bims-conane Biomed News
on Congenital anemias
Issue of 2024–12–22
ten papers selected by
João Conrado Khouri dos Santos, Universidade de São Paulo
  1. Mutations in AMBRA1 aggravate β-thalassemia by impairing autophagy-mediated clearance of free α-globin.
  2. Reframing thalassaemia syndrome as a benign haematopoietic stem cell disorder.
  3. Hb Monza: A novel extensive HBB duplication with preserved α-β subunit interaction and unstable hemoglobin phenotype.
  4. DNA phenotyping and mapping intragenic deletion mutations in Fanconi anemia: Patterns and diagnostic inferences.
  5. Preclinical development of lentiviral vector gene therapy for Diamond-Blackfan anemia syndrome.
  6. Evaluation of Three Mutations in Codon 385 of Glucose-6-Phosphate Dehydrogenase via Biochemical and In Silico Analysis.
  7. New insights into the mechanisms of red blood cell enucleation: From basics to clinical applications.
  8. Luspatercept in β-thalassemia: Who and when. Strengths and weaknesses points of a real-world evidence.
  9. Diagnosis of Diamond-Blackfan anemia in adulthood: case series and review of the literature.
  10. Interplay Between Metabolic Pathways and Increased Oxidative Stress in Human Red Blood Cells.
  1. Blood. 2024 Dec 18. pii: blood.2023022688. [Epub ahead of print]
    Mutations in AMBRA1 aggravate β-thalassemia by impairing autophagy-mediated clearance of free α-globin.
    Yong Long, Qianqian Zhang, Ling Ling, Yuan Zhuang, Xiaolei Wei, Haoyang Huang, Zhanping Lu, Yushan Huang, Xianming Chen, Yuhua Ye, Xiaoqin Feng, Haokun Hao Zhang, Binbin Huang, Yueyan Huang, Yidan Liang, Mingyan Fang, Yukio Nakamura, Bin Lin, Xinhua Zhang, Daru Lu, Xin Jin, Xiangmin Xu.
      Accumulation of free α-globin is a critical factor in the pathogenesis of β-thalassemia. Autophagy plays a crucial role in clearing toxic free α-globin, thereby reducing disease severity. However, the impact of natural mutations in autophagy-related genes (ATGs) on the phenotypic variability of β-thalassemia remains unclear. In this study, we systematically investigated the relationship between variants in ATGs and disease phenotypes in a cohort of 1,022 patients with β-thalassemia, identifying four missense mutations in the autophagy and beclin 1 regulator 1 (AMBRA1) gene. Disruption of the Ambra1 gene in β-thalassemic mice was found to reduce autophagic clearance of α-globin in red blood cell precursors, exacerbating disease phenotypes. Functional characterization of the AMBRA1 gene and these mutations in patient-derived CD34+ cells, edited HUDEP-2 cells, and engineered HUDEP-2 β-thalassemic cells confirmed that AMBRA1 facilitates the autophagic clearance of free α-globin in human erythroid cells. Functional studies demonstrated that AMBRA1 missense mutants destabilize ULK1 protein, inhibit LC3 lipidation, and subsequently hinder autophagic flux, leading to increased α-globin deposition. Additionally, these mutations were associated with erythrotoxic effects in vitro, including increased intracellular reactive oxygen species levels, higher apoptosis rates, and impaired erythroid differentiation and maturation. This study sheds light on the molecular association between mutations in ATGs and the exacerbation of β-thalassemia, highlighting the potential role of the AMBRA1 gene as a promising diagnostic and therapeutic target for β-hemoglobinopathies.
    DOI:  https://doi.org/10.1182/blood.2023022688
  2. Br J Haematol. 2024 Dec 15.
    Reframing thalassaemia syndrome as a benign haematopoietic stem cell disorder.
    Aurelio Maggio, Mariasanta Napolitano, Ali T Taher, Rayan Bou-Fakhredin, Mariano Anibal Ostuni.
      Thalassaemia, caused by over 250 mutations in the beta globin gene, changes the haematopoietic stem cell (HSC) differentiation, leading to ineffective erythropoiesis. This Wider Perspective article overlooks its underlying nature as a benign HSC disorder with a significant impact on the erythroid cell lineage. The simplicity of managing symptoms through transfusions and iron chelation therapy has shifted the focus away from the development of cell-based treatments. The identification of the beta039 mutation by Chang and Kan in 1979 marked a turning point, suggesting as main approach the molecular level by correcting the beta globin chain imbalances through gene insertion and editing. However, challenges of technology have delayed the implementation of these strategies for over four decades. In contrast, the past two decades have witnessed significant advances in the treatment of HSC disorders of the myeloid clone which are driven by a 'target cell strategy'. Many current and innovative treatments for thalassaemia are now adopting this approach, highlighting the importance of identifying suitable candidates through risk stratification. This manuscript explores the evolving understanding of thalassaemia syndromes as congenital HSC disorders of the erythroid clone and examines the implications of this perspective for the development of future treatments.
