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Key Points Severe bone marrow failure was primarily observed in early childhood in children with biallelic SBDS mutations.Absolute neutrophil counts were positively associated with age (P < .0001) in patients with biallelic SBDS mutations., Visual Abstract, Shwachman-Diamond syndrome (SDS) is an inherited bone marrow failure syndrome with leukemia predisposition. An understanding of the hematologic complications of SDS with age could guide clinical management, but data are limited for this rare disease. We conducted a cohort study of 153 subjects from 143 families with confirmed biallelic SBDS mutations enrolled on the North American Shwachman Diamond Registry or Bone Marrow Failure Registry. The SBDS c.258 + 2T>C variant was present in all but 1 patient. To evaluate the association between blood counts and age, 2146 blood counts were analyzed for 119 subjects. Absolute neutrophil counts were positively associated with age (P < .0001). Hemoglobin was also positively associated with age up to 18 years (P < .0001), but the association was negative thereafter (P = .0079). Platelet counts and marrow cellularity were negatively associated with age (P < .0001). Marrow cellularity did not correlate with blood counts. Severe marrow failure necessitating transplant developed in 8 subjects at a median age of 1.7 years (range, 0.4-39.5), with 7 of 8 requiring transplant prior to age 8 years. Twenty-six subjects (17%) developed a myeloid malignancy (16 myelodysplasia and 10 acute myeloid leukemia) at a median age of 12.3 years (range, 0.5-45.0) and 28.4 years (range, 14.4-47.3), respectively. A lymphoid malignancy developed in 1 patient at the age of 16.9 years. Hematologic complications were the major cause of mortality (17/20 deaths; 85%). These data inform surveillance of hematologic complications in SDS.

We follow a patient with Diamond-Blackfan anemia (DBA) mosaic for a pathogenic RPS19 haploinsufficiency mutation with persistent transfusion-dependent anemia. Her anemia remitted on eltrombopag (EPAG), but surprisingly, mosaicism was unchanged, suggesting that both mutant and normal cells responded. When EPAG was withheld, her anemia returned. In addition to expanding hematopoietic stem/progenitor cells, EPAG aggressively chelates iron. Because DBA anemia, at least in part, results from excessive intracellular heme leading to ferroptotic cell death, we hypothesized that the excess heme accumulating in ribosomal protein-deficient erythroid precursors inhibited the growth of adjacent genetically normal precursors, and that the efficacy of EPAG reflected its ability to chelate iron, limit heme synthesis, and thus limit toxicity in both mutant and normal cells. To test this, we studied Rpl11 haploinsufficient (DBA) mice and mice chimeric for the cytoplasmic heme export protein, FLVCR. Flvcr1-deleted mice have severe anemia, resembling DBA. Mice transplanted with ratios of DBA to wild-type marrow cells of 50:50 are anemic, like our DBA patient. In contrast, mice transplanted with Flvcr1-deleted (unable to export heme) and wild-type marrow cells at ratios of 50:50 or 80:20 have normal numbers of red cells. Additional studies suggest that heme exported from DBA erythroid cells might impede the nurse cell function of central macrophages of erythroblastic islands to impair the maturation of genetically normal coadherent erythroid cells. These findings have implications for the gene therapy of DBA and may provide insights into why del(5q) myelodysplastic syndrome patients are anemic despite being mosaic for chromosome 5q deletion and loss of RPS14.

Diamond Blackfan anemia (DBA) is predominantly an autosomal dominant inherited red cell aplasia primarily caused by pathogenic germline variants in ribosomal protein genes. DBA due to pathogenic RPL35A variants has been associated with large 3q29 deletions and phenotypes not common in DBA. We conducted a multi-institutional genotype-phenotype study of 45 patients with DBA associated with pathogenic RPL35A germline variants and curated the variant data on 21 additional cases from the literature. Genotype-phenotype analyses were conducted comparing patients with large deletions versus all other pathogenic variants in RPL35A. Twenty-two of the 45 cases had large deletions in RPL35A. After adjusting for multiple tests, a statistically significant association was observed between patients with a large deletion and steroid-resistant anemia, neutropenia, craniofacial abnormalities, chronic gastrointestinal problems, and intellectual disabilities (p

Anemia of inflammation, also known as anemia of chronic disease, is refractory to erythropoietin (EPO) treatment, but the mechanisms underlying the EPO refractory state are unclear. Here, we demonstrate that high mobility group box-1 protein (HMGB1), a damage-associated molecular pattern molecule recently implicated in anemia development during sepsis, leads to reduced expansion and increased death of EPO-sensitive erythroid precursors in human models of erythropoiesis. HMGB1 significantly attenuates EPO-mediated phosphorylation of the Janus kinase 2/STAT5 and mTOR signaling pathways. Genetic ablation of receptor for advanced glycation end products, the only known HMGB1 receptor expressed by erythroid precursors, does not rescue the deleterious effects of HMGB1 on EPO signaling, either in human or murine precursors. Furthermore, surface plasmon resonance studies highlight the ability of HMGB1 to interfere with the binding between EPO and the EPOR. Administration of a monoclonal anti-HMGB1 antibody after sepsis onset in mice partially restores EPO signaling in vivo. Thus, HMGB1-mediated restriction of EPO signaling contributes to the chronic phase of anemia of inflammation.

Adults infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have had high rates of thrombosis. A novel condition in children infected with SARS-CoV-2, multisystem inflammatory syndrome in children (MIS-C), has limited data on their prothrombotic state or need for thromboprophylaxis.We aimed to analyze the prothrombotic state using coagulation profiles, rotational thromboelastometry (ROTEM) parameters and clinical outcomes, to determine if this could aid in risk stratification for thromboprophylaxis.This analysis included patients (21 years of age) with a diagnosis of MIS-C (n = 40) and controls (presenting with suspicion of MIS-C but later ruled out; n = 26).MIS-C patients had higher levels of inflammatory markers including D-dimer (p .0001), compared with controls, along with evidence of hypercoagulability on ROTEM with elevated evaluation of fibrinogen activity (FIBTEM) maximum clot firmness (MCF) (p .05). For MIS-C patients with D-dimers1000 ng/ml, there was a significant correlation of FIBTEM MCF (p .0001) with a mean value of 37.4 (standard deviation 5.1). D-dimer2144 ng/ml was predictive of intensive care unit admission (area under the curve [AUC] 0.80; 95% confidence interval, 0.60-0.99; p .01; sensitivity: 82%, specificity: 75%), and elevated FIBTEM MCF (AUC 1 for2500 ng/ml). MIS-C patients (50%) received enoxaparin thromboprophylaxis (in addition to aspirin) with significant improvement in their inflammatory and ROTEM parameters upon outpatient follow-up; none developed symptomatic thrombosis.Despite an observed prothrombotic state, none of the MIS-C patients (on aspirin alone or in combination with enoxaparin) developed symptomatic thrombosis. ROTEM, in addition to coagulation profiles, may be helpful to tailor thromboprophylaxis in critically ill MIS-C patients.

To understand the mechanisms that mediate germline genetic leukemia predisposition, we studied the inherited ribosomopathy Shwachman-Diamond syndrome (SDS), a bone marrow failure disorder with high risk of myeloid malignancies at an early age. To define the mechanistic basis of clonal hematopoiesis in SDS, we investigate somatic mutations acquired by patients with SDS followed longitudinally. Here we report that multiple independent somatic hematopoietic clones arise early in life, most commonly harboring heterozygous mutations in EIF6 or TP53. We show that germline SBDS deficiency establishes a fitness constraint that drives selection of somatic clones via two distinct mechanisms with different clinical consequences. EIF6 inactivation mediates a compensatory pathway with limited leukemic potential by ameliorating the underlying SDS ribosome defect and enhancing clone fitness. TP53 mutations define a maladaptive pathway with enhanced leukemic potential by inactivating tumor suppressor checkpoints without correcting the ribosome defect. Subsequent development of leukemia was associated with acquisition of biallelic TP53 alterations. These results mechanistically link leukemia predisposition to germline genetic constraints on cellular fitness, and provide a rational framework for clinical surveillance strategies., Understanding the molecular basis of leukaemia predisposition is essential for intervention. The authors here investigate germline genetic leukaemia predisposition by studying Shwachman-Diamond syndrome and report compensatory inactivating mutations in EIF6 and transforming biallelic TP53 alterations.

Diamond Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome characterized by red cell failure, congenital anomalies, poor linear growth, and cancer predisposition. Two previous analyses from the Diamond Blackfan Anemia Registry have quantified DBA as a cancer predisposition syndrome of moderate cancer penetrance. Patients with DBA have a 4.8-fold higher relative risk of developing cancer with an overall cumulative incidence of 13.7% by age 45 years. The two most prevalent solid tumors are colorectal cancer (CRC) and osteogenic sarcoma. Current and evolving data support the institution of cancer screening and surveillance strategies for CRC in DBA.

