. In particular, a significantly increased number of elliptocytes was observed. Interestingly, iron overload, detected in erythrocytes from β-Thal⁺ subjects, provoked a significant production of reactive oxygen species (ROS), overactivation of the endogenous antioxidant enzymes catalase and superoxide dismutase, and glutathione depletion, resulting in (a) increased lipid peroxidation, (b) protein sulfhydryl group (-SH) oxidation. Iron overload-related oxidative stress affected Na⁺/K⁺-ATPase activity, which in turn may have contributed to impaired β-Thal⁺ erythrocyte deformability. As a result, alterations in the distribution of cytoskeletal proteins, including α/β-spectrin, protein 4.1, and α-actin, in erythrocytes from β-Thal⁺ subjects have been detected. Significantly, oxidative stress was also associated with increased phosphorylation and altered band 3 ion transport activity, as well as increased oxidized hemoglobin, which led to abnormal clustering and redistribution of band 3 on the plasma membrane. Taken together, these findings contribute to elucidating potential oxidative stress-related perturbations of ion transporters and associated cytoskeletal proteins, which may affect erythrocyte and systemic homeostasis in β-Thal⁺ subjects. Full article

Hemoglobin is an oxygen-transport protein in red blood cells that interacts with multiple ligands, e.g., oxygen, carbon dioxide, carbon monoxide, and nitric oxide. Genetic variations in hemoglobin chains, such as those underlying sickle cell disease and thalassemias, present substantial clinical challenges. Here, we review the progress in research, including the use of allosteric modulators, pharmacological chaperones, and antioxidant treatments, which has begun to improve hemoglobin stability and oxygen affinity. According to UniProt (as of 7 August 2024), 819 variants of the α-hemoglobin subunit and 771 variants of the β-hemoglobin subunit have been documented, with over 116 classified as unstable. These data demonstrate the urgent need to develop variant-specific stabilizing options. Beyond small-molecule drugs/binders, novel protein-based strategies—such as engineered hemoglobin-binding proteins (including falcilysin, llama-derived nanobodies, and α-hemoglobin-stabilizing proteins)—offer promising new options. As our understanding of hemoglobin’s structural and functional diversity grows, so does the potential for genotype-driven approaches. Continued research into hemoglobin stabilization and ligand-binding modification may yield more precise, effective treatments and pave the way toward effective strategies for hemoglobinopathies. Full article

Thalassemias and hemoglobinopathies are among the most common genetic diseases worldwide and have a significant impact on public health. The decreasing cost of next-generation sequencing (NGS) has quickly enabled the development of new assays that allow for the simultaneous analysis of small nucleotide variants (SNVs) and copy number variants (CNVs) as deletions/duplications of α- and β-globin genes. Background/Objectives: This study highlighted the efficacy and rapid identification of all types of mutations in the α- and β-globin genes, including silent variants, using the Devyser Thalassemia NGS kit. Furthermore, we report the frequency of mutations identified in a total population of 2649 individuals recruited from four Italian Medical Genetics Laboratories. Methods: All samples were first hematologically characterized, and sequence analysis was conducted by using the Devyser Thalassemia NGS kit. All variants were also validated in an independent sample by a conventional molecular test. Results: A total of 1789 subjects were identified with genetic variants in the globin genes, of which 966 (53.9%) had variations in the β-gene, 480 (26.8%) had variations in the α-gene; and 307 (17.1%) had variations in both α- and β-genes. Variant analysis evidenced a heterogeneous mutation spectrum enriched with variants not usually observed in the Italian population. Conclusions: This study showed the high effectiveness and the rapid identification of all mutation types in both α- and β-globin genes, including silent variants. It should be emphasized that the NGS approach greatly shortens turnaround reporting times, overcoming the classic diagnostic flowchart which envisages multistep, subsequent, diagnostic approaches, often requiring long resolution times. Full article

