Base editing of haematopoietic stem cells rescues sickle cell disease in mice.

Sickle cell disease (SCD) is caused by a mutation in the ß-globin gene HBB1. We used a custom adenine base editor (ABE8e-NRCH)2,3 to convert the SCD allele (HBBS) into Makassar ß-globin (HBBG), a non-pathogenic variant4,5. Ex vivo delivery of mRNA encoding the base editor with a targeting guide RNA into haematopoietic stem and progenitor cells (HSPCs) from patients with SCD resulted in 80% conversion of HBBS to HBBG. Sixteen weeks after transplantation of edited human HSPCs into immunodeficient mice, the frequency of HBBG was 68% and hypoxia-induced sickling of bone marrow reticulocytes had decreased fivefold, indicating durable gene editing. To assess the physiological effects of HBBS base editing, we delivered ABE8e-NRCH and guide RNA into HSPCs from a humanized SCD mouse6 and then transplanted these cells into irradiated mice. After sixteen weeks, Makassar ß-globin represented 79% of ß-globin protein in blood, and hypoxia-induced sickling was reduced threefold. Mice that received base-edited HSPCs showed near-normal haematological parameters and reduced splenic pathology compared to mice that received unedited cells. Secondary transplantation of edited bone marrow confirmed that the gene editing was durable in long-term haematopoietic stem cells and showed that HBBS-to-HBBG editing of 20% or more is sufficient for phenotypic rescue. Base editing of human HSPCs avoided the p53 activation and larger deletions that have been observed following Cas9 nuclease treatment. These findings point towards a one-time autologous treatment for SCD that eliminates pathogenic HBBS, generates benign HBBG, and minimizes the undesired consequences of double-strand DNA breaks.

Erythroblastic islands foster granulopoiesis in parallel to terminal erythropoiesis.

The erythroblastic island (EBI), composed of a central macrophage surrounded by maturing erythroblasts, is the erythroid precursor niche. Despite numerous studies, its precise composition is still unclear. Using multispectral imaging flow cytometry, in vitro island reconstitution, and single-cell RNA sequencing of adult mouse bone marrow (BM) EBI-component cells enriched by gradient sedimentation, we present evidence that the CD11b+ cells present in the EBIs are neutrophil precursors specifically associated with BM EBI macrophages, indicating that erythro-(myelo)-blastic islands are a site for terminal granulopoiesis and erythropoiesis. We further demonstrate that the balance between these dominant and terminal differentiation programs is dynamically regulated within this BM niche by pathophysiological states that favor granulopoiesis during anemia of inflammation and favor erythropoiesis after erythropoietin stimulation. Finally, by molecular profiling, we reveal the heterogeneity of EBI macrophages by cellular indexing of transcriptome and epitope sequencing of mouse BM EBIs at baseline and after erythropoietin stimulation in vivo and provide a searchable online viewer of these data characterizing the macrophage subsets serving as hematopoietic niches. Taken together, our findings demonstrate that EBIs serve a dual role as niches for terminal erythropoiesis and granulopoiesis and the central macrophages adapt to optimize production of red blood cells or neutrophils.

Red cell abnormalities characterized by ektacytometry in children with cholestasis.

