Publications by authors named "Richard van Wijk"

101 Publications

Editorial: New Methods for Red Blood Cell Research and Diagnosis.

Front Physiol 2021 29;12:755664. Epub 2021 Sep 29.

Central Diagnostic Laboratory-Research, University Medical Center Utrecht, Utrecht University, Utrecht, Netherlands.

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http://dx.doi.org/10.3389/fphys.2021.755664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8513634PMC
September 2021

Recommendations for diagnosis and treatment of methemoglobinemia.

Am J Hematol 2021 Sep 1. Epub 2021 Sep 1.

Hematology, University of Utah & Huntsman Cancer Center, Salt Lake City, Utah, USA.

Methemoglobinemia is a rare disorder associated with oxidization of divalent ferro-iron of hemoglobin (Hb) to ferri-iron of methemoglobin (MetHb). Methemoglobinemia can result from either inherited or acquired processes. Acquired forms are the most common, mainly due to the exposure to substances that cause oxidation of the Hb both directly or indirectly. Inherited forms are due either to autosomal recessive variants in the CYB5R3 gene or to autosomal dominant variants in the globin genes, collectively known as HbM disease. Our recommendations are based on a systematic literature search. A series of questions regarding the key signs and symptoms, the methods for diagnosis, the clinical management in neonatal/childhood/adulthood period, and the therapeutic approach of methemoglobinemia were formulated and the relative recommendations were produced. An agreement was obtained using a Delphi-like approach and the experts panel reached a final consensus >75% of agreement for all the questions.
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http://dx.doi.org/10.1002/ajh.26340DOI Listing
September 2021

Comparisons of oxygen gradient ektacytometry parameters between sickle cell patients with or without α-thalassaemia.

Br J Haematol 2021 Aug 15. Epub 2021 Aug 15.

Team 'Vascular Biology and Red Blood Cell', Laboratoire Interuniversitaire de Biologie de la Motricité (LIBM) EA7424, Université Claude Bernard Lyon 1, Université de Lyon, Villeurbanne, France.

The present study tested the impact of α-thalassaemia on oxygen gradient ektacytometry in sickle cell anaemia (SCA). Three SCA groups were compared: (i) no α-thalassaemia (four α-genes, n = 62), (ii) silent α-thalassaemia (three α-genes, n = 35) and (iii) homozygous α-thalassaemia (two α-genes, n = 12). Red blood cell (RBC) deformability measured in normoxia was not different between the three groups. The lowest RBC deformability reached at low oxygen partial pressure (pO ) was greater and the pO at which RBC started to sickle was lower in the two α-genes group compared to the other groups. Our present study showed an effect of α-thalassaemia on oxygen gradient ektacytometry in SCA.
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http://dx.doi.org/10.1111/bjh.17777DOI Listing
August 2021

Rare Anemias: Are Their Names Just Smoke and Mirrors?

Front Physiol 2021 10;12:690604. Epub 2021 Jun 10.

Experimental Physics, Dynamics of Fluids Group, Saarland University, Saarbrücken, Germany.

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http://dx.doi.org/10.3389/fphys.2021.690604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8222994PMC
June 2021

Dried blood spot metabolomics reveals a metabolic fingerprint with diagnostic potential for Diamond Blackfan Anaemia.

Br J Haematol 2021 06 17;193(6):1185-1193. Epub 2021 May 17.

Department of Paediatric Haematology, University Medical Center Utrecht, Utrecht University, Utrecht, the Netherlands.

The diagnostic evaluation of Diamond Blackfan Anaemia (DBA), an inherited bone marrow failure syndrome characterised by erythroid hypoplasia, is challenging because of a broad phenotypic variability and the lack of functional screening tests. In this study, we explored the potential of untargeted metabolomics to diagnose DBA. In dried blood spot samples from 18 DBA patients and 40 healthy controls, a total of 1752 unique metabolite features were identified. This metabolic fingerprint was incorporated into a machine-learning algorithm, and a binary classification model was constructed using a training set. The model showed high performance characteristics (average accuracy 91·9%), and correct prediction of class was observed for all controls (n = 12) and all but one patient (n = 4/5) from the validation or 'test' set (accuracy 94%). Importantly, in patients with congenital dyserythropoietic anaemia (CDA) - an erythroid disorder with overlapping features - we observed a distinct metabolic profile, indicating the disease specificity of the DBA fingerprint and underlining its diagnostic potential. Furthermore, when exploring phenotypic heterogeneity, DBA treatment subgroups yielded discrete differences in metabolic profiles, which could hold future potential in understanding therapy responses. Our data demonstrate that untargeted metabolomics in dried blood spots is a promising new diagnostic tool for DBA.
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http://dx.doi.org/10.1111/bjh.17524DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8251760PMC
June 2021

