HEATR3 variants impair nuclear import of uL18 (RPL5) and drive Diamond-Blackfan anemia.

The congenital bone marrow failure syndrome Diamond-Blackfan anemia (DBA) is typically associated with variants in ribosomal protein (RP) genes impairing erythroid cell development. Here we report multiple individuals with biallelic HEATR3 variants exhibiting bone marrow failure, short stature, facial and acromelic dysmorphic features, and intellectual disability. These variants destabilize a protein whose yeast homolog is known to synchronize the nuclear import of RPs uL5 (RPL11) and uL18 (RPL5), which are both critical for producing ribosomal subunits and for stabilizing the p53 tumor suppressor when ribosome biogenesis is compromised. Expression of HEATR3 variants or repression of HEATR3 expression in primary cells, cell lines of various origins, and yeast models impairs growth, differentiation, pre-ribosomal RNA processing, and ribosomal subunit formation reminiscent of DBA models of large subunit RP gene variants. Consistent with a role of HEATR3 in RP import, HEATR3-depleted cells or patient-derived fibroblasts display reduced nuclear accumulation of uL18. Hematopoietic progenitor cells expressing HEATR3 variants or small-hairpin RNAs knocking down HEATR3 synthesis reveal abnormal acceleration of erythrocyte maturation coupled to severe proliferation defects that are independent of p53 activation. Our study uncovers a new pathophysiological mechanism leading to DBA driven by biallelic HEATR3 variants and the destabilization of a nuclear import protein important for ribosome biogenesis.

HAX1-related congenital neutropenia: Long-term observation in paediatric and adult patients enrolled in the European branch of the Severe Chronic Neutropenia International Registry (SCNIR).

HAX1-related congenital neutropenia (HAX1-CN) is a rare autosomal recessive disorder caused by pathogenic variants in the HAX1 gene. HAX1-CN patients suffer from bone marrow failure as assessed by a maturation arrest of the myelopoiesis revealing persistent severe neutropenia from birth. The disorder is strongly associated with severe bacterial infections and a high risk of developing myelodysplastic syndrome or acute myeloid leukaemia. This study aimed to describe the long-term course of the disease, the treatment, outcome and quality of life in patients with homozygous HAX1 mutations reported to the European branch of the Severe Chronic Neutropenia International Registry. We have analysed a total of 72 patients with different types of homozygous (n = 68), compound heterozygous (n = 3), and digenic (n = 1) HAX1 mutations. The cohort includes 56 paediatric (<18 years) and 16 adult patients. All patients were initially treated with G-CSF with a sufficient increase in absolute neutrophil counts. Twelve patients required haematopoietic stem cell transplantation for leukaemia (n = 8) and non-leukaemic indications (n = 4). While previous genotype-phenotype reports documented a striking correlation between two main transcript variants and clinical neurological phenotypes, our current analysis reveals novel mutation subtypes and clinical overlaps between all genotypes including severe secondary manifestations, e.g., high incidence of secondary ovarian insufficiency.

Nationwide study of eculizumab in paroxysmal nocturnal hemoglobinuria: Evaluation of treatment indications and outcomes.

Eculizumab is an effective treatment for paroxysmal nocturnal hemoglobinuria (PNH). However, considering the risk of life-threatening meningococcal disease, life-long duration and costs, there are strict criteria for initiation of therapy. To evaluate the application and real-world effectiveness of eculizumab in the Netherlands, a multicenter retrospective cohort study was conducted: indications and treatment outcomes were collected for 105 Dutch PNH patients. In all patients, eculizumab was initiated conforming to indications as formulated in the Dutch PNH guideline. According to recently published response criteria, 23.4% of the patients had reached a complete hematological response, 53.2% a good or partial response, and 23.4% a minor response after 12 months of therapy. In the majority of patients the response remained stable during long-term follow-up. The degree and relevance of extravascular hemolysis significantly differed between response groups (p = 0.002). Improvements of EORTC-QLQc30 and FACIT-fatigue scores were observed, however patients reported lower scores than the general population. A detailed evaluation of 18 pregnancies during eculizumab showed no maternal or fetal deaths, and no thromboembolic events during pregnancy. This study demonstrates that the majority of patients benefit from eculizumab when adhering to the indications as formulated in the Dutch PNH guideline. However, novel therapies are needed to further improve real-world outcomes, such as hematological responses and quality of life.

Organized Sports Activities Are Safe for Children With Sickle Cell Disease: A Pilot Intervention Study.

