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Introduction:Sickle cell disease(SCD) is caused by a mutation in the beta globin gene that causes formation of abnormal hemoglobin S (HbS). Red blood cells (RBCs) from patients with SCD are less deformable and take on the shape of sickle upon deoxygenation. This results in multiple downstream effects including RBC adhesion, microvascular occlusion and chronic hemolysis. RBCs rely solely on anaerobic glycolysis to produce adenosine triphosphate (ATP), which is needed to maintain the cell's structure and function. Pyruvate kinase (PK) is a key enzyme involved in the last step of glycolysis. We have previously demonstrated a decrease in PK activity and stability as a novel pathophysiological feature of SCD. Currently there are several clinical trials ongoing investigating the efficacy of PK activation by small molecules such as mitapivat (NL8517, NCT04000165, NCT05031780), AG-946 (NCT04536792) and etavopivat (NCT04624659, NCT04987489). In this study we aim to investigate how PK properties correlate to sickle RBC properties in order to explore if impaired PK function is associated with specific features of the complex SCD pathophysiology.

Background

Background: Red blood cells (RBCs) are biconcave-shaped cells with viscoelastic membranes that are optimally adapted for oxygen delivery and gas exchange during their normal lifespan of 120 days. Reactive oxygen species (ROS) continuously challenge the viability of RBCs. Despite an arsenal of compounds to protect the RBC, including Glutathione (GSH), Vitamin E, superoxide dismutase (SOD) and Catalase, damage can easily be inflicted that will disrupt cellular deformability, structure, and membrane properties. The susceptibility to oxidative stress in individuals with Sickle Cell Disease (SCD) is believed to be linked to multiple factors. These include the release of heme and iron during hemolysis, inflammation processes, and a reduction in antioxidant defenses.

Background:Hereditary spherocytosis (HS) is caused by pathogenic variants in genes encoding red blood cell (RBC) cytoskeletal and (trans)membrane proteins. This ultimately leads to instability of the RBC membrane and subsequent membrane loss, resulting in the transformation of the biconcave RBC into a poorly deformable spherocyte, which is susceptible to premature splenic clearance. The diagnosis of HS has substantially improved over the last years and new advanced techniques have been established, including osmotic gradient ektacytometry, eosin-5'-maleimide (EMA) binding test and next-generation sequencing (NGS). However, there is little knowledge on the applicability of these diagnostic outcome parameters as biomarkers for clinical severity in HS.

Background:Myelodysplastic syndromes (MDS) consist of a heterogenous group of clonal myeloid malignancies, characterized by dysplasia, ineffective hematopoiesis and cytopenias. MDS patients frequently suffer from anemia, directly affecting quality of life. The limited therapeutic options often do not result in the desired clinical improvement. Interestingly, in MDS the activity of the red blood cell (RBC) enzyme pyruvate kinase (PK), a key regulatory enzyme of glycolysis, may be decreased. The activation of PK via small molecules is hypothesized to be beneficial in a wide range of anemias. Currently, a phase 2a, open-label, proof of concept trial is running on the use of the PK activator AG-946 in low-risk MDS patients (NCT05490446). In light of these developments, we investigated the effect of ex vivotreatment of MDS RBCs with AG-946.

