Biallelic hexokinase 1 (HK1) variants causative of non-spherocytic haemolytic anaemia: A case series with emphasis on the HK1 promoter variant and literature review.

The hexokinase (HK) enzyme plays a key role in red blood cell energy production. Hereditary non-spherocytic haemolytic anaemia (HNSHA) caused by HK deficiency is a rare disorder with only 12 different disease-associated variants identified. Here, we describe the clinical features and genotypes of four previously unreported patients with hexokinase 1 (HK1)-related HNSHA, yielding two novel truncating HK1 variants. The patients' phenotypes varied from mild chronic haemolytic anaemia to severe infantile-onset transfusion-dependent anaemia. Three of the patients had mild haemolytic disease caused by the common HK1 promoter c.-193A>G variant combined with an intragenic HK1 variant, emphasizing the importance of including this promoter variant in the haemolytic disease gene panels. HK activity was normal in a severely affected patient with a homozygous HK1 c.2599C>T, p.(His867Tyr) variant, but the affinity for ATP was reduced, hampering the HK function. In cases of HNSHA, kinetic studies should be considered in the functional studies of HK. We reviewed the literature of previously published patients to provide better insight into this rare disease and add to the understanding of genotype-phenotype correlation.

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).

Omega-3 supplementation changes the physical properties of leukocytes but not erythrocytes in healthy individuals: An exploratory trial.

n3-PUFA impact health in several ways, including cardiovascular protection and anti-inflammatory effects, but the underlying mechanisms are not fully understood. In this exploratory study involving 31 healthy subjects, we aimed to investigate the effects of 12 weeks of fish-oil supplementation (1500 mg EPA+DHA/day) on the physical properties of multiple blood cell types. We used deformability cytometry (DC) for all cell types and Laser-assisted Optical Rotational Red Cell Analysis (Lorrca) to assess red blood cell (RBC) deformability. We also investigated the correlation between changes in the physical properties of blood cells and changes in the Omega-3 Index (O3I), defined as the relative content of EPA+DHA in RBCs. Following supplementation, the mean±SD O3I increased from 5.3 %±1.5 % to 8.3 %±1.4 % (p < 0.001). No significant changes in RBC properties were found by both techniques. However, by DC we observed a consistent pattern of physical changes in lymphocytes, neutrophils and monocytes. Among these were significant increases in metrics correlated with the cells' deformability resulting in less stiff cells. The results suggest that leukocytes become softer and have an increased ability to deform under induced short-term physical stress such as hydrodynamic force in the circulation. These changes could impact immune function since softer leukocytes can potentially circulate more easily and could facilitate a more rapid response to systemic inflammation or infection. In conclusion, fish-oil supplementation modulates some physical properties of leukocyte-subfractions, potentially enhancing their biological function. Further studies are warranted to explore the impact of n3-PUFA on blood cell biology, particularly in disease states associated with leukocyte dysregulation.

Designing a single-arm phase 2 clinical trial of mitapivat for adult patients with erythrocyte membranopathies (SATISFY): a framework for interventional trials in rare anaemias - pilot study protocol.

INTRODUCTION: Membranopathies encompass haemolytic disorders arising from genetic variants in erythrocyte membrane proteins, including hereditary spherocytosis and stomatocytosis. Congenital dyserythropoietic anaemia type II (CDA II) is associated with the SEC23B gene and can exhibit phenotypic similarities to membranopathies. Current treatment options for these conditions, apart from splenectomy, are primarily supportive. Mitapivat, a novel pyruvate kinase (PK) activator, has demonstrated efficacy in increasing haemoglobin levels and reducing haemolysis in patients with PK deficiency, thalassemia, sickle cell disease and a mouse model of hereditary spherocytosis. METHODS AND ANALYSES: Safety and efficacy of mitapivat sulfate in adult patients with erythrocyte membranopathies (SATISFY) is a prospective, multicentre, single-arm phase two trial involving approximately 25 adult patients (≥18 years) diagnosed with a membranopathy or CDA II. During the 8-week dose escalation period, subjects will receive an initial dose of 50 mg mitapivat two times per day and may increase to 100 mg two times per day at week 4 based on the safety and changes in haemoglobin levels. Patients tolerating mitapivat well may be eligible to continue in two consecutive 24-week fixed dose periods.The primary objective of this study is to evaluate the safety of mitapivat, assessed through the occurrence of treatment-emergent adverse events. Secondary objectives include assessing the effects of mitapivat on haemoglobin levels, haemolysis, erythropoiesis, patient-reported outcome measures and spleen size.SATISFY aims to assess the safety and efficacy of mitapivat in adult patients with red blood cell membranopathies and CDA II, with the aim of establishing proof-of-concept in patients living with these rare conditions. ETHICS AND DISSEMINATION: NCT05935202/CTIS:2023-503271-24-01. Findings will be published in peer-reviewed journals. TRIAL REGISTRATION NUMBER: Clinicaltrials.gov, NCT05935202. CTIS:2023-503271-24-01. Registered 07-July-2023. Protocol number: 2.1. https://clinicaltrials.gov/study/NCT05935202.

