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Purpose This study aimed at gaining insight into parents' perceptions towards health behaviour in parenting and the impact of the school environment. Healthcare professionals experience barriers to discuss health behaviour in families where levels of overweight and obesity are elevated. There is a need to understand parents' perception and perceived significance of health behaviour. Design and methods Sixty-three semi-structured interviews were conducted with parents of children from three primary schools located in two different neighbourhoods in Zwolle, the Netherlands. Parents were asked to formulate their top three priority goals in parenting, additionally supplemented with 15 predetermined goals, including a health behaviour related goal. Parents ranked the goals in order of importance and gave an explanation. Finally, parents shared their opinions regarding the school environment in promoting health behaviour, according to the EnrG framework. Results In 8 out of 63 interviews, health was reported in the top three self-formulated goals. Other goals considered important were happiness and being respectful to others. When health was ranked as less important in parenting, routine of health behaviour, own beliefs and religion were given as explanations. The physical school environment and teachers were mentioned as important factors in promoting health behaviour. Conclusions Parents indicate health behaviour as a key-element in parenting, even when health behaviour is not considered as the most important priority. Practice Implications: Since school is perceived as a logical and powerful environment, healthcare professionals should collaborate with school staff to empower their own, teachers' and parents' roles to address health promotion.

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. ispartof: Blood vol:139 issue:21 pages:3111-3126 ispartof: location:United States status: Published online

Diamond–Blackfan anemia (DBA) is one of the inherited bone marrow failure syndromes marked by erythroid hypoplasia. Underlying variants in ribosomal protein (RP) genes account for 80% of cases, thereby classifying DBA as a ribosomopathy. In addition to RP genes, extremely rare variants in non-RP genes, including GATA1, the master transcription factor in erythropoiesis, have been reported in recent years in patients with a DBA-like phenotype. Subsequently, a pivotal role for GATA-1 in DBA pathophysiology was established by studies showing the impaired translation of GATA1 mRNA downstream of the RP haploinsufficiency. Here, we report on a patient from the Dutch DBA registry, in which we found a novel hemizygous variant in GATA1 (c.220+2T>C), and an Iranian patient with a previously reported variant in the initiation codon of GATA1 (c.2T>C). Although clinical features were concordant with DBA, the bone marrow morphology in both patients was not typical for DBA, showing moderate erythropoietic activity with signs of dyserythropoiesis and dysmegakaryopoiesis. This motivated us to re-evaluate the clinical characteristics of previously reported cases, which resulted in the comprehensive characterization of 18 patients with an inherited GATA-1 defect in exon 2 that is presented in this case-series. In addition, we re-investigated the bone marrow aspirate of one of the previously published cases. Altogether, our observations suggest that DBA caused by GATA1 defects is characterized by distinct phenotypic characteristics, including dyserythropoiesis and dysmegakaryopoiesis, and therefore represents a distinct phenotype within the DBA disease spectrum, which might need specific clinical management.

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.

Diamond-Blackfan anemia (DBA) is a rare congenital erythroblastopenia and inherited bone marrow failure syndrome that affects approximately seven individuals in every million live births. In addition to anemia, about 50% of all DBA patients suffer from various physical malformations of the face, hands, heart, or urogenital region. The disorder is almost exclusively driven by haploinsufficient mutations in one of several ribosomal protein (RP) genes, although for similar to 30% of diagnosed patients no mutation is found in any of the known DBA-linked genes. Because DBA is such a rare disease with a particularly wide range of clinical phenotypes and molecular signatures, the development of collaborative efforts such as the ERARE-funded European DBA consortium (EuroDBA) has become imperative for DBA research. EuroDBA was founded in 2012 and brings together dedicated clinical and biological researchers of DBA from France, Italy, the Netherlands, Germany, Israel, Poland, and Turkey to achieve a number of goals including the consolidation of data in patient registries, establishment of minimal diagnostic criteria, and projects aimed at more fully describing the different mutations linked to DBA. This review will cover the history of the EuroDBA registries, the methods used by EuroDBA in the diagnosis of DBA, and how the consortium has successfully worked together towards the discovery of new DBA-linked genes and the better understanding their pathophysiological effects.