Background Inflammation and leukocyte infiltration are hallmarks of atherosclerosis. Clinically routine hematology analyzers mostly perform an entire differential blood count by default, irrespective of the requested parameter. We hypothesize that these normally unreported leukocyte characteristics associate with coronary artery disease (CAD) severity and can improve prediction of mortality in coronary angiography patients.Methods We studied coronary angiography patients suspected of CAD (n= 1015) from the Utrecht Coronary Biobank cohort. Leukocyte characteristics were routinely assessed in blood drawn directly prior to angiography using an automated hematology analyzer and extracted from the Utrecht patient oriented database (UPOD) database. Patients were followed up for a median duration of 805 days, during which 65 patients died. We evaluated the association of leukocyte characteristics with synergy between PCI with taxus and cardiac surgery (SYNTAX) score as a measure of CAD severity, all-cause and cardiovascular mortality and major adverse cardiovascular events (MACEs). In order to determine the improvement of risk prediction, we calculated continuous net reclassification improvement (cNRI) and integrated discrimination improvement (IDI).Results Monocyte percentage showed strong independent predictive value for all-cause mortality (hazard ratio (HR) 1.44 (1.19–1.74), p< 0.001), and the monocyte-to-lymphocyte ratio performed best for cardiovascular mortality (HR 1.42 (1.11–1.81), p= 0.005). The cNRIs and IDIs of leukocyte characteristics for all-cause mortality confirmed the improvement in mortality risk prediction. No significantly predictive leukocyte characteristics were found for MACEs.Conclusion Readily available yet unreported leukocyte characteristics from routine hematology analyzers significantly improved prediction of mortality in coronary angiography patients on top of clinical characteristics.

Patients who underwent carotid endarterectomy (CEA) still have a residual risk of 13% of developing a major adverse cardiovascular event (MACE) within 3 years. Inflammatory processes leading up to MACE are not fully understood. Therefore, we examined blood cell characteristics (BCCs), possibly reflecting inflammatory processes, in relation to MACE to identify BCCs that may contribute to an increased risk.

•We established longer term proof of concept of PK activation as a potential novel therapy in SCD.•More than 1-year treatment with mitapivat, a PK activator, improved hemolytic anemia and reduced vaso-occlusive events in patients with SCD.

Context: Current laboratory parameters provide limited information about the prognosis of severely injured patients; therefore, novel laboratory parameters are needed.Materials and methods: We compared the morphological parameters of neutrophils and lymphocytes, and white blood cell (WBC) differential count between survivors and nonsurvivors within 7 days after admission. Multiple logistic regression analysis was conducted to identify independent prognostic factors for 7-day in-hospital mortality.Results: Neutrophil cell size was significantly different between survivors and nonsurvivors (p= 0.04), whereas WBC count and differential were not significantly different. Multiple logistic regression showed that neutrophil cell size was a significant predictor of poor outcome.Conclusions: Neutrophil cell size at admission is a prognostic factor for 7-day in-hospital mortality in severely injured trauma patients, whereas conventional WBC count and differential have no prognostic value.

Context: A biomarker for discriminating mechanisms of chemotherapy-induced thrombocytopenia (CIT) (i.e. increased platelet destruction and decreased platelet production) would be valuable in managing treatment.Objective: We explored the discriminating value of platelet size indices for this purpose in a population of adult oncology patients.Materials and methods: Mean platelet volume (MPV) and platelet distribution width (PDW) were compared between patients with (i) thrombocytopenia possibly due to increased platelet destruction; (ii) thrombocytopenia possibly due to decreased platelet destruction; and (iii) no thrombocytopenia.Results and conclusions: We obtained negative results, suggesting that these indices are not useful for discriminating different CIT mechanisms.

Inflammation has been identified as an important factor for disease exacerbation in obstructive lung disease. In this study, we used neutrophil and eosinophil counts as biomarkers for exacerbation in obstructive lung disease. We conducted a case–control study within a cohort of patients frequenting an outpatient clinic of Respiratory Medicine using data from the Utrecht Patient Oriented Database (UPOD). Cases were patients with a hospital admission for obstructive lung disease in 2005. For each case, one control patient was sampled from the same study base. We identified 143 cases (118 patients with chronic obstructive pulmonary disease and 25 asthma patients) and 143 controls. Admission was associated with both neutrophilia (adjusted odds ratio (OR) 4.3; 95% confidence interval (CI) 2.2–8.5), and eosinophilia (adjusted OR 2.6; 95% CI 1.1–6.2). The association with eosinophilia was only seen in asthma patients. In conclusion, neutrophil and eosinophil counts seem to be useful biomarkers for identifying exacerbations in pharmacoepidemiological studies on obstructive lung disease.

