Defective Erythropoiesis Research Articles

Whole Blood Transcriptome Analysis in Congenital Anemia Patients.

Congenital anemias include a broad range of disorders marked by inherent abnormalities in red blood cells. These abnormalities include enzymatic, membrane, and congenital defects in erythropoiesis, as well as hemoglobinopathies such as sickle cell disease and thalassemia. These conditions range in presentation from asymptomatic cases to those requiring frequent blood transfusions, exhibiting phenotypic heterogeneity and different degrees of severity. Despite understanding their different etiologies, all of them have a common pathophysiological origin with congenital defects of erythropoiesis. We can find different types, from congenital sideroblastic anemia (CSA), which is a bone marrow failure anemia, to hemoglobinopathies as sickle cell disease and thalassemia, with a higher prevalence and clinical impact. Recent efforts have focused on understanding erythropoiesis dysfunction in these anemias but, so far, deep gene sequencing analysis comparing all of them has not been performed. Our study used Quant 3' mRNA-Sequencing to compare transcriptomic profiles of four sickle cell disease patients, ten thalassemia patients, and one rare case of SLC25A38 CSA. Our results showed clear differentiated gene map expressions in all of them with respect to healthy controls. Our study reveals that genes related to metabolic processes, membrane genes, and erythropoiesis are upregulated with respect to healthy controls in all pathologies studied except in the SLC25A38 CSA patient, who shows a unique gene expression pattern compared to the rest of the congenital anemias studied. Our analysis is the first that compares gene expression patterns across different congenital anemias to provide a broad spectrum of genes that could have clinical relevance in these pathologies.

Biallelic variation in the choline and ethanolamine transporter FLVCR1 underlies a severe developmental disorder spectrum

PurposeFLVCR1 encodes a solute carrier protein implicated in heme, choline, and ethanolamine transport. Although Flvcr1−/− mice exhibit skeletal malformations and defective erythropoiesis reminiscent of Diamond-Blackfan anemia (DBA), biallelic FLVCR1 variants in humans have previously only been linked to childhood or adult-onset ataxia, sensory neuropathy, and retinitis pigmentosa. MethodsWe identified individuals with undiagnosed neurodevelopmental disorders and biallelic FLVCR1 variants through international data sharing and characterized the functional consequences of their FLVCR1 variants. ResultsWe ascertained 30 patients from 23 unrelated families with biallelic FLVCR1 variants and characterized a novel FLVCR1-related phenotype: severe developmental disorders with profound developmental delay, microcephaly (z-score −2.5 to −10.5), brain malformations, epilepsy, spasticity, and premature death. Brain malformations ranged from mild brain volume reduction to hydranencephaly. Severely affected patients share traits, including macrocytic anemia and skeletal malformations with Flvcr1−/− mice and DBA. FLVCR1 variants significantly reduce choline and ethanolamine transport and/or disrupt mRNA splicing. ConclusionThese data demonstrate a broad FLVCR1-related phenotypic spectrum ranging from severe multiorgan developmental disorders resembling DBA to adult-onset neurodegeneration. Our study expands our understanding of Mendelian choline and ethanolamine disorders and illustrates the importance of anticipating a wide phenotypic spectrum for known disease genes and incorporating model organism data into genome analysis to maximize genetic testing yield.

Exploring the bone marrow micro environment in thalassemia patients: potential therapeutic alternatives.

Genetic mutations in the β-globin gene lead to a decrease or removal of the β-globin chain, causing the build-up of unstable alpha-hemoglobin. This condition is referred to as beta-thalassemia (BT). The present treatment strategies primarily target the correction of defective erythropoiesis, with a particular emphasis on gene therapy and hematopoietic stem cell transplantation. However, the presence of inefficient erythropoiesis in BT bone marrow (BM) is likely to disturb the previously functioning BM microenvironment. This includes accumulation of various macromolecules, damage to hematopoietic function, destruction of bone cell production and damage to osteoblast(OBs), and so on. In addition, the changes of BT BM microenvironment may have a certain correlation with the occurrence of hematological malignancies. Correction of the microenvironment can be achieved through treatments such as iron chelation, antioxidants, hypoglycemia, and biologics. Hence, This review describes damage in the BT BM microenvironment and some potential remedies.

