Search Results (55)

Sickle cell disease (SCD) is the most common hemoglobinopathy, caused by a mutation in the β-globin gene of hemoglobin. It predisposes patients to painful Vaso-occlusive crises (VOC) and multi-organ dysfunctions. The disease exhibits significant phenotypic variability, making it challenging to predict severity and outcomes. This study aimed to characterize the whole blood gene expression profile of Bahraini SCD patients, identifying differentially expressed genes during steady-state (n = 10) and VOC (n = 10) compared to healthy controls (n = 8). Analysis revealed 2073 and 3363 dysregulated genes during steady-state and VOC, respectively, compared to controls, with 1078 genes differentially expressed during VOC versus steady-state. Gene Ontology (GO) enrichment analysis highlighted significant deregulation in immune and hematopoietic pathways, including down-regulation of critical genes for immune modulation and hematopoietic balance. Notably, the transcription factor RUNX3, involved in immune cell differentiation and inflammation, was among the 668 down-regulated genes. RUNX3 was four-fold down-regulated in microarray analysis, three-fold in PCR, and showed a mean protein concentration of 11.13 pg/mL during VOC compared to 457.93 pg/mL during steady-state (p < 0.01). These findings suggest that RUNX3 may serve as a potential biomarker for VOC. Future large-scale validation, additional proteomic studies, and functional investigations are recommended to confirm its clinical utility and significance. 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

Elevated fetal hemoglobin (HbF), which is partly controlled by genetic modifiers, ameliorates disease severity in β hemoglobinopathies. Understanding the genetic basis of this trait holds great promise for personalized therapeutic approaches. PubMed, MedRxiv, and the GWAS Catalog were searched up to May 2024 to identify eligible GWAS studies following PRISMA guidelines. Four independent reviewers screened, extracted, and synthesized data using narrative and descriptive methods. Study quality was assessed using a modified version of the Q-Genie tool. Pathway enrichment analysis was conducted on gene lists derived from the selected GWAS studies. Out of 113 initially screened studies, 62 underwent full-text review, and 16 met the inclusion criteria for quality assessment and data synthesis. A total of 939 significant SNP-trait associations (p-value < 1 × 10⁻⁵) were identified, mapping to 133 genes (23 with overlapping variant positions) and 103 intergenic sequences. Most SNP-trait associations converged around BCL11A (chr.2), HBS1L-MYB, (chr.6), olfactory receptor and beta globin (HBB) gene clusters (chr.11), with less frequent loci including FHIT (chr.3), ALDH8A1, BACH2, RPS6KA2, SGK1 (chr.6), JAZF1 (chr.7), MMP26 (chr.11), COCH (chr.14), ABCC1 (chr.16), CTC1, PFAS (chr.17), GCDH, KLF1, NFIX, and ZBTB7A (chr.19). Pathway analysis highlighted Gene Ontology (GO) terms and pathways related to olfaction, hemoglobin and haptoglobin binding, and oxygen carrier activity. This systematic review confirms established genetic modifiers of HbF level, while highlighting less frequently associated loci as promising areas for further research. Expanding research across ethnic populations is essential for advancing personalized therapies and enhancing outcomes for individuals with sickle cell disease or β-thalassemia. 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

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

In recent years, clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) protein have emerged as a revolutionary gene editing tool to treat inherited disorders affecting different organ systems, such as blood and muscles. Both hematological and neuromuscular genetic disorders benefit from genome editing approaches but face different challenges in their clinical translation. The ability of CRISPR/Cas9 technologies to modify hematopoietic stem cells ex vivo has greatly accelerated the development of genetic therapies for blood disorders. In the last decade, many clinical trials were initiated and are now delivering encouraging results. The recent FDA approval of Casgevy, the first CRISPR/Cas9-based drug for severe sickle cell disease and transfusion-dependent β-thalassemia, represents a significant milestone in the field and highlights the great potential of this technology. Similar preclinical efforts are currently expanding CRISPR therapies to other hematologic disorders such as primary immunodeficiencies. In the neuromuscular field, the versatility of CRISPR/Cas9 has been instrumental for the generation of new cellular and animal models of Duchenne muscular dystrophy (DMD), offering innovative platforms to speed up preclinical development of therapeutic solutions. Several corrective interventions have been proposed to genetically restore dystrophin production using the CRISPR toolbox and have demonstrated promising results in different DMD animal models. Although these advances represent a significant step forward to the clinical translation of CRISPR/Cas9 therapies to DMD, there are still many hurdles to overcome, such as in vivo delivery methods associated with high viral vector doses, together with safety and immunological concerns. Collectively, the results obtained in the hematological and neuromuscular fields emphasize the transformative impact of CRISPR/Cas9 for patients affected by these debilitating conditions. As each field suffers from different and specific challenges, the clinical translation of CRISPR therapies may progress differentially depending on the genetic disorder. Ongoing investigations and clinical trials will address risks and limitations of these therapies, including long-term efficacy, potential genotoxicity, and adverse immune reactions. This review provides insights into the diverse applications of CRISPR-based technologies in both preclinical and clinical settings for monogenic blood disorders and muscular dystrophy and compare advances in both fields while highlighting current trends, difficulties, and challenges to overcome. Full article

