A Proposed Concept for Defective Mitophagy Leading to Late Stage Ineffective Erythropoiesis in Pyruvate Kinase Deficiency

Pyruvate kinase deficiency (PKD) is a rare congenital hemolytic anemia caused by mutations in the PKLR gene. Here, we review pathophysiological aspects of PKD, focusing on the interplay between pyruvate kinase (PK)-activity and reticulocyte maturation in the light of ferroptosis, an iron-dependent process of regulated cell death, and in particular its key player glutathione peroxidase 4 (GPX4). GPX4 plays an important role in mitophagy, the key step of peripheral reticulocyte maturation and GPX4 deficiency in reticulocytes results in a failure to fully mature. Mitophagy depends on lipid oxidation, which is under physiological conditions controlled by GPX4. Lack of GPX4 leads to uncontrolled auto-oxidation, which will disrupt autophagosome maturation and thereby perturb mitophagy. Based on our review, we propose a model for disturbed red cell maturation in PKD. A relative GPX4 deficiency occurs due to glutathione (GSH) depletion, as cytosolic L-glutamine is preferentially used in the form of α-ketoglutarate as fuel for the tricarboxylic acid (TCA) cycle at the expense of GSH production. The relative GPX4 deficiency will perturb mitophagy and, subsequently, results in failure of reticulocyte maturation, which can be defined as late stage ineffective erythropoiesis. Our hypothesis provides a starting point for future research into new therapeutic possibilities, which have the ability to correct the oxidative imbalance due to lack of GPX4

Rare Anemias: Are Their Names Just Smoke and Mirrors?

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Activation of pyruvate kinase as therapeutic option for rare hemolytic anemias:Shedding new light on an old enzyme

Novel developments in therapies for various hereditary hemolytic anemias reflect the pivotal role of pyruvate kinase (PK), a key enzyme of glycolysis, in red blood cell (RBC) health. Without PK catalyzing one of the final steps of the Embden-Meyerhof pathway, there is no net yield of adenosine triphosphate (ATP) during glycolysis, the sole source of energy production required for proper RBC function and survival. In hereditary hemolytic anemias, RBC health is compromised and therefore lifespan is shortened. Although our knowledge on glycolysis in general and PK function in particular is solid, recent advances in genetic, molecular, biochemical, and metabolic aspects of hereditary anemias have improved our understanding of these diseases. These advances provide a rationale for targeting PK as therapeutic option in hereditary hemolytic anemias other than PK deficiency. This review summarizes the knowledge, rationale, (pre)clinical trials, and future advances of PK activators for this important group of rare diseases.</p

Liver Iron Retention Estimated from Utilization of Oral and Intravenous Radioiron in Various Anemias and Hemochromatosis in Humans

Patients with hereditary hemochromatosis and non-transfusion-dependent hereditary anemia develop predominantly liver iron-overload. We present a unique method allowing quantification of liver iron retention in humans during first-pass of 59Fe-labeled iron through the portal system, using standard ferrokinetic techniques measuring red cell iron uptake after oral and intravenous 59Fe administration. We present data from patients with iron deficiency (ID; N = 47), hereditary hemochromatosis (HH; N = 121) and non-transfusion-dependent hereditary anemia (HA; N = 40). Mean mucosal iron uptake and mucosal iron transfer (±SD) were elevated in patients with HH (59 ± 18%, 80 ± 15% respectively), HA (65 ± 17%, 74 ± 18%) and ID (84 ± 14%, 94 ± 6%) compared to healthy controls (43 ± 19%, 64 ± 18%) (p < 0.05) resulting in increased iron retention after 14 days compared to healthy controls in all groups (p < 0.01). The fraction of retained iron utilized for red cell production was 0.37 ± 0.17 in untreated HA, 0.55 ± 0.20 in untreated HH and 0.99 ± 0.22 in ID (p < 0.01). Interestingly, compared to red blood cell iron utilization after oral iron administration, red blood cell iron utilization was higher after injection of transferrin-bound iron in HA and HH. Liver iron retention was considerably higher in HH and HA compared to ID. We hypothesize that albumin serves as a scavenger of absorbed Fe(II) for delivering albumin-bound Fe(III) to hepatocytes

Glucose phosphate isomerase deficiency demasked by whole-genome sequencing: a case report