Hemoglobinopathies, namely β-thalassemia and sickle cell disease (SCD), are hereditary diseases, characterized by molecular genetic aberrations in the beta chains of hemoglobin. These defects affect the normal production of hemoglobin with severe anemia due to less or no amount of beta globins in patients with β-thalassemia (quantitative disorder), while SCD is a serious disease in which a mutated form of hemoglobin distorts the red blood cells into a crescent shape at low oxygen levels (qualitative disorder). Despite the revolutionary progress in recent years with the approval of gene therapy and gene editing for specific patients, there is an unmet need for highlighting the mechanisms influencing hemoglobin production and for the development of novel drugs and targeted therapies. The identification of the transcription factors and other genetic modifiers of hemoglobin expression is of utmost importance for discovering novel therapeutic approaches for patients with hemoglobinopathies. The aim of this review is to describe these complex molecular mechanisms and pathways affecting hemoglobin expression and to highlight the relevant investigational approaches or pharmaceutical interventions focusing on restoring the hemoglobin normal function by linking the molecular background of the disease with the clinical perspective. All the associated drugs increasing the hemoglobin expression in patients with hemoglobinopathies, along with gene therapy and gene editing, are also discussed. Full article

Hemoglobin H/Constant Spring (Hb H/CS) disease represents a form of non-deletional Hb H disease characterized by chronic hemolytic anemia that ranges from moderate to severe and may lead to transfusion-dependent thalassemia. To study the underlying mechanisms of this disease, we conducted an analysis of erythropoiesis and gene expression in erythroid progenitor cells derived from CD34+ hematopoietic stem/progenitor cells from patients with Hb H/CS disease and normal controls. Twelve patients with Hb H/CS disease and five normal controls were enrolled. Peripheral blood samples were collected to isolate CD34+ hematopoietic stem/progenitor cells for the analysis of cell proliferation and differentiation. Six samples from patients with Hb H/CS disease and three controls were subsequently studied for gene expression by next generation sequencing analysis. Erythroid progenitor cells derived from patients with Hb H/CS disease exhibited a trend towards increased rates of erythroid proliferation and decreased cell viability compared to those from controls. Moreover, erythroid progenitor cells derived from patients with Hb H/CS disease demonstrated delayed terminal differentiation. Gene expression profiling revealed elevated levels of genes encoding molecular chaperones, including the heat shock protein genes (HSPs) and the chaperonin containing TCP-1 subunit genes (CCTs) in the Hb H/CS disease group. In summary, erythroid progenitor cells derived from patients with Hb H/CS disease exhibit a trend towards heightened erythroid proliferation, diminished cell viability, and delayed terminal differentiation. Additionally, the increased expression of genes encoding molecular chaperones was observed, providing information on potential underlying pathophysiological mechanisms. Full article

Thalassemia represents a diverse group of inherited hematological disorders characterized by defective globin chain synthesis, leading to chronic anemia and associated complications. The complicated pathophysiology of beta-thalassemia involves genetic mutations or rarely deletions of the beta-globin gene on chromosome 11 whereas alpha-thalassemia involves deletions in the HBA1 and HBA2 genes or occasionally alterations to the DNA sequence in or around these genes. These mutation and deletion effects disrupt the balance of α/β-globin chain production, resulting in ineffective erythropoiesis, hemolysis, and a cascade of clinical manifestations including anemia, bone deformities, and iron overload. Advances in diagnostic techniques have enhanced our ability to detect and characterize these mutations, facilitating early and accurate diagnoses. Current management strategies encompass regular blood transfusions, the use of hydroxyurea to improve hemoglobin levels, and iron chelation therapy to prevent iron-related organ damage. Moreover, other therapeutics such as thalidomide for those not responding to hydroxyurea, Sirolimus for patients with immunodeficiencies, and use of vitamin E as an antioxidant have proven to be effective. Innovative therapies such as gene therapy and bone marrow transplantation offer promising curative potential, opening a new era in the treatment of thalassemia. This review focuses on pathophysiological mechanisms underlying thalassemia, explores the diagnostic methodologies, and highlights recent advancements in therapeutic approaches. Full article