Historically, children with sickle cell disease (SCD) are advised to refrain from sports activities, based on the assumption that physical exercise can trigger vaso-occlusive episodes. This pilot intervention study examined the safety (ie, no vaso-occlusive episodes) of a 10-week organized sports program for children with SCD. Eight children with SCD (5 boys/3 girls), aged 7 to 12 years old, received 10 training sessions (each 90 min) once a week. Training sessions were performed by a professional soccer club under the supervision of a medical team from the Wilhelmina Children's Hospital. During the study period, one child experienced a vaso-occlusive crisis, which could not be directly related to the organized sports program. None of the other children experienced vaso-occlusive episodes. The results of this study indicate that children with SCD can participate safely in moderate-intensity organized sports activities when personalized medical background and practical training information is shared with the trainer beforehand. All children continued their sports participation after the study period.

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

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.

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

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.

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

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.

How I manage children with Diamond-Blackfan anaemia.

Diamond-Blackfan anaemia (DBA) is a rare inherited marrow failure disorder, characterized by hypoplastic anaemia, congenital anomalies and a predisposition to cancer as a result of ribosomal dysfunction. Historically, treatment is based on glucocorticoids and/or blood transfusions, which is accompanied by significant toxicity and long-term sequelae. Currently, stem cell transplantation is the only curative option for the haematological DBA phenotype. Whereas this procedure has been quite successful in the last decade in selected patients, novel therapies and biological insights are still warranted to improve clinical care for all DBA patients. In addition to paediatric haematologists, other physicians (e.g. endocrinologist, gynaecologist) should ideally be involved in the care of this chronic condition from an early age, to improve lifelong management of haematological and non-haematological symptoms, and screen for DBA-associated malignancies. Here we provide an overview of current knowledge and recommendations for the day-to-day care of DBA patients.

Rapid and reproducible characterization of sickling during automated deoxygenation in sickle cell disease patients.

In sickle cell disease (SCD), sickle hemoglobin (HbS) polymerizes upon deoxygenation, resulting in sickling of red blood cells (RBCs). These sickled RBCs have strongly reduced deformability, leading to vaso-occlusive crises and chronic hemolytic anemia. To date, there are no reliable laboratory parameters or assays capable of predicting disease severity or monitoring treatment effects. We here report on the oxygenscan, a newly developed method to measure RBC deformability (expressed as Elongation Index - EI) as a function of pO2 . Upon a standardized, 22 minute, automated cycle of deoxygenation (pO2 median 16 mmHg ± 0.17) and reoxygenation, a number of clinically relevant parameters are produced in a highly reproducible manner (coefficients of variation <5%). In particular, physiological modulators of oxygen affinity, such as, pH and 2,3-diphosphoglycerate showed a significant correlation (respectively R = -0.993 and R = 0.980) with Point of Sickling (PoS5% ), which is defined as the pO2 where a 5% decrease in EI is observed during deoxygenation. Furthermore, in vitro treatment with antisickling agents, including GBT440, which alter the oxygen affinity of hemoglobin, caused a reproducible left-shift of the PoS, indicating improved deformability at lower oxygen tensions. When RBCs from 21 SCD patients were analyzed, we observed a significantly higher PoS in untreated homozygous SCD patients compared to treated patients and other genotypes. We conclude that the oxygenscan is a state-of-the-art technique that allows for rapid analysis of sickling behavior in SCD patients. The method is promising for personalized treatment, development of new treatment strategies and could have potential in prediction of complications.

Epigenetic drug screen identifies the histone deacetylase inhibitor NSC3852 as a potential novel drug for the treatment of pediatric acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) is a heterogeneous disease regarding morphology, immunophenotyping, genetic abnormalities, and clinical behavior. The overall survival rate of pediatric AML is 60% to 70%, and has not significantly improved over the past two decades. Children with Down syndrome (DS) are at risk of developing acute megakaryoblastic leukemia (AMKL), which can be preceded by a transient myeloproliferative disorder during the neonatal period. Intensification of current treatment protocols is not feasible due to already high treatment-related morbidity and mortality. Instead, more targeted therapies with less severe side effects are highly needed. PROCEDURE: To identify potential novel therapeutic targets for myeloid disorders in children, including DS-AMKL and non-DS-AML, we performed an unbiased compound screen of 80 small molecules targeting epigenetic regulators in three pediatric AML cell lines that are representative for different subtypes of pediatric AML. Three candidate compounds were validated and further evaluated in normal myeloid precursor cells during neutrophil differentiation and in (pre-)leukemic pediatric patient cells. RESULTS: Candidate drugs LMK235, NSC3852, and bromosporine were effective in all tested pediatric AML cell lines with antiproliferative, proapoptotic, and differentiation effects. Out of these three compounds, the pan-histone deacetylase inhibitor NSC3852 specifically induced growth arrest and apoptosis in pediatric AML cells, without disrupting normal neutrophil differentiation. CONCLUSION: NSC3852 is a potential candidate drug for further preclinical testing in pediatric AML and DS-AMKL.

Pediatric Diamond-Blackfan anemia in the Netherlands: An overview of clinical characteristics and underlying molecular defects.