Background: Summaries of product characteristics (SPCs) and clinical guideline recommendations are available for monitoring the platelet count for heparin-induced thrombocytopenia (HIT) in patients receiving low-molecular-weight heparin (LMWH). Testing for the presence of heparin-platelet factor 4 antibodies (HPF4-Ab) and starting alternative anticoagulation is recommended when HIT is suspected.Objective: To investigate the frequency of compliance with recommendations for platelet count monitoring and management of possible HIT in hospitalized patients receiving prophylaxis and treatment dosing of LMWH for at least 5 consecutive days.Methods: A retrospective cohort study within the Utrecht Patient Oriented Database (UPOD) was conducted. For all inpatients, all episodes of exposure to dalteparin or nadroparin for at least 5 consecutive days in 2004–2005 were selected. In 4 different nonexclusive groups of patients (all pts. receiving dalteparin, all pts. receiving nadroparin, surgical pts. with a prophylactic dose of either dalteparin or nadroparin, and pts. exposed to unfractionated heparin [UFH] within 100 days before receiving either dalteparin or nadroparin), compliance with recommendations for platelet count monitoring from SPCs and a clinical guideline was studied. The frequency of compliance with these recommendations was determined. In addition, it was determined whether patient and treatment characteristics were associated with regular platelet count monitoring. Finally, the frequency of testing for HPF4-Ab and the initiation of danaparoid treatment in patients with a drop of at least 50% in platelet count were investigated.Results: A total of 6804 patients, with 7770 episodes of LMWH treatment, were included in the analysis. The frequency of compliance with platelet count monitoring recommendations was 26.3% for all patients receiving dalteparin, 35.6% for all patients receiving nadroparin, 23.0% for surgical patients receiving prophylactic dosing of either dalteparin or nadroparin, and 41.5% for patients exposed to UFH within 100 days before the start of either dalteparin or nadroparin treatment. Regular platelet count monitoring was strongly positively associated with medical patients (relative risk [RR] 2.33), surgical patients (RR 2.03), critically ill patients (RR 2.60), and those with recent exposure to UFH (RR 2.19). The frequency of testing for HPF4-Ab was 5.4% and the initiation of alternative anticoagulation with danaparoid in patients with a 50% drop in platelet count was 0%.Conclusions: The results suggest that compliance with recommendations for platelet count monitoring and management of possible HIT is low at our institution. Policies and tools to improve compliance with recommended laboratory monitoring should be developed to secure the safe use of LMWH and other medications.

Wijk: Agios Pharmaceuticals Inc.: Research Funding; RR mechatronics: Research Funding.

Background:

The red blood cell depends solely on the anaerobic conversion of glucose by the Embden-Meyerhof pathway for the generation and storage of high-energy phosphates, which is necessary for the maintenance of a number of vital functions. Many red blood cell enzymopathies have been described that disturb the erythrocyte's integrity, shorten its cellular survival, and result in hemolytic anemia. By far the majority of these enzymopathies are hereditary in nature. In this review, we summarize the current knowledge regarding the genetic, biochemical, and structural features of clinically relevant red blood cell enzymopathies involved in the Embden-Meyerhof pathway and the Rapoport-Luebering shunt.

The red blood cell depends solely on the anaerobic conversion of glucose by the Embden-Meyerhof pathway for the generation and storage of high-energy phosphates, which is necessary for the maintenance of a number of vital functions. Many red blood cell enzymopathies have been described that disturb the erythrocyte's integrity, shorten its cellular survival, and result in hemolytic anemia. By far the majority of these enzymopathies are hereditary in nature. In this review, we summarize the current knowledge regarding the genetic, biochemical, and structural features of clinically relevant red blood cell enzymopathies involved in the Embden-Meyerhof pathway and the Rapoport-Luebering shunt.