    Keywords:  HSC disorder; haematopoietic stem cells; thalassaemia
    DOI:  https://doi.org/10.1111/bjh.19919
  3. Med. 2024 Dec 14. pii: S2666-6340(24)00448-3. [Epub ahead of print]
    Hb Monza: A novel extensive HBB duplication with preserved α-β subunit interaction and unstable hemoglobin phenotype.
    Ivan Civettini, Arianna Zappaterra, Paola Corti, Amedeo Messina, Andrea Aroldi, Andrea Biondi, Fabrizio Cavalca, Valentina Crippa, Francesca Crosti, Giulia Maria Ferrari, Federica Malighetti, Luca Mologni, Alberto Piperno, Daniele Ramazzotti, Chiara Scollo, Alfonso Zambon, Fabio Rossi, Carlo Gambacorti-Passerini.
       BACKGROUND: Unstable hemoglobins are caused by single amino acid substitutions in the HBB gene, often affecting key histidine residues, leading to protein destabilization and hemolytic crises. In contrast, long HBB variants, exceeding 20 bp, are rare and associated with a β-thalassemia phenotype due to disrupted α-β chain interactions. We describe a family wherein four of six members carry a novel 23-amino-acid in-frame duplication of HBB (c.176_244dup), named hemoglobin (Hb) Monza. Despite its length, this duplication manifests as an unstable hemoglobin variant rather than a β-thalassemia phenotype.
    METHODS: A static 3D model of the Hb Monza β chain was generated using AlphaFold and SWISS-MODEL. Molecular dynamics (MD) simulations were performed with the Generalized Born implicit solvent model. After energy minimization and heating to 311 K (38°C), a 40 ns production run was conducted.
    FINDINGS: 3D modeling of Hb Monza revealed minimal structural changes in the Hb β chain, particularly in the key histidine residues and their interaction with the iron atom. Additionally, the static 3D model showed a preserved α-β interaction, explaining the absence of a β-thalassemia clinical phenotype. MD simulations under thermal stress revealed a notable increase in root-mean-square deviation compared to the wild-type β subunit, along with a loss of contacts with the heme, explaining the hemolytic crises during febrile episodes.
    CONCLUSION: Despite the long duplication in HBB, Hb Monza retains functional α-β interaction while demonstrating instability under stressful conditions. This unique variant presents with an unstable Hb phenotype rather than a β-thalassemia phenotype.
    FUNDING: No financial funding was received.
    Keywords:  3D protein; AlphaFold; HBB; Hb Monza; Translation to patients; beta-thalassemia; hemoglobin; hemoglobin Monza; hemolyitic anemia; molecular dynamics; unstable hemoglobin
    DOI:  https://doi.org/10.1016/j.medj.2024.11.007
  4. J Genet Eng Biotechnol. 2024 Dec;pii: S1687-157X(24)00138-0. [Epub ahead of print]22(4): 100435
    DNA phenotyping and mapping intragenic deletion mutations in Fanconi anemia: Patterns and diagnostic inferences.
    Rehab Mosaad, Ghada El-Kamah, Maha Eid, Khalda Amr.
       BACKGROUND: Fanconi anemia is a genetically heterogeneous recessive disorder distinguished by cytogenetic instability, hypersensitivity to DNA crosslinking agents, increased chromosomal breakage, and disturbed DNA repair. To date, Fanconi anemia complementation group (FANC) includes 23 FANC genes identified of which, FANCA gene is the most commonly mutated. The mutation spectrum of the FANCA gene is highly heterogeneous with large intragenic deletions due to Alu elements-mediated recombination. The study aimed to identify different deletion mutations on FANCA gene in Egyptian Fanconi anemia patients by multiplex ligation-dependent probe amplification (MLPA) technique to define the spectrum of FA molecular pathology as a step for disease control. The study included 80 FA patients (36 females and 44 males) whose ages ranged from 4 months to 17 years descending from unrelated consanguineous families referred to the Hereditary Blood Disorders Clinic, National Research Centre (NRC), Egypt. Patients were diagnosed with classical clinical presentation of FA and were confirmed by chromosomal breakage using Diepoxybutane (DEB).