Background: Glucose-6-phosphate isomerase deficiency is a rare genetic disorder causing hereditary nonspherocytic hemolytic anemia. It is the second most common glycolytic enzymopathy in red blood cells. About 90 cases are reported worldwide, with symptoms including chronic hemolytic anemia, jaundice, splenomegaly, gallstones, cholecystitis, and in severe cases, neurological impairments, hydrops fetalis, and neonatal death. Case presentation: This paper details the case of the first Danish patient diagnosed with glucose-6-phosphate isomerase deficiency. The patient, a 27-year-old white female, suffered from lifelong anemia of unknown origin for decades. Diagnosis was established through whole-genome sequencing, which identified two GPI missense variants: the previously documented variant p.(Thr224Met) and a newly discovered variant p.(Tyr341Cys). The pathogenicity of these variants was verified enzymatically. Conclusions: Whole-genome sequencing stands as a potent tool for identifying hereditary anemias, ensuring optimal management strategies

The RoxyScan is a novel measurement of red blood cell deformability under oxidative and shear stress

Exposure to both oxidative and shear stress, a condition that the red blood cell (RBC) continuously experiences in the circulation in vivo can be mimicked in a Couette type viscometer and monitored by ektacytometry. RBCs maintain their deformation and orientation under shear stress and oxidative stress until a threshold is reached at which these conditions appear to overwhelm the elaborate and complex pathways that maintain a proper redox environment in the cell. Oxidative stress under shear alters the ability of the cell to deform, changes cell morphology, its orientation in the shear stress field, and appears to alter intracellular and membrane characteristics. The application of the RoxyScan technology allows the comparison of oxidant effects and the role of antioxidant systems. This provides the opportunity to study the ability of RBC to deal with oxidative stress in various conditions, including RBC disorders such as sickle cell disease (SCD)

Interplay of erythropoietin, fibroblast growth factor 23, and erythroferrone in patients with hereditary hemolytic anemia

Recently, erythropoietin (EPO) was identified as regulator of fibroblast growth factor 23 (FGF23). Proteolytic cleavage of biologically active intact FGF23 (iFGF23) results in the formation of C-terminal fragments (cFGF23). An increase in cFGF23 relative to iFGF23 suppresses FGF receptor signaling by competitive inhibition. EPO lowers the i:cFGF23 ratio, thereby overcoming iFGF23-mediated suppression of erythropoiesis. We investigated EPO-FGF23 signaling and levels of erythroferrone (ERFE) in 90 patients with hereditary hemolytic anemia (www.trialregister.nl [NL5189]). We show, for the first time, the importance of EPO-FGF23 signaling in hereditary hemolytic anemia: there was a clear correlation between total FGF23 and EPO levels (r = +0.64; 95% confidence interval [CI], 0.09-0.89), which persisted after adjustment for iron load, inflammation, and kidney function. There was no correlation between iFGF23 and EPO. Data are consistent with a low i:cFGF23 ratio. Therefore, as expected, we report a correlation between EPO and ERFE in a diverse set of hereditary hemolytic anemias (r = +0.47; 95% CI, 0.14-0.69). There was no association between ERFE and total FGF23 or iFGF23, which suggests that ERFE does not contribute to the connection between FGF23 and EPO. These findings open a new area of research and might provide potentially new druggable targets with the opportunity to ameliorate ineffective erythropoiesis and the development of disease complications in hereditary hemolytic anemias

Rapid diagnosis of hereditary haemolytic anaemias using automated rheoscopy and supervised machine learning

Haemolytic anaemias arise when red blood cell (RBC) integrity is compromised, eventually resulting in premature clearance or lysis and leading to anaemia when these effects cannot be sufficiently compensated by the capacity of the bone marrow to produce new cells. Hereditary anaemia occurs as a consequence of genetic mutation (e.g. affecting membrane complex or cytoskeletal proteins, haemoglobin or metabolic enzymes), and diagnosing affected patients is a complex process since, given the wide variety of possible genetic causes, multiple examinations must be performed and an unambiguous result is usually reached only after DNA sequencing. Furthermore, phenotypic severity can vary widely not just among individuals with different mutations but also among individuals suffering from the same mutation, thereby complicating diagnosis