In this short review, we presented and discussed studies on the expression of globin genes in β-thalassemia, focusing on the impact of α-globin gene expression and α-globin modifiers on the phenotype and clinical severity of β-thalassemia. We first discussed the impact of the excess of free α-globin on the phenotype of β-thalassemia. We then reviewed studies focusing on the expression of α-globin-stabilizing protein (AHSP), as a potential strategy of counteracting the effects of the excess of free α-globin on erythroid cells. Alternative processes controlling α-globin excess were also considered, including the activation of autophagy by β-thalassemia erythroid cells. Altogether, the studies reviewed herein are expected to have a potential impact on the management of patients with β-thalassemia and other hemoglobinopathies for which reduction in α-globin excess is clinically beneficial. Full article

Hemoglobinopathies are monogenic disorders affecting hemoglobin synthesis. Thalassemia and sickle cell disease (SCD) are considered the two major hemoglobinopathies. Thalassemia is a genetic disorder and one of the major hemoglobinopathies determined by an impairment of globin chain production, which causes an alteration of erythropoiesis, an improvement in hemolysis, and an alteration of iron homoeostasis. In SCD, the mutations are on the β-globin chain of hemoglobin which results in a substitution of glutamic acid by valine with consequent formation of Hemoglobin S (HbS). Several factors are involved in bone metabolism alteration in patients with hemoglobinopathies, among them hormonal deficiency, bone marrow hyperplasia, iron overload, inflammation, and increased bone turnover. Bone metabolism is the result of balance maintenance between bone deposition and bone resorption, by osteoblasts (OBs) and osteoclasts (OCs). An impairment of this balance is responsible for the onset of bone diseases, such as osteoporosis (OP). Therefore, here we will discuss the alteration of bone metabolism in patients with hemoglobinopathies and the possible therapeutic strategies to contain and/or counteract bone health impairment in these patients, taking into consideration not only the pharmacological treatments already used in the clinical armamentarium, but also the new possible therapeutic strategies. Full article

Therapy via the gene addition of the anti-sickling β^AS3-globin transgene is potentially curative for all β-hemoglobinopathies and therefore of particular clinical and commercial interest. This study investigates GLOBE-based lentiviral vectors (LVs) for β^AS3-globin addition and evaluates strategies for an increased β-like globin expression without vector dose escalation. First, we report the development of a GLOBE-derived LV, GLV2-βAS3, which, compared to its parental vector, adds anti-sickling action and a transcription-enhancing 848-bp transcription terminator element, retains high vector titers and allows for superior β-like globin expression in primary patient-derived hematopoietic stem and progenitor cells (HSPCs). Second, prompted by our previous correction of HBB^{IVSI−110(G>A)} thalassemia based on RNApol(III)-driven shRNAs in mono- and combination therapy, we analyzed a series of novel LVs for the RNApol(II)-driven constitutive or late-erythroid expression of HBB^{IVSI−110(G>A)}-specific miRNA30-embedded shRNAs (shRNAmiR). This included bifunctional LVs, allowing for concurrent β^AS3-globin expression. LVs were initially compared for their ability to achieve high β-like globin expression in HBB^{IVSI−110(G>A)}-transgenic cells, before the evaluation of shortlisted candidate LVs in HBB^{IVSI−110(G>A)}-homozygous HSPCs. The latter revealed that β-globin promoter-driven designs for monotherapy with HBB^{IVSI−110(G>A)}-specific shRNAmiRs only marginally increased β-globin levels compared to untransduced cells, whereas bifunctional LVs combining miR30-shRNA with β^AS3-globin expression showed disease correction similar to that achieved by the parental GLV2-βAS3 vector. Our results establish the feasibility of high titers for LVs containing the full HBB transcription terminator, emphasize the importance of the HBB terminator for the high-level expression of HBB-like transgenes, qualify the therapeutic utility of late-erythroid HBB^{IVSI−110(G>A)}-specific miR30-shRNA expression and highlight the exceptional potential of GLV2-βAS3 for the treatment of severe β-hemoglobinopathies. Full article

Hemoglobinopathies, including β-thalassemia and sickle cell disease (SCD), are common genetic blood disorders. Endocrine disorders are frequent manifestations of organ damage observed mainly in patients with β-thalassemia and rarely in SCD. Iron overload, oxidative stress-induced cellular damage, chronic anemia, and HCV infection contribute to the development of endocrinopathies in β-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 β-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. Full article