The coagulopathy of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is well documented in adults, with increases in D-dimer and prothrombin time found to be strong predictors of mortality, and anticoagulation shown to decrease this mortality. Viscoelastic parameters such as elevations in maximum clot firmness (MCF) on rotational thromboelastometry (ROTEM) have correlated with a hypercoagulable state in adults with SARS-CoV-2. We report our experience in children infected with SARS-CoV-2, with noted elevations in D-dimer and MCF on ROTEM (indicating hypercoagulability). Exploration of viscoelastic testing to provide additional laboratory-based evidence for pediatric-specific risk assessment for thromboprophylaxis in SARS-CoV-2 is warranted.

Background Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by anemia, short stature, congenital anomalies, and cancer predisposition. Most cases are due to mutations in genes encoding ribosomal proteins (RP) leading to RP haploinsufficiency. Effective treatments for the anemia of DBA include chronic red cell transfusions, long-term corticosteroid therapy, or hematopoietic stem cell transplantation. In a small patient series and in animal models, there have been hematologic responses to L-leucine with amelioration of anemia. The study objectives of this clinical trial were to determine feasibility, safety, and efficacy of L-leucine in transfusion-dependent patients with DBA. Procedure Patients ≥2 years of age received L-leucine 700 mg/m2 orally three times daily for nine months to determine a hematologic response and any improvement in growth (NCT01362595). Results This multicenter, phase I/II study enrolled 55 subjects; 43 were evaluable. There were 21 males; the median age at enrollment was 10.4 years (range, 2.5-46.1 years). No significant adverse events were attributable to L-leucine. Two subjects had a complete erythroid response and five had a partial response. Nine of 25, and 11 of 25, subjects experienced a positive weight and height percentile change, respectively, at the end of therapy. Conclusions L-leucine is safe, resulted in an erythroid response in 16% of subjects with DBA, and led to an increase in weight and linear growth velocity in 36% and 44% of evaluable subjects, respectively. Further studies will be critical to understand the role of L-leucine in the management of patients with DBA.

Background Recent data show survival after matched unrelated donor (MUD) bone marrow transplantation (BMT) is similar to matched sibling procedures for young patients with severe aplastic anemia (SAA). Donor delays, risk of transplant-related mortality (TRM), and concern about chronic graft versus host disease raise questions about whether MUD BMT or immune suppression therapy (IST) should be preferred initial therapy for young patients lacking matched sibling donors. Procedure We performed a pilot trial to assess the feasibility of randomizing patients under age 26 with newly diagnosed SAA to receive IST versus MUD BMT. Primary aims assessed the acceptability of randomization and timing of BMT. Secondary aims measured toxicities, response, and survival. Results Sixty-seven patients with possible SAA were screened at nine centers. Of 57 with confirmed SAA, 23 underwent randomization and received therapy with a median follow-up of 18 months. Of 12 randomized to BMT, 10 started BMT as initial therapy at a median of 36 days after randomization. One BMT recipient experienced secondary graft failure, requiring a second procedure. Six of 11 randomized to IST responded, whereas five with refractory disease underwent successful salvage BMT. One patient achieving complete response relapsed after discontinuation of immune suppression and died of infection after salvage BMT. Conclusions This feasibility study showed that a high percentage of patients underwent randomization and received up-front MUD BMT. Our study lays the groundwork for a larger randomized trial that will define best initial therapy for young patients with SAA who have an available MUD.

Background Hemophagocytic lymphohistiocytosis (HLH) can be familial or secondary, which is often triggered by infection or malignancy. HLH therapy includes dexamethasone and etoposide. However, therapy is associated with significant morbidity and mortality. Anakinra, a recombinant interleukin-1 receptor antagonist, has been reported to treat macrophage activation syndrome (MAS), rheumatic sHLH. We report our experience with anakinra to treat patients with nonrheumatic secondary HLH (sHLH). Procedure Six children were diagnosed with HLH from December 2014 to August 2016 and were treated with subcutaneous anakinra (6-10 mg/kg/day divided over four doses) with or without dexamethasone (10 mg/m2 /day). Therapy was either escalated or weaned based on clinical and laboratory response. Results Five of six patients were treated with anakinra and dexamethasone, and one with anakinra alone due to active cytomegalovirus (CMV) pneumonitis. The median age of diagnosis was 1.8 years (range 0.8-14.9 years). No pathogenic mutations associated with HLH were identified, but three of six possessed genetic variants of unknown significance. Infectious triggers were identified for four patients and two patients had malignancies. The average treatment duration was 8 weeks with 3.5-5.5 years of follow up. No patient needed escalation of therapy to include etoposide. All patients achieved remission. Anakinra was well tolerated without significant adverse effects. Conclusion Initial treatment with anakinra (with or without dexamethasone) is a feasible treatment alternative for patients with secondary HLH and may allow for avoidance of etoposide. We recommend early initiation of anakinra when HLH is suspected. A broader investigation of the use of anakinra as a first-line agent for HLH is ongoing.

Despite the effective clinical use of steroids for the treatment of Diamond Blackfan anemia (DBA), the mechanisms through which glucocorticoids regulate human erythropoiesis remain poorly understood. We report that the sensitivity of erythroid differentiation to dexamethasone is dependent on the developmental origin of human CD34(+) progenitor cells, specifically increasing the expansion of CD34(+) progenitors from peripheral blood (PB) but not cord blood (CB). Dexamethasone treatment of erythroid-differentiated PB, but not CB, CD34(+) progenitors resulted in the expansion of a newly defined CD34(+)CD36(+)CD71(hi)CD105(med) immature colony-forming unit–erythroid (CFU-E) population. Furthermore, proteomics analyses revealed the induction of distinct proteins in dexamethasone-treated PB and CB erythroid progenitors. Dexamethasone treatment of PB progenitors resulted in the specific upregulation of p57(Kip2), a Cip/Kip cyclin–dependent kinase inhibitor, and we identified this induction as critical; shRNA-mediated downregulation of p57(Kip2), but not the related p27(Kip1), significantly attenuated the impact of dexamethasone on erythroid differentiation and inhibited the expansion of the immature CFU-E subset. Notably, in the context of DBA, we found that steroid resistance was associated with dysregulated p57(Kip2) expression. Altogether, these data identify a unique glucocorticoid-responsive human erythroid progenitor and provide new insights into glucocorticoid-based therapeutic strategies for the treatment of patients with DBA.

TO THE EDITOR: Inherited bone marrow failure syndromes (IBMFSs), characterized by bone marrow failure and developmental anomalies, are among a group of rare cancer predisposition syndromes. As illustrated by Fanconi anemia (FA),[1][1] the unique biology of this IBMFS has provided important insights