We established the molecular basis for pyruvate kinase (PK) deficiency in a white male patient with severe nonspherocytic hemolytic anemia. The paternal allele exhibited the commonPKLR cDNA sequence (c.) 1529G>A mutation, known to be associated with PK deficiency. On the maternal allele, 3 in cis mutations were identified in the erythroid-specific promoter region of the gene: one deletion of thymine −248 and 2 single nucleotide substitutions, nucleotide (nt) −324T>A and nt −83G>C. Analysis of the patient's RNA demonstrated the presence of only the 1529A allele, indicating severely reduced transcription from the allele linked to the mutated promoter region. Transfection of promoter constructs into erythroleukemic K562 cells showed that the most upstream −324T>A and −248delT mutations were nonfunctional polymorphisms. In contrast, the −83G>C mutation strongly reduced promoter activity. Site-directed mutagenesis of the promoter region revealed the presence of a putative regulatory element (PKR-RE1) whose core binding motif, CTCTG, is located between nt −87 and nt −83. Electrophoretic mobility shift assay using K562 nuclear extracts indicated binding of an as-yet-unidentified trans-acting factor. This novel element mediates the effects of factors necessary for regulation of pyruvate kinase gene expression during red cell differentiation and maturation.

We established the molecular basis for pyruvate kinase (PK) deficiency in a white male patient with severe nonspherocytic hemolytic anemia. The paternal allele exhibited the commonPKLRcDNA sequence (c.) 1529G>A mutation, known to be associated with PK deficiency. On the maternal allele, 3 in cis mutations were identified in the erythroid-specific promoter region of the gene: one deletion of thymine −248 and 2 single nucleotide substitutions, nucleotide (nt) −324T>A and nt −83G>C. Analysis of the patient's RNA demonstrated the presence of only the 1529A allele, indicating severely reduced transcription from the allele linked to the mutated promoter region. Transfection of promoter constructs into erythroleukemic K562 cells showed that the most upstream −324T>A and −248delT mutations were nonfunctional polymorphisms. In contrast, the −83G>C mutation strongly reduced promoter activity. Site-directed mutagenesis of the promoter region revealed the presence of a putative regulatory element (PKR-RE1) whose core binding motif, CTCTG, is located between nt −87 and nt −83. Electrophoretic mobility shift assay using K562 nuclear extracts indicated binding of an as-yet-unidentified trans-acting factor. This novel element mediates the effects of factors necessary for regulation of pyruvate kinase gene expression during red cell differentiation and maturation.

Coagulation factor V (FV) plays an important role in maintaining the hemostatic balance in both the formation of thrombin in the procoagulant pathway as well as in the protein C anticoagulant pathway. FV deficiency is a rare bleeding disorder with variable phenotypic expression. Little is known about the molecular basis underlying this disease. This study identified 5 novel mutations associated with FV deficiency in 3 patients with severe FV deficiency but different clinical expression and 2 unaffected carriers. Four mutations led to a premature termination codon either by a nonsense mutation (single-letter amino acid codes): A1102T, K310Term. (FV Amersfoort) and C2491T, Q773Term. (FV Casablanca) or a frameshift: an 8–base pair deletion between nucleotides 1130 and 1139 (FV Seoul1) and a 1–base pair deletion between nucleotides 4291 and 4294 (FV Utrecht). One mutation was a novel missense mutation: T1927C, C585R (FV Nijkerk), resulting in the absence of mutant protein despite normal transcription to RNA. Most likely, an arginine at this position disrupts the hydrophobic interior of the FV A2 domain. The sixth detected mutation was a previously reported missense mutation: A5279G, Y1702C (FV Seoul2). In all cases, the presence of the mutation was associated with type I FV deficiency. Identifying the molecular basis of mutations underlying this rare coagulation disorder will help to obtain more insight into the mechanisms involved in the variable clinical phenotype of patients with FV deficiency.