A novel composition of endogenous metabolic modulators improves red blood cell properties in sickle cell disease.

The most common forms of sickle cell disease (SCD) are sickle cell anemia (SCA; HbSS) and HbSC disease. In both, especially the more dense, dehydrated and adherent red blood cells (RBCs) with reduced deformability are prone to hemolysis and sickling, and thereby vaso-occlusion. Based on plasma amino acid profiling in SCD, a composition of 10 amino acids and derivatives (RCitNacQCarLKHVS; Axcella Therapeutics, USA), referred to as endogenous metabolic modulators (EMMs), was designed to target RBC metabolism. The effects of ex vivo treatment with the EMM composition on different RBC properties were studied in SCD (n = 9 SCA, n = 5 HbSC disease). Dose-dependent improvements were observed in RBC hydration assessed by hemocytometry (MCV, MCHC, dense RBCs) and osmotic gradient ektacytometry (Ohyper). Median (interquartile range [IQR]) increase in Ohyper compared to vehicle was 4.9% (4.0%-5.5%), 7.5% (6.9%-9.4%), and 12.8% (11.5%-14.0%) with increasing 20×, 40×, and 80X concentrations, respectively (all p < 0.0001). RBC deformability (EImax using oxygen gradient ektacytometry) increased by 8.1% (2.2%-12.1%; p = 0.0012), 9.6% (2.9%-15.1%; p = 0.0013), and 13.3% (5.7%-25.5%; p = 0.0007), respectively. Besides, RBC adhesion to subendothelial laminin decreased by 43% (6%-68%; p = 0.4324), 58% (48%-72%; p = 0.0185), and 71% (49%-82%; p = 0.0016), respectively. Together, these results provide a rationale for further studies with the EMM composition targeting multiple RBC properties in SCD.

Osmotic gradient ektacytometry - a novel diagnostic approach for neuroacanthocytosis syndromes.

Introduction: The unique red blood cell (RBC) properties that characterize the rare neuroacanthocytosis syndromes (NAS) have prompted the exploration of osmotic gradient ektacytometry (Osmoscan) as a diagnostic tool for these disorders. In this exploratory study, we assessed if Osmoscans can discriminate NAS from other neurodegenerative diseases. Methods: A comprehensive assessment was conducted using Osmoscan on a diverse group of patients, including healthy controls (n = 9), neuroacanthocytosis syndrome patients (n = 6, 2 VPS13A and 4 XK disease), Parkinson's disease patients (n = 6), Huntington's disease patients (n = 5), and amyotrophic lateral sclerosis patients (n = 4). Concurrently, we collected and analyzed RBC indices and patients' characteristics. Results: Statistically significant changes were observed in NAS patients compared to healthy controls and other conditions, specifically in osmolality at minimal elongation index (Omin), maximal elongation index (EImax), the osmolality at half maximal elongation index in the hyperosmotic part of the curve (Ohyper), and the width of the curve close to the osmolality at maximal elongation index (Omax-width). Discussion: This study represents an initial exploration of RBC properties from NAS patients using osmotic gradient ektacytometry. While specific parameters exhibited differences, only Ohyper and Omax-width yielded 100% specificity for other neurodegenerative diseases. Moreover, unique correlations between Osmoscan parameters and RBC indices in NAS versus controls were identified, such as osmolality at maximal elongation index (Omax) vs. mean cellular hemoglobin content (MCH) and minimal elongation index (EImin) vs. red blood cell distribution width (RDW). Given the limited sample size, further studies are essential to establish diagnostic guidelines based on these findings.

Metabolic blood profile and response to treatment with the pyruvate kinase activator mitapivat in patients with sickle cell disease.