Diamond Blackfan anemia syndrome (DBAS) is a rare, heritable bone marrow failure syndrome characterized by severe macrocytic anemia, congenital anomalies and predisposition to cancer, most often diagnosed during infancy. More than 98% of DBAS patients with a molecular diagnosis have mutations in a gene encoding one of the ~80 ribosomal proteins (RP) leading to haploinsufficiency. A molecular diagnosis in a patient with DBAS is critical for a definitive diagnosis, the identification of compatible related transplant donors, and developing reproductive strategies for families. Targeted sequencing of RP genes, single nucleotide polymorphism comparative genome hybridization (SNP array) to detect >30 kb deletions (Farrar et al. Blood. 2011) and exome sequencing (WES) (Ulrisch et al. Am J Hum Genet. 2018) has identified RP mutations in ~80% of patients, leaving ~20% of patients with DBAS without a molecular diagnosis. Targeted sequencing and WES focus on only coding sequences. We hypothesized that remaining 20% of DBAS mutations were in the non-coding regions of RP genes, such as promoters or introns. To test this hypothesis, we collected DNA with informed consent for whole genome sequencing (WGS) analysis from 14 patients with no molecular diagnosis after targeted sequencing, SNP array or WES. On average, we aligned ~3.2x10 7 paired end reads of 250 base pairs for each patient (~65X coverage). We focused our analysis on the sequences in and around the RP genes. To identify deletions, we used a suite of detection tools: DELLY, GRIDSS, MANTA, and LUMPY. More than 90% of deletions identified by any 2 of these tools were confirmed by PCR. We identified 5 deletions in the introns of RP genes, ranging from 11 to 467 base pairs in length, which we hypothesized disrupted splicing of the nascent RNA transcript. To test this, we created minigenes in which we replaced exon 2 of a gamma globin gene with either the WT or mutant RP exon. All wild type exons spliced normally. A 467 base pair deletion in RPL27 exon 3 was sufficient to prevent the correct splicing of that intron. Examination of the eCLIP data for RNA binding proteins revealed that spliceosome complex proteins (including SF3B1, SF3B4 and EFTUD2) and Dead-box RNA helicases bind in the deleted region. A 28 base pair deletion in exon 3 of RPL6 removes a polypyrimidine tract that is a critical part of the 3' splice junction consensus sequence, which we presume is also deleterious. The other 3 intronic deletions did not disrupt splicing. We also identified 2 causative point mutations. A point mutation 5 bases into intron 1 of the RPS26 gene changes a base in the 5' splice donor consensus sequence, which activated a cryptic splice donor in the 5' untranslated region. This aberrant splice removes the ATG initiation codon causing an untranslatable RNA. In another patient, we identified a mutation in exon 1 of the RPS27 gene, judged to be a benign amino acid change. This mutation disrupted splicing.by activating a cryptic splice donor site in the 5' untranslated region which removes the ATG initiation codon and causes a frame shift. We were referred two patients with possible duplications of the RPL35a gene. To identify duplications, we employed MinION long read single molecule sequencing. We had an average read length of ~ 6-10kb with the longest read being 1.3Mb. Overall coverage was >85X. We used minimap2 to align the reads to the reference human genome and used SNIFFLES to call the variants. One patient was the parent of DBAS-affected patient with no history of anemia. In this patient, we identified a duplication of 400 kb that included the entire RPL35a region along with genes on either side. We conclude that this duplication is not likely to cause DBA. The second patient was diagnosed with DBAS. In this patient, we identified a duplication of 4 kb including exons 1 and 2 of RPL35a We conclude that this duplication disrupts the RPL35a gene and is a likely cause of DBA. Whole genome sequencing of 15 DBAS patients identified 5 likely causative mutations in RP genes, confirming that most genetically undiagnosed cases of DBAS will involve known genes encoding RP. We conclude that the pipeline for obtaining a molecular diagnosis for DBAS from targeted sequencing, SNP array, and exome sequencing to whole genome sequencing. Disclosures Vlachos: Novartis: Membership on an entity's Board of Directors or advisory committees. Lipton: Celgene: Membership on an entity's Board of Directors or advisory committees.

Background: Diamond Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome characterized by anemia, congenital anomalies and a predisposition to cancer. Patients usually present during infancy or early childhood, but can also be diagnosed as adults. In the vast majority of cases DBA is due to a mutation in a gene encoding a small or large subunit-associated ribosomal protein (RP) leading to RP haploinsufficiency. In a study of 702 patients enrolled in the DBA Registry (DBAR), the observed to expected ratio for acute myeloid leukemia (AML) was 28.8 and for myelodysplastic syndrome (MDS), 352.1 (Vlachos et al, Blood, 2018). The average age of onset for MDS in the DBA cohort was 26 years, compared to 60-70 years in the general population. Evolving clonal hematopoiesis (CH) with age has been observed as a precursor to MDS, with CH rarely observed in individuals younger than 40 years of age. Thus we hypothesized that the young age at the development of MDS in DBA would be presaged by evolving CH. Objective: The primary objective was to perform whole exome sequencing (WES) specifically screening for previously reported somatic mutations in 56 genes associated with CH (Jaiswal et al, NEJM, 2014). Design/Method: A total of 69 samples were analyzed from 65 patients, mostly targeting patients older than 18 years (median age 30 years). Multiple samples were run on patients who had available samples in the DBAR Biorepository to determine rate of acquisition of mutations. 468 age- and sex-matched healthy controls were made available from GeneDx who performed the WES for the study. We used a threshold for variant calling of minimum 5% with a minimum of 2 variant reads. Results: Three of the 65 DBA patients (5%) were found to have somatic mutations in STAG1, U2AF1, SF3B1, and DNMT3A at 8, 20, 41, and 70 years, respectively (Table 1). The patient who was 20 years of age had a sample in the DBAR biorepository from when he was age 8 years which was found to have a different somatic mutation (STAG1) than was found at present (U2AF1). This patient did go on to develop MDS at the age of 21 years. In comparison, of the 468 controls, 4 (0.8 %) had a somatic mutation in SF3B1, LUC7L2, DNMT3A, and LUC7L2 at ages 12, 31, 33 and 40 years, respectively. Conclusion: Patients with DBA show more somatic mutations as compared to controls (p Disclosures No relevant conflicts of interest to declare.

Inherited bone marrow failure syndromes (IBMFS) are a heterogenous group of disorders characterized by dysregulated hematopoiesis across various lineages, predisposition to malignancy, and diverse syndromic features. The scat (severe combined anemia and thrombocytopenia) mouse model has been characterized as a unique model of IBMFS. Scat carries an autosomal recessive missense mutation in the Rasa3 gene that results in RASA3 mislocalization and loss of function. RASA3 functions as a Ras-GTPase activating protein, and its loss of function in scat results in increased erythroid Ras activity, increased reactive oxygen species, and altered cell cycle progression, all culminating in delayed terminal erythroid differentiation. However, the precise mechanism of RASA3 regulation of erythroid differentiation remains undefined, and elucidation of this mechanism is crucial to identifying new therapeutic targets in inherited anemia. Considering the role of RASA3 as regulator of Ras signaling and cell cycle progression, and the importance of these processes to erythroid differentiation, we sought to first characterize the coordination of Ras signaling pathways and cell cycle progression in normal murine erythropoiesis, then observe how loss of RASA3 function alters this regulatory axis. We observed that wild type (WT) erythroblasts demonstrate population-specific influence of ERK and PI3K/AKT signaling in regulating cell cycle progression. Inhibition of both pathways with increasing doses of U0126 and LY294002, respectively, induced accumulation in G0/G1 from the proerythroblast stage until the late basophilic/polychromatic stage (U0126 vehicle vs. 1uM p= 0.0023; LY294002 vehicle vs 1uM p=0.0389). At these later stages, ERK and PI3K/AKT inhibition led to a decrease in G0/G1 percentages, suggesting a stage-specific switch in signaling mediated cell cycle regulation, with PI3K inhibition demonstrating more potent and consistent effects (U0126 vehicle vs 1uM p=0.0406, 0.0481; LY294002 vehicle vs 1uM p=0.0003, 0.0197, 0.0086, n=3). These patterns suggest that ERK and AKT may facilitate cell cycle progression past the G0/G1 checkpoint in early erythropoiesis while inducing cell cycle exit or accumulation in G0/G1 in late erythropoiesis. In scat, we previously characterized increased active Ras in erythroid cells and a delay in terminal erythroid differentiation with accumulation at the polychromatic stage. We therefore next sought to examine the potential mechanistic contribution of altered PI3K/AKT signaling and cell cycle progression to the differentiation delay seen in scat. Phospho-flow analyses demonstrate that scat bone marrow-derived basophilic and polychromatic erythroblasts have increased AKT activation compared to WT (p=0.0402, p=0.0559, respectively; n=4), with similar trends evident in scat spleen basophilic erythroblasts (p=0.064; n=4). These results are consistent with increased Ras activation in scat. Ex vivo EdU/PI analyses revealed that scat bone marrow-derived polychromatic erythroblasts demonstrate G0/G1 accumulation (p=0.0466) and decreased progression to S-phase (0.0414; n=6), also with similar trends in scat spleen basophilic erythroblasts (p=0.004; n=6). These results correlate with the observed differentiation delay in scat and indicate that RASA3 regulates stage-specific signaling and cell cycle progression during erythropoiesis. To study if cell cycle dysregulation in scat begins at an earlier stage of erythroid differentiation, we analyzed murine hematopoietic and erythroid progenitors as Ter119-, cKit+ cells expressing increasing levels of CD71 and found that both CD71lo and CD71med bone marrow-derived scat progenitors present with G0/G1 accumulation (p=0.0015, p=0.0073, respectively) and decreased progression to S-phase (p=0.0014, p=0.0241; n=7). This suggests a dynamic relationship between RASA3, Ras signaling, and cell cycle progression throughout early and late erythroid differentiation. Together, these findings support the role of RASA3 as a regulator of the signaling networks governing erythropoiesis and reveal a new targetable axis in a model of inherited bone marrow failure. Disclosures No relevant conflicts of interest to declare.

Objective Diamond Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome. The mainstays of treatment involve chronic red cell transfusions, long-term glucocorticoid therapy, and stem cell transplantation. Systematic data concerning endocrine function in DBA are limited. We studied patients in the DBA Registry (DBAR) of North America to assess the prevalence of various endocrinopathies.