Lysophosphatidic Acid Receptor 3 Activation Is Involved in the Regulation of Erythrocyte Differentiation Through Ferroptosis

Ferroptosis, a unique form of programmed cell death trigged by lipid peroxidation and iron accumulation, has been implicated in embryonic erythropoiesis and aging. Our previous research demonstrated that lysophosphatidic acid receptor 3 (LPA3) activation mitigated oxidative stress in progeria cells and accelerate the recovery of acute-anemia in mice. Given that both processes involve iron metabolism, we hypothesized that LPA3 activation might mediate cellular ferroptosis. In this study, we used LPA3 agonists 1‐Oleoyl‐2‐O‐methyl‐rac‐glycerophosphothionate (OMPT) to activate LPA3 and examine its effects on the ferroptosis process. OMPT treatment elevated anti-ferroptosis genes protein expression, including solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4), heme oxygenase-1 (HO-1), and ferritin heavy chain (FTH1) in erastin-induced cells. Furthermore, OMPT reduced lipid peroxidation and intracellular ferrous iron accumulation, as evidenced by C11 BODIPYTM 581/591 Lipid Peroxidation Sensor and FerroOrange staining. These observations were validated by applying LPAR3 siRNA in the experiments mentioned above. In addition, the protein expression level of nuclear factor erythroid 2-related factor (NRF2), a key regulator of oxidative stress regulator, was also enhanced in OMPT treated cells. Lastly, we verified that LPA3 plays a critical role in erastin-induced ferroptotic human 25 erythroleukemia K562 cells. OMPT rescued the erythropoiesis defect caused by erastin in K562 cells through using Gly A promoter luciferase assay. Taken together, our findings suggest that LPA3 activation inhibits cell ferroptosis by suppressing lipid oxidation and iron accumulation, indicating that ferroptosis could potentially serve as a link between LPA3, aging and erythropoiesis. MOST 110-2311-B-002-026 MOST 111-2311-B-002-007. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Enhancement of PRMT6 binding to a novel germline GATA1 mutation associated with congenital anemia.

Mutations in the master hematopoietic transcription factor GATA1 are often associated with functional defects in erythropoiesis and megakaryopoiesis. In this study, we identified a novel GATA1 germline mutation (c.1162delGG, p.Leu387Leufs*62) in a patient with congenital anemia and occasional thrombocytopenia. The C-terminal GATA1, a rarely studied mutational region, undergoes frameshifting translation as a consequence of this double-base deletion mutation. To investigate the specific function and pathogenic mechanism of this mutant, in vitro mutant models of stable re-expression cells were generated. The mutation was subsequently validated to cause diminished transcriptional activity of GATA1 and defective differentiation of erythroid and megakaryocytes. Using proximity labeling and mass spectrometry, we identified selective alterations in the proximal protein networks of the mutant, revealing decreased binding to a set of normal GATA1-interaction proteins, including the essential co-factor FOG1. Notably, our findings further demonstrated enhanced recruitment of the protein arginine methyltransferase PRMT6, which mediates histone modification at H3R2me2a and represses transcription activity. We also found an enhanced binding of this mutant GATA1/PRMT6 complex to the transcriptional regulatory elements of GATA1's target genes. Moreover, treatment of the PRMT6 inhibitor MS023 could partially rescue the inhibited transcriptional and impaired erythroid differentiation caused by the GATA1 mutation. Taken together, our results provide molecular insights into erythropoiesis in which mutation leads to partial loss of GATA1 function, and the role of PRMT6 and its inhibitor MS023 in congenital anemia, highlighting PRMT6 binding as a negative factor of GATA1 transcriptional activity in aberrant hematopoiesis.

Lysophosphatidic Acid Receptor 3 Activation Is Involved in the Regulation of Ferroptosis.

Ferroptosis, a unique form of programmed cell death trigged by lipid peroxidation and iron accumulation, has been implicated in embryonic erythropoiesis and aging. Our previous research demonstrated that lysophosphatidic acid receptor 3 (LPA3) activation mitigated oxidative stress in progeria cells and accelerated the recovery of acute anemia in mice. Given that both processes involve iron metabolism, we hypothesized that LPA3 activation might mediate cellular ferroptosis. In this study, we used an LPA3 agonist, 1-Oleoyl-2-O-methyl-rac-glycerophosphothionate (OMPT), to activate LPA3 and examine its effects on the ferroptosis process. OMPT treatment elevated anti-ferroptosis gene protein expression, including solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4), heme oxygenase-1 (HO-1), and ferritin heavy chain (FTH1), in erastin-induced cells. Furthermore, OMPT reduced lipid peroxidation and intracellular ferrous iron accumulation, as evidenced by C11 BODIPY™ 581/591 Lipid Peroxidation Sensor and FerroOrange staining. These observations were validated by applying LPAR3 siRNA in the experiments mentioned above. In addition, the protein expression level of nuclear factor erythroid 2-related factor (NRF2), a key regulator of oxidative stress, was also enhanced in OMPT-treated cells. Lastly, we verified that LPA3 plays a critical role in erastin-induced ferroptotic human erythroleukemia K562 cells. OMPT rescued the erythropoiesis defect caused by erastin in K562 cells based on a Gly A promoter luciferase assay. Taken together, our findings suggest that LPA3 activation inhibits cell ferroptosis by suppressing lipid oxidation and iron accumulation, indicating that ferroptosis could potentially serve as a link among LPA3, erythropoiesis, and aging.