BACKGROUND: Spur-cell anemia sometimes accompanies cholestasis. We postulated that even in the absence of spur-cells, cholestasis might alter red blood cell (RBC) osmotic fragility and deformability. Therefore, we assessed these RBC measures by ektacytometry in pediatric patients. METHODS: We conducted a single center, prospective, cross-sectional investigation of RBC membrane characteristics by ektacytometry in pediatric patients with intra- and extrahepatic cholestasis followed at Cincinnati Children's Hospital Medical Center. We measured red cell membrane fragility and deformability in 17 patients with cholestasis and 17 age-matched controls without cholestasis. RESULTS: Patients with cholestasis had decreased RBC osmotic fragility compared to controls, with a significant left shift in Omin, indicating increased RBC surface-to-volume ratio. One showed spur cell morphology. However, the other 16 had no spurring, indicating that ektacytometry is a sensitive method to detect RBC membrane abnormalities. Left shift of Omin positively correlated with serum conjugated bilirubin levels and even more negatively with serum vitamin E concentration. CONCLUSIONS: This study suggests that subclinical red blood cell membrane abnormalities exist in most pediatric patients with cholestasis, increasing risk for hemolysis when subjected to oxidative stress. Hence minimizing pro-oxidants exposure and maximizing antioxidant exposure is advisable for this group. GOV IDENTIFIER: NCT05582447 https://clinicaltrials.gov/ct2/show/NCT05582447?cond=Cholestasis&cntry=US&state=US%3AOH&city=Cincinnati&draw=2&rank=2 . IMPACT: Spur cell anemia due to decreased red cell osmotic fragility and decreased deformability has been reported among patients with cholestasis. Ektacytometry is a reliable, reproducible method to measure red cell osmotic fragility and deformability. Few data describe red cell osmotic fragility or deformability in patients with cholestasis who may or may not have spur cell anemia. Ektacytometry shows that red cell osmotic fragility and deformability are decreased in many children with cholestasis even when spur cell anemia has not yet occurred. Factors associated with decreased osmotic fragility include elevated serum bilirubin, elevated serum bile acids, and decreased serum vitamin E.

Rapid measurement of hemoglobin-oxygen dissociation by leveraging Bohr effect and Soret band bathochromic shift.

Oxygen (O2) binds to hemoglobin (Hb) in the lungs and is then released (dissociated) in the tissues. The Bohr effect is a physiological mechanism that governs the affinity of Hb for O2 based on pH, where a lower pH results in a lower Hb-O2 affinity and higher Hb-O2 dissociation. Hb-O2 affinity and dissociation are crucial for maintaining aerobic metabolism in cells and tissues. Despite its vital role in human physiology, Hb-O2 dissociation measurement is underutilized in basic research and in clinical laboratories, primarily due to the technical complexity and limited throughput of existing methods. We present a rapid Hb-O2 dissociation measurement approach by leveraging the Bohr effect and detecting the optical shift in the Soret band that corresponds to the light absorption by the heme group in Hb. This new method reduces Hb-O2 dissociation measurement time from hours to minutes. We show that Hb deoxygenation can be accelerated chemically at the optimal pH of 6.9. We show that time and pH-controlled deoxygenation of Hb results in rapid and distinct conformational changes in its tertiary structure. These molecular conformational changes are manifested as significant, detectable shifts in Hb's optical absorption spectrum, particularly in the characteristic Soret band (414 nm). We extensively validated the method by testing human blood samples containing normal Hb and Hb variants. We show that rapid Hb-O2 dissociation can be used to screen for and detect Hb-O2 affinity disorders and to evaluate the function and efficacy of Hb-modifying therapies. The ubiquity of optical absorption spectrophotometers positions this approach as an accessible, rapid, and accurate Hb-O2 dissociation measurement method for basic research and clinical use. We anticipate this method's broad adoption will democratize the diagnosis and prognosis of Hb disorders, such as sickle cell disease. Further, this method has the potential to transform the research and development of new targeted and genome-editing-based therapies that aim to modify or improve Hb-O2 affinity.

Crosstalk between terminal erythropoiesis and granulopoiesis within their common niche: the erythromyeloblastic island.

PURPOSE OF REVIEW: The identity of the erythroblastic island (EBI) macrophage (Mϕ) has been under investigation for decades since it was recognized as the first hematopoietic niche 'nursing' terminal erythropoiesis. This review will focus on the current insights to the characteristics and the role of the EBI Mϕ balancing terminal erythropoiesis and granulopoiesis. RECENT FINDINGS: While the EBI has long been known as the niche for erythroid precursors, significant advancements in biology research technologies, including optimization of EBI enrichment protocols, single-cell ribonucleic acid sequencing, and imaging flow cytometry, have recently revealed that granulocytic precursors co-exist in this niche, termed erythromyeloblastic island (EMBI). More importantly, the balance noted at baseline between terminal granulopoiesis and erythropoiesis within EBIs/EMBIs is altered with diseases affecting hematopoiesis, such as stress erythropoiesis and inflammatory conditions causing anemia of inflammation. The role of the EMBI niche has yet to be fully investigated mechanistically, however, a notable degree of transcriptional and cell surface marker heterogeneity has been identified for the EMBI Mϕ, implicating its plasticity and diverse function. SUMMARY: Terminal erythropoiesis and granulopoiesis are regulated within the EMBI. Investigations of their balance within this niche in health and disease may reveal new targets for treatment of diseases of terminal hematopoiesis.