Background Diamond Blackfan anemia (DBA) is a genetically and clinically heterogeneous ribosomopathy and inherited bone marrow failure syndrome characterized by anemia, reticulocytopenia, and decreased erythroid precursors in the bone marrow with an increased risk of malignancy and, in approximately 50%, physical abnormalities. Methods We retrospectively analyzed clinical data from 77 patients with DBA born in the Russian Federation from 1993 to 2014. In 74 families there was one clinically affected individual; in only three instances a multiplex family was identified. Genomic DNA from 57 DBA patients and their first-degree relatives was sequenced for mutations in RPS19, RPS10, RPS24, RPS26, RPS7, RPS17, RPL5, RPL11, RPL35a, and GATA1. Results Severe anemia presented before 8 months of age in all 77 patients; before 2 months in 61 (78.2%); before 4 months in 71 (92.2%). Corticosteroid therapy was initiated after 1 year of age in the majority of patients. Most responded initially to steroids, while 5 responses were transient. Mutations in RP genes were detected in 35 of 57 patients studied: 15 in RPS19, 6 in RPL5, 3 in RPS7, 3 each in RPS10, RPS26, and RPL11 and 1 each in RPS24 and RPL35a; 24 of these mutations have not been previously reported. One patient had a balanced chromosomal translocation involving RPS19. No mutations in GATA1 were found. Conclusion In our cohort from an ethnically diverse population the distribution of mutations among RP genes was approximately the same as was reported by others, although within genotypes most of the mutations had not been previously reported. Pediatr Blood Cancer 2015;62:1597–1600. © 2015 Wiley Periodicals, Inc.

Patients with inherited bone marrow failure syndromes (IBMFS), such as Fanconi anemia (FA), dyskeratosis congenita (DC), or Diamond Blackfan anemia (DBA), can have hematologic manifestations cured through hematopoietic cell transplantation (HCT). Subsequent late effects seen in these patients arise from a combination of the underlying disease, the pre-HCT therapy, and the HCT process. During the international consensus conference sponsored by the Pediatric Blood and Marrow Transplant Consortium on late effects screening and recommendations following allogeneic hematopoietic cell transplantation for immune deficiency and nonmalignant hematologic diseases held in Minneapolis, Minnesota in May 2016, a half-day session was focused specifically on the unmet needs for these patients with IBMFS. This multidisciplinary group of experts in rare diseases and transplantation late effects has already published on the state of the science in this area, along with discussion of an agenda for future research. This companion article outlines consensus disease-specific long-term follow-up screening guidelines for patients with IMBFS.

Biogenesis of mammalian red blood cells requires nuclear expulsion by orthochromatic erythoblasts late in terminal differentiation (enucleation), but the mechanism is largely unexplained. Here, we employed high-resolution confocal microscopy to analyze nuclear morphology and F-actin rearrangements during the initiation, progression, and completion of mouse and human erythroblast enucleation in vivo. Mouse erythroblast nuclei acquire a dumbbell-shaped morphology during enucleation, whereas human bone marrow erythroblast nuclei unexpectedly retain their spherical morphology. These morphological differences are linked to differential expression of Lamin isoforms, with primary mouse erythroblasts expressing only Lamin B and primary human erythroblasts only Lamin A/C. We did not consistently identify a continuous F-actin ring at the cell surface constriction in mouse erythroblasts, nor at the membrane protein-sorting boundary in human erythroblasts, which do not have a constriction, arguing against a contractile ring-based nuclear expulsion mechanism. However, both mouse and human erythroblasts contain an F-actin structure at the rear of the translocating nucleus, enriched in tropomodulin 1 (Tmod1) and nonmuscle myosin IIB. We investigated Tmod1 function in mouse and human erythroblasts both in vivo and in vitro and found that absence of Tmod1 leads to enucleation defects in mouse fetal liver erythroblasts, and in CD34+ hematopoietic stem and progenitor cells, with increased F-actin in the structure at the rear of the nucleus. This novel structure, the "enucleosome," may mediate common cytoskeletal mechanisms underlying erythroblast enucleation, notwithstanding the morphological heterogeneity of enucleation across species.

Fanconi anemia (FA), dyskeratosis congenita (DC), and Diamond Blackfan anemia (DBA) are 3 of the most common inherited bone marrow failure syndromes (IBMFS), in which the hematologic manifestations can be cured with hematopoietic cell transplantation (HCT). Later in life, these patients face a variety of medical conditions, which may be a manifestation of underlying disease or due to pre-HCT therapy, the HCT, or a combination of all these elements. Very limited long-term follow-up data exist in these populations, with FA the only IBMFS that has specific published data. During the international consensus conference sponsored by the Pediatric Blood and Marrow Transplant Consortium entitled "Late Effects Screening and Recommendations following Allogeneic Hematopoietic Cell Transplant (HCT) for Immune Deficiency and Nonmalignant Hematologic Disease" held in Minneapolis, Minnesota in May of 2016, a half-day session was focused specifically on the unmet needs for these patients with IBMFS. A multidisciplinary group of experts discussed what is currently known, outlined an agenda for future research, and laid out long-term follow-up guidelines based on a combination of evidence in the literature as well as expert opinion. This article addresses the state of science in that area as well as consensus regarding the agenda for future research, with specific screening guidelines to follow in the next article from this group.

Diamond-Blackfan anemia (DBA) is a congenital bone marrow failure syndrome characterized by erythroid hypoplasia, usually without perturbation of other hematopoietic lineages. Approximately 65% of DBA patients with autosomal dominant inheritance have heterozygous mutations or deletions in ribosomal protein (RP) genes while

Diamond Blackfan anemia (DBA), a syndrome primarily characterized by anemia and physical abnormalities, is one among a group of related inherited bone marrow failure syndromes (IBMFS) which share overlapping clinical features. Heterozygous mutations or single-copy deletions have been identified in 12 ribosomal protein genes in approximately 60% of DBA cases, with the genetic etiology unexplained in most remaining patients. Unlike many IBMFS, for which functional screening assays complement clinical and genetic findings, suspected DBA in the absence of typical alterations of the known genes must frequently be diagnosed after exclusion of other IBMFS. We report here a novel deletion in a child that presented such a diagnostic challenge and prompted development of a novel functional assay that can assist in the diagnosis of a significant fraction of patients with DBA. The ribosomal proteins affected in DBA are required for pre-rRNA processing, a process which can be interrogated to monitor steps in the maturation of 40S and 60S ribosomal subunits. In contrast to prior methods used to assess pre-rRNA processing, the assay reported here, based on capillary electrophoresis measurement of the maturation of rRNA in pre-60S ribosomal subunits, would be readily amenable to use in diagnostic laboratories. In addition to utility as a diagnostic tool, we applied this technique to gene discovery in DBA, resulting in the identification of RPL31 as a novel DBA gene.

Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome. As with the other rare inherited bone marrow failure syndromes, the study of these disorders provides important insights into basic biology and, in the case of DBA, ribosome biology; the disruption of which characterizes the disorder. Thus DBA serves as a paradigm for translational medicine in which the efforts of clinicians to manage DBA have informed laboratory scientists who, in turn, have stimulated clinical researchers to utilize scientific discovery to provide improved care. In this review we describe the clinical syndrome Diamond Blackfan anemia and, in particular, we demonstrate how the study of DBA has allowed scientific inquiry to create opportunities for progress in its understanding and treatment.