Potential Association of Serum Hepcidin, hs-CRP and Iron Status Levels in Hemodialytic Patients

Patients on hemodialysis (HD) are usually anemic because of defective erythropoiesis. Hepcidin is a polypeptide that regulates iron homeostasis and could indicate functional iron deficiency in patients with end-stage renal disease (ESRD); this may also aid in assessing a patient's response to erythropoietin (EPO). The present study was directed to investigate serum levels of hepcidin, iron status, and inflammation markers such as C-reactive protein (CRP) in patients with ESRD on maintenance HD and to observe the correlation of serum hepcidin with conventional iron and inflammatory markers. Forty-two patients of both sexes on maintenance HD and EPO therapy were enrolled; 42 age and sex-matched healthy subjects were included as controls. Laboratory tests were performed, including complete blood count, serum hepcidin, total iron binding capacity (TIBC), serum ferritin, serum iron, and CRP. Serum hepcidin levels were significantly higher in patients with ESRD than in the control group (18.2 \(\pm\) 2.8 ng/mL and 8.5 \(\pm\) 2.3 ng/mL, respectively P = 0.000). The hemoglobin, hematocrit, serum iron, TIBC, and transferrin saturation levels in the patient group were significantly lower than in the control group. Higher hepcidin levels were found in EPO non-responders (19.6 (\pm) 2.4 ng/mL), while lower levels (16.9 \(\pm\) 2.5 ng/mL) were seen in responders (P = 0.001). A positive and significant correlation was observed between the values of serum hepcidin and CRP. Our study indicates that higher hepcidin levels are found in ESRD patients on HD and in those not responding to EPO. Our findings suggest that hepcidin might play a role in the pathophysiology of anemia associated with chronic diseases and EPO resistance.

Hemolysis-driven IFNα production impairs erythropoiesis by negatively regulating EPO signaling in sickle cell disease

AbstractDisordered erythropoiesis is a feature of many hematologic diseases, including sickle cell disease (SCD). However, very little is known about erythropoiesis in SCD. Here, we show that although bone marrow (BM) erythroid progenitors and erythroblasts in Hbbth3/+ thalassemia mice were increased more than twofold, they were expanded by only ∼40% in Townes sickle mice (SS). We further show that the colony-forming ability of SS erythroid progenitors was decreased and erythropoietin (EPO)/EPO receptor (EPOR) signaling was impaired in SS erythroid cells. Furthermore, SS mice exhibited reduced responses to EPO. Injection of mice with red cell lysates or hemin, mimicking hemolysis in SCD, led to suppression of erythropoiesis and reduced EPO/EPOR signaling, indicating hemolysis, a hallmark of SCD, and could contribute to the impaired erythropoiesis in SCD. In vitro hemin treatment did not affect Stat5 phosphorylation, suggesting that hemin-induced erythropoiesis suppression in vivo is via an indirect mechanism. Treatment with interferon α (IFNα), which is upregulated by hemolysis and elevated in SCD, led to suppression of mouse BM erythropoiesis in vivo and human erythropoiesis in vitro, along with inhibition of Stat5 phosphorylation. Notably, in sickle erythroid cells, IFN-1 signaling was activated and the expression of cytokine inducible SH2–containing protein (CISH), a negative regulator of EPO/EPOR signaling, was increased. CISH deletion in human erythroblasts partially rescued IFNα-mediated impairment of cell growth and EPOR signaling. Knocking out Ifnar1 in SS mice rescued the defective BM erythropoiesis and improved EPO/EPOR signaling. Our findings identify an unexpected role of hemolysis on the impaired erythropoiesis in SCD through inhibition of EPO/EPOR signaling via a heme-IFNα-CISH axis.