Hemoglobin Shaare Zedek (Hb SZ) is a rare structural α-Hb variant. Characterizing its genotype–phenotype relationship and genetic origin enhances diagnostic and clinical management insights. We studied a proband and six family members using high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), PCR, and sequencing to analyze α- and β-globin genes and α-globin haplotypes. Pathogenicity predictions and a rapid diagnostic method were developed. The proband, his father, grandfather, and aunt had Hb migrating to the HbH-zone on CE and elevated fetal hemoglobin (HbF) on HPLC. Direct sequencing identified an A to G mutation at codon 56 of the α2-globin gene, characteristic of Hb SZ. Additionally, the proband carried a β-globin gene mutation [HBB.52A>T]. Mild thalassemia-like changes were observed in the proband, whereas individuals with only the Hb SZ variant did not exhibit these changes. Pathogenicity predictions indicated that Hb SZ is benign. The variant can be identified using restriction fragment length polymorphism (RFLP) and allele-specific PCR. The Thai variant of Hb SZ is associated with the haplotype [- - M - - - -]. Hb SZ is a non-pathological variant that minimally affects red blood cell parameters, even when it coexists with β⁰-thalassemia. HPLC and CE systems cannot distinguish it from other Hbs, necessitating DNA analysis for accurate diagnosis. Full article

Introduction: The NPRL3 gene is a critical component of the GATOR1 complex, which negatively regulates the mTORC1 pathway, essential for neurogenesis and brain development. Located on chromosome 16p13.3, NPRL3 is situated near the α-globin gene cluster. Haploinsufficiency of NPRL3, either by deletion or a pathogenic variant, is associated with a variable phenotype of focal epilepsy, with or without malformations of cortical development, with known decreased penetrance. Case Description: This work details the diagnostic odyssey of a neurotypical 10-year-old boy who presented at age 2 with unusual nocturnal episodes and a history of microcytic anemia, as well as a review of the existing literature on NPRL3-related epilepsy, with an emphasis on individuals with deletions who also present with α-thalassemia trait. The proband’s episodes were mistaken for gastroesophageal reflux disease for several years. He had molecular testing for his α-thalassemia trait and was noted to carry a deletion encompassing the regulatory region of the α-thalassemia gene cluster. Following the onset of overt focal motor seizures, genetic testing revealed a heterozygous loss of NPRL3, within a 106 kb microdeletion on chromosome 16p13.3, inherited from his mother. This deletion encompassed the entire NPRL3 gene, which overlaps the regulatory region of the α-globin gene cluster, giving him the dual diagnosis of NPRL3-related epilepsy and α-thalassemia trait. Brain imaging postprocessing showed left hippocampal sclerosis and mid-posterior para-hippocampal focal cortical dysplasia, leading to the consideration of epilepsy surgery. Conclusions: This case underscores the necessity of early and comprehensive genetic assessments in children with epilepsy accompanied by systemic features, even in the absence of a family history of epilepsy or a developmental delay. Recognizing phenotypic overlaps is crucial to avoid diagnostic delays. Our findings also highlight the impact of disruptions in regulatory regions in genetic disorders: any individual with full gene deletion of NPRL3 would have, at a minimum, α-thalassemia trait, due to the presence of the major regulatory element of α-globin genes overlapping the gene’s introns. Full article

The blood counts of α thalassemia carriers (α-thal) are similar to those of β thalassemia carriers, except for Hemoglobin A₂ (Hb A₂), which is not elevated. The objective of this study was to determine whether mathematical formulas are effective for detecting suspected α-thal. The data were obtained from the database of the prevention program for detecting couples at risk for having a child with hemoglobinopathy. Red Blood Cells (RBC) indices were analyzed using mathematical formulas, and the sensitivity and negative predictive value (NPV) were calculated. Among 1334 blood counts suspected of α-thal analyzed, only the Shine and Lal and the Support Vector Machine formulas revealed high sensitivity and NPV. Sensitivity was 85.54 and 99.33%, and NPV was 98.93 and 99.93%, respectively. Molecular defects were found in 291, and 81 had normal α genes. Molecular analysis was not performed in 962 of the samples. Based on these results, mathematical formulas incorporating one of these reliable formulas for detecting suspected α or β thalassemia carriers in the program of the automatic analyzers can flag these results, increase the awareness of the primary physicians about the carrier risk, and send an alert with a recommendation for further testing. Full article