Diamond Blackfan Anemia Syndrome (DBA) is a rare, congenital bone marrow failure syndrome characterized by severe macrocytic anemia, most often diagnosed during infancy. Congenital anomalies and predisposition to cancer are also important features of DBA. Establishment of a molecular diagnosis in a patient with DBA is critical to determine treatment strategies (i.e. the identification of compatible related transplant donors), as well as developing reproductive strategies for genetically at risk families. The overwhelming majority (>98.75%) of DBA patients with a molecular diagnosis have mutations in a Ribosomal Protein (RP) gene. Targeted and exome sequencing (WES) strategies can identify RP mutations in >70% of DBA patients (Ulrisch et al. Am J Hum Genet. 2018). Single Nucleotide Polymorphism Comparative Genome Hybridization (SNP array) detects >30 kb deletions of RP genes (which cannot be identified by sequencing) in ~10% of DBA patients (Farrar et al. Blood. 2011), leaving ~20% of DBA patients without a molecular diagnosis. We hypothesized that smaller copy number variants (CNVs - either insertions or deletions) in RP genes that are below the limit of detection of SNP array are responsible for the remaining 20%. To test this hypothesis we collected DNA with informed consent for whole genome sequencing (WGS) analysis from 6 patients who had no mutations detected by WES or SNP array. On average, we aligned ~1x1010paired end reads of 250 base pairs for each patient (~83X coverage of the genome). The aligned sequences were analyzed for CNVs using two independent software packages. Delly analyzes the two ends of each sequence read and maps them to the current human reference genome. Read ends that map further apart than expected are flagged as potential CNVs. CNVkit estimates regions of copy loss by changes in average sequencing depth. Using relatively relaxed thresholds in Delly and CNVkit we identified ~100 candidate CNVs in each patient. We filtered out CNVs present in public databases and focused on those CNVs in the region of the RP genes. This analysis identified 2-5 potential RP gene associated CNVs in each patient. We designed PCR primers that flanked each putative CNV and confirmed at least one RP CNV in all 6 patient DNAs. At this time, the CNVs in two patients are in the process of evaluation. We have validated causative RP CNVs in the other 4 patients, representing one known and three novel DBA genes. One patient had a 464 bp deletion in 3rdintron of the RPL27 gene, which is mutated in other DBA patients. We hypothesized that the deletion caused a splicing defect. Using a mini gene in which the second intron of the gamma globin gene was replaced with the 3rdintron of either the wild type or mutant RPL27 gene, we showed that the mutant exon was not spliced. An alternative hypothesis, that the deletion removed an enhancer element, was also tested, but no enhancer activity was detected. We conclude that the RPL27exon deletion causes aberrant splicing leading to an unstable RPL27 mRNA and haploinsufficiency of RPL27. A second patient had a 3.5 kb deletion at the 3' end of the RPS5 gene, including the stop codon and poly A addition site. We hypothesized that the lack of the 3' processing signals would lead to an unstable mRNA. To test this hypothesis we generated MYC and FLAG tagged wildtype and 3' deleted RPS5 genes and co-transfected them into 293T cells. Regardless of the tag used, RT-PCR analysis showed a severe reduction in the mutant mRNA levels. Western Blot analysis demonstrated that only the wild type protein was expressed, leading to the conclusion that the RPS5 truncation led to an unstable RPS5 mRNA and haploinsufficiency of RPS5. A third patient had a 28 kb deletion that removes the RPS9 gene. shRNA knockdown of RPS9 mRNA in normal CD34+ cells inhibited erythroid differentiation, leading to the conclusion that RPS9 deficiency causes DBA. Finally, we observed a 3 bp insertion in exon 6 of the RPL14 gene. The deletion adds an alanine residue to a string of 10 alanines in the wild type allele. We confirmed the insertion by targeted sequence analysis of patient DNA. Our data show that WGS can identify small CNVs that cause DBA in at least 2/3 of patients who do not have a mutation detectable by other methods. We believe that WGS analysis following targeted sequencing, SNP array and WES can identify virtually all DBA mutations. With declining WGS costs, we recommend adding WGS to the molecular diagnostic pipeline for genetic testing of DBA. Disclosures Farrar: Novartis: Research Funding. Vlachos:Novartis: Other: Steering committee member.

Glucocorticoids are widely used to increase red cell production in a number of hematological disorders including Diamond Blackfan anemia (DBA). Despite their clinical effectiveness we do not fully understand the mechanistic basis for the regulation of human erythropoiesis by glucocorticoids. To improve the clinical management of these patients, we studied the mechanisms of dexamethasone (Dex) on human erythroid progenitors. We employed an erythroid culture system to differentiate primary human CD34+ cells isolated from patients with DBA and healthy controls as well as cord blood samples. We found that Dex increases the proliferation of CD34+ cells derived from peripheral blood but surprisingly not of CD34+ cells from cord blood. Furthermore, by mass spectrometry proteomic analysis, we found that Dex specifically altered the induction of several targets unique to peripheral blood including the transcription factor and cell cycle regulator NR4A1. When examining the cell populations expanded with Dex treatment by immunophenotyping, we detected a specific expansion of CFU-Es. Dex increased the average size of CFU-E colonies in methylcellulose colony forming assays in the presence of Epo alone, suggesting that CFU-Es are the most Dex responsive progenitor in humans. In support of this hypothesis, reticulocyte counts from patients with DBA treated with prednisone were found to increase approximately one week after treatment, consistent with the time course of differentiation of CFU-E to reticulocytes in bone marrow. In order to further resolve the progenitor target of steroid responsive progenitor we developed a new gating scheme that allows for increased resolution of BFU-E and CFU-E subpopulations by monitoring surface expression of CD71 and CD105. Using this system, we found that Dex treatment led to the maintenance of a novel immature transitional progenitor population in peripheral blood-derived cells that we term immature CFU-E. In methylcellulose colony forming assays, these peripheral blood-derived immature CFU-E treated with Dex produced larger colonies than the untreated controls and mature CFU-E. The expansion and proliferation of Dex treated immature CFU-Es derived from peripheral blood was associated with a decreased percentage of cells in the S phase of the cell cycle. No change was noted for either Dex treated BFU-E from peripheral blood or for BFU-E or CFU-E from cord blood. Importantly, Dex responsive peripheral blood-derived CFU-Es divided at a faster rate than untreated CFU-Es, revealing a dissociation in the link between differentiation and proliferation. Mechanistically, we found that Dex treated peripheral blood CFU-E exhibited increased expression of the negative cell cycle regulator p57Kip2. Moreover, this protein was also found to be dysregulated in CD34+ cells from transfusion-dependent patients with DBA, wherein p57Kip2 expression was not significantly altered following Dex treatment, indicating a role for p57Kip2 in the erythroid progenitor defects seen in these patients. To further evaluate the role of p57Kip2 in the Dex dependent alteration in CFU-E proliferation, peripheral blood-derived CD34+ cells were transduced with p57Kip2-specific shRNA lentiviral constructs. shRNA knockdown of p57Kip2 abrogated the effects of Dex on growth and induced a premature EPO-mediated differentiation. In summary, our findings provide new insights into functional heterogeneity of human erythroid progenitors and reveal mechanistic insights into the action of Dex in normal and disordered erythropoiesis. Disclosures Vlachos: Novartis: Other: Steering committee member.

Background Randomized clinical trials in pediatric aplastic anemia (AA) are rare and data to guide standards of care are scarce. Procedure Eighteen pediatric institutions formed the North American Pediatric Aplastic Anemia Consortium to foster collaborative studies in AA. The initial goal of NAPAAC was to survey the diagnostic studies and therapies utilized in AA. Results Our survey indicates considerable variability among institutions in the diagnosis and treatment of AA. There were areas of general consensus, including the need for a bone marrow evaluation, cytogenetic and specific fluorescent in situ hybridization assays to establish diagnosis and exclude genetic etiologies with many institutions requiring results prior to initiation of immunosuppressive therapy (IST); uniform referral for hematopoietic stem cell transplantation as first line therapy if an HLA-identical sibling is identified; the use of first-line IST containing horse anti-thymocyte globulin and cyclosporine A (CSA) if an HLA-identical sibling donor is not identified; supportive care measures; and slow taper of CSA after response. Areas of controversy included the need for telomere length results prior to IST, the time after IST initiation defining a treatment failure; use of hematopoietic growth factors; the preferred rescue therapy after failure of IST; the use of specific hemoglobin and platelet levels as triggers for transfusion support; the use of prophylactic antibiotics; and follow-up monitoring after completion of treatment. Conclusions These initial survey results reflect heterogeneity in diagnosis and care amongst pediatric centers and emphasize the need to develop evidence-based diagnosis and treatment approaches in this rare disease. Pediatr Blood Cancer 2014;61:869–874. © 2013 Wiley Periodicals, Inc.

Classical 5q- syndrome is an acquired macrocytic anemia of the elderly. Similar to Diamond Blackfan anemia (DBA), an inherited red cell aplasia, the bone marrow is characterized by a paucity of erythroid precursors. RPS14 deletions in combination with other deletions in the region have been implicated as causative of the 5q- syndrome phenotype. We asked whether smaller, less easily detectable deletions could account for a syndrome with a modified phenotype. We employed single-nucleotide polymorphism array genotyping to identify small deletions in patients diagnosed with DBA and other anemias lacking molecular diagnoses. Diminutive mosaic deletions involving RPS14 were identified in a 5-year-old patient with nonclassical DBA and in a 17-year-old patient with myelodysplastic syndrome. Patients with nonclassical DBA and other hypoproliferative anemias may have somatically acquired 5q deletions with RPS14 haploinsufficiency not identified by fluorescence in situ hybridization or cytogenetic testing, thus refining the spectrum of disorders with 5q- deletions.

Current therapeutic strategies for sickle cell anemia are aimed at reactivating fetal hemoglobin. Pomalidomide, a third-generation immunomodulatory drug, was proposed to induce fetal hemoglobin production by an unknown mechanism. Here, we report that pomalidomide induced a fetal-like erythroid differentiation program, leading to a reversion of γ-globin silencing in adult human erythroblasts. Pomalidomide acted early by transiently delaying erythropoiesis at the burst-forming unit-erythroid/colony-forming unit-erythroid transition, but without affecting terminal differentiation. Further, the transcription networks involved in γ-globin repression were selectively and differentially affected by pomalidomide including BCL11A, SOX6, IKZF1, KLF1, and LSD1. IKAROS (IKZF1), a known target of pomalidomide, was degraded by the proteasome, but was not the key effector of this program, because genetic ablation of IKZF1 did not phenocopy pomalidomide treatment. Notably, the pomalidomide-induced reprogramming was conserved in hematopoietic progenitors from individuals with sickle cell anemia. Moreover, multiple myeloma patients treated with pomalidomide demonstrated increased in vivo γ-globin levels in their erythrocytes. Together, these data reveal the molecular mechanisms by which pomalidomide reactivates fetal hemoglobin, reinforcing its potential as a treatment for patients with β-hemoglobinopathies.