DDX41 Dissolves G-Quadruplexes to Maintain Erythroid Genomic Integrity and Prevent Genome DNA Leakage-Induced Cgas Activation

DDX41 is one of the most frequently mutated genes that are associated with familial myeloid neoplasms. To understand the pathogenesis of DDX41 mutations, which often lead to loss of function, we developed various mouse models with lineage-specific deletion of Ddx41. We found embryonical lethality with the hematopoietic specific knockout (KO) of Ddx41 ( VavCre:Ddx41 fl/fl). Examination of E13.5 fetuses showed that Ddx41 deficient fetal livers are pale with marked apoptosis, indicating defects in erythropoiesis. Indeed, depletion of Ddx41 in the early stage of erythropoiesis ( EpoRCre:Ddx41 fl/fl) also led to embryonic death. However, mice with Ddx41 KO in the late-stage erythropoiesis ( HBBCre:Ddx41 fl/fl) survived with normal complete blood count, indicating a stage-specific requirement of Ddx41 in erythropoiesis. We further revealed that mice with Ddx41 KO in monocytes ( LysMCre:Ddx41 fl/fl), granulocytes ( MRP8Cre:Ddx41 fl/fl), and dendritic cells ( CD11cCre:Ddx41 fl/fl) also showed no obvious phenotypes, suggesting that Ddx41 is dispensable in other myeloid cells in mice. We further discovered that Ddx41 KO in erythroid cells induces the accumulation of G-quadruplexes (G4), a noncanonical DNA structure that is often associated with active replication. We found that erythroid cells contain significantly higher G4 signals. G4 accumulation reaches its peak level in the CFU-Es and proerythroblasts, which is likely due to replication stress at this stage of erythropoiesis and consistent with the lethality of EpoRCre:Ddx41 fl/flmice. A CUT&Tag analysis revealed genome-wide colocalization of Ddx41 and G4 in hematopoietic stem and progenitor cells, which became further enhanced in erythroid cells. We confirmed the direct binding of Ddx41 to G4, which is sequence independent. A fluorescence resonance energy transfer (FRET) assay demonstrated that Ddx41 binds to and dose-dependently dissolves G4. These findings indicate a critical role of DDX41 in erythropoiesis by maintaining the level of G4 and genomic integrity. Consistent with the knowledge that increased level of G4 induces genomic instability, we found an increased level of γ-H2AX in Ddx41 KO erythroid cells or cells treated with pyridostatin (PDS), a well-known G4 stabilizer. One unique feature of developing erythroblasts is the dynamic nuclear opening with the release of genomic materials, which is critical for nuclear condensation. However, this process also poses potential risks of triggering the cGAS-STING pathway with the increased damage-associated molecular patterns (DAMPs), particularly under situations with increased genomic instability. Indeed, we found an increased level of cGAS and STING in Ddx41 KO and PDS-treated erythroid cells. Consistently, erythroblasts from cGAS KO mice are resistant to PDS treatment mediated cell death. The significance of the cGAS pathway was genetically confirmed in that cGAS KO completely rescued the lethality of VavCre:Ddx41 fl/fl mice. In addition, we demonstrated that Ddx41 KO and PDS treatment-mediated activation of the cGAS-STING pathway induces an increased level of interferon, which results in ribosome biogenesis defect, p53 upregulation, and cell apoptosis. Notably, cGAS KO in VavCre:Ddx41 fl/fl mice counteracted these effects and rescued erythroid ribosomopathy, although the level of G4 remains upregulated. Overall, our study reveals a critical role of DDX41 in dissolving G4 and maintaining the genomic integrity of the erythroid cells. Our data also indicate that defect in the erythroid lineage through ribosomopathy is one of the major pathologic features in DDX41 mutated myeloid neoplasms, which is important for the development of novel therapies.

Ncrna-a3 Epigenetically Regulates TAL1 and Its Target Genes to Drive Erythroid Differentiation