Background Sickle cell disease (SCD) is one of the most common and devastating inherited blood disorders characterized by a single nucleotide mutation in the beta-globin chain leading to the production of mutant hemoglobin S (HbS). HbS polymerizes upon deoxygenation causing red blood cells (RBC) to sickle which results in extremely painful episodes of vaso-occlusive crisis (VOC), severe hemolytic anemia, chronic multiorgan failure and a reduced life span. An important metabolic feature directly associated with RBC sickling is increased intracellular levels of the glycolytic intermediate 2,3-diphosphyglycerate (2,3-DPG) which promotes deoxygenation by lowering the oxygen affinity of hemoglobin (Hb). Pyruvate kinase R (PKR) is a key enzyme in RBC metabolism, generating adenosine triphosphate (ATP) to maintain energy homeostasis, membrane integrity and deformability, and modulates 2,3-DPG levels. Mitapivat (AG-348) is an oral, small molecule allosteric activator of PKR and shows promise as an anti-sickling agent in addition to its effect in PK deficiency and thalassemia. The safety and efficacy of mitapivat in subjects with SCD was evaluated in the dose finding period of this ongoing phase 2 study. Methods The ESTIMATE study is a phase 2, open-label study in which subjects ≥16 years with SCD (HbSS, HbS/β0, HbS/β+) with a baseline hemoglobin >6.1 g/dL and ≤11.1 g/dL, no chronic transfusion and adequate organ function were eligible. In the 8-week Dose Finding Period, initial dosing of mitapivat was 20 mg twice daily (BID). Subjects received a maximum of two sequential dose escalations of mitapivat (i.e. from 20 mg BID to 50 mg BID and 100 mg BID) depending on safety. The primary endpoints were safety, evaluated by frequency and severity of adverse events (AEs), and efficacy of mitapivat on RBC sickling. RBC sickling was evaluated by change in Point of Sickling (PoS), the pO2 at which sickling occurs as measured by oxygen gradient ektacytrometry on the Lorrca (RR Mechatronics). Secondary endpoints included changes in hematological parameters, levels of 2,3-DPG and ATP, Hb-oxygen affinity (p50) and surrogate markers of organ damage and mortality. Subjects who safely tolerated mitapivat and showed evidence of clinical improvement, were eligible to continue a 52-week follow-up period (Fixed Dose Extension Period). Results Six subjects have been enrolled as of September 2020 and completed the Dose Finding Period. All had homozygous HbSS except one patient who had HbS/β0-thalassemia. Baseline characteristics were: median age of 36 years (range 20-59 years), 4 (66.7%) were female and 5 (83.3%) were on stable-dose hydroxyurea. All subjects received dose escalation to a maximum dose of 100 mg BID. No serious adverse events (SAEs) occurred. Adverse events (AEs) were mild and often transient, with the most common treatment emergent AEs: transaminase increase (n=3 [50.0%], Grade 1), gastrointestinal disorders including dyspepsia, diarrhea and abdominal discomfort (n=3 [50.0%], Grade 1) and headache (n=2 [33.3%], Grade 1). One VOC occurred without hospital admission and did not require dose reduction or discontinuation. Table 1 summarizes the anti-sickling effect as well as the hematological and biochemical response to mitapivat treatment. Sickling occurred at lower pO2 levels in all 6 patients during the Dose Finding Period reflected by a significant decrease in treatment week 8 mean PoS compared to baseline. 5/6 subjects (83.3%) achieved a Hb increase of ≥1 g/dL during this period, which was accompanied by a decrease in hemolytic markers. Consistent with activation of PKR, 2,3-DPG levels decreased and ATP levels increased. Additional results including biomarker data will be presented. Conclusion Mitapivat demonstrated an adequate safety profile during the 8-week Dose Finding Period in patients with SCD. The data show promising efficacy in terms of a decrease in the pO2 at which RBCs start to sickle, as well as increase in Hb from baseline and a concomitant decrease in markers reflecting hemolysis. The observed changes in 2,3-DPG and ATP levels are consistent with the proposed mechanism of the drug. The study is ongoing and further data including follow-up data, patient-reported outcomes, PKR activity and thermostability will be reported at a later stage. Figure 1 Figure 1. Disclosures 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.