In this issue of Blood , Seo et al demonstrate that the extensive characterization of the inherited bone marrow failure syndromes (IBMFS) can lead to important insights into the biology of hematopoiesis. 1

Diamond-Blackfan anemia (DBA) is a congenital BM failure syndrome characterized by hypoproliferative anemia, associated physical abnormalities, and a predisposition to cancer. Perturbations of the ribosome appear to be critically important in DBA; alterations in 9 different ribosomal protein genes have been identified in multiple unrelated families, along with rarer abnormalities of additional ribosomal proteins. However, at present, only 50% to 60% of patients have an identifiable genetic lesion by ribosomal protein gene sequencing. Using genome-wide single-nucleotide polymorphism array to evaluate for regions of recurrent copy variation, we identified deletions at known DBA-related ribosomal protein gene loci in 17% (9 of 51) of patients without an identifiable mutation, including RPS19, RPS17, RPS26, and RPL35A. No recurrent regions of copy variation at novel loci were identified. Because RPS17 is a duplicated gene with 4 copies in a diploid genome, we demonstrate haploinsufficient RPS17 expression and a small subunit ribosomal RNA processing abnormality in patients harboring RPS17 deletions. Finally, we report the novel identification of variable mosaic loss involving known DBA gene regions in 3 patients from 2 kindreds. These data suggest that ribosomal protein gene deletion is more common than previously suspected and should be considered a component of the initial genetic evaluation in cases of suspected DBA.

Background Diversity is necessary for the survival and success of both biological and social systems including societies. There is a lack of diversity, particularly the proportion of women and minorities in leadership positions, within medicine.1;2 In 2009 a group of ASPHO members recognized the need to support the career advancement of women and minority members. This article reports the results of a survey designed to characterize the comparative career pathway experience of women and minority ASPHO members.

von Hippel-Lindau (VHL) protein is the principal negative regulator of hypoxia sensing mediated by transcription factors. Mutations in exon 3 of the VHL gene lead to Chuvash (VHL(R200W)) and Croatian (VHL(H191D)) polycythemias. Here, we describe an infant of Bangladesh ethnicity with a novel homozygous VHL(D126N) mutation with congenital polycythemia and dramatically elevated erythropoietin (EPO) levels, who developed severe fatal pulmonary hypertension. In contrast to Chuvash polycythemia, erythroid progenitors (BFU-Es) did not reveal a marked EPO hypersensitivity. Further, NF-E2 and RUNX1 transcripts that correlate with BFU-Es EPO hypersensitivity in polycythemic mutations were not elevated.

Background: Diamond Blackfan anemia (DBA) is a congenital anemia characterized by failure of adequate erythrocyte expansion from hematopoietic precursors. The genetic basis of DBA is largely established, with mutation or deletion of at least 19 structural ribosomal protein (RP) genes, a RP chaperone (TSR2), or a pivotal erythroid transcription factor (GATA1) identifiable in most DBA cases. However, the marked clinical variability in DBA-including varying ages of presentation, severity of anemia, responsiveness to corticosteroids, and sporadic hematologic remissions-remains unexplained by genotype and may be modulated by epigenetic factors. Further understanding of this variability is of potential therapeutic relevance for biomarkers of steroid response and remission as well as in application to novel treatment approaches. Aim: We characterized genome-wide methylation and chromatin accessibility of erythroid progenitors from normal controls and DBA patients during erythroid commitment in order to identify the epigenetic features associated with erythroid failure in DBA, steroid response, and remission. Methods: We expanded and sorted CD71+/CD235- (transferrin receptor/glycophorin A) and CD71+/CD235+ erythroid cell fractions from DBA patients and controls after isolation of primary circulating CD34+ cells from peripheral blood (O'Brien et al, Blood 129(23):3111, 2017). We performed DNA methylation analysis using Illumina Epic arrays in 9 control and 22 DBA subjects (11 transfusion-dependent, 6 steroid responsive, 5 remission), characterizing differentially methylated probes and regions among groups. To define broad chromatin domains, we identified A/B chromatin compartments (representing open/closed chromatin) using long-range correlations in methylation data as previously described (Fortin et al. Genome Biol 16:180, 2015). To identify discrete local changes in chromatin accessibility, we performed ATAC-sequencing in 9 controls and 17 DBA patients (10 transfusion, 6 steroid, 1 remission). Results: Global DNA methylation falls during erythroid commitment, with 258,618 differentially hypermethylated CpG sites in normal control GlyA- cells compared to their more differentiated GlyA+ counterparts. This pattern is exaggerated in DBA samples, with 297,926 sites hypermethylated in GlyA- cells. We identified 17,392 CpC sites that distinguish GlyA- DBA progenitors from normal progenitors (16,953 hyper- and 439 hypomethylated). We identified 1,749 differentially methylated sites in comparison of transfusion dependent and remission DBA, as well as 79 sites different between transfusion dependent and steroid responsive DBA. Using genome-wide methylation data, we evaluated A/B compartment organization among these groups to identify large regions of open and closed chromatin during normal and DBA early erythroid differentiation. We observe significant shifts in A/B compartments in normal cells concurrent with the acquisition of GlyA surface expression. At genome scale, transfusion dependent and steroid resistant DBA samples are generally similar to each other, with thousands of regions where A/B identity are closely matched in DBA, but diametrically opposed to the configuration in stage-matched normal controls. Intriguingly, remission samples generally matched A/B compartments of other DBA samples in GlyA- fractions but GlyA+ compartments more closely resemble those of the controls, indicating that normalization of chromatin structural maturation accompanies hematologic remission. We generated a uniform set of 8,877 enriched ATAC-seq peaks on autosomes for differential chromatin accessibility analysis. As with methylation data, a large proportion (25%; 1085 up and 1114 down, B-H adj. P < 0.1) showed differential accessibility in normal control GlyA- vs GlyA+ cells. Steroid-responsive cases showed additional regions of differential accessibility during early maturation, with 31% of regions (1515 up and 1248 down) differentially accessible. Among transfusion dependent DBA patients, this count was much higher, with over half of peak regions (52%, 2400 up and 2216 down, B-H adj. P < 0.1) showing differential accessibility. Conclusion: Epigenetic maturation is broadly altered in DBA erythroid progenitors compared to stage matched normal controls, with specific changes identifiable in patients responding to steroids and in remission. Disclosures No relevant conflicts of interest to declare.

Diamond Blackfan Anemia (DBA) is an inherited bone marrow failure syndrome that is frequently managed with glucocorticoids to increase red cell mass. Despite sustained effective clinical use for many decades in DBA and other anemias, we still do not fully understand the mechanistic basis for the regulation of human erythropoiesis by glucocorticoids. In order to improve the clinical management of patients with DBA, we studied the mechanisms of action of dexamethasone (Dex) on erythroid progenitors. To study the effects of Dex on erythroid progenitor biology, we employed a serum-free erythroid culture system to differentiate primary human CD34+ cells isolated from the peripheral blood of adults or DBA patients as well as cord blood samples. We found that Dex increases the total proliferation of CD34+ cells from adult peripheral blood but not in cord blood over 14 days in culture. Dex treatment also led to an acceleration of the BFU-E to CFU-E transition in peripheral blood with a minimal effect in cord blood. In methylcellulose colony forming assays, peripheral blood derived CFU-E treated with Dex for 24hrs produced more colonies or larger colonies than the untreated controls while a lesser effect was seen in cord blood derived CFU-E. Both assays revealed that Dex treatment enhanced self-renewal of peripheral blood derived CFU-E. We also noted that BFU-E and CFU-E differentially express the glucocorticoid receptor (GR) isoforms, GRα and GRβ. BFU-E expressed comparable levels of GRα and the dominant-negative GRβ, whereas only the GRα transcript was identified in CFU-E. We observed similar expression patterns for GR between peripheral and cord blood derived progenitors. Importantly, following Dex treatment, GRα was shown to translocate to the nucleus of erythroid progenitors derived from peripheral blood derived CD34+ cells but not in the cord blood derived progenitors. When we examined the cell cycle progression of sorted human erythroid progenitors, CFU-E and BFU-E, we observed a G0/G1 arrest of peripheral blood CFU-E treated with Dex compared to untreated CFU-E. In marked contrast, no such change was noted for either Dex treated BFU-E from peripheral blood or for CFU-E or BFU-E from cord blood. In addition, we found an increase in the expression of cell cycle regulators p57Kip2 and p27Kip1 in Dex treated peripheral blood CFU-E but no such increased expression was seen in cord blood CFU-E. When peripheral blood derived erythroid progenitors were treated with olomoucine (Olo), a small molecule CDK inhibitor, we observed an acceleration of the BFU-E to CFU-E transition similar to that induced by Dex. Upon co-treatment with Dex and Olo, there was a synergistic effect in the accelerated formation of CFU-E population from BFU-Es. To validate this finding of the dependence of CFU-E on cell cycle regulators, we transduced peripheral blood derived CD34+ cells with a lentiviral shRNA construct to knockdown p57 expression. Following successful knockdown of the expression levels of p57, the numbers of CFU-E indeed decreased implying a role for cell cycle in defective transition of BFU-E to CFU-E. Erythroid progenitors derived from cultures of primary CD34+ cells from DBA patients were also studied. Importantly, we noted increased baseline expression of p57Kip2 and p27Kip1 in erythroid progenitors from steroid resistant DBA patients and in contrast to controls, their expression did not increase following Dex treatment. Our findings provide novel mechanistic insights into the regulation of human erythropoiesis by Dex. We have shown that alterations in cell cycle regulation of erythroid progenitors plays a key role in the effects of glucocorticoids on red blood cells. Furthermore, this work has direct relevance for the development of improved therapeutic treatment strategies for patients with bone marrow failure syndromes such as DBA. Disclosures No relevant conflicts of interest to declare.