INTRODUCTION: Diamond-Blackfan anemia (DBA) is characterized by hypoplastic anemia, congenital anomalies, and a predisposition for malignancies. Most of our understanding of this disorder stems from molecular studies combined with extensive data input from international patient registries. OBJECTIVES: To create an overview of the pediatric DBA population in the Netherlands. METHODS: Forty-three patients diagnosed with DBA from all Dutch university pediatric hospitals were included in this study, and their clinical and genetic characteristics were collected from patient records. RESULTS: Congenital malformations were present in 24 of 43 patients (55.8%). An underlying genetic defect was identified in 26 of 43 patients (60.5%), the majority of which were found in the RPS19 gene (12 of 43, 27.9%) with 1 patient carrying a mutation in a novel DBA candidate gene, RPL9. In 31 of 35 (88.6%) patients, an initial response to glucocorticoid treatment was observed. Six patients (14.0%) underwent hematopoietic stem cell transplantation, and eleven patients (11 of 43, 25.6%) became treatment-independent spontaneously. CONCLUSION: In agreement with previous reports, the Dutch pediatric DBA population is both clinically and genetically heterogeneous. National and international registries, together with more extensive genetic testing, are crucial to increase our understanding of genotype and phenotype correlations of this intriguing disorder.

Acetylation of C/EBPε is a prerequisite for terminal neutrophil differentiation.

C/EBPε, a member of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors, is exclusively expressed in myeloid cells and regulates transition from the promyelocytic stage to the myelocytic stage of neutrophil development, being indispensable for secondary and tertiary granule formation. Knowledge concerning the functional role of C/EBPε posttranslational modifications is limited to studies concerning phosphorylation and sumoylation. In the current study, using ectopic expression and ex vivo differentiation of CD34(+) hematopoietic progenitor cells, we demonstrate that C/EBPε is acetylated, which was confirmed by mass spectrometry analysis, identifying 4 acetylated lysines in 3 distinct functional domains. Regulation of C/EBPε acetylation levels by the p300 acetyltransferase and the sirtuin 1 deacetylase controls transcriptional activity, which can at least in part be explained by modulation of DNA binding. During neutrophil development, acetylation of lysines 121 and 198 were found to be crucial for terminal neutrophil differentiation and the expression of neutrophil-specific granule proteins, including lactoferrin and collagenase. Taken together, our data illustrate a critical role for acetylation in the functional regulation of C/EBPε activity during terminal neutrophil development.

Understanding chronic neutropenia: life is short.

The pathophysiological mechanisms underlying chronic neutropenia are extensive, varying from haematopoietic stem cell disorders resulting in defective neutrophil production, to accelerated apoptosis of neutrophil progenitors or circulating mature neutrophils. While the knowledge concerning genetic defects associated with congenital neutropenia or bone marrow failure is increasing rapidly, the functional role and consequences of these genetic alterations is often not well understood. In addition, there is a large group of diseases, including primary immunodeficiencies and metabolic diseases, in which chronic neutropenia is one of the symptoms, while there is no clear bone marrow pathology or haematopoietic stem cell dysfunction. Altogether, these disease entities illustrate the complexity of normal neutrophil development, the functional role of the (bone marrow) microenvironment and the increased propensity to undergo apoptosis, which is typical for neutrophils. The large variety of disorders associated with chronic neutropenia makes classification almost impossible and possibly not desirable, based on the clinical phenotypes. However, a better understanding of the regulation of normal myeloid differentiation and neutrophil development is of great importance in the diagnostic evaluation of unexplained chronic neutropenia. In this review we propose insights in the pathophysiology of chronic neutropenia in the context of the functional role of key players during normal neutrophil development, neutrophil release and neutrophil survival.

Novel Homozygous Mutation of the Internal Translation Initiation Start Site of VHL is Exclusively Associated with Erythrocytosis: Indications for Distinct Functional Roles of von Hippel-Lindau Tumor Suppressor Isoforms.

Congenital secondary erythrocytosis is a rare disorder characterized by increased red blood cell production. An important cause involves defects in the oxygen sensing pathway, in particular the PHD2-VHL-HIF axis. Mutations in VHL are also associated with the von Hippel-Lindau tumor predisposition syndrome. The differences in phenotypic expression of VHL mutations are poorly understood. We report on three patients with erythrocytosis, from two unrelated families. All patients show exceptionally high erythropoietin (EPO) levels, and are homozygous for a novel missense mutation in VHL: c.162G>C p.(Met54Ile). The c.162G>C mutation is the most upstream homozygous VHL mutation described so far in patients with erythrocytosis. It abolishes the internal translational start codon, which directs expression of VHLp19, resulting in the production of only VHLp30. The exceptionally high EPO levels and the absence of VHL-associated tumors in the patients suggest that VHLp19 has a role for regulating EPO levels that VHLp30 does not have, whereas VHLp30 is really the tumor suppressor isoform.