Navigating the marrow sea towards erythromyeloblastic islands under normal and inflammatory conditions.

PURPOSE OF REVIEW: Terminal erythroid differentiation occurs in specialized niches called erythroblastic islands. Since their discovery in 1958, these niches have been described as a central macrophage surrounded by differentiating erythroblasts. Here, we review the recent advances made in the characterization of these islands and the role they could play in anaemia of inflammation. RECENT FINDINGS: The utilization of multispectral imaging flow cytometry (flow cytometry with microscopy) has enabled for a more precise characterization of the niche that revealed the presence of maturing granulocytes in close contact with the central macrophage. These erythromyeloblastic islands (EMBIs) can adapt depending on the peripheral needs. Indeed, during inflammation wherein inflammatory cytokines limit erythropoiesis and promote granulopoiesis, EMBIs present altered structures with increased maturing granulocytes and decreased erythroid precursors. SUMMARY: Regulation of the structure and function of the EMBI in the bone marrow emerges as a potential player in the pathophysiology of acute and chronic inflammation and its associated anaemia.

VPS4A Mutations in Humans Cause Syndromic Congenital Dyserythropoietic Anemia due to Cytokinesis and Trafficking Defects.

The Congenital Dyserythropoietic Anemia (CDA) Registry was established with the goal to facilitate investigations of natural history, biology, and molecular pathogenetic mechanisms of CDA. Three unrelated individuals enrolled in the registry had a syndrome characterized by CDA and severe neurodevelopmental delay. They were found to have missense mutations in VPS4A, a gene coding for an ATPase that regulates the ESCRT-III machinery in a variety of cellular processes including cell division, endosomal vesicle trafficking, and viral budding. Bone marrow studies showed binucleated erythroblasts and erythroblasts with cytoplasmic bridges indicating abnormal cytokinesis and abscission. Circulating red blood cells were found to retain transferrin receptor (CD71) in their membrane, demonstrating that VPS4A is critical for normal reticulocyte maturation. Using proband-derived induced pluripotent stem cells (iPSCs), we have successfully modeled the hematologic aspects of this syndrome in vitro, recapitulating their dyserythropoietic phenotype. Our findings demonstrate that VPS4A mutations cause cytokinesis and trafficking defects leading to a human disease with detrimental effects to erythropoiesis and neurodevelopment.

Rapid degradation of protein tyrosine phosphatase 1B in sickle cells: Possible contribution to sickle cell membrane weakening.

Although both protein tyrosine phosphatases and kinases are constitutively active in healthy human red blood cells (RBCs), the preponderance of phosphatase activities maintains the membrane proteins in a predominantly unphosphorylated state. We report here that unlike healthy RBCs, proteins in sickle cells are heavily tyrosine phosphorylated, raising the question regarding the mechanism underpinning this tyrosine phosphorylation. Upon investigating possible causes, we observe that protein tyrosine phosphatase 1B (PTP1B), the major erythrocyte tyrosine phosphatase, is largely digested to a lower molecular weight fragment in sickle cells. We further find that the resulting truncated form of PTP1B is significantly less active than its intact counterpart, probably accounting for the intense tyrosine phosphorylation of Band 3 in sickle erythrocytes. Because this tyrosine phosphorylation of Band 3 promotes erythrocyte membrane weakening that causes release of both membrane vesicles and cell free hemoglobin that in turn initiates vaso-occlusive events, we conclude that cleavage of PTP1B could contribute to the symptoms of sickle cell disease. We further posit that methods to inhibit proteolysis of PTP1B could mitigate symptoms of the disease.

Etavopivat, a Pyruvate Kinase Activator in Red Blood Cells, for the Treatment of Sickle Cell Disease.