    RESULTS: The common clinical presentation in our FA patients were the presence of café au lait spots with hyperpigmentation in 65/80 (81%) followed by skeletal defects in 40/80 (50%). MLPA revealed a total of five different intragenic homozygous deletions of FANCA gene in 16 /80 (20%) patients, among them two deletion patterns were novel.
    CONCLUSION: Molecular analysis using MLPA could detect pathogenic mutations in 20% of FA patients, our study generated considerable data on causative mutations that was used for genetic counseling and prenatal diagnosis.
    Keywords:  Deletion mutation; FANCA gene; Fanconi anemia; MLPA
    DOI:  https://doi.org/10.1016/j.jgeb.2024.100435
  5. Mol Ther. 2024 Dec 12. pii: S1525-0016(24)00819-0. [Epub ahead of print]
    Preclinical development of lentiviral vector gene therapy for Diamond-Blackfan anemia syndrome.
    Senthil Velan Bhoopalan, Thiyagaraj Mayuranathan, Nana Liu, Kalin Mayberry, Yu Yao, Jingjing Zhang, Jean-Yves Métais, Koon-Kiu Yan, Robert E Throm, Steven R Ellis, Yan Ju, Lei Han, Shruthi Suryaprakash, Lance E Palmer, Sheng Zhou, Jiyang Yu, Yong Cheng, Jonathan S Yen, Stephen Gottschalk, Mitchell J Weiss.
      Diamond-Blackfan anemia syndrome (DBAS) is an inherited bone marrow failure disorder caused by haploinsufficiency of ribosomal protein genes, most commonly RPS19. Limited access to patient hematopoietic stem/progenitor cells (HSPCs) is a major roadblock to developing novel therapies for DBAS. We developed a novel self-inactivating third-generation RPS19-encoding lentiviral vector (LV), termed "SJEFS-S19", for DBAS gene therapy. To facilitate LV design, optimize transduction and assess potential therapeutic efficacy, we leveraged a human cellular model of DBAS based on heterozygous disruption of RPS19 in healthy donor CD34+ HSPCs. We show that SJEFS-S19 LV can rescue DBAS-associated defects in ribosomal RNA processing, erythropoiesis and competitive bone marrow repopulation. Transduction of RPS19+/- CD34+ HSPCs with SJEFS-S19 LV followed by xenotransplantation into immunodeficient mice generated a polyclonal HSPC population with normal multi-lineage differentiation and a diverse integration site profile resembling that of clinically proven LVs. Overall, these preclinical studies demonstrate the safety and efficacy of SJEFS-S19, a novel LV for future DBAS gene therapy.
    DOI:  https://doi.org/10.1016/j.ymthe.2024.12.020
  6. Int J Mol Sci. 2024 Nov 22. pii: 12556. [Epub ahead of print]25(23):
    Evaluation of Three Mutations in Codon 385 of Glucose-6-Phosphate Dehydrogenase via Biochemical and In Silico Analysis.
    Adriana Gálvez-Ramírez, Abigail González-Valdez, Beatriz Hernández-Ochoa, Luis Miguel Canseco-Ávila, Alexander López-Roblero, Roberto Arreguin-Espinosa, Verónica Pérez de la Cruz, Elizabeth Hernández-Urzua, Noemi Cárdenas-Rodríguez, Sergio Enríquez-Flores, Ignacio De la Mora-De la Mora, Abraham Vidal-Limon, Saúl Gómez-Manzo.
      Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an enzymopathy that affects approximately 500 million people worldwide. A great number of mutations in the G6PD gene have been described. However, three class A G6PD variants known as G6PD Tomah (C385R), G6PD Kangnam (C385G), and G6PD Madrid (C385W) have been reported to be clinically important due to their associations with severe clinical manifestations such as hemolytic anemia. Therefore, this work aimed to perform, for the first time, biochemical and functional characterizations of these variants. The G6PD variants were cloned and purified for this purpose, followed by analyses of their kinetic parameters and thermal stability, as well as in silico studies. The results showed that the mutations induced changes in the proteins. Regarding the kinetic parameters, it was observed that the three variants showed lower affinities for G6P and NADP+, as well as lower thermal stability compared to WT-G6PD. Molecular dynamics simulations showed that C385 mutations induced changes around neighboring amino acids. Metadynamics simulations showed that most remarkable changes account for the binding pocket volumes, particularly in the structural NADP+ binding site, with a concomitant loss of affinity for catalytic processes.
    Keywords:  G6PD; molecular dynamics simulations; mutations
    DOI:  https://doi.org/10.3390/ijms252312556
  7. EJHaem. 2024 Dec;5(6): 1301-1311
    New insights into the mechanisms of red blood cell enucleation: From basics to clinical applications.