Mitapivat is an investigational, oral, small-molecule allosteric activator of pyruvate kinase (PK). PK is a regulatory glycolytic enzyme that is key in providing the red blood cell (RBC) with sufficient amounts of adenosine triphosphate (ATP). In sickle cell disease (SCD), decreased 2,3-DPG levels increase the oxygen affinity of hemoglobin, thereby preventing deoxygenation and polymerization of sickle hemoglobin. The PK activator mitapivat has been shown to decrease levels of 2,3-DPG and increase levels of ATP in RBCs in patients with SCD. In this phase 2, investigator-initiated, open-label study (https://www.clinicaltrialsregister.eu/ NL8517; EudraCT 2019-003438-18), untargeted metabolomics was used to explore the overall metabolic effects of 8-week treatment with mitapivat in the dose-finding period. In total, 1773 unique metabolites were identified in dried blood spots of whole blood from ten patients with SCD and 42 healthy controls (HCs). The metabolic phenotype of patients with SCD revealed alterations in 139/1773 (7.8%) metabolites at baseline when compared to HCs (false discovery rate-adjusted p < 0.05), including increases of (derivatives of) polyamines, purines, and acyl carnitines. Eight-week treatment with mitapivat in nine patients with SCD altered 85/1773 (4.8%) of the total metabolites and 18/139 (12.9%) of the previously identified altered metabolites in SCD (unadjusted p < 0.05). Effects were observed on a broad spectrum of metabolites and were not limited to glycolytic intermediates. Our results show the relevance of metabolic profiling in SCD, not only to unravel potential pathophysiological pathways and biomarkers in multisystem diseases but also to determine the effect of treatment.

DAHEAN: A Danish nationwide study ensuring quality assurance through real-world data for suspected hereditary anemia patients.

BACKGROUND: Hereditary anemias are a group of genetic diseases prevalent worldwide and pose a significant health burden on patients and societies. The clinical phenotype of hereditary anemias varies from compensated hemolysis to life-threatening anemia. They can be roughly categorized into three broad categories: hemoglobinopathies, membranopathies, and enzymopathies. Traditional therapeutic approaches like blood transfusions, iron chelation, and splenectomy are witnessing a paradigm shift with the advent of targeted treatments. However, access to these treatments remains limited due to lacking or imprecise diagnoses. The primary objective of the study is to establish accurate diagnoses for patients with hereditary anemias, enabling optimal management. As a secondary objective, the study aims to enhance our diagnostic capabilities. RESULTS: The DAHEAN study is a nationwide cohort study that collects advanced phenotypic and genotypic data from patients suspected of having hereditary anemias from all pediatric and hematological departments in Denmark. The study deliberates monthly by a multidisciplinary anemia board involving experts from across Denmark. So far, fifty-seven patients have been thoroughly evaluated, and several have been given diagnoses not before seen in Denmark. CONCLUSIONS: The DAHEAN study and infrastructure harness recent advancements in diagnostic tools to offer precise diagnoses and improved management strategies for patients with hereditary anemias.

Diagnosis and management of pyruvate kinase deficiency: international expert guidelines.

Pyruvate kinase (PK) deficiency is the most common cause of chronic congenital non-spherocytic haemolytic anaemia worldwide, with an estimated prevalence of one in 100 000 to one in 300 000 people. PK deficiency results in chronic haemolytic anaemia, with wide ranging and serious consequences affecting health, quality of life, and mortality. The goal of the International Guidelines for the Diagnosis and Management of Pyruvate Kinase Deficiency was to develop evidence-based guidelines for the clinical care of patients with PK deficiency. These clinical guidelines were developed by use of GRADE methodology and the AGREE II framework. Experts were invited after consideration of area of expertise, scholarly contributions in PK deficiency, and country of practice for global representation. The expert panel included 29 expert physicians (including adult and paediatric haematologists and other subspecialists), geneticists, laboratory specialists, nurses, a guidelines methodologist, patients with PK deficiency, and caregivers from ten countries. Five key topic areas were identified, the panel prioritised key questions, and a systematic literature search was done to generate evidence summaries that were used in the development of draft recommendations. The expert panel then met in person to finalise and vote on recommendations according to a structured consensus procedure. Agreement of greater than or equal to 67% among the expert panel was required for inclusion of a recommendation in the final guideline. The expert panel agreed on 31 total recommendations across five key topics: diagnosis and genetics, monitoring and management of chronic complications, standard management of anaemia, targeted and advanced therapies, and special populations. These new guidelines should facilitate best practices and evidence-based PK deficiency care into clinical practice.