Etavopivat is an investigational, oral, small molecule activator of erythrocyte pyruvate kinase (PKR) in development for the treatment of sickle cell disease (SCD) and other hemoglobinopathies. PKR activation is proposed to ameliorate the sickling of SCD red blood cells (RBCs) through multiple mechanisms, including reduction of 2,3-diphosphoglycerate (2,3-DPG), which consequently increases hemoglobin (Hb)-oxygen affinity; increased binding of oxygen reduces sickle hemoglobin polymerization and sickling. In addition, PKR activation increases adenosine triphosphate (ATP) produced via glycolytic flux, which helps preserve membrane integrity and RBC deformability. We evaluated the pharmacodynamic response to etavopivat in nonhuman primates (NHPs) and in healthy human subjects and evaluated the effects in RBCs from patients with SCD after ex vivo treatment with etavopivat. A single dose of etavopivat decreased 2,3-DPG in NHPs and healthy subjects. Hb-oxygen affinity was significantly increased in healthy subjects after 24 hours. After daily dosing of etavopivat over 5 consecutive days in NHPs, ATP was increased by 38% from baseline. Etavopivat increased Hb-oxygen affinity and reduced sickling in RBCs collected from patients with SCD with either homozygous hemoglobin S or hemoglobin S and C disease. Collectively, these results demonstrate the ability of etavopivat to decrease 2,3-DPG and increase ATP, resulting in increased Hb-oxygen affinity and improved sickle RBC function. Etavopivat is currently being evaluated in clinical trials for the treatment of SCD. SIGNIFICANCE STATEMENT: Etavopivat, a small molecule activator of the glycolytic enzyme erythrocyte pyruvate kinase, decreased 2,3-diphosphoglycerate in red blood cells (RBCs) from nonhuman primates and healthy subjects and significantly increased hemoglobin (Hb)-oxygen affinity in healthy subjects. Using ex vivo RBCs from donors with sickle cell disease (SCD) (homozygous hemoglobin S or hemoglobin S and C genotype), etavopivat increased Hb-oxygen affinity and reduced sickling under deoxygenation. Etavopivat shows promise as a treatment for SCD that could potentially reduce vaso-occlusion and improve anemia.

Red cell membrane disorders: structure meets function.

The mature red blood cell (RBC) lacks a nucleus and organelles characteristic of most cells, but it is elegantly structured to perform the essential function of delivering oxygen and removing carbon dioxide from all other cells while enduring the shear stress imposed by navigating small vessels and sinusoids. Over the past several decades, the efforts of biochemists, cell and molecular biologists, and hematologists have provided an appreciation of the complexity of RBC membrane structure, while studies of the RBC membrane disorders have offered valuable insights into structure-function relationships. Within the last decade, advances in genetic testing and its increased availability have made it possible to substantially build upon this foundational knowledge. Although disorders of the RBC membrane due to altered structural organization or altered transport function are heterogeneous, they often present with common clinical findings of hemolytic anemia. However, they may require substantially different management depending on the underlying pathophysiology. Accurate diagnosis is essential to avoid emergence of complications or inappropriate interventions. We propose an algorithm for laboratory evaluation of patients presenting with symptoms and signs of hemolytic anemia with a focus on RBC membrane disorders. Here, we review the genotypic and phenotypic variability of the RBC membrane disorders in order to raise the index of suspicion and highlight the need for correct and timely diagnosis.

Altered erythropoiesis in newborns with congenital heart disease.