Erythropoiesis is the development of enucleated red blood cells from multipotent hematopoietic stem cells. This complex process is tightly regulated by the dynamic changes in occupancy of tissue specific transcription factors (TFs) that activate and repress genes appropriately. TAL1, a critical regulator is expressed throughout the development of mature erythroblasts from multi-progenitors. Loss of TAL1 leads to defective erythropoiesis. As opposed to TAL1's role in erythropoiesis, itis an extraneous factor in developing lymphoid cells and aberrant expression of TAL1 in T-cells is associated with T-ALL. The differential regulation during normal and malignant hematopoiesis remains largely unclear. We found that a distil RNA - ncRNA-a3, is transcribed from erythroid specific enhancer, 50kbp downstream of the TAL1 TSS. We hypothesized that ncRNA-a3 plays an important role in regulating TAL1 and its target gene transcription in an erythroid specific manner. To test this hypothesis, we examine the regulatory potential of ncRNA-a3 towards the activation of TAL1 expression in the context of hematopoiesis and leukemogenesis. We used RNA interference to deplete ncRNA-a3 expression across several hematopoietic and leukemic cells. The decrease in ncRNA-a3 resulted in significant reduction of TAL1 transcript and protein levels in hematopoietic cells while TAL1 transcripts showed little or no reduction in leukemic cells. Subsequently, transcriptomic profile changes in erythroleukemic K562 cells showed downregulation of erythroid specific targets of TAL1. To ascertain the role of ncRNA-a3 in erythropoiesis, we used siRNA against ncRNA-a3 in primary CD34+ cells and subjected cells to differentiation. Quantification of early and late erythroblasts by colony forming assay and flow cytometry indicated impaired erythroid differentiation and maturation in orthochromatic and polychromatic stages. Further, RNA-seq analysis of ncRNA-a3 knockdown cells revealed defects in globin gene function, heme biosynthesis and nucleosome condensation compared to control CD34+ cells during time course EPO induced erythroid differentiation. Mechanistically, we observed ncRNA-a3s occupancy at TAL1 regulatory regions from ChIRP-qPCR across TAL1 locus. NG Capture-C data depicting changes in interaction frequencies indicated that ncRNA-a3 maintains canonical interactions at the TAL1 locus. Knockdown of ncRNA-a3 negatively impacted the chromatin accessibility in TAL1 and TAL1 target loci. Perturbation of chromatin landscape affected TAL1 and TAL1 binding partner - P300 and BRG1 occupancy at TAL1 target sites. To distinguish reduced TAL1 binding as a consequence of lower levels of TAL1, from RNA dependent binding of TAL1, we examined TAL1, P300 and BRG1 chromatin occupancy through Cut&Run in conjunction with RNase treatment. Effective depletion of global RNAs resulted in reduced enrichment of TAL1, P300 and BRG1 bound fragments indicating possible RNA dependent binding of TAL1 and its cofactors. TAL1 coimmunoprecipitation using RNase treatment (1) and ncRNA-a3 KD (2), demonstrated that TAL1s interaction with P300 and BRG1 is RNA dependent, but ncRNA-a3 independent. RNA-IP results further confirmed that ncRNA-a3 is associated with TAL1 through its interacting epigenetic cofactors P300 and BRG1. Together, our data indicate that although RNA dependent binding of TAL1 and its epigenetic cofactors is cell type or target locus specific, ncRNA-a3 modulates the occupancy of TAL1, P300 and BRG1 at the TAL1 locus during erythroid specific TAL1 transcription.

Human cellular model systems of β-thalassemia enable in-depth analysis of disease phenotype

β-thalassemia is a prevalent genetic disorder causing severe anemia due to defective erythropoiesis, with few treatment options. Studying the underlying molecular defects is impeded by paucity of suitable patient material. In this study we create human disease cellular model systems for β-thalassemia by gene editing the erythroid line BEL-A, which accurately recapitulate the phenotype of patient erythroid cells. We also develop a high throughput compatible fluorometric-based assay for evaluating severity of disease phenotype and utilize the assay to demonstrate that the lines respond appropriately to verified reagents. We next use the lines to perform extensive analysis of the altered molecular mechanisms in β-thalassemia erythroid cells, revealing upregulation of a wide range of biological pathways and processes along with potential novel targets for therapeutic investigation. Overall, the lines provide a sustainable supply of disease cells as research tools for identifying therapeutic targets and as screening platforms for new drugs and reagents.

Next generation sequencing (NGS) interest in deciphering erythrocyte molecular defects' association in red cell disorders: Clinical and erythrocyte phenotypes of patients with mutations inheritance in PIEZO1, Spectrin ß1, RhAG and SLC4A1

We report here an instructive case referred at 16 months-old for exploration of hemolysis without anemia (compensated anemia with reticulocytosis). The biology tests confirmed the hemolysis with increased total and indirect bilirubin. The usual hemolysis diagnosis tests were normal (DAT, G6PD, PK, Hb electrophoresis) except cytology and ektacytometry suggesting an association of multiple red blood cell (RBC) membrane disorders. This led us to propose a molecular screening analysis using targeted-Next Generation Sequencing (t-NGS) with a capture technique on 93 genes involved in RBC and erythropoiesis defects. We identified 4 missense heterozygous allelic variations, all of them were described without any significance (VUS) in the SLC4A1, RhAG, PIEZO1 and SPTB genes. The study of the familial cosegregation and research functional tests allowed to decipher the role of at least two by two genes in the phenotype and the hemolytic disease of this young patient. Specialized t-NGS panel (or virtual exome/genome sequencing) in a disease-referent laboratory and the motivated collaboration of clinicians, biologists and scientists should be the gold standard for improving the diagnosis of the patients affected with RBC diseases or rare inherited anemias.