Background-Aim: Infection from SARS-CoV-2 has emerged as new pathological entity within the global medical community. One of the earliest questions was in relation to the ability of the immunocompromised patients to clear the infection. In COST EuNet-INNOCHRON we were interested in the impact of SARS-CoV-2 infection in patients with different types of chronic neutropenia (CNP). The aim of the current study is to understand the impact of SARS-CoV-2 infection and to identify any possible characteristic patterns of the clinical course in patients with CNP. Patients and Methods: The COST EuNet-INNOCHRON Action in collaboration with the European Haematology Association - Scientific Working Group (EHA-SWG) on Granulocytes and Constitutional Marrow Failure Syndromes has conducted an online survey on SARS-CoV-2 infection in patients with CNP. The EuNet-INNOCHRON participants from different countries got access to an on-line platform fulfilling the General Data Protection Regulation (GDPR) and could register adult and paediatric CNP patients who had been infected by SARS-CoV-2 from March 2020 to June 2021. Data on demographic characteristics, type of CNP, patients' background and SARS-CoV-2 infection history (symptoms, laboratory features, radiological appearance, therapeutic approach and outcome) were collected. Results: Twenty-six patients with diagnosis of CNP, 7 males and 19 females were registered. Patient age distribution as follows: 16 patients >18 years old (y.o.)5 patients 5-18 y.o, 4 patients < 5 y.o whereas age was not available for one of the patients. Nine of the patients were diagnosed with idiopathic CNP, 7 patients with congenital neutropenia (6 of them with severe congenital neutropenia), 3 with secondary CNP, 2 with suspected autoimmune neutropenia of infancy (although antineutrophil Ab were negative), one with autoimmune neutropenia, one with drug induced neutropenia and 3 with other types of CNP. Twelve patients were on treatment with G-CSF and 6 patients had a history of previous viral or bacterial infections. Clonal Cytopenia(s) of Undetermined Significance (CCUS) was excluded in the eight patients who were investigated. Twenty-four out of 26 patients had positive PCR and one was found incidentally with positive antibodies for SARS-CoV-2. One more patient was symptomatic with history of close contact with SARS-CoV-2 infected family members. The commonest observed symptoms were fever >38 oC (19 patients), cough (10 patients), rhinorrhoea (10 patients), sore throat (6 patients), musculoskeletal pains (7 patients), taste/smell loss (5 patients), headache (5 patients), dyspnoea (4 patients), chest pain (one patient) and none of them had gastrointestinal symptoms. No other associated respiratory viral or bacterial infections were reported. Four patients who had one or more underlying conditions (immune deficiency, heart/respiratory/kidney disease) were admitted in hospital and needed anti SARS-CoV-2 treatment. Two of them had non-invasive ventilation and one of them needed admission in intensive care unit (ICU); both recovered. Another patient with Fallot's tetralogy needed mechanical ventilation in ICU and sadly passed away. No other deaths were observed. Deterioration of the pre-existing neutropenia was seen in two patients, two patients developed thrombocytopenia, one patient developed worsening lymphopenia and one anaemia. Twelve patients had chest X-ray and consolidation was found in two of them. All three patients who had chest CT scans were found with ground-glass changes. During the observation period (up to two months), no re-infection from SARS-CoV-2 was found. The Stockholm, Sweden experience is similar to the above data. One hundred fifty-four patients with CNP were followed up, for 10 months (March 1 to December 31, 2020) for SARS-CoV-2. Seventeen of these (i.e. 11 %) were infected. None needed hospitalization and there were no fatalities. Conclusion: Although the relative susceptibility of neutropenic patients to contract SARS-CoV-2 needs to be assessed with further studies, the clinical course and severity of SARS-CoV-2 infection doesn't seem to be worse in CNP patients (regardless the type of neutropenia and the need for GCSF treatment) compared to the general population. Also, like what has been observed in non-neutropenic patients, underlying comorbidities is a significant risk factor for severe disease and adverse outcome. Disclosures Dale: X4 Pharmaceuticals: Consultancy, Honoraria, Research Funding. Palmblad: Chiesi Ltd Sweden: Honoraria; Roche Sweden: Speakers Bureau; Chiesi Ltd Candada,: Honoraria.