The mutation HBA2:c.*94A>G (AATAAA>AATAAG; rs63751269) is a 3′-UTR (3 prime untranslated region) single-nucleotide substitution in the polyadenylation (PA) signal of HBA2 (α^PA:A→G). This pathogenic (CADD score, 14.92) variant is sporadic in the Arabian Peninsula. It results in inefficient mRNA processing, transcription termination, and possibly using an alternate cryptic downstream polyadenylation signal. As a result, the allele α^T (or α^T-Saudi) poses challenges in premarital counseling with respect to fetal risk of hemoglobin H disease. Homozygous HBA2:c.*94A>G (α^Tα/α^Tα) results in moderate-to-severe microcytosis (mean red cell volume, MCV, 55 to 65 fL), reflecting markedly impaired hemoglobin synthesis (hemoglobin H disease). Homozygous rightward −α^3.7 (a 3804-neocleotide deletion allele, NM_000517.4:c.[-2_-3delAC; −α^3.7]), on the other hand, results in mild microcytosis (MCV, 70 to 75 fL, alpha-thalassemia trait). Thus, HBA2:c.*94A>G is more damaging than −α^3.7. Consistently, the value of MCV in compound heterozygosity, HBA2:c.*94A>G and −α^3.7, is 65 to 70 fL. We report here a healthy couple who presented for premarital counseling on their hemoglobinopathy. The man has homozygous HBA2:c.*94A>G (α^Tα/α^Tα), and the woman has compound heterozygous (−α^3.7/α^Tα, also annotated as: ^−3.7α/α^Tα). As a result, the genotype of their offspring would be that of the father (α^Tα/α^Tα) or the mother (−α^3.7/α^Tα). The counseling was mainly based on the benign phenotypes of the parents. As both were asymptomatic and their anemia was clinically insignificant, they proceeded with the marriage. Full article

The β-thalassemias are inherited genetic disorders affecting the hematopoietic system. In β-thalassemias, more than 350 mutations of the adult β-globin gene cause the low or absent production of adult hemoglobin (HbA). A clinical parameter affecting the physiology of erythroid cells is the excess of free α-globin. Possible experimental strategies for a reduction in excess free α-globin chains in β-thalassemia are CRISPR-Cas9-based genome editing of the β-globin gene, forcing “de novo” HbA production and fetal hemoglobin (HbF) induction. In addition, a reduction in excess free α-globin chains in β-thalassemia can be achieved by induction of the autophagic process. This process is regulated by the Unc-51-like kinase 1 (Ulk1) gene. The interplay with the PI3K/Akt/TOR pathway, with the activity of the α-globin stabilizing protein (AHSP) and the involvement of microRNAs in autophagy and Ulk1 gene expression, is presented and discussed in the context of identifying novel biomarkers and potential therapeutic targets for β-thalassemia. Full article

Background/Objectives: in β-thalassemia, important clinical complications are caused by the presence of free α-globin chains in the erythroid cells of β-thalassemia patients. These free α-globin chains are present in excess as a result of the lack of β-globin chains to bind with; they tend to aggregate and precipitate, causing deleterious effects and overall cytotoxicity, maturation arrest of the erythroid cells and, ultimately, ineffective erythropoiesis. The chaperone protein α-hemoglobin-stabilizing protein (AHSP) reversibly binds with free α-globin; the resulting AHSP-αHb complex prevents aggregation and precipitation. Sirolimus (rapamycin) has been previously demonstrated to induce expression of fetal hemoglobin and decrease the excess of free α-globin chain in the erythroid cells of β-thalassemia patients. The objective of this study was to verify whether sirolimus is also able to upregulate AHSP expression in erythroid precursor cells (ErPCs) isolated from β-thalassemia patients. Methods: the expression of AHSP genes was analyzed by measuring the AHSP mRNA content by real-time quantitative PCR (RT-qPCR) and the AHSP protein production by Western blotting. Results: AHSP gene expression was found to be higher in ErPCs of β-thalassemia patients in comparison to ErPCs isolated from healthy subjects. In addition, AHSP expression was further induced by treatment of β-thalassemia ErPCs with sirolimus. Finally, AHSP mRNA was expressed at an increased level in ErPCs of sirolimus-treated β-thalassemia patients participating in the NCT03877809 Sirthalaclin clinical trial. Conclusions: this exploratory study suggests that AHSP expression should be considered as an endpoint in clinical trials based on sirolimus. Full article