Red blood cell phenotyping from 3D confocal images using artificial neural networks.

PLoS Comput Biol 2021 05 13;17(5):e1008934. Epub 2021 May 13.

Department of Experimental Physics, Saarland University, Campus E2.6, Saarbrücken, Germany.

The investigation of cell shapes mostly relies on the manual classification of 2D images, causing a subjective and time consuming evaluation based on a portion of the cell surface. We present a dual-stage neural network architecture for analyzing fine shape details from confocal microscopy recordings in 3D. The system, tested on red blood cells, uses training data from both healthy donors and patients with a congenital blood disease, namely hereditary spherocytosis. Characteristic shape features are revealed from the spherical harmonics spectrum of each cell and are automatically processed to create a reproducible and unbiased shape recognition and classification. The results show the relation between the particular genetic mutation causing the disease and the shape profile. With the obtained 3D phenotypes, we suggest our method for diagnostics and theragnostics of blood diseases. Besides the application employed in this study, our algorithms can be easily adapted for the 3D shape phenotyping of other cell types and extend their use to other applications, such as industrial automated 3D quality control.
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http://dx.doi.org/10.1371/journal.pcbi.1008934DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8118337PMC
May 2021

Effects of Genotypes and Treatment on Oxygenscan Parameters in Sickle Cell Disease.

Cells 2021 04 5;10(4). Epub 2021 Apr 5.

Laboratoire Interuniversitaire de Biologie de la Motricité (LIBM) EA7424, Team « Vascular Biology and Red Blood Cell », Université Claude Bernard Lyon 1, Université de Lyon, 69008 Lyon, France.

(1) Background: The aim of the present study was to compare oxygen gradient ektacytometry parameters between sickle cell patients of different genotypes (SS, SC, and S/β) or under different treatments (hydroxyurea or chronic red blood cell exchange). (2) Methods: Oxygen gradient ektacytometry was performed in 167 adults and children at steady state. In addition, five SS patients had oxygenscan measurements at steady state and during an acute complication requiring hospitalization. (3) Results: Red blood cell (RBC) deformability upon deoxygenation (EImin) and in normoxia (EImax) was increased, and the susceptibility of RBC to sickle upon deoxygenation was decreased in SC patients when compared to untreated SS patients older than 5 years old. SS patients under chronic red blood cell exchange had higher EImin and EImax and lower susceptibility of RBC to sickle upon deoxygenation compared to untreated SS patients, SS patients younger than 5 years old, and hydroxyurea-treated SS and SC patients. The susceptibility of RBC to sickle upon deoxygenation was increased in the five SS patients during acute complication compared to steady state, although the difference between steady state and acute complication was variable from one patient to another. (4) Conclusions: The present study demonstrates that oxygen gradient ektacytometry parameters are affected by sickle cell disease (SCD) genotype and treatment.
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http://dx.doi.org/10.3390/cells10040811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8067408PMC
April 2021

Impaired Cytoskeletal and Membrane Biophysical Properties of Acanthocytes in Hypobetalipoproteinemia - A Case Study.

Front Physiol 2021 23;12:638027. Epub 2021 Feb 23.

CELL Unit & PICT Imaging Platform, de Duve Institute, UCLouvain, Brussels, Belgium.