Coagulation factor V (FV) plays an important role in maintaining the hemostatic balance in both the formation of thrombin in the procoagulant pathway as well as in the protein C anticoagulant pathway. FV deficiency is a rare bleeding disorder with variable phenotypic expression. Little is known about the molecular basis underlying this disease. This study identified 5 novel mutations associated with FV deficiency in 3 patients with severe FV deficiency but different clinical expression and 2 unaffected carriers. Four mutations led to a premature termination codon either by a nonsense mutation (single-letter amino acid codes): A1102T, K310Term. (FV Amersfoort) and C2491T, Q773Term. (FV Casablanca) or a frameshift: an 8–base pair deletion between nucleotides 1130 and 1139 (FV Seoul1) and a 1–base pair deletion between nucleotides 4291 and 4294 (FV Utrecht). One mutation was a novel missense mutation: T1927C, C585R (FV Nijkerk), resulting in the absence of mutant protein despite normal transcription to RNA. Most likely, an arginine at this position disrupts the hydrophobic interior of the FV A2 domain. The sixth detected mutation was a previously reported missense mutation: A5279G, Y1702C (FV Seoul2). In all cases, the presence of the mutation was associated with type I FV deficiency. Identifying the molecular basis of mutations underlying this rare coagulation disorder will help to obtain more insight into the mechanisms involved in the variable clinical phenotype of patients with FV deficiency.

Background

van Dijk: Agios Pharmaceuticals: Research Funding; Axcella Health: Research Funding. Rab: Axcella Health: Research Funding; Agios Pharmaceuticals: Research Funding. Rijneveld: Servier: Research Funding; Amgen: Research Funding. Nur: Celgene: Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Roche: Speakers Bureau. Schutgens: CSL Behring: Research Funding; Novo Nordisk: Research Funding; OctaPharma: Research Funding; Pfizer: Research Funding; Shire/Takeda: Research Funding; Bayer: Research Funding. Wijk: Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Axcella health: Research Funding; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding. Van Beers: Pfizer: Research Funding; Novartis: Research Funding; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; RR Mechatronics: Research Funding.

We present a novel G1091 to A mutation in the human liver and red blood cell (RBC) pyruvate kinase (PK) gene causing severe hemolytic anemia. In two families, three children were severely PK-deficient compound heterozygotes exhibiting the G1091 to A mutation and a common G1529 to A mutation on the other allele. In one family, the mother, a G1091 to A heterozygote, later had a second baby with a new husband, also a G1091 to A carrier. The baby was homozygous for the G1091 to A mutation and died 6 weeks after birth from severe hemolysis. Both mutant alleles were expressed at the RNA level. The G1091 to A mutation results in the substitution of a conserved glycine by an aspartate in domain A of RBC PK, whereas the G1529 to A mutation leads to the substitution of a conserved arginine residue with glutamine in the C-domain. Molecular modelling of human RBC PK, based on the crystal structure of cat muscle PK, shows that both mutations are located outside the catalytic site at the interface of domains A and C. The mutations are likely to disrupt the critical conformation of the interface by introducing alternative salt bridges. In this way the Gly364 to Asp and Arg510 to Gln substitutions may cause PK deficiency by influencing the allosteric properties of the enzyme.

Background:To evaluate the hypothesis that gastrin is a local growth factor in colonic carcinomas, the expression of gastrin messenger RNA (mRNA) and peptides were examined in five human colon carcinoma cell lines, 12 solid colon carcinomas, and normal colonic tissue. Methods:Northern analysis, reverse-transcription PCR, and a library of sequence-specific radioimmunoassays were the principal methods. Results:Cell lines, tumors, and normal tissue all expressed a gastrin mRNA of 0.7 kilobases, and all cell lines contained incompletely processed progastrin (range, 17–54 fmol/106cells). Two cell lines secreted progastrin into the media (LoVo, 25 ± 3 pmol/L; HCT 116; 12 ± 2 pmol/L). Normal colonic tissue and all the solid tumors also contained progastrin, the concentration being higher in tumors (range, 0.4–2 pmol/g) than in normal tissue (range, 0.1–0.2 pmol/g). Only one tumor contained carboxyamidated gastrins. Conclusions:Normal and neoplastic colonic mucosa both express the gastrin gene, but the posttranslational phase of expression is attenuated. The incomplete processing and low level of expression suggest that autocrine gastrin secretion has only minor significance for normal adult and most neoplastic colonic tissue.