BACKGROUND: Fetal hypoxia has been implicated in fetal growth restriction in congenital heart disease (CHD) and leads to stress erythropoiesis in utero. The objective is to assess erythropoiesis and its association with growth in newborns with CHD. METHODS: Fetuses with prenatally diagnosed CHD from 2013 to 2018 were retrospectively reviewed. Pregnancies with multiple gestation, genetic abnormalities, major extra-cardiac anomalies, and placental abruption were excluded. Complete blood count tests at birth were compared to published normative values. Spearman correlation assessed associations of red blood cell (RBC) indices with birth anthropometrics and prenatal Doppler measures. RESULTS: A total of 160 newborns were included. Median gestational age was 38.3 (37.3, 39.0) weeks. Infants ≥37 weeks gestation had lower hemoglobin (Hgb), hematocrit, and elevated nucleated RBC (nRBC), mean corpuscular volume, and mean corpuscular hemoglobin compared to reference. No differences in RBC indices were observed in infants <34 and 34-37 weeks gestation. There was no difference in Hgb and nRBC between CHD subgroups. Neither Hgb nor nRBC were associated with birth anthropometrics or Doppler patterns. CONCLUSIONS: Term infants with CHD demonstrated multiple alterations in erythrocyte indices suggesting ineffective stress erythropoiesis in late gestation resulting in lower Hgb at birth. Altered erythropoiesis was not correlated to growth or Doppler patterns. IMPACT: Newborns with congenital heart disease (CHD) born at term gestation demonstrated altered erythropoiesis. Term newborns with CHD have decreased hemoglobin levels despite having red blood cell indices consistent with stress erythropoiesis, suggesting an incomplete compensatory response to in utero physiologic disturbances associated with CHD. The etiology is unknown; however, it may be influenced by multiple risk factors during pregnancy in the maternal-fetal dyad. Alterations in red blood cell indices were not associated with outcomes of fetal growth.

Early initiation of hydroxyurea (hydroxycarbamide) using individualised, pharmacokinetics-guided dosing can produce sustained and nearly pancellular expression of fetal haemoglobin in children with sickle cell anaemia.

Hydroxyurea (hydroxycarbamide) is an effective treatment for sickle cell anaemia (SCA), but clinical responses depend primarily upon the degree of fetal haemoglobin (HbF) induction and the heterogeneity of HbF expression across erythrocytes. The number and characteristics of HbF-containing cells (F-cells) are not assessed by traditional HbF measurements. Conventional hydroxyurea dosing (e.g. fixed doses or low starting doses with stepwise escalation) produces a moderate heterocellular HbF induction, but haemolysis and clinical complications continue. Robust, pancellular HbF induction is needed to minimise or fully inhibit polymerisation of sickle haemoglobin. We treated children with hydroxyurea using an individualised, pharmacokinetics-guided regimen starting at predicted maximum tolerated dose (MTD). We observed sustained HbF induction (mean >30%) for up to 6 years, which was not dependent on genetic determinants of HbF expression. Nearly 70% of patients had ≥80% F-cells (near-pancellular), and almost half had ≥90% F-cells (pancellular). The mean HbF/F-cell content was ~12 pg. Earlier age of initiation and better medication adherence were associated with high F-cell responses. In summary, early initiation of hydroxyurea using pharmacokinetics-guided starting doses at predicted MTD can achieve sustained near-pancellular or pancellular HbF expression and should be considered an achievable goal for children with SCA treated with hydroxyurea at optimal doses. Clinical trial registration number: NCT02286154 (clinicaltrials.gov).

Rasa3 regulates stage-specific cell cycle progression in murine erythropoiesis.

Inherited bone marrow failure syndromes (IBMFS) are heterogeneous disorders characterized by dysregulated hematopoiesis in various lineages, developmental anomalies, and predisposition to malignancy. The scat (severe combined anemia and thrombocytopenia) mouse model is a model of IBMFS with a phenotype of pancytopenia cycling through crises and remission. Scat carries an autosomal recessive missense mutation in Rasa3 that results in RASA3 mislocalization and loss of function. RASA3 functions as a Ras-GTPase activating protein (GAP), and its loss of function in scat results in increased erythroid RAS activity and reactive oxygen species (ROS) and altered erythroid cell cycle progression, culminating in delayed terminal erythroid differentiation. Here we sought to further resolve the erythroid cell cycle defect in scat through ex vivo flow cytometric analyses. These studies revealed a specific G0/G1 accumulation in scat bone marrow (BM) polychromatophilic erythroblasts and scat BM Ter119-/c-KIT+/CD71lo/med progenitors, with no changes evident in equivalent scat spleen populations. Systematic analyses of RNAseq data from megakaryocyte-erythroid progenitors (MEPs) in scat crisis vs. scat partial remission reveal altered expression of genes involved in the G1-S checkpoint. Together, these data indicate a precise, biphasic role for RASA3 in regulating the cell cycle during erythropoiesis with relevance to hematopoietic disease progression.

Congenital dyserythropoietic anemia type I: First report from the Congenital Dyserythropoietic Anemia Registry of North America (CDAR).