Familial hypobetalipoproteinemia is a metabolic disorder mainly caused by mutations in the gene. In its homozygous form it can lead without treatment to severe ophthalmological and neurological manifestations. In contrast, the heterozygous form is generally asymptomatic but associated with a low risk of cardiovascular disease. Acanthocytes or thorny red blood cells (RBCs) are described for both forms of the disease. However, those morphological changes are poorly characterized and their potential consequences for RBC functionality are not understood. Thus, in the present study, we asked whether, to what extent and how acanthocytes from a patient with heterozygous familial hypobetalipoproteinemia could exhibit altered RBC functionality. Acanthocytes represented 50% of the total RBC population and contained mitoTracker-positive surface patches, indicating the presence of mitochondrial fragments. While RBC osmotic fragility, calcium content and ATP homeostasis were preserved, a slight decrease of RBC deformability combined with an increase of intracellular free reactive oxygen species were observed. The spectrin cytoskeleton was altered, showing a lower density and an enrichment in patches. At the membrane level, no obvious modification of the RBC membrane fatty acids nor of the cholesterol content were detected but the ceramide species were all increased. Membrane stiffness and curvature were also increased whereas transversal asymmetry was preserved. In contrast, lateral asymmetry was highly impaired showing: (i) increased abundance and decreased functionality of sphingomyelin-enriched domains; (ii) cholesterol enrichment in spicules; and (iii) ceramide enrichment in patches. We propose that oxidative stress induces cytoskeletal alterations, leading to increased membrane stiffness and curvature and impaired lipid lateral distribution in domains and spicules. In addition, ceramide- and spectrin-enriched patches could result from a RBC maturation defect. Altogether, the data indicate that acanthocytes are associated with cytoskeletal and membrane lipid lateral asymmetry alterations, while deformability is only mildly impaired. In addition, familial hypobetalipoproteinemia might also affect RBC precursors leading to disturbed RBC maturation. This study paves the way for the potential use of membrane biophysics and lipid vital imaging as new methods for diagnosis of RBC disorders.
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http://dx.doi.org/10.3389/fphys.2021.638027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7940373PMC
February 2021

The Interplay between Drivers of Erythropoiesis and Iron Homeostasis in Rare Hereditary Anemias: Tipping the Balance.

Int J Mol Sci 2021 Feb 23;22(4). Epub 2021 Feb 23.

Van Creveldkliniek, University Medical Center Utrecht, 3584 CX Utrecht, The Netherlands.

Rare hereditary anemias (RHA) represent a group of disorders characterized by either impaired production of erythrocytes or decreased survival (i.e., hemolysis). In RHA, the regulation of iron metabolism and erythropoiesis is often disturbed, leading to iron overload or worsening of chronic anemia due to unavailability of iron for erythropoiesis. Whereas iron overload generally is a well-recognized complication in patients requiring regular blood transfusions, it is also a significant problem in a large proportion of patients with RHA that are not transfusion dependent. This indicates that RHA share disease-specific defects in erythroid development that are linked to intrinsic defects in iron metabolism. In this review, we discuss the key regulators involved in the interplay between iron and erythropoiesis and their importance in the spectrum of RHA.
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http://dx.doi.org/10.3390/ijms22042204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7927117PMC
February 2021

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

Front Physiol 2020 20;11:609103. Epub 2021 Jan 20.

Central Diagnostic Laboratory-Research, University Medical Center Utrecht, Utrecht University, Utrecht, Netherlands.

Pyruvate kinase deficiency (PKD) is a rare congenital hemolytic anemia caused by mutations in the 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 -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.
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http://dx.doi.org/10.3389/fphys.2020.609103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854701PMC
January 2021

Mechanical Stress Induces Ca-Dependent Signal Transduction in Erythroblasts and Modulates Erythropoiesis.

Int J Mol Sci 2021 Jan 19;22(2). Epub 2021 Jan 19.

Department of Hematopoiesis, Sanquin Research and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, 1012 WX Amsterdam, The Netherlands.