The mechanisms underlying the development of erythropoietin (EPO)-refractory anemia in the setting of chronic inflammatory states are largely unknown. Elevated levels of the classical inflammatory mediators decrease red cell output. However, pathologic concentrations of many of these molecules do not persist beyond the acute phase, indicating that specific mediators are likely to play a role in the anemia associated with chronic inflammation. High mobility group box protein 1 (HMGB1) is a potent alarmin able to induce tissue injury during the acute and chronic phases of inflammation, and recently, shown to contribute to anemia in a murine model of sepsis. Here, we show that HMGB1 directly inhibits erythropoiesis by modulating EPO signal transduction in human erythroid cells through a newly identified HMGB1 receptor, which is surprisingly the erythropoietin receptor (EPOR). Surface plasmon resonance (SPR) reveals that HMGB1 binds the extracellular domain of EPOR (Kd = 130nM) with an affinity comparable to that of EPO. Cysteine residues contained within the A- and B-box domains of HMGB1 that have previously been shown to mediate HMGB1-receptor interactions are also responsible for the EPOR-HMGB1 interaction since a mutant form of HMGB1 lacking these cysteine residues (i.e. 3S HMGB1) fails to bind the EPOR. Cell-based assays suggest that the direct binding of HMGB1 to the EPOR and the subsequent degradation of EPOR accounts for altered EPO signaling by HMGB1. Biologically, HMGB1 reduces the phosphorylation of intracellular EPO effectors including JAK2 (2-fold reduction), STAT5 (4-fold), and ERK1/2 (4-fold). Decreased effector phosphorylation is not due to the increased activity of SHP1/2 phosphatases further implicating inhibition at the receptor level. Loss of EPO signaling due to HMGB1 binding results in decreased erythroid proliferation of differentiated CD34+ cells at the EPO-dependent stages of erythropoiesis: Day 14: 1.03x108 ± 4.67x107 cells/mL vs 1.87x106 ± 9.70x105 cells/mL, vehicle vs HMGB1, respectively. In addition, HMGB1 decreases the numbers of colony forming unit-erythroid (CFU-E) progenitors by 60%, and these progenitors fail to undergo terminal erythroid differentiation with a block at the basophilic erythroblast stage and apoptosis of late-stage erythroblasts as determined by flow cytometric analysis of annexin V staining. To understand the consequences of HMGB1-EPOR interactions on the EPO-induced transcriptome, RNA-sequencing was performed on purified human CFU-E dosed with HMGB1 and EPO. HMGB1 reduces the expression of known EPO target genes (ERFE, CISH, EGR1), and concomitantly, upregulates a number of unique transcripts (ETS2, VMP1, NFKBIZ) suggesting that HMGB1-EPOR interactions may alter receptor conformation in manner that differentially activates the EPOR and consequently, gene expression. Finally, in a mouse model of sepsis survival, bone marrow-derived erythroid precursor cells contain diminished phosphorylated STAT5 levels at a time when elevated HMGB1 plasma concentrations are observed, thereby demonstrating that the loss of EPO signal transduction also occurs in vivo. Taken together, our work identifies HMGB1 as a novel inhibitor of EPO signaling through its interaction with the EPOR, and strongly implicates HMGB1 as a previously undiscovered effector of EPO-refractory anemia associated with chronic inflammation. Disclosures No relevant conflicts of interest to declare.

Diamond Blackfan anemia (DBA) is a rare, inherited bone marrow failure syndrome characterized by anemia, congenital anomalies and a predisposition to cancer. The patients usually present during infancy or early childhood, but can also present in adulthood. In the majority of cases DBA is due to a mutation in a small or large ribosomal protein (RP) subunit leading to RP haploinsufficiency. The only treatments for the anemia of DBA are red cell transfusions (accompanied by iron chelation), oral corticosteroid therapy or stem cell transplantation. Pospisilova et al. (Haematologica 2007; 92(5):e66-67) reported one complete and two partial erythroid responses after the use of the branched chain amino acid L-leucine in 6 select patients. In skeletal muscle, leucine supplementation can upregulate components of the protein synthetic machinery, including ribosomal proteins, promoting protein translation. Mouse and fish models of DBA respond with amelioration of anemia to L-leucine. We therefore proposed to study the effect of L-leucine on transfusion dependence and growth in subjects with DBA. Methods: The primary objectives were to determine the feasibility of administering L-leucine in subjects with DBA who are red cell transfusion-dependent and to determine the efficacy of L-leucine to produce a hematologic and growth response. The secondary objective was to determine the safety profile of L-leucine. Twelve study sites were involved in this multi-center, Phase I/II study with an anticipated accrual of 50 subjects. A dose of 700 mg/M2 orally three times per day for 9 months was used. Inclusion criteria included age > 2 years, the diagnosis of DBA and transfusion dependence with adequate kidney and liver function. Response was evaluated at 9 months with Complete Response (CR) defined as no further transfusions required and Hb >9; Partial Response (PR): Hb < 9 gm/dL with an increase in reticulocyte count and transfusion interval; and No Response (NR): no change in transfusion needs, Hb or reticulocyte count . Growth percentiles were evaluated at baseline and at completion of 9 months of treatment and the growth velocity change was calculated. Results: The study opened July 2014 and closed February 2017; 55 subjects were consented; 12 were screen failures; 43 subjects were evaluable. There were 21 males; the median age was 9 years 1 month (2 years 5 months - 46 years 1 month). There were no untoward side effects experienced by any subject that were attributable to the L-leucine. Two subjects had an erythroid CR and 1 subject had a PR. The CRs occurred at 1 month and 3 months after start of L-leucine. The subject with PR had an elevated reticulocyte count but was not able to maintain a Hb >9 gm/dL without a transfusion and thus was not transfusion independent. Of the 30 subjects with growth potential who received L-leucine 10 experienced a positive growth velocity change at 9 months of therapy compared to baseline. At a median age of 7.5 years, the mean pre-leucine height percentile was 27 +/- 17.9 and the post-leucine height percentile was 35 +/- 19.9 (p Conclusions: The administration of L-leucine is safe. L-leucine administration resulted in an erythroid response in 7% of subjects and an increased growth velocity in 33% of growing subjects. Based upon extrapolation from animal models, it is likely that this dose was suboptimal. We hypothesize that higher doses of L-leucine will lead to hematologic responses in more subjects who are transfusion dependent. The potential benefit of added growth in children with DBA may improve final adult height. Disclosures Glader: Agios: Consultancy, Research Funding.