Congenital dyserythropoietic anemias (CDAs) are characterized by ineffective erythropoiesis and distinctive erythroblast abnormalities; the diagnosis is often missed or delayed due to significant phenotypic heterogeneity. We established the CDA Registry of North America (CDAR) to study the natural history of CDA and create a biorepository to investigate the pathobiology of this heterogeneous disease. Seven of 47 patients enrolled so far in CDAR have CDA-I due to biallelic CDAN1 mutations. They all presented with perinatal anemia and required transfusions during infancy. Anemia spontaneously improved during infancy in three patients; two became transfusion-independent rapidly after starting interferon-α2; and two remain transfusion-dependent at last follow-up at ages 5 and 30 y.o. One of the transfusion-dependent patients underwent splenectomy at 11 y.o due to misdiagnosis and returned to medical attention at 27 y.o with severe hemolytic anemia and pulmonary hypertension. All patients developed iron overload even without transfusions; four were treated with chelation. Genetic testing allowed for more rapid and accurate diagnosis; the median age of confirmed diagnosis in our cohort was 3 y.o compared to 17.3 y.o historically. In conclusion, CDAR provides an organized research network for multidisciplinary clinical and research collaboration to conduct natural history and biologic studies in CDA.

Implementation of near-universal hydroxyurea uptake among children with sickle cell anemia: A single-center experience.

BACKGROUND: Without early initiation of disease-modifying therapy, the acute and chronic complications of sickle cell anemia (SCA) begin early in childhood and progress throughout life. Hydroxyurea is a safe and effective medication that reduces or prevents most SCA-related complications. Despite recommendations to prescribe hydroxyurea for all children with SCA as young as 9 months, utilization remains low. PROCEDURE: We completed a retrospective review of hydroxyurea-prescribing practices and associated clinical outcomes at our institution over a 10-year period before and after the 2014 National Heart, Lung, and Blood Institute (NHLBI) recommendations to use hydroxyurea for all children with SCA. RESULTS: Hydroxyurea use more than doubled within our pediatric SCA population from 43% in 2010 to 95% in 2019. The age of hydroxyurea initiation was significantly younger during 2014-2019 compared to 2010-2013 (median 2 years vs. 6 years, p ≤ .001). With this change in clinical practice, nearly all (69/71 = 97%) children born after 2013 received disease-modifying therapy by the end of 2019, primarily hydroxyurea (93%). Concurrently, the number of SCA-related admissions significantly decreased from 67/100 patient-years in 2010 to 39/100 patient-years in 2019 (p < .001). CONCLUSION: The early and universal prescription of hydroxyurea for children with SCA is the standard of care. Here, we demonstrate that a careful and deliberate commitment to follow this guideline in clinical practice is feasible and results in measurable improvements in clinical outcomes. Our approach and improved outcomes can serve as a model for other programs to expand their hydroxyurea use for more children with SCA.

Inhibition of Band 3 tyrosine phosphorylation: a new mechanism for treatment of sickle cell disease.

Many hypotheses have been proposed to explain how a glutamate to valine substitution in sickle haemoglobin (HbS) can cause sickle cell disease (SCD). We propose and document a new mechanism in which elevated tyrosine phosphorylation of Band 3 initiates sequelae that cause vaso-occlusion and the symptoms of SCD. In this mechanism, denaturation of HbS and release of heme generate intracellular oxidants which cause inhibition of erythrocyte tyrosine phosphatases, thus permitting constitutive tyrosine phosphorylation of Band 3. This phosphorylation in turn induces dissociation of the spectrin-actin cytoskeleton from the membrane, leading to membrane weakening, discharge of membrane-derived microparticles (which initiate the coagulation cascade) and release of cell-free HbS (which consumes nitric oxide) and activates the endothelium to express adhesion receptors). These processes promote vaso-occlusive events which cause SCD. We further show that inhibitors of Syk tyrosine kinase block Band 3 tyrosine phosphorylation, prevent release of cell-free Hb, inhibit discharge of membrane-derived microparticles, increase sickle cell deformability, reduce sickle cell adhesion to human endothelial cells, and enhance sickle cell flow through microcapillaries. In view of reports that imatinib (a Syk inhibitor) successfully treats symptoms of sickle cell disease, we suggest that Syk tyrosine kinase inhibitors warrant repurposing as potential treatments for SCD.

How I approach hereditary hemolytic anemia and splenectomy.

Hereditary hemolytic anemias (HHA) are a heterogeneous group of anemias associated with decreased red cell survival. While there can be clinical benefit of splenectomy in many cases, splenectomy is not appropriate for all types of HHA. Additionally, there are significant risks during and following splenectomy including surgical risks, postsplenectomy sepsis, and thrombotic complications. This review discusses the diagnostic approach to HHA as well as the role of splenectomy in the management. Surgical approaches and outcomes for total and partial splenectomy are discussed.

RGL2 Deficiency Impairs Human Erythropoiesis By Altering Terminal Erythroid Differentiation and Apoptosis.

DISCLOSURES: No relevant conflicts of interest to declare.

Novel mechanisms of PIEZO1 dysfunction in hereditary xerocytosis.

Mutations in PIEZO1 are the primary cause of hereditary xerocytosis, a clinically heterogeneous, dominantly inherited disorder of erythrocyte dehydration. We used next-generation sequencing-based techniques to identify PIEZO1 mutations in individuals from 9 kindreds referred with suspected hereditary xerocytosis (HX) and/or undiagnosed congenital hemolytic anemia. Mutations were primarily found in the highly conserved, COOH-terminal pore-region domain. Several mutations were novel and demonstrated ethnic specificity. We characterized these mutations using genomic-, bioinformatic-, cell biology-, and physiology-based functional assays. For these studies, we created a novel, cell-based in vivo system for study of wild-type and variant PIEZO1 membrane protein expression, trafficking, and electrophysiology in a rigorous manner. Previous reports have indicated HX-associated PIEZO1 variants exhibit a partial gain-of-function phenotype with generation of mechanically activated currents that inactivate more slowly than wild type, indicating that increased cation permeability may lead to dehydration of PIEZO1-mutant HX erythrocytes. In addition to delayed channel inactivation, we found additional alterations in mutant PIEZO1 channel kinetics, differences in response to osmotic stress, and altered membrane protein trafficking, predicting variant alleles that worsen or ameliorate erythrocyte hydration. These results extend the genetic heterogeneity observed in HX and indicate that various pathophysiologic mechanisms contribute to the HX phenotype.

Robust clinical and laboratory response to hydroxyurea using pharmacokinetically guided dosing for young children with sickle cell anemia.

Hydroxyurea is FDA-approved and now increasingly used for children with sickle cell anemia (SCA), but dosing strategies, pharmacokinetic (PK) profiles, and treatment responses for individual patients are highly variable. Typical weight-based dosing with step-wise escalation to maximum tolerated dose (MTD) leads to predictable laboratory and clinical benefits, but often takes 6 to 12 months to achieve. The Therapeutic Response Evaluation and Adherence Trial (TREAT, NCT02286154) was a single-center study designed to prospectively validate a novel personalized PK-guided hydroxyurea dosing strategy with a primary endpoint of time to MTD. Enrolled participants received a single oral 20 mg/kg dose of hydroxyurea, followed by a sparse PK sampling approach with three samples collected over three hours. Analysis of individual PK data into a population PK model generated a starting dose that targets the MTD. The TREAT cohort (n = 50) was young, starting hydroxyurea at a median age of 11 months (IQR 9-26 months), and PK-guided starting doses were high (27.7 ± 4.9 mg/kg/d). Time to MTD was 4.8 months (IQR 3.3-9.3), significantly shorter than comparison studies (p < 0.0001), thus meeting the primary endpoint. More remarkably, the laboratory response for participants starting with a PK-guided dose was quite robust, achieving higher hemoglobin (10.1 ± 1.3 g/dL) and HbF (33.3 ± 9.1%) levels than traditional dosing. Though higher than traditional dosing, PK-guided doses were safe without excess hematologic toxicities. Our data suggest early initiation of hydroxyurea, using a personalized dosing strategy for children with SCA, provides laboratory and clinical response beyond what has been seen historically, with traditional weight-based dosing.