Background:To elucidate the hypothesis that gastrin and Cholecystokinin (CCK) are local growth factors for colorectal mucosa, we have examined the peptide gene expression in rat colon during development. Methods:Northern analysis, reverse transcription PCR, and sequence-specific radioimmunoassays were the essential methods. Results:High concentrations of gastrin and CCK messenger RNA were found in the fetal colon. At birth, gastrin and CCK mRNA's were both undetectable but increased subsequently towards adult life. The fetal colon contained 5.5 and 4.2 pmol/ g tissue gastrin and CCK, respectively. After birth, carboxyamidated gastrin disappeared from the colon, whereas the concentration of CCK remained at 1 pmol/g. Glycine-extended gastrin and CCK were also present in the fetal colon, but towards adult life they decreased below 0.2 pmol/g. In contrast, progastrin and proCCK were detectable at all ages. Conclusions:Rat colon expresses the gastrin and CCK genes throughout life. The posttranslational maturation of progastrin, however, ceases shortly after birth, indicating that gastrin may play a role in the developing colon. Whether CCK influences the development remains to be shown.

So far, no cells have been found to synthesize both of the homologous horomones, cholecystokinin and gastrin. Northern analysis and reverse transcription PCR showed, however, that the human gastric carcinoma cell line (AGS) expresses both a gastrin mRNA or 0.7 kb and a cholecystokinin transcript of 0.8 kb. A library of sequence-specific radioimmunoassays, cleavage with processing-like enzymes and chromatography subsequently revealed that the gastrin mRNA was translated into progastrin that was constitutively secreted into the medium (45 ± 3 pmol/1). Neither procholecystokinin nor any of its processing products were detectable in cells and media. The result suggest that differentiation into gastrin- or cholecystokinin-producing cells may be regulated at the translational level. The gastric cell line. AGS, provides a model for studies of translational regulation of cell differentiation.

Studies of transgenic mice have shown that transcriptional control of the gastrin gene exhibits significant species differences. Transfection of the human gastrin promoter in murine cells have depicted proximal Sp1, E‐box and CACC elements as the major determinants of transcription. We have examined cis‐regulatory elements of the human promoter in a human gastrin expressing cell line and find that a distal −135 to −142 Sp1 element is necessary for maximal activity. Alignment of the mouse and human promoters shows that the proximal human Sp1 and CACC elements are not conserved, whereas the E‐box element is retained. The distal Spl element is present in mouse but exhibits a C to T transition in the core that is likely to reduce binding affinity of Sp1. We conclude that gastrin gene transcription is regulated by distinct elements in man and rodents.

Studies of transgenic mice have shown that transcriptional control of the gastrin gene exhibits significant species differences. Transfection of the human gastrin promoter in murine cells have depicted proximal Sp1, E-box and CACC elements as the major determinants of transcription. We have examined cis-regulatory elements of the human promoter in a human gastrin expressing cell line and find that a distal −135 to −142 Sp1 element is necessary for maximal activity. Alignment of the mouse and human promoters shows that the proximal human Sp1 and CACC elements are not conserved, whereas the E-box element is retained. The distal Spl element is present in mouse but exhibits a C to T transition in the core that is likely to reduce binding affinity of Sp1. We conclude that gastrin gene transcription is regulated by distinct elements in man and rodents.