Introduction: Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome (IBMFS) characterized by hypoplastic anemia, congenital malformations and an increased risk to develop malignancies.Until now, treatment of DBA consists of red blood cell (RBC) transfusions, glucocorticoids (GC) and allogeneic hematopoietic stem cell transplantation in a selection of patients. Whereas RBC transfusions are the main cause of IO, elevated iron parameters have also been reported in non-transfusion-dependent DBA patients. Here we investigated the incidence and severity of IO in a well-described cohort of transfusion-dependent and non-transfusion-dependent DBA patients in order to gain more insight in the regulation of iron metabolism in DBA, and to provide clinical guiding to improve the diagnosis and management of IO in DBA. Methods: In this retrospective, observational study we have included twenty-nine pediatric and adult DBA patients for whom at least one serum ferritin level and/or MRI result was available. Ten patients (34%) were classified as transfusion-dependent (TD) (ten or more transfusions during the twelve months prior to evaluation). Non-transfusion-dependent (NTD) patients (66%) were treated with either GC, incidental transfusions or received no treatment. Transfusion burden (transfusion history) was assessed via medical records. Serum ferritin levels ≥250 ng/mL in males and ≥150 ng/mL in females were considered to be elevated. Results of MRI were expressed as liver iron content (LIC) and as cardiac T2* in milliseconds (ms). LIC ≥3 mg/g indicates significant hepatic IO, and LIC ≥7 mg/g is associated with clinical morbidity. Cardiac T2* ≤20 ms indicates significant cardiac IO. Results: In 15/29 (52%) MRI analysis of IO was performed. Hepatic IO (LIC >3 mg/g) was present in 9/29 (31%) of DBA patients, of which 8/9 (89%) had moderate to severe IO (LIC>7mg/g), despite the fact that all but one were treated with chelation therapy. Overall serum ferritin levels and LIC correlated significantly (r=0.7907, p1000 ng/mL (Figure 1A). Interestingly, in the NTD group, hepatic IO was present in 2/7 patients (29%), who both only had mildly elevated serum ferritin levels (263 ng/mL and 277 ng/mL) and were not treated with iron chelation therapy. Based on total transfusion burden since birth, patients were classified in distinct groups: nine patients who received ³10 transfusions during life (9/10) were diagnosed with hepatic IO, whilst none of the patients who received Discussion: We demonstrate that IO is common in DBA yet can be easily overlooked in NTD patients that were treated with transfusions in the past. While serum ferritin levels significantly correlated with LIC values, this parameter cannot be used exclusively to screen for IO or titrate iron chelation therapy. We conclude that in clinical practice, biochemical parameters in combination with transfusion history justify a low-threshold to perform an MRI-based evaluation of IO, and to start adequate chelation therapy. Figure 1 Figure 1. Disclosures Van Beers: Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; Novartis: Research Funding; RR Mechatronics: Research Funding. Wijk: Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Axcella health: Research Funding; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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 24/43 patients (55.8%). An underlying genetic defect was identified in 26/43 patients (60.5%), the majority of which were found in the RPS19 gene (12/43, 27.9%) with one patient carrying a mutation in a novel DBA candidate gene, RPL9. In 31/35 (88.6%) patients an initial response to glucocorticoid treatment was observed. Six patients (14.0%) underwent hematopoietic stem cell transplantation, and eleven patients (11/43, 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 in order to increase our understanding of genotype and phenotype correlations of this intriguing disorder. This article is protected by copyright. All rights reserved.