#3777 ACTIVATION OF HYPOXIA-INDUCIBLE FACTORS IN ERYTHROID PROGENITOR CELLS RESULTS IN DEFECTIVE ERYTHROPOIESIS

Abstract Background and Aims Hypoxia-inducible factor prolyl hydroxylase inhibitor (HIF-PHI) activates HIF in renal pericytes and fibroblasts to promote erythropoietin production and erythropoiesis. However, the effects of HIF stabilization in the erythroid lineage are not clear. We aim to study the effects of erythroid lineage-specific stabilization of HIF on erythropoiesis by preclinical models. Method EporGFP-Cre/+;VhlF/F mice were used to achieve erythroid lineage-specific stabilization of HIF. Surface expression of TER-119, CD44, and forward scatter (FSC) were used to define erythroblast, reticulocyte, and red blood cell in the bone marrow. Expression of propidium iodide and surface annexin V were used to define apoptosis. Stress erythropoiesis was induced by subcutaneous administration of phenylhydrazine. Results Compared with littermate VhlF/F mice, EporGFP-Cre/+;VhlF/F mice had decreased hematocrit, decreased percentage of erythroblasts in the bone marrow, and increased apoptosis of erythroblasts in the bone marrow under steady state. These phenotypes of defective erythropoiesis were normalized in mice harboring concomitant Vhl, Hif1a, and Hif2a deletion (EporGFP-Cre/+;VhlF/F;Hif1aF/F;Hif2aF/Fmice). Although macrophages in the bone marrow also express Epor, macrophage-specific Vhl deletion in either Tg(Csf1r-CreESR1);VhlF/F or Lyz2Cre/+;VhlF/F mice did not result in defective erythropoiesis. During stress erythropoiesis, compared with littermate VhlF/F mice, EporGFP-Cre/+;VhlF/F mice had similar hematocrit, lower percentage of erythroblast in the bone marrow, and increased percentage of erythroblast in the spleen. Conclusion Vhl deletion in erythroid progenitor cells impairs erythropoiesis in murine bone marrow in the steady state. The phenotypes of defective erythropoiesis were partially reversed during stress erythropoiesis.

Haploinsufficiency of the essential gene Rps12 causes defects in erythropoiesis and hematopoietic stem cell maintenance.

Ribosomal protein (Rp) gene haploinsufficiency can result in Diamond-Blackfan Anemia (DBA), characterized by defective erythropoiesis and skeletal defects. Some mouse Rp mutations recapitulate DBA phenotypes, although others lack erythropoietic or skeletal defects. We generated a conditional knockout mouse to partially delete Rps12. Homozygous Rps12 deletion resulted in embryonic lethality. Mice inheriting the Rps12KO/+ genotype had growth and morphological defects, pancytopenia, and impaired erythropoiesis. A striking reduction in hematopoietic stem cells (HSCs) and progenitors in the bone marrow (BM) was associated with decreased ability to repopulate the blood system after competitive and non-competitive BM transplantation. Rps12KO/+ lost HSC quiescence, experienced ERK and MTOR activation, and increased global translation in HSC and progenitors. Post-natal heterozygous deletion of Rps12 in hematopoietic cells using Tal1-Cre-ERT also resulted in pancytopenia with decreased HSC numbers. However, post-natal Cre-ERT induction led to reduced translation in HSCs and progenitors, suggesting that this is the most direct consequence of Rps12 haploinsufficiency in hematopoietic cells. Thus, RpS12 has a strong requirement in HSC function, in addition to erythropoiesis.

Long noncoding RNA HIKER regulates erythropoiesis in Monge's disease via CSNK2B.

Excessive erythrocytosis (EE) is a major hallmark of patients suffering from chronic mountain sickness (CMS, also known as Monge's disease) and is responsible for major morbidity and even mortality in early adulthood. We took advantage of unique populations, one living at high altitude (Peru) showing EE, with another population, at the same altitude and region, showing no evidence of EE (non-CMS). Through RNA-Seq, we identified and validated the function of a group of long noncoding RNAs (lncRNAs) that regulate erythropoiesis in Monge's disease, but not in the non-CMS population. Among these lncRNAs is hypoxia induced kinase-mediated erythropoietic regulator (HIKER)/LINC02228, which we showed plays a critical role in erythropoiesis in CMS cells. Under hypoxia, HIKER modulated CSNK2B (the regulatory subunit of casein kinase 2). A downregulation of HIKER downregulated CSNK2B, remarkably reducing erythropoiesis; furthermore, an upregulation of CSNK2B on the background of HIKER downregulation rescued erythropoiesis defects. Pharmacologic inhibition of CSNK2B drastically reduced erythroid colonies, and knockdown of CSNK2B in zebrafish led to a defect in hemoglobinization. We conclude that HIKER regulates erythropoiesis in Monge's disease and acts through at least one specific target, CSNK2B, a casein kinase.