So far, no cells have been found to synthesize both of the homologous horomones, cholecystokinin and gastrin. Northern analysis and reverse transcription PCR showed, however, that the human gastric carcinoma cell line (AGS) expresses both a gastrin mRNA or 0.7 kb and a cholecystokinin transcript of 0.8 kb. A library of sequence‐specific radioimmunoassays, cleavage with processing‐like enzymes and chromatography subsequently revealed that the gastrin mRNA was translated into progastrin that was constitutively secreted into the medium (45 ± 3 pmol/1). Neither procholecystokinin nor any of its processing products were detectable in cells and media. The result suggest that differentiation into gastrin‐ or cholecystokinin‐producing cells may be regulated at the translational level. The gastric cell line. AGS, provides a model for studies of translational regulation of cell differentiation.

We present a novel G1091to A mutation in the human liver and red blood cell (RBC) pyruvate kinase (PK) gene causing severe hemolytic anemia. In two families, three children were severely PK-deficient compound heterozygotes exhibiting the G1091to A mutation and a common G1529to A mutation on the other allele. In one family, the mother, a G1091to A heterozygote, later had a second baby with a new husband, also a G1091to A carrier. The baby was homozygous for the G1091to A mutation and died 6 weeks after birth from severe hemolysis. Both mutant alleles were expressed at the RNA level. The G1091to A mutation results in the substitution of a conserved glycine by an aspartate in domain A of RBC PK, whereas the G1529to A mutation leads to the substitution of a conserved arginine residue with glutamine in the C-domain. Molecular modelling of human RBC PK, based on the crystal structure of cat muscle PK, shows that both mutations are located outside the catalytic site at the interface of domains A and C. The mutations are likely to disrupt the critical conformation of the interface by introducing alternative salt bridges. In this way the Gly364to Asp and Arg510to Gln substitutions may cause PK deficiency by influencing the allosteric properties of the enzyme.

Hexokinase deficiency is a rare autosomal recessive disease with a clinical phenotype of severe hemolysis. We report a novel homozygous missense mutation in exon 15 (c.2039C>G, HK [hexokinase] Utrecht) ofHK1, the gene that encodes red blood cell–specific hexokinase-R, in a patient previously diagnosed with hexokinase deficiency. The Thr680Ser substitution predicted by this mutation affects a highly conserved residue in the enzyme's active site that interacts with phosphate moieties of adenosine diphosphate, adenosine triphosphate (ATP), and inhibitor glucose-6-phosphate. We correlated the molecular data to the severe clinical phenotype of the patient by means of altered enzymatic properties of partially purified hexokinase from the patient, notably with respect to Mg2+-ATP binding. These kinetic properties contradict those obtained from a recombinant mutant brain hexokinase-I with the same Thr680Ser substitution. This contradiction thereby stresses the valuable contribution of studying patients with hexokinase deficiency to achieve a better understanding of hexokinase's key role in glycolysis.

Hexokinase deficiency is a rare autosomal recessive disease with a clinical phenotype of severe hemolysis. We report a novel homozygous missense mutation in exon 15 (c.2039C>G, HK [hexokinase] Utrecht) of HK1, the gene that encodes red blood cell–specific hexokinase-R, in a patient previously diagnosed with hexokinase deficiency. The Thr680Ser substitution predicted by this mutation affects a highly conserved residue in the enzyme's active site that interacts with phosphate moieties of adenosine diphosphate, adenosine triphosphate (ATP), and inhibitor glucose-6-phosphate. We correlated the molecular data to the severe clinical phenotype of the patient by means of altered enzymatic properties of partially purified hexokinase from the patient, notably with respect to Mg2+-ATP binding. These kinetic properties contradict those obtained from a recombinant mutant brain hexokinase-I with the same Thr680Ser substitution. This contradiction thereby stresses the valuable contribution of studying patients with hexokinase deficiency to achieve a better understanding of hexokinase's key role in glycolysis.