Background: In sickle cell disease (SCD), hemoglobin S (HbS) polymerizes upon deoxygenation, reducing red blood cell (RBC) deformability. RBC deformability can be measured over a gradient of oxygen tensions (pO2) with the Laser Optical Rotational Red Cell Analyzer (Lorrca) ektacytometer (RR Mechatronics). Oxygen gradient ektacytometry generates 3 key parameters: 1) EImax, RBC deformability at normoxia; 2) EImin, minimum RBC deformability upon deoxygenation; and 3) the point of sickling (PoS): the oxygen tension at which a 5% decrease in deformability is observed during deoxygenation, reflecting the patient-specific pO2 at which sickling begins (Figure 1A). Previously we showed that oxygen gradient ektacytometry-derived biomarkers correlate with measures of SCD disease severity and hemolytic rate (Rab et al. Blood 2018), and is associated with vaso-occlusive crisis (VOC) frequency (Rab et al, Blood 2019). In this study, we confirm these observations in 2 independent cohorts and extend it to occurrence of acute chest syndrome (ACS), stroke including silent cerebral infarction (SCI), and transcranial Doppler (TCD) outcome. Methods: We analyzed 2 cohorts of SCD patients; an adult patient cohort of 53 SCD patients, enrolled at either University Medical Center Utrecht, The Netherlands (UMCU, n=25) or Hospital Lyon France (LIBM, n=28), and a pediatric patient cohort of 190 SCD patients enrolled at Texas Children's Hospital, USA (TCH). Subjects were HbSS or HbS/β-thalassemia, with a substantial number of subjects on hydroxyurea (HU) therapy (adult cohort 66% and pediatric cohort 86%), and not on chronic transfusion therapy. Correlations between oxygen gradient ektacytometry-derived biomarkers and the clinical complications of stroke or silent infarcts (SCI), ACS, VOC were assessed in both pediatric and adult patients. Patient groups generally did not significantly differ significantly by age, gender or HU treatment in the adult cohort except for age, which was lower in the ACS+ group (25.3years (y) compared to 32.0y) and also lower in the VOC+ group (27.1y compared to 35.8y). In the pediatric cohort, patient groups differed significantly in the ACS+ group compared to the ACS- group by age (ACS- group 8.37, ACS+ group 10.9y) and HU treatment (ACS- group 76%, ACS+ group 93%). Similarly, age was significantly higher in the Stroke+ group compared to the Stroke- group (14.0y compared to 9.3y), which was also found when studying VOC (VOC+ group 11.6y, VOC- group 8.2y). Results: In the pediatric cohort, PoS was significantly higher in patients with ACS (mean 40.3 compared to 34.9 mmHg, p=0.0001, Figure 1B). In the adult cohort, PoS was also higher in those with ACS although this did not reach significance (p=0.053, Figure 1C). In the pediatric cohort, PoS was higher in patients with stroke or SI (mean 43.0 mmHg compared to mean 37.3 mmHg, p In the adult and pediatric patient cohort, PoS was higher in patients with recent VOC (both p Differences in mean PoS between pediatric and adult patient cohorts could be due to differences in treatment, age, genetic background or technical differences between Lorrca devices. Lower significance levels found in the adult patient cohort could be due to smaller sample size. Conclusion: We show that oxygen gradient ektacytometry provides functional, clinically relevant next generation biomarkers that are associated with ACS, stroke and VOC. This study therefore further validates the clinical usefulness of these biomarkers, in particular in relation to cerebral vasculopathy. Since our results merely describe an association and not causality further validation is warranted to establish how well oxygen gradient ektacytometry can assess disease severity. However, its parameters could already provide the clinician with information about patient RBC characteristics and sickling propensity that could aid in clinical decision making. Our results provide a rationale for further development of these biomarkers in the evaluation of novel therapies as part of clinical care, or clinical trial endpoints. Disclosures Rab: RR Mechatronics: Research Funding. Bos:RR Mechatronics: Research Funding. Cnossen:Takeda: Research Funding; Shire: Research Funding; Baxter: Research Funding; Bayer: Research Funding; Sobi: Research Funding; CSL behring: Research Funding; Nordic Pharma: Research Funding; Novo Nordisk: Research Funding; Pfizer: Research Funding. Wijk:Agios Pharmaceuticals Inc.: Research Funding; RR mechatronics: Research Funding. Sheehan:Emmaus: Research Funding; Global Blood Therapeutics: Research Funding; Novartis: Research Funding. van Beers:Pfizer: Research Funding; RR mechatronics: Research Funding; Agios: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding.

Background: Diamond-Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome (IBMFS) marked by erythroid hypoplasia, reticulocytopenia and macrocytosis, and associated with congenital malformations and an increased risk of developing malignancies. From a clinical and molecular perspective this disease is highly heterogeneous, and no clear genotype-phenotype correlations can be found. Since the majority of molecular defects have been found in ribosomal protein (RP) genes, DBA is regarded a "ribosomopathy". While the molecular basis has been studied intensively, the pathophysiology of DBA is still not fully understood. One of the major unresolved issues is how RP gene mutations result in the specific erythroid defect seen in DBA, with macrocytic erythrocytes and increased adenosine deaminase activity. In addition, the highly heterogeneous and variable clinical presentation and disease course, even in patients with similar molecular defects, remains enigmatic. Hence, in order to investigate the cellular defect, and increase our understanding of disease pathophysiology and clinical heterogeneity, novel tools are needed. In this study, we explore the potential of untargeted metabolomics on dried blood spots and report for the first time a metabolic fingerprint for DBA. Aims: Defining a metabolic signature for DBA in order to: 1. Increase our understanding of cellular determinants of impaired ribosome biogenesis, and 2. Extend the toolbox for diagnostic evaluation. Methods: Untargeted metabolic profiling was performed on DBS samples obtained from 18 DBA patients and 45 healthy controls using direct infusion high resolution mass spectrometry following a previously established approach1. Statistical analysis was performed in MetaboAnalyst and predictive modeling was executed within R-software. Results: In total, 1917 unique metabolite features were identified in DBS samples from patients and controls. Multivariate analysis yielded distinct metabolic profiles, reflected by natural separation detected by principal component analysis (Figure 1A), and emphasized by clear distinction with partial least square discriminant analysis (Figure 1B). This 'metabolic fingerprint' was incorporated into a machine learning algorithm, and subsequently a binary classification (or prediction) model was constructed by randomly dividing patient and controls into 'training' (32 HC, 13 DBA) or 'test' set (13 HC, 5 DBA). Accurate class assignment was achieved for all patients and controls in the training set (Figure 1C). Prominent metabolites in the fingerprint and classification algorithm were a.o. menadione (a vitamin K precursor), 4-hydroxyproline (collagen component) and methylmalonylcarnitine (an acylcarnitine). In addition a large number of interesting metabolites were identified that could provide novel starting points for studying/understanding downstream effects of RP defects in DBA and therapeutic mechanisms (Figure 1D). Conclusion: In this study we performed untargeted metabolomics on dried blood spots from a substantial cohort of DBA patients and report for the first time a metabolic fingerprint of this disease. By incorporating this fingerprint in a machine learning algorithm we underlined the diagnostic potential of this approach. Moreover, the metabolites identified in this fingerprint, provide promising starting points for further studies to increase our insights in disease pathophysiology, including the mechanism involved in elevated eADA activity, as well as the development of new therapeutic strategies. References: 1. de Sain-van der Velden MGM, van der Ham M, Gerrits J, et al. Quantification of metabolites in dried blood spots by direct infusion high resolution mass spectrometry. Anal Chim Acta. 2017;979:45-50. Disclosures Wijk: Agios Pharmaceuticals Inc.: Research Funding; RR mechatronics: Research Funding.