Bioreactors are increasingly implemented for large scale cultures of various mammalian cells, which requires optimization of culture conditions. Such upscaling is also required to produce red blood cells (RBC) for transfusion and therapy purposes. However, the physiological suitability of RBC cultures to be transferred to stirred bioreactors is not well understood. PIEZO1 is the most abundantly expressed known mechanosensor on erythroid cells. It is a cation channel that translates mechanical forces directly into a physiological response. We investigated signaling cascades downstream of PIEZO1 activated upon transitioning stationary cultures to orbital shaking associated with mechanical stress, and compared the results to direct activation of PIEZO1 by the chemical agonist Yoda1. Erythroblasts subjected to orbital shaking displayed decreased proliferation, comparable to incubation in the presence of a low dose of Yoda1. Epo (Erythropoietin)-dependent STAT5 phosphorylation, and Calcineurin-dependent NFAT dephosphorylation was enhanced. Phosphorylation of ERK was also induced by both orbital shaking and Yoda1 treatment. Activation of these pathways was inhibited by intracellular Ca chelation (BAPTA-AM) in the orbital shaker. Our results suggest that PIEZO1 is functional and could be activated by the mechanical forces in a bioreactor setup, and results in the induction of Ca-dependent signaling cascades regulating various aspects of erythropoiesis. With this study, we showed that Yoda1 treatment and mechanical stress induced via orbital shaking results in comparable activation of some Ca-dependent pathways, exhibiting that there are direct physiological outcomes of mechanical stress on erythroblasts.
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http://dx.doi.org/10.3390/ijms22020955DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7835781PMC
January 2021

Methodological aspects of oxygen gradient ektacytometry in sickle cell disease: Effects of sample storage on outcome parameters in distinct patient subgroups.

Clin Hemorheol Microcirc 2021 ;77(4):391-394

"Vascular Biology and Red Blood Cell" Team, Laboratory LIBM EA7424, University of Lyon 1, Lyon, France.

Sickle cell disease (SCD) is a genetic disorder characterized by the production of an abnormal hemoglobin (Hb), which, under deoxygenation, may polymerize and cause a mechanical distortion of red blood cell (RBC) into a crescent-like shape. Recently a method, using ektacytometry principle, has been developed to assess RBC deformability as a function of oxygen tension (pO2) and is called oxygen gradient ektacytometry (oxygenscan). However, standardization of this test is needed to properly assess the tendency of sickling of RBCs under deoxygenation and to allow comparisons between different laboratories. The study compared the oxygenscan responses during blood storage between distinct populations of SCD patients. Blood from 40 non-transfused homozygous SCD patients (HbSS), 16 chronically transfused HbSS patients, and 14 individuals with compound heterozygous hemoglobin SC disease (HbSC) at steady-state was collected in EDTA tubes. Measurements were performed within 4 hours after collection and after 24 hours of storage at 4°C. We showed that storage affected the minimum RBC deformability reached during deoxygenation (EImin) in both non-transfused HbSS and HbSC patients and the maximum RBC deformability (EImax) measured before deoxygenation (i.e., in normoxia) in the three groups. In contrast, the tendency of RBCs to sickle under deoxygenation (i.e., the point of sickling; PoS) remained rather stable between the two time of measurements. Collectively, since the time between blood sampling and analysis affects some key oxygen gradient ektacytometry-derived parameters we recommend that each laboratory performs oxygenscan measurements at a standardized time point.
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http://dx.doi.org/10.3233/CH-201037DOI Listing
May 2021

Infantile Pyknocytosis in a Premature Dichorionic Diamniotic Twin.

J Pediatr Hematol Oncol 2021 Oct;43(7):e1037-e1039

Clinical Chemistry, Deventer Hospital, Deventer.

Infantile pyknocytosis is a rare and self-limiting cause of hemolytic anemia in neonates. It can result in severe anemia and hyperbilirubinemia. The pathogenesis is unknown: a genetic origin has been discussed; however, based on the current literature it is not clear which genetic mutations should be considered. We present a case of a premature twin, in whom genetic screening was performed. Genetic mutations in 46 genes associated with hereditary hemolytic anemia and dyserythropoietic anemia were tested. No mutations were found. In infantile pyknocytosis, a genetic defect in these genes is unlikely.
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http://dx.doi.org/10.1097/MPH.0000000000002004DOI Listing
October 2021

Editorial: Pathophysiology of Rare Hemolytic Anemias.

Front Physiol 2020 22;11:601746. Epub 2020 Oct 22.

Hematology Unit, Phatophysiology of Anemias Unit, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy.

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http://dx.doi.org/10.3389/fphys.2020.601746DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642987PMC
October 2020

Untargeted metabolic profiling in dried blood spots identifies disease fingerprint for pyruvate kinase deficiency.

Haematologica 2021 10 1;106(10):2720-2725. Epub 2021 Oct 1.

Central Diagnostic Laboratory-Research, University Medical Center Utrecht, Utrecht.

The diagnostic evaluation and clinical characterization of rare hereditary anemia (RHA) is to date still challenging. In particular, there is little knowledge on the broad metabolic impact of many of the molecular defects underlying RHA. In this study we explored the potential of untargeted metabolomics to diagnose a relatively common type of RHA: Pyruvate Kinase Deficiency (PKD). In total, 1903 unique metabolite features were identified in dried blood spot samples from 16 PKD patients and 32 healthy controls. A metabolic fingerprint was identified using a machine learning algorithm, and subsequently a binary classification model was designed. The model showed high performance characteristics (AUC 0.990, 95%CI 0.981-0.999) and an accurate class assignment was achieved for all newly added control (13) and patient samples (6), with the exception of one patient (accuracy 94%). Important metabolites in the metabolic fingerprint included glycolytic intermediates, polyamines and several acyl carnitines. In general, the application of untargeted metabolomics in dried blood spots is a novel functional tool that holds promise for diagnostic stratification and studies on disease pathophysiology in RHA.
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http://dx.doi.org/10.3324/haematol.2020.266957DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8485668PMC
October 2021

The variable manifestations of disease in pyruvate kinase deficiency and their management.

Haematologica 2020 09 1;105(9):2229-2239. Epub 2020 Sep 1.

Dana/Farber Boston Children's Cancer and Blood Disorders Center, Harvard Medical School, Boston, MA, USA.

Pyruvate kinase deficiency (PKD) is the most common cause of chronic hereditary non-spherocytic hemolytic anemia and results in a broad spectrum of disease. The diagnosis of PKD requires a high index of suspicion and judicious use of laboratory tests that may not always be informative, including pyruvate kinase enzyme assay and genetic analysis of the PKLR gene. A significant minority of patients with PKD have occult mutations in non-coding regions of PKLR which are missed on standard genetic tests. The biochemical consequences of PKD result in hemolytic anemia due to red cell pyruvate and ATP deficiency while simultaneously causing increased red cell 2,3-diphosphoglycerate, which facilitates oxygen unloading. This phenomenon, in addition to numerous other factors such as genetic background and differences in splenic function result in a poor correlation between symptoms and degree of anemia from patient to patient. Red cell transfusions should, therefore, be symptom-directed and not based on a hemoglobin threshold. Patients may experience specific complications, such as paravertebral extramedullary hematopoiesis and chronic debilitating icterus, which require personalized treatment. The decision to perform splenectomy or hematopoietic stem cell transplantation is nuanced and depends on disease burden and long-term outlook given that targeted therapeutics are in development. In recognition of the complicated nature of the disease and its management and the limitations of the PKD literature, an international working group of ten PKD experts convened to better define the disease burden and manifestations. This article summarizes the conclusions of this working group and is a guide for clinicians and investigators caring for patients with PKD.
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http://dx.doi.org/10.3324/haematol.2019.240846DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556504PMC
September 2020

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

Blood Adv 2020 04;4(8):1678-1682

Van Creveldkliniek, Division of Internal Medicine and Dermatology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands.

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.
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http://dx.doi.org/10.1182/bloodadvances.2020001595DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7189297PMC
April 2020

Genotype-phenotype correlation and molecular heterogeneity in pyruvate kinase deficiency.

Am J Hematol 2020 05 6;95(5):472-482. Epub 2020 Mar 6.

Department of Pediatrics, University of Würzburg, Würzburg, Germany.