Global profile of anemia during pregnancy versus country income overview: 19 years estimative (2000–2019)

Anemia is a worldwide public health problem that is worst in low- and middle-income countries (LMICs), reaching 60% of prevalence. The etiology of anemia is diverse and multifactorial, with iron deficiency being the most prevalent, and often found in pregnant women. Iron is indispensable for the production of red blood cells and approximately 80% of the available heme iron is used for hemoglobin synthesis in mature erythroblasts. Iron deficiency affects oxygen transport, compromising energy and muscle metabolism and can occur with depletion of iron storage, defective erythropoiesis, and low hemoglobin levels. We analyzed anemia prevalence in pregnant women from 2000 to 2019 worldwide correlating them with current (2022) country income, with especial attention to LMICs using WHO dataset. Our analysis indicates that pregnant women from LMICs had a higher probability (40%) of anemia during pregnancy especially those from Africa and South Asia. Africa and the Americas showed a higher decrease in the prevalence of anemia from 2000 to 2019. The Americas and Europe have a lower prevalence, concentrated in 57% of most upper-middle- and high-income countries. Black women are also more prone to develop anemia during pregnancy, especially if they are from LMICs. However, the prevalence of anemia appears to decrease with an increase in educational level. In conclusion, anemia prevalence fluctuated from 5.2 to 65.7% worldwide in 2019, validating it as a public health problem.

Analysis of Immunophenotypic Changes during Ex Vivo Human Erythropoiesis and Its Application in the Study of Normal and Defective Erythropoiesis.

Erythropoiesis is a highly regulated process and undergoes several genotypic and phenotypic changes during differentiation. The phenotypic changes can be evaluated using a combination of cell surface markers expressed at different cellular stages of erythropoiesis using FACS. However, limited studies are available on the in-depth phenotypic characterization of progenitors from human adult hematopoietic stem and progenitor cells (HSPCs) to red blood cells. Therefore, using a set of designed marker panels, in the current study we have kinetically characterized the hematopoietic, erythroid progenitors, and terminally differentiated erythroblasts ex vivo. Furthermore, the progenitor stages were explored for expression of CD117, CD31, CD41a, CD133, and CD45, along with known key markers CD36, CD71, CD105, and GPA. Additionally, we used these marker panels to study the stage-specific phenotypic changes regulated by the epigenetic regulator; Nuclear receptor binding SET Domain protein 1 (NSD1) during erythropoiesis and to study ineffective erythropoiesis in myelodysplastic syndrome (MDS) and pure red cell aplasia (PRCA) patients. Our immunophenotyping strategy can be used to sort and study erythroid-primed hematopoietic and erythroid precursors at specified time points and to study diseases resulting from erythroid dyspoiesis. Overall, the current study explores the in-depth kinetics of phenotypic changes occurring during human erythropoiesis and applies this strategy to study normal and defective erythropoiesis.

Abstract A46: Heterozygous mutations in DDX41 cause erythroid progenitor cell defects and cooperate with p53 mutations to cause hematologic malignancy

Abstract Germline mutations in the RNA Helicase gene DDX41 are the most common cause of inherited susceptibility to adult MDS and AML. These mutations are always heterozygous and are typically frameshifts, causing loss of functional protein. We recently reported that at least one functional copy of DDX41 is essential for hematopoiesis, and that DDX41 is required for ribosome biogenesis (Chlon et al., Cell Stem Cell 2021). While biallelic DDX41 mutations cause dramatic defects in hematopoiesis, the role of heterozygous mutations in MDS pathogenesis is not yet understood. DDX41 mutation carriers frequently experience idiopathic cytopenias of unknown significance (ICUS) prior to MDS onset, suggesting that underlying hematopoietic defects contribute to MDS/AML (Choi et al., Haemotologica 2021). The majority of DDX41-mutant MDS patients have refractory anemia, indicating that the erythroid lineage is particularly affected in these patients (Sebert et al., Blood 2019). Since ribosome defects are a common cause of inherited anemias and also contribute to MDS pathogenesis, we characterized the effect of heterozygous DDX41 mutations on erythropoiesis in murine and human models. Mice that were transplanted with Ddx41+/− bone marrow develop anemia at 12-15 months post-transplant. At younger ages, these mice have normal hematopoietic indices, but when stress erythropoiesis is induced by Pheylhydrazine-treatment, the Ddx41+/− mice have prolonged anemia. These observations indicate that heterozygous loss of DDX41 causes defects in erythropoiesis during stress and aging. We further characterized the effect of Ddx41-hetrozyogisty on erythroid progenitor function in vitro. In colony assays, we found that Ddx41+/− HSPC form fewer BFU-E but comparable numbers of myeloid colonies. In liquid culture erythroid differentiation cultures, we found that Ddx41+/− HSPC produce fewer CD71+ Ter119+ progenitors than controls. Mechanistically, we found that in vitro-derived erythroid progenitors from both mice and cell line models had decreased protein translation, suggesting that ribosome defects underlie the observed inefficiency in erythropoiesis. In congenital ribosomopathy diseases, ribosome defects lead to p53 activation which contributes to defects in erythropoiesis. Interestingly, TP53 mutations are common in DDX41-mutant MDS/AML (Sebert et al., Blood 2019), suggesting that the ribosome deficiency selects for these mutations. To assess the role of p53 mutations in the development of DDX41-mutant MDS, we generated Ddx41+/−;p53+/− mice and transplanted the bone marrow into lethally-irradiated recipients and followed the mice for 1 year. These mice developed a lethal MDS-like phenotype at 7-12 months post-transplant while control mice lived until the end of the study period. The sick mice had anemia and other cytopenias accompanied by enlarged spleens and dysplastic myeloid cells. Collectively, these results indicate that p53 mutations cooperate with Ddx41-heterozygosity to promote hematologic malignancy. Citation Format: Timothy M Chlon, Emily Stepanchick, Analise Sulentic, Andrew Wilson, Daniel T Starczynowski. Heterozygous mutations in DDX41 cause erythroid progenitor cell defects and cooperate with p53 mutations to cause hematologic malignancy [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A46.

Crosstalk between miR-144/451 and Nrf2 during Recovery from Acute Hemolytic Anemia.

miR-144/451 and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) regulate two antioxidative systems that have been identified to maintain redox homeostasis in erythroid cells by removing excess reactive oxygen species (ROS). Whether these two genes coordinate to affect ROS scavenging and the anemic phenotype, or which gene is more important for recovery from acute anemia, has not been explored. To address these questions, we crossed miR-144/451 knockout (KO) and Nrf2 KO mice and examined the phenotype change in the animals as well as the ROS levels in erythroid cells either at baseline or under stress condition. Several discoveries were made in this study. First, Nrf2/miR-144/451 double-KO mice unexpectedly exhibit similar anemic phenotypes as miR-144/451 single-KO mice during stable erythropoiesis, although compound mutations of miR-144/451 and Nrf2 lead to higher ROS levels in erythrocytes than single gene mutations. Second, Nrf2/miR-144/451 double-mutant mice exhibit more dramatic reticulocytosis than miR-144/451 or Nrf2 single-KO mice during days 3 to 7 after inducing acute hemolytic anemia using phenylhydrazine (PHZ), indicating a synergistic effect of miR-144/451 and Nrf2 on PHZ-induced stress erythropoiesis. However, the coordination does not persist during the whole recovery stage of PHZ-induced anemia; instead, Nrf2/miR-144/451 double-KO mice follow a recovery pattern similar to miR-144/451 single-KO mice during the remaining period of erythropoiesis. Third, the complete recovery from PHZ-induced acute anemia in miR-144/451 KO mice takes longer than in Nrf2 KO mice. Our findings demonstrate that complicated crosstalk between miR-144/451 and Nrf2 does exist and the crosstalk of these two antioxidant systems is development-stage-dependent. Our findings also demonstrate that miRNA deficiency could result in a more profound defect of erythropoiesis than dysfunctional transcription factors.

Duodenal macrophages control dietary iron absorption via local degradation of transferrin

AbstractIron is an essential cellular metal that is important for many physiological functions including erythropoiesis and host defense. It is absorbed from the diet in the duodenum and loaded onto transferrin (Tf), the main iron transport protein. Inefficient dietary iron uptake promotes many diseases, but mechanisms regulating iron absorption remain poorly understood. By assessing mice that harbor a macrophage-specific deletion of the tuberous sclerosis complex 2 (Tsc2), a negative regulator of mechanistic target of rapamycin complex 1 (mTORC1), we found that these mice possessed various defects in iron metabolism, including defective steady-state erythropoiesis and a reduced saturation of Tf with iron. This iron deficiency phenotype was associated with an iron import block from the duodenal epithelial cells into the circulation. Activation of mTORC1 in villous duodenal CD68+ macrophages induced serine protease expression and promoted local degradation of Tf, whereas the depletion of macrophages in mice increased Tf levels. Inhibition of mTORC1 with everolimus or serine protease activity with nafamostat restored Tf levels and Tf saturation in the Tsc2-deficient mice. Physiologically, Tf levels were regulated in the duodenum during the prandial process and Citrobacter rodentium infection. These data suggest that duodenal macrophages determine iron transfer to the circulation by controlling Tf availability in the lamina propria villi.