Supplemental Digital Content is available in the text, The clinical use of histone deacetylase inhibitors (HDACi) for the treatment of bone marrow failure and hematopoietic malignancies has increased dramatically over the last decades. Nonetheless, their effects on normal myelopoiesis remain poorly evaluated. Here, we treated cord blood derived CD34+ progenitor cells with two chemically distinct HDACi inhibitors MS-275 or SAHA and analyzed their effects on the transcriptome (RNA-seq), epigenome (H3K27ac ChIP-seq) and functional and morphological characteristics during neutrophil development. MS-275 (entinostat) selectively inhibits class I HDACs, with a preference for HDAC1, while SAHA (vorinostat) is a non-selective class I/II HDACi. Treatment with individual HDACi resulted in both overlapping and distinct effects on both transcriptome and epigenome, whereas functional effects were relatively similar. Both HDACi resulted in reduced expansion and increased apoptosis in neutrophil progenitor cells. Morphologically, HDACi disrupted normal neutrophil differentiation what was illustrated by decreased percentages of mature neutrophils. In addition, while SAHA treatment clearly showed a block at the promyelocytic stage, MS-275 treatment was characterized by dysplastic features and skewing towards the monocytic lineage. These effects could be mimicked using shRNA-mediated knockdown of HDAC1. Taken together, our data provide novel insights into the effects of HDAC inhibition on normal hematopoietic cells during neutrophil differentiation. These findings should be taken into account when considering the clinical use of MS-275 and SAHA, and can be potentially utilized to tailor more specific, hematopoietic-directed HDACi in the future.

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.

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.

Summary 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.

[Figure][1] A 16-year-old boy was referred by his general practitioner to a pediatrician because of thrombocytopenia (30 × 109/L) discovered in a routine blood count, which was performed before initiation of treatment with isotretinoin. He had no physical complaints or bleeding tendency.

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.

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.