Pyruvate kinase (PK) deficiency is a rare recessive congenital hemolytic anemia caused by mutations in the PKLR gene. This study reports the molecular features of 257 patients enrolled in the PKD Natural History Study. Of the 127 different pathogenic variants detected, 84 were missense and 43 non-missense, including 20 stop-gain, 11 affecting splicing, five large deletions, four in-frame indels, and three promoter variants. Within the 177 unrelated patients, 35 were homozygous and 142 compound heterozygous (77 for two missense, 48 for one missense and one non-missense, and 17 for two non-missense variants); the two most frequent mutations were p.R510Q in 23% and p.R486W in 9% of mutated alleles. Fifty-five (21%) patients were found to have at least one previously unreported variant with 45 newly described mutations. Patients with two non-missense mutations had lower hemoglobin levels, higher numbers of lifetime transfusions, and higher rates of complications including iron overload, extramedullary hematopoiesis, and pulmonary hypertension. Rare severe complications, including lower extremity ulcerations and hepatic failure, were seen more frequently in patients with non-missense mutations or with missense mutations characterized by severe protein instability. The PKLR genotype did not correlate with the frequency of complications in utero or in the newborn period. With ICCs ranging from 0.4 to 0.61, about the same degree of clinical similarity exists within siblings as it does between siblings, in terms of hemoglobin, total bilirubin, splenectomy status, and cholecystectomy status. Pregnancy outcomes were similar across genotypes in PK deficient women. This report confirms the wide genetic heterogeneity of PK deficiency.
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http://dx.doi.org/10.1002/ajh.25753DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8127999PMC
May 2020

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

Int J Mol Sci 2020 Feb 6;21(3). Epub 2020 Feb 6.

Departments of Haematology and Internal Medicine, University Medical Center Utrecht, Utrecht University, 3584 CX Utrecht, The Netherlands.

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 Fe-labeled iron through the portal system, using standard ferrokinetic techniques measuring red cell iron uptake after oral and intravenous Fe 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%) ( < 0.05) resulting in increased iron retention after 14 days compared to healthy controls in all groups ( < 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 ( < 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.
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http://dx.doi.org/10.3390/ijms21031077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7037197PMC
February 2020

AG-348 (Mitapivat), an allosteric activator of red blood cell pyruvate kinase, increases enzymatic activity, protein stability, and ATP levels over a broad range of PKLR genotypes.

Haematologica 2021 01 1;106(1):238-249. Epub 2021 Jan 1.

Laboratory of Clinical Chemistry and Haematology, University Medical Center Utrecht.

Pyruvate kinase (PK) deficiency is a rare hereditary disorder affecting red cell (RBC) glycolysis, causing changes in metabolism including a deficiency in ATP. This affects red cell homeostasis, promoting premature removal of RBCs from the circulation. In this study we characterized and evaluated the effect of AG-348, an allosteric activator of PK that is currently in clinical trials for treatment of PK deficiency, on RBCs and erythroid precursors from PK-deficient patients. In 15 patients ex vivo treatment with AG-348 resulted in increased enzymatic activity in all patient cells after 24 hours (mean increase 1.8-fold, range 1.2-3.4). ATP levels increased (mean increase 1.5-fold, range 1.0-2.2) similar to control cells (mean increase 1.6-fold, range, 1.4-1.8). Generally, PK thermostability was strongly reduced in PK-deficient RBCs. Ex vivo treatment with AG-348 increased residual activity 1.4 to >10-fold than residual activity of vehicle-treated samples. Protein analyses suggests that a sufficient level of PK protein is required for cells to respond to AG-348 treatment ex-vivo, as treatment effects were minimal in patient cells with very low or undetectable levels of PK-R. In half of the patients, ex vivo treatment with AG-348 was associated with an increase in RBC deformability. These data support the hypothesis that drug intervention with AG-348 effectively upregulates PK enzymatic activity and increases stability in PK-deficient RBCs over a broad range of PKLR genotypes. The concomitant increase in ATP levels suggests that glycolytic pathway activity may be restored. AG-348 treatment may represent an attractive way to correct the underlying pathologies of PK deficiency. (AG-348 is currently in clinical trials for the treatment of PK deficiency. ClinicalTrials.gov: NCT02476916, NCT03853798, NCT03548220, NCT03559699).
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http://dx.doi.org/10.3324/haematol.2019.238865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7776327PMC
January 2021

Characterization of Sickling During Controlled Automated Deoxygenation with Oxygen Gradient Ektacytometry.

J Vis Exp 2019 11 5(153). Epub 2019 Nov 5.

Laboratory of Clinical Chemistry and Hematology, University Medical Center Utrecht, Utrecht University.

In sickle cell disease (SCD), a single point mutation in the gene coding for beta-globin causes the production of abnormal hemoglobin S (HbS). When deoxygenated, HbS can polymerize, forming rigid rods of hemoglobin, resulting in the sickling of red blood cells (RBCs). These sickled RBCs have significantly reduced deformability, causing vaso-occlusion, which leads to numerous SCD-related clinical complications, including pain, stroke, and organ damage. RBC deformability is also reduced by RBC dehydration, resulting in dense red blood cells that are more likely to sickle. To date, there is not a single widely available, rapid, and reproducible laboratory assay capable of predicting the disease severity or directly monitoring the treatment effects for novel, non-fetal hemoglobin inducing therapies. In this study, we describe a protocol to measure RBC deformability as a function of pO2 that allows for the quantitation of sickling behavior in SCD patients. Oxygen gradient ektacytometry measures RBC deformability, expressed as the elongation index (EI), as a function of pO2. RBCs are exposed to a fixed shear stress of 30 Pa during one round of deoxygenation and reoxygenation. Six readout parameters are produced. Of these, the point of sickling (PoS), defined as the pO2 at which maximum EI (EImax) shows a 5% decrease, and minimum EI during deoxygenation (EImin) are the most informative, reflecting an individual patient's pO2 at which sickling starts and the minimal deformability of a patient's red blood cells, respectively. PoS is associated with an individual patient's hemoglobin affinity for oxygen, whereas EImin shows a strong correlation with fetal hemoglobin levels. We conclude that oxygen gradient ektacytometry is a promising technique to monitor the treatment of patients with SCD, as a biomarker for anti-sickling agents in clinical and preclinical trials, and an important tool to study sickling behavior of RBCs from individuals with SCD and sickle cell traits.
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http://dx.doi.org/10.3791/60213DOI Listing
November 2019

The Complexity of Genotype-Phenotype Correlations in Hereditary Spherocytosis: A Cohort of 95 Patients: Genotype-Phenotype Correlation in Hereditary Spherocytosis.

Hemasphere 2019 Aug 7;3(4):e276. Epub 2019 Aug 7.

Department of Clinical Chemistry and Haematology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands.

Hereditary spherocytosis (HS) is a phenotypically and genetically heterogeneous disease. With the increased use of Next Generation Sequencing (NGS) techniques in the diagnosis of red blood cell disorders, the list of unique pathogenic mutations underlying HS is growing rapidly. In this study, we aimed to explore genotype-phenotype correlation in 95 HS patients genotyped by targeted NGS as part of routine diagnostics (UMC Utrecht, Utrecht, The Netherlands). In 85/95 (89%) of patients a pathogenic mutation was identified, including 56 novel mutations. mutations were most frequently encountered (36%, 31/85 patients), primarily in patients with autosomal recessive forms of HS. Three (α-spectrin) mutations showed autosomal dominant inheritance. (ankyrin1) mutations accounted for 27% (23/85 patients) and (β-spectrin) mutations for 20% (17/85 patients). Moderate or severe HS was more frequent in patients with or mutations, reflected by lower hemoglobin concentrations and higher reticulocyte counts. Interestingly, mutations affecting spectrin association domains of , and resulted in more severe phenotypes. Additionally, we observed a clear association between phenotype and aspects of red cell deformability as determined by the Laser assisted Optical Rotational Cell Analyzer (LoRRca MaxSis). Both maximal deformability and area under the curve were negatively associated with disease severity (respectively r = -0.46, p < 0.01, and r = -0.39, p = 0.01). Genotype-phenotype prediction in HS facilitates insight in consequences of pathogenic mutations for the assembly and dynamic interactions of the red cell cytoskeleton. In addition, we show that measurements of red blood cell deformability are clearly correlated with HS severity.
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http://dx.doi.org/10.1097/HS9.0000000000000276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6745925PMC
August 2019
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