Background: RPL35A, a gene encoding a large ribosomal subunit protein located at the telomeric end of chromosome 3q (3q29-qter) is essential for rRNA processing, ribosomal biogenesis, cell proliferation, and apoptosis, and accounts for a subset of patients with Diamond Blackfan anemia (DBA). Reported pathogenic RPL35A mutations include single-nucleotide variants (SNVs), small insertion/deletions (indels), and large contiguous gene deletions associated with 3q29 microdeletion syndrome. 3q29 deletion syndrome is an overlapping syndrome that consists of developmental and intellectual disability with or without dysmorphic features, and other congenital anomalies but no anemia or cytopenia. The clinical phenotype and disease severity of patients with RPL35A-related DBA may be influenced by other genes deleted within 3q29 and be different in patients with large deletions compared to those with SNVs or small indels. Objectives: To determine whether DBA patients with large deletions of the 3q29 region have a more severe disease phenotype than those with SNVs or small indels in RPL35A, and whether other genes deleted within the 3q29 region might contribute to some of the features. Methods and Results: We identified 40 patients in a multi-institutional, international collaborative study of patients with DBA with RPL35A haploinsufficiency: 21 had deletion of RPL35A as part of 3q29 contiguous gene deletion, ranging in size from 0.012 Mb to 11 Mb in 16 patients; the extent of the deletion beyond RPL35A in either direction was unknown in 5 patients. Nineteen patients had SNVs or small indels (7 missense, 1 nonsense, 3 splice site, 6 indels and 2 unclear pathogenicity). Thirty-nine of 40 patients had severe anemia, 32 had neutropenia at some time and 3 had thrombocytopenia. Compared to the patients with SNVs or small indels, a significantly higher proportion of patients with RPL35A haploinsufficiency due to 3q29 deletion had steroid-resistant anemia (17 vs 7; p=0.009), severe chronic or intermittent neutropenia requiring treatment with G-CSF (7 vs 0; p=0.009), and humoral and/or cellular immunodeficiency (7 vs 1; p=0.046) diagnosed in some patients due to recurrent infections requiring hospitalizations (10 vs 2; p=0.03). Learning difficulties (12 vs 2; p=0.003), craniofacial abnormalities (11 vs 3; p=0.02), skeletal and limb defects (9 vs 2; p=0.03) or multiple physical anomalies (≥3) were also more frequent in patients with large deletions than in those with SNVs or indels (11 vs 3; p=0.02). Microcephaly (28%), short stature (33%), cardiac defects (28%) and/or urogenital abnormalities (23%) were equally distributed. The potential genes of interest near RPL35A that may be associated with immune dysregulation and/or neutropenia are RNF168, TFRC, PAK2, PIGZ, DLG1 and LMLN. One or more of these genes were deleted in at least 6 of 7 patients with neutropenia or immunodeficiency. Eight of 9 patients with malformations involving extremities, skeleton and ribs had deletions of TCTEX1D2 which is associated with rib/thoracic dysplasia and polydactyly. The genes of interest deleted in patients with developmental delay and learning disabilities included PAK2 and DLG1 in 9 patients, as well as RNF168, PPP1R2, TNK2 and q29 KIAA0226 in 8 of 12 patients in whom the extent of the deletion was known. Summary and Conclusion: Patients with DBA due to RPL35A contiguous gene deletions are clearly different from those with SNVs or small indels and have increased frequency of steroid-resistant transfusion-dependent anemia, severe neutropenia, immunodeficiency, learning/developmental delay, and craniofacial/skeletal/limb anomalies. Distinction of this subtype of DBA with RPL35A haploinsufficiency due to 3q29 contiguous gene deletion is relevant to their management, and evaluations should include a work-up for immunodeficiency. Further studies are needed to determine whether the complex phenotypes and severe disease manifestations in these patients are solely due to RPL35A haploinsufficiency or to the potential effect of other genes deleted in the 3q29 region. Genotype-phenotype characterization and comparison of DBA patients with 3q29 deletion with those due to deletions in other ribosomal protein genes may determine similarities or differences in disease phenotypes related to large gene deletions versus the influence of multigenic contiguous deletions. Supported by: AZV 16-32105A Disclosures Kattamis: Novartis: Consultancy, Honoraria; CELGENE: Consultancy, Honoraria; ApoPharma: Honoraria; Vifor Pharma: Consultancy. Niemeyer:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees.

W/W v and Sl/Sl d mice with macrocytic anemias are a potential model for human inherited pure red cell anemia, called Diamond-Blackfan anemia (DBA). The W mutation involves the gene for c-kit, and the Sl mutation the gene for the kit ligand, called mast cell growth factor, steel factor, or stem cell factor. Since many children with DBA respond to treatment with corticosterrids, we administered steroids to these genetically anemic mice, to determine whether they might provide a model for the human disease. There was no improvement in the murine anemia, consistent with other evidence suggesting that mutations in kit or steel may not be involved in Diamond-Blackfan anemia

Diamond Blackfan anemia (DBA) is a genetically and clinically heterogeneous disorder characterized by erythroid failure, congenital anomalies and a predisposition to cancer. Faulty ribosome biogenesis, resulting in pro-apoptotic erythropoiesis leading to erythroid failure, is hypothesized to be the underlying defect. The genes identified to date that are mutated in DBA all encode ribosomal proteins associated with either the small (RPS) or large (RPL) subunit and in these cases haploinsufficiency gives rise to the disease. Extraordinarily robust laboratory and clinical investigations have recently led to demonstrable improvements in clinical care for patients with DBA.

The combination of 2-chlorodeoxyadenosine (2-CDA) and cytosine arabinoside (Ara-C) has been shown to be effective in children with refractory Langerhans cell histiocytosis (LCH). We have treated 5 patients with recurrent LCH with 2-CDA/Ara-C chemotherapy and closely followed immune and hematopoietic function. These patients display a decline in the absolute CD4, CD8, and natural killer cell number, decrease in the CD4/CD8 ratio. Septic events, including pneumocystis infection were present after most of the treatment courses (15/21). These data suggest that 2-CDA /Ara-C, should be considered in resistant and relapsed pediatric patients with LCH with high-risk multiorgan involvement. Consequent profound prolonged combined immune deficiency and myelosupression should be anticipated.

Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by anemia, congenital abnormalities, and cancer predisposition. Small ribosomal subunit genes RPS19, RPS24, and RPS17 are mutated in approximately one-third of patients. We used a candidate gene strategy combining high-resolution genomic mapping and gene expression microarray in the analysis of 2 DBA patients with chromosome 3q deletions to identify RPL35A as a potential DBA gene. Sequence analysis of a cohort of DBA probands confirmed involvement RPL35A in DBA. shRNA inhibition shows that Rpl35a is essential for maturation of 28S and 5.8S rRNAs, 60S subunit biogenesis, normal proliferation, and cell survival. Analysis of pre-rRNA processing in primary DBA lymphoblastoid cell lines demonstrated similar alterations of large ribosomal subunit rRNA in both RPL35A-mutated and some RPL35A wild-type patients, suggesting additional large ribosomal subunit gene defects are likely present in some cases of DBA. These data demonstrate that alterations of large ribosomal subunit proteins cause DBA and support the hypothesis that DBA is primarily the result of altered ribosomal function. The results also establish that haploinsufficiency of large ribosomal subunit proteins contributes to bone marrow failure and potentially cancer predisposition.

Diamond Blackfan anemia (DBA) is a genetically and clinically heterogeneous disorder characterized by erythroid failure, congenital anomalies, and a predisposition to cancer. Faulty ribosome biogenesis is hypothesized to be the underlying defect, leading to erythroid failure due to accelerated apoptosis in affected erythroid progenitors/precursors. Since first observed in DBA, pro-apoptotic hematopoiesis has been recognized as a common mechanism for hematopoietic failure in virtually all of the inherited bone marrow failure syndromes. Inherited as an autosomal dominant trait, one of what appears to be multiple DBA genes, coding for ribosomal protein RPS19, has been cloned. The discovery of additional genes will no doubt clarify the molecular pathophysiology of this disorder. Even within families, individuals may vary dramatically as to the degree of anemia, treatment response, and the presence of congenital anomalies. The study of DBA has been facilitated by the creation of international patient registries that provide more reliable information regarding clinical presentation, genetics, and outcome, as well as descriptions of congenital malformations and cancer predisposition, than can be culled from the literature. Analysis of registry data has led to improvements in clinical care and provides patients and research specimens for clinical and laboratory investigations.

Summary The genetic basis of Diamond–Blackfan anaemia (DBA), a congenital erythroid hypoplasia that shows marked clinical heterogeneity, remains obscure. However, the fact that nearly one-quarter of patients harbour a variety of mutations in RPS19, a ribosomal protein gene, provides an opportunity to examine whether haplo-insufficiency of RPS19 protein can be demonstrated in certain cases. To that end, we identified 19 of 81 DBA index cases, both familial and sporadic, with RPS19 mutations. We found 14 distinct insertions, deletions, missense, nonsense and splice site mutations in the 19 probands, and studied mutations in 10 patients at the RNA level and in three patients at the protein level. Characterization of the mutations in 10 probands, including six with novel insertions, nonsense and splice site mutations, showed that the abnormal transcript was detectable in nine cases. The RPS19 mRNA and protein in CD34+ bone marrow cells identified haplo-insufficiency in three cases predicted to have one functional allele. Our data support the notion that, in addition to rare DBA patients with the deletion of one allele, the disease in certain other RPS19 mutant patients is because of RPS19 protein haplo-insufficiency.