Background: The group of rare hereditary anemias includes a large variety of intrinsic defects of the red blood cell, as well as erythropoiesis. They include hemolytic anemias (e.g. enzyme deficiencies), hemoglobinopathies, hypoplastic anemias (e.g. Diamond-Blackfan Anemia, DBA), and dyserythropoietic anemias. As a result of the rapid developments in genetic testing and the subsequent increased knowledge of molecular defects underlying hereditary anemias, our understanding of the pathophysiology of rare anemias has increased during the last decade. However, in a substantial number of patients, the clinical phenotype does not fit classical criteria of a disease, response to therapy is less than expected, or a molecular defect cannot be found. In addition, in patients with well-described molecular defects, there is often no clear genotype-phenotype correlation. In order to better understand the underlying pathophysiological mechanisms driving ineffective erythropoiesis in patients and to improve their classification and clinical evaluation, novel functional tests are needed. Metabolomics is the large-scale, unbiased study of metabolites and their interactions within a biological system, directly reflecting the underlying biochemical activity and state of cells. Metabolomics can be used to identify novel disease biomarkers, study deregulated cellular pathways, and to determine the cellular responses to therapeutic interventions. In this study we demonstrate that dried blood spots (DBS) can be used as a minimal invasive and validated technical approach to perform large scale metabolomics in a variety of rare hereditary anemias. Methods: DBS samples from >100 patients suffering from a variety of rare anemiaswere collected during regular hospital visits. Quantification of metabolites was performed by direct infusion high resolution mass spectrometry (DI-HRMS) followed by an untargetedmetabolomics pipeline. For annotation, the Human Metabolome Database (HMDB) was used. Results were compared with DBS samples of 70 healthy adult controls and 35 pediatric patients negatively screened for metabolic diseases Results: For each patient sample, Z-scores were calculated for all mass peaks annotated with metabolites (HMDB, 3930). Mass peak, intensity and corresponding Z-scores were compared with two distinct groups of controls (∆Z-scores): pediatric patients who were screened for metabolic diseases but were found negative, and healthy adult controls. For data interpretation, two strategies were used. First, by untargeted statistical analysis in Metabo-analyst, we identified metabolites (and/or isomers) that showed either increased or decreased intensity. For the second strategy we specifically focused on red blood cell metabolic pathways, including glycolysis, the pentose phosphate pathway, ascorbate and glutathione metabolism, arginine and polyamine metabolism, and erythrocyte membrane turnover and transport. We corrected for a potential hematocrit effect and performed subgroup analyses correcting for reticulocyte counts. Our preliminary data indicate potential biomarkers for distinct disease entities, including altered polyamine metabolism (DBA, SCD), glycolysis (DBA, HS), and aberrant arginine metabolism (SCD) (Figure 1). Further in-depth pathway analyses, and targeted validation of biomarker profiles are currently being performed. Conclusion: Untargeted metabolomics using dried blood spots provides a novel functional tool to identify disease biomarkers and common and distinct deregulated cellular pathways. This will improve diagnostic evaluation and clinical management of patients with rare hereditary anemias, contribute to a better understanding of disease pathophysiology, and aid in the development of therapeutic strategies. Disclosures van Beers: Agios Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Research Funding; RR Mechatronics: Research Funding. van Wijk:RR Mechatronics: Research Funding; Agios Pharmaceuticals: Consultancy, Research Funding.

Background: In sickle cell disease (SCD), hemoglobin S (HbS) polymerizes upon deoxygenation, reducing red blood cell (RBC) deformability. RBC deformability can be measured over a range of oxygen concentrations using a Laser Optical Rotational Red Cell Analyzer (Lorrca) ektacytometer (RR Mechatronics, Zwaag, the Netherlands) with Oxygenscan module. The Oxygenscan measures 3 key parameters: 1) EImax, RBC deformability at normoxia; 2) EImin, RBC deformability upon deoxygenation; and 3) the point of sickling (PoS): the oxygen tension at which a 5% decrease in normoxic EI is observed during deoxygenation, reflecting the patient-specific pO2 at which sickling begins (Figure 1A). Previously we showed that Oxygenscan parameters correlate with measures of SCD disease severity and hemolysis (absolute reticulocyte count, %fetal hemoglobin, %HbS, total Hb, %dense RBC, (Rab et al. Blood 2018). In this study, we investigated the relationship between oxygenscan parameters and incidence of vaso-occlusive crisis (VOC), and we tested the ability of the Oxygenscan parameters to assess response to hydroxyurea (HU) and chronic transfusion (CTF) in patients with SCD. Methods: We analyzed 2 cohorts: a European cohort (EUC) of 62 SCD patients (all HbSS or HbS/β-thalassemia), enrolled at either University Medical Center Utrecht (UMCU, n= 42) or Hospital Lyon (LIBM, n=20), and a US cohort (USC) of 97 SCD patients enrolled at Texas Children's Hospital (TCH). Differences in Oxygenscan parameters in SCD patients without/with VOC (requiring a doctor's evaluation) in the past 2 years were measured in 46 adult EUC SCD patients and 80 pediatric USC SCD patients. EUC patients without VOC (n=18, median age 40.6 years (y); 11 female (F), 44% on HU, 6% on CTF, 28% on both treatments), were compared to patients with a positive history of VOC (n=28, median age 23.9y, 13F, 39% on HU, 11% on CTF and 11% on both). Patients without VOC in the USC (n= 34, median age 8.0y, 15F, 53% on HU, 15% on CTF and 21% on both treatments) were compared to patients with VOC (n=46, median age 11.8y, 20F, 74% on HU, 13% on CTF and 11% on both treatments). To establish treatment related Oxygenscan parameter changes, we analyzed RBCs from 9 SCD patients from UMCU (median age 19y, 5F), before and during HU treatment (measurements performed at baseline, and 1, 3 and 6 months after starting HU), 7 SCD patients from UMCU (median age 26.7y; 6F) before and after transfusion and 17 SCD patients from TCH (median age 10.8y; 6F) on HU before and after transfusion. Results: In the EUC, PoS differed significantly between patients without VOC in the last 2 years (median 41.6mmHg) and patients with VOC in the last 2 years (median 53.7 mmHg, p Transfusion improved EImax, EImin, and PoS (USC: p Conclusion: Our results show that RBC from SCD patients without VOC in the last two years were able to tolerate lower oxygen concentrations before sickling (PoS). RBCs from patients without VOC were also more deformable when deoxygenated (EImin) compared to patients who had experienced one or more VOCs in the last two years. In contrast, RBC deformability, when oxygenated (EImax) was not different in patients with or without VOC in the last two years. All 3 Oxygenscan parameters significantly improved with standard of care SCD treatments, namely CTF and/or HU. We therefore conclude that the PoS, EImax and EImin are useful biomarkers of clinical severity and treatment response, and may be essential in monitoring novel SCD treatments as part of a clinical trial as a surrogate endpoint. Disclosures Rab: RR Mechatronics: Research Funding. van Wijk:Agios Pharmaceuticals: Consultancy, Research Funding; RR Mechatronics: Research Funding. Bos:RR Mechatronics: Research Funding. Nur:Novartis Pharmaceuticals: Consultancy. Cnossen:NWO: Other: Governmental grants , ZonMW, Innovation fund and Nationale Wetenschapsagenda 2018; Roche: Other: Travel Grants; Takeda: Other: Travel Grants, Research Funding; Shire: Other: Travel Grants, Research Funding; Baxter: Other: Travel Grants, Research Funding; Sobi: Research Funding; CSL Behring: Other: Travel Grants, Research Funding; Nordic Pharma: Research Funding; Novo Nordisk: Research Funding; Bayer: Other: Travel Grants, Research Funding; Pfizer: Other: Travel Grants, Research Funding. van Beers:Agios Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Research Funding; RR Mechatronics: Research Funding.