    Qianli Zhuo, Zhaojun Zhang, Xiangdong Fang.
       Background: Red blood cell (RBC) enucleation is a crucial step in the process of erythropoiesis. By removing the nucleus, RBCs gain greater flexibility, enabling them to traverse narrow capillaries with ease, thereby enhancing the efficiency of oxygen and carbon dioxide transport. This transformation underscores the intricate balance between cellular structure and function essential for maintaining homeostasis.
    Topic: This review delves into the multifaceted enucleation process, outlining its complex steps that encompass protein sorting, vesicle trafficking, cytoskeletal remodeling, and apoptosis regulation, while also exploring the potential of enhancing the enucleation rate of RBCs in vitro. We emphasize the intricate regulation of this process, which is orchestrated by multiple factors. This includes transcription factors that meticulously guide protein synthesis and sorting through the modulation of gene expression, as well as non-coding RNAs that play a pivotal role in post-transcriptional regulation during various stages of RBC enucleation. Additionally, macrophages participate in the enucleation process by engulfing and clearing the extruded nuclei, further ensuring the proper development of RBCs. Although many studies have deeply explored the molecular mechanisms of enucleation, the roles of apoptosis and anti-apoptotic processes in RBC enucleation remain incompletely understood.
    Implication: In this review, we aim to comprehensively summarize the RBC enucleation process and explore the progress made in ex vivo RBC generation. In the future, a deeper understanding of the enucleation process could provide significant benefits to patients suffering from anemia and other related conditions.
    Keywords:  RBCs; apoptosis; biological fundamentals; enucleation; erythropoiesis; methodology
    DOI:  https://doi.org/10.1002/jha2.1051
  8. EJHaem. 2024 Dec;5(6): 1354-1358
    Luspatercept in β-thalassemia: Who and when. Strengths and weaknesses points of a real-world evidence.
    Lorenza Torti, Nicolina Ardu, Laura Maffei, Paolo De Fabritiis, Francesco Sorrentino.
      
    Keywords:  adverse events; luspatercept; thalassemia; thrombocytosis; transfusional therapy
    DOI:  https://doi.org/10.1002/jha2.1032
  9. Orphanet J Rare Dis. 2024 Dec 19. 19(1): 470
    Diagnosis of Diamond-Blackfan anemia in adulthood: case series and review of the literature.
    Francesco Versino, Paola Bianchi, Elisa Fermo, Wilma Barcellini, Bruno Fattizzo.
      Diamond-Blackfan anemia (DBA) is a rare constitutional inherited bone marrow failure syndrome (iBMF) characterized by progressive severe non-regenerative anemia and congenital abnormalities. Diagnosis is made by identification of a DBA-causing variant, typically in a ribosomal protein gene. More than 99% of patients are diagnosed in the pediatric age, but clinical manifestation may be mild and severe anemia can occur later in the patient's life. Moreover, the expanding availability of molecular testing is increasing the ability to identify DBA variants also in adults with a non-canonical DBA phenotype. Therefore, adult hematologists must maintain a high clinical suspicion and awareness towards possible DBA diagnosis in adulthood. In this context, the most common differential diagnoses are acquired BMFs such as pure red cell aplasia (PRCA) or hypoplastic myelodysplastic syndrome (MDS). Here, we present three adult patients diagnosed with DBA, where the identification of the causative mutation occurred several years from PRCA misdiagnosis or was made after screening for an affected relative. We also provide a review of 16 cases available in the literature and give hints on possible treatment strategies.
    Keywords:  Anemia; DBA; Inherited bone marrow failure
    DOI:  https://doi.org/10.1186/s13023-024-03490-6
  10. Cells. 2024 Dec 07. pii: 2026. [Epub ahead of print]13(23):
    Interplay Between Metabolic Pathways and Increased Oxidative Stress in Human Red Blood Cells.
    Sara Spinelli, Angela Marino, Rossana Morabito, Alessia Remigante.
      Red blood cells (RBCs) are highly specialized cells with a limited metabolic repertoire. However, it has been demonstrated that metabolic processes are affected by the production of reactive oxygen species (ROS), and critical enzymes allied to metabolic pathways can be impaired by redox reactions. Thus, oxidative stress-induced alternations in the metabolic pathways can contribute to cell dysfunction of human RBCs. Herein, we aim to provide an overview on the metabolic pathways of human RBCs, focusing on their pathophysiological relevance and their regulation in oxidative stress-related conditions.
    Keywords:  antioxidant machinery; erythrocytes; intracellular pathways; metabolism; redox system
    DOI:  https://doi.org/10.3390/cells13232026
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