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

Background/Objective: Paroxysmal nocturnal hemoglobinuria (PNH) is an extremely rare disease and is even more rare in children; pediatric cases are estimated to account for Methods: The analysis included eculizumab-treated patients enrolled in the Registry as of May 2017. Patients were stratified by age at baseline (defined as the date of eculizumab initiation; age categories, Results: Of 4903 patients enrolled in the Registry as of May 1, 2017, 1725 had been treated with eculizumab and had data available on demographics and enrollment date (5 times the upper limit of normal at baseline to normal or near normal range at last follow-up in both age cohorts. There were no TEs reported in the pediatric cohort after baseline, but 2/43 patients (4.7%) with no history of MAVEs at baseline experienced non-TE MAVEs after baseline. Among adult patients, 19/1497 (1.3%) with no history of TEs and 7/1497 (0.5%) with history of TEs experienced TEs after baseline. Non-TE MAVEs after baseline in patients with no history of MAVEs were experienced by 17/1492 patients (1.1%) and non-TE MAVEs after baseline in patients with history of MAVEs were experienced by 15/1492 patients (1.0%). The proportion of patients requiring transfusion was lower at last follow-up in both cohorts compared with baseline (23.0% at last follow-up vs 49.3% at baseline for adults and 32.3% at last follow-up vs 48.4% at baseline for pediatric patients). A similar proportion of patients became transfusion-independent after treatment with eculizumab in both age cohorts (32.3% [10/31] in the pediatric cohort compared with 33.8% [370/1095] in the adult cohort). The proportion of patients with PNH-related symptoms decreased after baseline in both cohorts for most of the symptoms assessed, although fewer than 10 pediatric patients had symptom data available at both baseline and last follow-up. Conclusions: The effectiveness of eculizumab for reducing intravascular hemolysis and transfusion requirements, and preventing MAVEs (including TEs) was similar in these large cohorts of pediatric patients with PNH and adults with PNH. The higher proportion of patients transfusion-dependent among the pediatric cohort may be associated with the higher likelihood of underlying aplastic anemia in pediatric patients with PNH. Table. Table. Disclosures Urbano-Ispizua: Alexion Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees. Kulasekararaj:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Other: Travel Support . Bartels:Alexion Pharmaceuticals, Inc.: Honoraria, Other: Travel Support and is site investigator for clinical trials with the company, Research Funding. Patriquin:Ra Pharmaceuticals: Consultancy, Research Funding; Octapharma: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Other: Travel Support and is site investigator for clinical trials with the company. Hoechsmann:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding. Wilson:Alexion Pharmaceuticals, Inc.: Employment, Equity Ownership. Gustovic:Alexion Pharma GmbH: Employment, Equity Ownership. Schrezenmeier:Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding.