Congenital Hypoplastic Anemia Research Articles

Establishment of a Diamond-Blackfan anemia like model in zebrafish.

Anemia is defined as a lack of erythrocytes, low hemoglobin levels, or abnormal erythrocyte morphology. Diamond-Blackfan anemia (DBA) is a rare and severe congenital hypoplastic anemia that occurs due to the dominant inheritance of a ribosomal protein gene mutation. Even rarer is a case described as Diamond-Blackfan anemia like (DBAL), which occurs due to a loss-of-function EPO mutation recessive inheritance. The effective cures for DBAL are bone marrow transfusion and treatment with erythropoiesis-stimulating agents (ESAs). To effectively manage the condition, construction of DBAL models to identify new medical methods or screen drugs are necessary. Here, an epoa-deficient mutant zebrafish called epoaszy8 was generated to model DBAL. The epoa-deficiency in zebrafish caused developmental defects in erythroid cells, leading to severe congenital anemia. Using the DBAL model, we validated a loss-of-function EPO mutation using an in vivo functional analysis and explored the ability of ESAs to alleviate congenital anemia. Together, our study demonstrated that epoa deficiency in zebrafish leads to a phenotype resembling DBAL. The DBAL zebrafish model was found to be beneficial for the in vivo assessment of patient-derived EPO variants with unclear implications and for devising potential therapeutic approaches for DBAL.

Diamond-Blackfan anemia, the archetype of ribosomopathy: How distinct is it from the other constitutional ribosomopathies?

Diamond-Blackfan anemia (DBA) was the first ribosomopathy described in humans. DBA is a congenital hypoplastic anemia, characterized by macrocytic aregenerative anemia, manifesting by differentiation blockage between the BFU-e/CFU-e developmental erythroid progenitor stages. In 50 % of the DBA cases, various malformations are noted. Strikingly, for a hematological disease with a relative erythroid tropism, DBA is due to ribosomal haploinsufficiency in 24 different ribosomal protein (RP) genes. A few other genes have been described in DBA-like disorders, but they do not fit into the classical DBA phenotype (Sankaran et al., 2012; van Dooijeweert et al., 2022; Toki et al., 2018; Kim et al., 2017 [1–4]).Haploinsufficiency in a RP gene leads to defective ribosomal RNA (rRNA) maturation, which is a hallmark of DBA. However, the mechanistic understandings of the erythroid tropism defect in DBA are still to be fully defined. Erythroid defect in DBA has been recently been linked in a non-exclusive manner to a number of mechanisms that include: 1) a defect in translation, in particular for the GATA1 erythroid gene; 2) a deficit of HSP70, the GATA1 chaperone, and 3) free heme toxicity. In addition, p53 activation in response to ribosomal stress is involved in DBA pathophysiology. The DBA phenotype may thus result from the combined contributions of various actors, which may explain the heterogenous phenotypes observed in DBA patients, even within the same family.

Reduced Polycomb Repressor Complex 2 (PRC2) Activity and Increased TP53 Activity Mediate Hematopoietic Stem Cell Dysfunction in RPS19-Mutated Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA) is a congenital hypoplastic anemia caused by heterozygous loss-of-function mutations in one of 23 ribosomal protein (RP) genes, with RPS19 being mutated in approximately 25% of patients. While DBA typically manifests in infancy as isolated anemia with reticulocytopenia, older patients can develop multilineage cytopenias and bone marrow (BM) hypocellularity suggesting impaired hematopoietic stem cell (HSC) function. Defects in HSCs have been observed in Rps19-depleted mice but studies on human DBA HSCs have been inconclusive, largely because patient BM cells are difficult to obtain for research. To address this gap, we developed a new, robust, and scalable cellular model using CRISPR/Cas9 to create heterozygous RPS19 loss-of-function mutations in healthy donor CD34 + hematopoietic stem and progenitor cells (HSPCs). Transplantation of RPS19+/- HSPCs into NBSGW immunodeficient mice revealed a defect in BM repopulation that was partially rescued by co-disruption of the TP53 gene (Bhoopalan et al, JCI Insight, 2023). Here we provide further mechanistic insight into this HSC defect by showing that RP imbalance caused by RPS19 deficiency leads to the activation of TP53 and inhibition of Polycomb Repressor Complex 2 (PRC2), which has been previously shown to be required for HSC maintenance. Healthy donor CD34 + HSPC were electroporated with ribonucleoprotein (RNP) complex consisting of Cas9 and guide RNAs (gRNAs) targeting RPS19 or the AAVS1 safe harbor locus as a negative control. RPS19 disruption was verified by next-generation sequencing of the targeted region and by Western blot analysis showing reduction of RPS19 protein. RNA-Seq at 3 days after electroporation showed enrichment of TP53 pathway genes in RPS19-edited HSPCs compared to control cells, confirming our previous observations. Additionally, we noted an enrichment of HSPC genes that were previously shown to harbor the repressive histone mark H3K27me3 in normal HSPCs. Methylation of H3K27 is catalyzed by EZH2, the catalytic component of PRC2, which maintains HSCs by inhibiting transcription of genes that promote differentiation. Gene set enrichment analysis (GSEA) revealed significant induction of PRC2 target genes in RPS19-disrupted HSPCs and in induced pluripotent stem cells (iPSCs) generated from two different DBA patients with heterozygous RPS19 mutations. The “Cleavage under targets and release using nuclease (CUT&RUN)” assay indicated globally reduced EZH2 binding to chromatin in RPS19-disrupted HSPCs. These findings suggest that PRC2 activity is inhibited by RPS19 haploinsufficiency. Previous studies have shown that RPS19 haploinsufficiency can cause accumulation of large RP subunits, RPL5 and RPL11, which bind 5S ribosomal RNA to form 5S RNP. This complex can sequester the ubiquitin ligase MDM2 to inhibit its function. MDM2 ubiquitinates TP53, targeting it for proteasomal degradation. Additionally, MDM2 binds PRC2 to augment its activity. Thus, we theorized that RPS19 haploinsufficiency leads to the accumulation of 5S RNP complex which sequesters MDM2, causing increased TP53 activity and suppression of PRC2 activity, both of which can inhibit HSC maintenance. To test this, we inhibited formation of 5S RNP complex by co-disrupting RPL5 or RPL11 along with RPS19 in CD34 + HSPCs, followed by RNA-Seq analysis or transplantation into NBSGW mice. Remarkably, haploinsufficiency of either large RP gene rescued the BM repopulation defect of RPS19 +/- HSPC (p<0.05) ( Fig. 1A), reduced TP53 activity and improved PRC2 activity (p<0.001 and FDR q<0.01) ( Fig. 1B). In summary, our data support the model that RPS19 haploinsufficiency leads to increased TP53 activity and reduced PRC2 activity through 5S RNP-mediated sequestration of MDM2, causing impairment of HSC function. These studies provide insights into the molecular mechanisms of DBA and could potentially inform new therapies.

Establishment and characterization of CSCRi006-A: an induced pluripotent stem cell line generated from a patient with Diamond-Blackfan Anemia (DBA) carrying ribosomal protein S19 (RPS19) mutation.

Diamond-Blackfan anemia (DBA) is a congenital hypoplastic anemia characterized by ineffective erythropoiesis. DBA is majorly caused by mutations in the ribosomal protein (RP) genes (Gadhiya and Wills in Diamond-Blackfan Anemia, https://www.statpearls.com/ ; 2023). A suitable disease model that yields a continuous supply of erythroid cells is required to study disease pathogenesis and drug discovery. Toward this, we reprogrammed dermal fibroblasts from a DBA patient with a heterozygous mutation c.22-23delAG in the RPS19 gene identified through exome sequencing. To generate induced pluripotent stem cells (iPSCs), we induced episomal expression of the reprogramming factors OTC3/4, L-MYC, LIN28, SOX2, and KLF4, and a p53 shRNA2. The DBA-iPSC line CSCRi006-A generated during this study was extensively characterized for its pluripotency and genome stability. The clone retained normal karyotype and showed high expression levels of pluripotency markers, OCT4, NANOG, SOX2, TRA-I-60, TRA-I-81, and SSEA4. It could differentiate into cells originating from all three germ cell layers, as identified by immunostaining for SOX17 (endoderm), Brachyury (mesoderm), and PAX6 (ectoderm). IPSCs provide a renewable source of cells for in vitro disease modeling. CSCRi006-A, a thoroughly characterized iPSC line carrying heterozygous RPS19 c.22-23delAG mutation, is a valuable cell line for the disease modeling of DBA. This iPSC line can be differentiated into different blood cell types to study the mechanisms of disease development and identify potential treatments.

A Novel RPS19-Edited Hematopoietic Stem Cell Model of Diamond-Blackfan Anemia for Development of Lentiviral Vector Gene Therapy

Diamond-Blackfan anemia (DBA) is a congenital hypoplastic anemia that typically manifests in infancy as macrocytic anemia with reticulocytopenia. About 80% of DBA cases are caused by heterozygous loss-of-function mutations or deletions in one of 23 ribosomal proteins (RP) genes, with RPS19 being affected in ~25% of patients. Current therapies are suboptimal, and it is difficult to obtain DBA patient hematopoietic stem and progenitor cells (HSPCs) in sufficient quantity for preclinical development of new therapies. To address this gap, we used CRISPR/Cas9-edited healthy donor CD34 + HSPCs to create a novel model of RPS19-mutated DBA and used this model to develop an efficient RPS19-encoding lentiviral vector (LV) for gene therapy.Healthy donor CD34 + HSPCs were electroporated with ribonucleoprotein (RNP) complex consisting of Cas9 and guide RNAs (gRNAs) targeting RPS19 or the AAVS1 locus as a negative control, then grown in medium to support erythroid differentiation. All gRNAs analyzed generated high-frequency on-target insertion-deletion (indels) mutations (Fig. A). RPS19 indels specifically declined over time, suggesting that RPS19 disruption impairs cell proliferation and/or survival. To rescue the defect, we constructed a third-generation, self-inactivating LV expressing RPS19+GFP (RPS19/GFP LV). Transduction was optimized using poloxamer and prostaglandin E2 and linear transduction efficiency was noted even at high multiplicity of infection (MOI) (Fig. B). An MOI of 20 was used for subsequent experiments. In methylcellulose medium, RPS19 RNP-treated cells generated 72% fewer burst forming unit-erythroid (BFU-E) colonies compared to AAVS1 RNP-treated control cells. RPS19/GFP LVs with three different promoters (EF1α short, EF1α long and MND) partially restored BFU-E formation similarly (Fig. C); the EF1α short promoter was chosen for subsequent experiments due its track record for clinical use. We down-titrated the RNP concentration to generate a total indel frequency of ~25%, which resulted in approximately equal frequencies of RPS19 +/+ and RPS19 +/- BFU-E colonies. RPS19 -/- colonies were detected only after edited HSPCs were rescued by RPS19/GFP LV, due to lethality of this genotype (Fig. D). Transfection of CD34 + HSPCs with RPS19 RNP caused a 49% reduction in cell number after 14 days of liquid culture in erythroid differentiation medium compared to control HSPCs; this was corrected by treatment with RPS19/GFP LV (Fig. E). RPS19 RNP treatment of CD34 + HSPCs had no effect on the expansion of cells grown under myeloid differentiation conditions (Fig. F).We analyzed RNP-treated CD34 + cells further by transducing them with RPS19/GFP LV or control LV encoding GFPalone, transplanting them into immunodeficient NSGW mice and analyzing human donor cell progeny in mouse bone marrow after 16 weeks. In cells treated with AAVS1 RNP and GFP LV, the indel frequency dropped from 27.2±1.5% (SD) at 72 hours after editing (input) to 15.5±4.4% at 16 weeks post-transplant (43% reduction) (Fig. G). In HSPCs treated with RPS19 RNP and GFP LV the indel frequency dropped from 20.9±3.1% in input cells to 1.8±0.9% after 16 weeks (Fig. G) (92% reduction). In contrast, the indel frequency of donor HSPCs treated with RPS19 RNP and RPS19/GFP LV dropped from 23.6±2.7% in input cells to 8.4±1.6% (64% reduction), which represents a 5-fold increase in indel frequency compared to treatment with control GFP LV (p< 0.01)(Fig. G). In flow cytometry-purified, donor HSPC-derived myeloid, B-lymphocyte, HSPC and erythroid lineages at 16 weeks after xenotransplantation, the mean indel rates were 1.3% to 2.5% in cells derived from HSPCs treated with RPS19 RNP and GFP LV. Indel rates ranged from 6.9% to 9.2% in the progeny of input HSPCs that were rescued by RPS19/GFP LV, representing a 4-6-fold increase compared to transduction with control GFP LV (p<0.01) (Fig. H).In summary, our studies show that Cas9-mediated disruption of RPS19 in CD34 + HSPCs causes a selective erythroid defect in RPS19 +/- cells, recapitulating the canonical DBA defect. Additionally, deficient bone marrow repopulation by RPS19 +/- cells suggests an HSC defect, consistent with pancytopenia that is observed in many older DBA patients. The optimized RPS19 LV transduces HSPCs at high efficiency and alleviates both defects, supporting its potential utility for DBA therapy. [Display omitted] DisclosuresYen: Beam Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Weiss: Beam Therapeutics: Current holder of stock options in a privately-held company; Forma Therapeutics: Consultancy; Novartis: Consultancy; Cellarity Inc.: Consultancy.

An update on the pathogenesis and diagnosis of Diamond-Blackfan anemia.

Diamond–Blackfan anemia (DBA) is a rare congenital hypoplastic anemia characterized by a block in erythropoiesis at the progenitor stage, although the exact stage at which this occurs remains to be fully defined. DBA presents primarily during infancy with macrocytic anemia and reticulocytopenia with 50% of cases associated with a variety of congenital malformations. DBA is most frequently due to a sporadic mutation (55%) in genes encoding several different ribosomal proteins, although there are many cases where there is a family history of the disease with varying phenotypes. The erythroid tropism of the disease is still a matter of debate for a disease related to a defect in global ribosome biogenesis. Assessment of biological features in conjunction with genetic testing has increased the accuracy of the diagnosis of DBA. However, in certain cases, it continues to be difficult to firmly establish a diagnosis. This review will focus on the diagnosis of DBA along with a description of new advances in our understanding of the pathophysiology and treatment recommendations for DBA.

Fankoni Aplastik Anemili Çocuklarda Genitoüriner Sistem Anomalileri

Objective: Fanconi aplastic anemia (FAA) is a rare genetic disease that causes bone marrow failure. It is the most common bone marrow failure syndrome among the hereditary bone marrow failure syndromes. Genitourinary system anomalies occur in about one third of patients with FAA. Renal malformations that can lead to end stage chronic kidney disease can be one of the major causes of morbidity and mortality in these patients and requires detailed review. Material and Methods: In order to evaluate genitourinary system malformations in FAA patients, eleven FAA patients who were diagnosed with bone marrow aspiration and mutation analysis, accompanied by genitourinary system anomalies, and followed at Cukurova University Department of Pediatric Hematology were retrospectively analyzed, The presence of genitourinary system anomalies were detected by renal ultrasonography, dimercaptosuccinic acid (DMSA) scintigraphy and voiding cystourethrogram. Results: Five of the eleven patients with FAA were girls (45.5%) and six of them were boys (54.5%). Seven patients (63.6%) had bilateral vesicoureteral reflux (VUR) and one patient (9%) had unilateral VUR. Four patients (36.4%) had unilateral renal agenesis and two patients (18.2%) had an ectopic kidney. Three patients (27.2%) had neurogenic bladder requiring clean intermittent catheterization. One of the patients had a penile deformity and urethral stricture. Six of the patients had an estimated glomerular filtration rate lower than 90 ml/min/1.73 m2. One of them had end stage chronic kidney disease and was receiving chronic peritoneal dialysis treatment. Conclusion: FAA is a constitutional aplastic anemia and is often seen in the first decade of life. Among the congenital defects associated with FAA, genitourinary malformations are quite common. In our study, the most common genitourinary anomaly was vesicoureteral reflux and found in 72.7% of the 11 FAA patients. The presence of a renal abnormality in these patients is also a risk factor after bone marrow transplantation and may lead to acute kidney injury and chronic kidney disease. These patients should therefore be carefully assessed.

Patient features and survival of pediatric aplastic anemia in the USA: a large institution experience.

We performed the first epidemiologic investigation to examine association of demographics and clinical characteristics at diagnosis, as well as health care expense coverage, with survival of US children with aplastic anemia (AA). We obtained electronic medical record data of 1140 children aged 0-19 years diagnosed with AA followed at a pediatric health system between 2004 and 2014. Kaplan-Meier curve and Cox proportional hazards regressions were used. Self-pay patients had a mortality risk five times higher than that of those insured by publicly funded insurance (hazards ratio, 95% CI: 6.0, 3.7-9.8). Other features associated with higher mortality risk include pancytopenia (hazards ratio, referent: 4.2, constitutional AA); underweight (2.0, normal-weight); platelet count <50 × 109/l (1.3, ≥50 × 109/l); male sex (1.3, female); and ages at diagnosis 6-11, 11-16 and 16-19 years (1.6, 1.9, 2.3, 1-3 years), respectively. Self-pay was the strongest prognostic factor for pediatric AA mortality. Older age, pancytopenia, underweight, male sex and lower platelet count were also associated with increased risk of mortality. These findings may be useful for providers, researchers and policymakers to ensure effective health care delivery to this population and to motivate future etiologic research and establishment of a surveillance registry.

Nontransplant therapy for bone marrow failure.

Nontransplant therapeutic options for acquired and constitutional aplastic anemia have significantly expanded during the last 5 years. In the future, transplant may be required less frequently. That trilineage hematologic responses could be achieved with the single agent eltrombopag in refractory aplastic anemia promotes new interest in growth factors after years of failed trials using other growth factor agents. Preliminary results adding eltrombopag to immunosuppressive therapy are promising, but long-term follow-up data evaluating clonal evolution rates are required before promoting its standard use in treatment-naive disease. Danazol, which is traditionally less preferred for treating cytopenias, is capable of preventing telomere attrition associated with hematologic responses in constitutional bone marrow failure resulting from telomere disease.

Discovery of the First Pathogenic Human EPO Mutation Provides Mechanistic Insight into Cytokine Signaling

Congenital hypoplastic or Diamond-Blackfan anemia (DBA) is a rare bone marrow failure disorder characterized by a paucity of red blood cells and their precursors in the bone marrow. The majority of cases of DBA are due to haploinsufficient mutations in ribosomal protein genes and in rare cases result from GATA1 mutations. However, nearly half of the DBA cases do not have an identified genetic etiology. While analyzing whole exome sequencing data from a cohort of over 450 patients with a clinical diagnosis of DBA, we encountered the case of a male child of a first cousin consanguineous union who was diagnosed with DBA as an infant and remained transfusion dependent. The patient responded to corticosteroid therapy for a year as a toddler, but this treatment was discontinued due to side effects. The patient subsequently remained transfusion dependent and at 6 years of age an allogeneic bone marrow transplant from a matched maternal aunt was performed. Surprisingly, despite achievement of robust donor chimerism, the patient remained transfusion dependent. Unfortunately the patient developed severe graft-versus-host disease and died of resultant complications. Both the potential recessive nature of the mutation, given parental consanguinity, and the lack of anemia correction following transplant made this case extremely unusual. Thus we evaluated this patient’s whole exome sequencing data. We identified a homozygous recessive mutation in the erythropoietin gene (EPO), which resulted in an R150Q substitution in the mature EPO protein. This mutation was absent from a cohort of 60,706 individuals depleted for Mendelian disease and fit the model of complete penetrance in the family. The R150Q mutation was expected to disrupt the high-affinity binding site to the EPO receptor (EPOR). However, we found by producing recombinant proteins that the EPO R150Q mutation only reduced the EPOR binding affinity by 3-fold. Surprisingly, the patient had an over 100-fold elevation in their serum EPO levels, suggesting that this mutation did not cause disease through altered affinity. Rather we observed altered EPOR binding kinetics by this mutant ligand. There was a slightly increased on-rate with a much faster dissociation rate (t1/2 of 10 seconds for the mutant vs. 6 minutes for the wild type). Using human erythroid cells and primary hematopoietic stem and progenitor cells, we could show that this mutant ligand never reached the same efficacy as the wild type (WT) EPO in promoting erythroid differentiation and proliferation. To better characterize this abnormal activity, we examined downstream signaling responses. We found identical phosphorylation of STAT5 at maximally potent concentrations of the WT (1 nM) and R150Q mutant (100 nM) EPO. By surveying a broad array of >120 phosphorylation events using intracellular flow cytometry, we demonstrated that maximal levels of STAT3 and STAT1 phosphorylation were reduced by 30% and 25%, respectively, with the R150Q (100 nM) compared to WT (1 nM) EPO. To determine the mechanistic basis for variation in downstream effector activation by the R150Q mutant ligand, we used inhibitors of both the JAK2 kinase and the SHP1/2 phosphatases that are respectively up- and downstream of STAT phosphorylation. While SHP1/2 inhibition did not alter STAT phosphorylation, JAK2 inhibition by ruxolitinib more potently inhibited STAT1/3 phosphorylation compared to STAT5. Interestingly, treatment with a low dose of ruxolitinib (40 nM) reduced erythroid proliferation to the same extent at maximally potent concentrations of the WT or R150Q EPO, demonstrating that the impairment in signaling by the R150Q EPO was primarily due to reduced JAK2 activity. Finally, we utilized single molecule fluorescent imaging of EPOR dimerization at the intact cell surface to directly show that the kinetically-biased R150Q EPO has a reduced ability to promote productive dimerization as compared to the WT EPO, even at maximally potent concentrations. Collectively, our results demonstrate how the R150Q mutant EPO - the first pathogenic mutation in EPO identified in humans - results in biased agonism of EPOR signaling through reduced receptor dimerization and consequently impaired JAK2 activation. More broadly our findings reveal how variation of cytokine-receptor binding kinetics can be used to tune downstream responses, which has broad implications for modulating the activity of numerous hematopoietic cytokines. DisclosuresNo relevant conflicts of interest to declare.

Pluripotent stem cells reveal erythroid-specific activities of the GATA1 N-terminus.

Germline GATA1 mutations that result in the production of an amino-truncated protein termed GATA1s (where s indicates short) cause congenital hypoplastic anemia. In patients with trisomy 21, similar somatic GATA1s-producing mutations promote transient myeloproliferative disease and acute megakaryoblastic leukemia. Here, we demonstrate that induced pluripotent stem cells (iPSCs) from patients with GATA1-truncating mutations exhibit impaired erythroid potential, but enhanced megakaryopoiesis and myelopoiesis, recapitulating the major phenotypes of the associated diseases. Similarly, in developmentally arrested GATA1-deficient murine megakaryocyte-erythroid progenitors derived from murine embryonic stem cells (ESCs), expression of GATA1s promoted megakaryopoiesis, but not erythropoiesis. Transcriptome analysis revealed a selective deficiency in the ability of GATA1s to activate erythroid-specific genes within populations of hematopoietic progenitors. Although its DNA-binding domain was intact, chromatin immunoprecipitation studies showed that GATA1s binding at specific erythroid regulatory regions was impaired, while binding at many nonerythroid sites, including megakaryocytic and myeloid target genes, was normal. Together, these observations indicate that lineage-specific GATA1 cofactor associations are essential for normal chromatin occupancy and provide mechanistic insights into how GATA1s mutations cause human disease. More broadly, our studies underscore the value of ESCs and iPSCs to recapitulate and study disease phenotypes.

An SCF-FBXW7 Ubiquitin Ligase Mediated Feedback Loop Facilitates GATA Factor Switching and Reinforces Commitment to Terminal Erythroid Maturation

The GATA family transcription factors GATA2 and GATA1 play reciprocal roles during terminal erythroid and megakaryocytic maturation. GATA2 is expressed early in hematopoiesis and is required for early progenitor cell proliferation and survival. It must be down regulated in order for terminal maturation to occur. In contrast, GATA1 increases in expression during erythropoiesis and megakaryopoiesis, and is required for terminal maturation of these lineages. During this process, GATA1 displaces GATA2 at a large number of chromatin loci, typically leading to repression of early progenitor genes and activation of terminal maturation genes. This “GATA factor switch” is facilitated by the considerably shorter half-life of GATA2 (~30 min) compared to GATA1 (>4-6 hours). GATA2’s short half-life is mediated by ubiquitin-proteosomal degradation mechanisms. Yet the ubiquitin complex(es) responsible for GATA2-specific clearance has not been identified. In this study, we show that the Skip1-Cullin-Fbox (SCF) substrate recognition factor Fbxw7 is involved in GATA2 ubiquitin-mediated degradation. Proteomic and co-immunoprecipitation experiments show physical association of Fbxw7 with GATA2, but not GATA1. CRISPR/Cas9 deletion of Fbxw7 in G1ER4 cells results in elevated GATA2 steady-state protein levels, a prolonged GATA2 half-life, delayed GATA2 clearance upon estradiol induction, and impaired erythroid terminal maturation. Importantly, Fbxw7 mRNA and protein levels normally increase during terminal erythroid maturation and this occurs in a GATA1 dependent manner. We identified a key cis-regulatory element upstream of the Fbxw7 gene that is occupied by GATA2 during early erythropoiesis, but becomes bound by GATA1/TAL1 during late erythroid maturation in induced G1-ER4 cells and human CD34+ in vitro differentiated erythroblasts. This is accompanied by the acquisition of active enhancer histone marks at this site. Deletion of this regulatory element in G1ER4 cells blocks the GATA1 dependent increase in Fbxw7 mRNA transcript levels during erythroid cell maturation. Lastly, we identified a family with congenital hypoplastic anemia (lacking mutations in all known Diamond Blackfan Anemia genes) who harbor a germline Fbxw7missense mutation. Collectively, these findings identify GATA2 as a novel Fbxw7 substrate and uncover an ubiquitin-mediated post-transcriptional GATA factor feedback loop that reinforces GATA factor switching and commitment to terminal erythroid maturation. DisclosuresCantor:Amgen: Membership on an entity’s Board of Directors or advisory committees.

Telomerase Enzyme Activity In Egyptian Children With Bone Marrow Failure

Background Telomeres are structural elements that seal the ends of chromosomes protecting them from recombination and end to end fusion. Maintenance of the integrity of telomeres requires the telomerase ribonucleoprotein complex. Abnormal telomere maintenance is a feature of a variety of human diseases including constitutional aplastic anemia. Predisposition to the development of marrow failure has been conferred by genetic alternations results in low telomerase activity. Short telomeres in leukocytes and reduced hematopoietic function. Aim To evaluate the telomerase functional activity in Egyptian children with inherited and acquired bone marrow failure and its relation to the phenotypic features of the acquired disease and its response to immunosuppressive therapy. Patients and Methods This was a case-control study conducted on unrelated children (n=40) with bone marrow failure syndromes and forty healthy subjects age and sex-matched as controls. The diagnosis of bone marrow failure was based on the bone marrow biopsy and blood-count results. Patients with acquired aplastic anemia (AAA) were considered severe if at least 2 of the following were noted: neutrophil count &lt; 0.5×109/L; platelet count &lt; 20×109/L with hypocellular marrow. Response to immunosuppressive therapy (IST) was evaluated after 6 months of initiation of therapy. Assessment of Telomerase Activity Telomerase activity was measured in mononuclear cells utilizing the Telomeric Repeat Amplification Protocol (TRAP )using the TeloTAGGG Telomerase PCR ELISA. Results Forty patients were included in the study (30 AAA , 6 Fanconi Anemia (FA), 2 Pure Red Cell Aplasia (PRCA), one case with Dyskeratosis Congenita (DCK) and one case with constitutional aplastic anemia. The Mean age was 11.1±4.9 years (range 3.5 to 18 years, median 11 years ) and the duration of follow-up mean; 5.14 (±3.84) years (range 1-13 years). Patients with AAA (n=30) received treatment with cyclosporine A( n =27) and ATG( n=3). Telomerase level The median telomerase level was significantly lower in inherited BMF syndromes when compared to controls [5.05 (4.60 – 8.70 IQR) Vs 11.15 (5.90-16.60 IQR), p=0.04]. In AAA the median telomerase level was insignificantly lower than controls [5.4 (2.3 – 21.0 IQR) Vs 11.15 (5.90-16.60 IQR), p=0.228]. A good inverse correlation was detected between the telomerase level and age of the patients (r=-0.39, p=0.026). No correlation was found between the telomerase level and disease duration in hereditary or AAA (r= -0.303, p=0.111 and r=0.305, p=0.156 respectively). Telomerase activity and clinical variables We classified our cases into two groups according to the median value of the control group (11.5): Low telomerase level group: (n=27): included 18 AAA (66.7%) and 9 hereditary aplastic Anemia (33.3%). Cases with AAA 14/18 (77.8%) with low telomerase activity were severe cases with bone marrow cellularity less than 10 % Vs 6/12 (50%) with normal telomerase. Fifty per cent of cases (9/18) with low telomerase responded partially or completely to IST Vs. 83.4% of the normal telomerase (10/12) group. Patients of AAA who received cyclosporine therapy (n=27), 19 cases (70.4%) were responders versus zero % of patients who received ATG (n=3). The median telomerase of responders was 16.5 + 4.7 Vs 11.6 + 3.8 of none responders. Conclusion • Telomerase activity was affected in hereditary and acquired bone marrow failure. • Evaluation of telomerase activity is essential for therapeutic and prognostic aspects of these diseases. Cyclosporin A can be used as a monotherapy in the treatment of acquired aplastic anemia even in the presence of decreased telomerase activity Disclosures: No relevant conflicts of interest to declare.

DIAMOND BLACKFAN ANEMIA: A TERTIARY CARE CENTRE EXPERIENCE

Diamond Blackfan Anemia (DBA) is a rare hypoplastic anemia that presents in infancy with macrocytic anemia and reticulocytopenia. It is a ribosomopathy with autosomal dominant inheritance.In our series of 10 patients with DBA, congenital malformations were observed in 50% of the cases. Age at symptom onset ranged from 0–12 months. Age at diagnosis ranged from 4 months to 96 months. Male: female ratio was 9:1. Response to prednisolone was observed in 4 out of the 10 patients (either during initial treatment or during re-challenge). Response to cyclosporine was found to be poor. Bone marrow transplantation was successful in attaining remission in one patient. Malignancies were not reported in any patient possibly due to a short follow up period.

Eritroblastopenia Transitoria de la Infancia: Presentación de Tres Casos Clínicos

Transient childhood erythroblastopenia is a benign hematological condition, characterized by non-regenerative anemia and transient marrow aplasia. It is seen among post-viral infected children 1 through 14 years of age. Incidence is 4 in 100,000, and recovery is shown in 2-4 weeks. Objective: Three cases are presented, as a reminder due to low incidence, limited national literature and need for review of this subject. Clinical cases: Three patients, 1-4 years old, with similar symptoms including severe anemia, reticulocytopenia, and myelogram showing erythroblastopenia (2 cases) and one in recovery phase. No infectious etiology was identified. Hemogram, reactive protein C, hepatic and renal function studies, myelogram, bone marrow parvovirus B19 PCR, and Epstein-Barr IgM. Conclusions: Our experience is similar to that shown in the literature, with unidentified etiology, benign course, and emphasize on the differential diagnosis from congenital hypoplastic anemia.

Gene therapy of Diamond Blackfan anemia CD34 + cells leads to improved erythroid development and engraftment following transplantation

Diamond-Blackfan anemia (DBA) is a rare congenital hypoplastic anemia caused by mutations in ribosomal protein (RP) genes. Our aim is to develop gene therapy for DBA patients with mutations in RPS19. We previously demonstrated that RPS19 gene transfer partially corrects erythroid development in vitro. In this study, we asked if RPS19 gene transfer corrects erythroid development in unsorted cells transplanted to immunodeficient mice and if the RPS19-corrected fraction has a proliferative advantage after transplantation. We further determined if high level of RPS19 expression is required for correction. Mobilized peripheral blood CD34(+) cells were transduced by oncoretroviral vector particles pseudotyped with the feline endogenous retrovirus envelope. Vectors containing two different promoters with different RPS19 transgene expression levels were compared. Transduced cells were transplanted to immunocompromised nonobese diabetic/severe combined immunodeficient-beta2 microglobulin null mice in order to assess therapeutic effects of RPS19 gene transfer in vivo. We show that correction of erythroid development requires high RPS19 expression. The corrected fraction of unselected DBA cells have a survival advantage in vivo, suggesting that successful gene therapy may only require correction of a fraction of the patient cells. Our findings are fundamental for development of clinical gene therapy for DBA because they demonstrate increased engraftment of RPS19-transduced cells without selection of gene-corrected cells prior to transplantation, an essential prelude to studying long-term therapeutic effects in emerging animal models for DBA.

Missense mutations associated with Diamond–Blackfan anemia affect the assembly of ribosomal protein S19 into the ribosome

RPS19 has been identified as the first gene associated with Diamond-Blackfan anemia (DBA), a rare congenital hypoplastic anemia that includes variable physical malformations. It is mutated in approximately 25% of the patients although doubts remain as to whether DBA clinical phenotype depends on the ribosomal function of RPS19 or on an extra-ribosomal role or on both. RPS19 mRNAs with mutations that introduce premature stop codons or eliminate it are rapidly turned over by the surveillance mechanisms possibly causing a decrease in the RPS19 protein level. A decrease in RPS19 level has been shown to cause a defect in the maturation of 18S ribosomal RNA. Less clear is the effect of missense mutations in RPS19. With the aim of analyzing the functional features of mutated RPS19, we prepared cDNA constructs expressing RPS19 containing 11 missense mutations and a trinucleotide insertion found in DBA patients. After transfection, we analyzed the following properties of the mutated proteins: (i) protein stability, (ii) subcellular localization and (iii) assembly into ribosomes. Our results indicate that some RPS19 mutations alter the capacity of the protein to localize in nucleolar structure and these mutated RPS19 are very unstable. Moreover, none of the mutated RPS19 analyzed in this study, including those proteins that appear localized into the nucleolus, is able to be assembled into mature ribosome.

Transient red cell aplasia in siblings: a common environmental or a common hereditary factor?

During the years 1987-89, transient erythroblastopenia of childhood was diagnosed in 52 previously healthy Swedish children aged less than 4 y. Among these children there were four pairs of siblings, including one pair of identical female twins. This is a much higher familial occurrence than expected. The probability of finding 4 pairs of siblings with this disease in 50 families was estimated to be considerably less than 10–6. In the retrospectively analysed material, no environmental factor was implicated and no association with human leucocyte antigen could be proven. The twins demonstrated the disease simultaneously. Their anaemia was transient and did not recur, but showed certain features usually seen in congenital hypoplastic anaemia. The other pairs of siblings fulfilled the criteria for transient erythroblastopenia of childhood and several years elapsed between the development of the disease in siblings. Two of the fathers were reported to have had transient anaemia during their childhood. Our findings indicate that transient erythroblastopenia of childhood may involve hereditary factors, eventually demonstrating an autosomal dominant inheritance.

Genetic Variation in TERF1 but Not TERF2 Is Associated with Aplastic Anemia Risk.

Approximately one-third of individuals with acquired aplastic anemia (AA) have abnormally short leukocyte telomere lengths. Mutations in genes responsible for maintaining telomere length and stability have been identified in constitutional AA (DKC1) and acquired AA (TERC and TERT). Telomerase, a reverse transcriptase, uses an RNA template (TERC) to extend nucleotide repeats. Telomeric repeat binding factor 1 (TERF1) binds to telomeric DNA, inhibits telomerase and induces bending, looping and pairing of duplex telomeric DNA. Telomeric repeat binding factor 2 (TERF2) binds to the telomere and protects it from degradation and fusion. There are few single nucleotide polymorphisms (SNPs) in TERF1 and TERF2 and nucleotide diversity in these genes is limited. We hypothesized that mutations and/or SNPs in TERF1 and/or TERF2 may be associated with acquired AA. Bi-directional sequence analysis was performed in 47 AA patients who failed to respond to immunosuppressive therapy, had family history of hematologic abnormalities without physical characteristics of dyskeratosis congenita, or short telomeres in leukocytes, on all exons and proximal promoter regions of TERF1 and TERF2. Regions with variation in the first 47 patients were sequenced in an additional 95 patients and 289 healthy controls. SNP frequencies were determined and case-control genotype data analyzed using additive and mutation dominant (MD) genetic models and SAS v8.02. Haplotypes were estimated and a case-control permutation test performed using PHASEv2.1. HaploStats was used to construct haplotypes, determine a global score p value, haplotype frequencies and odds ratios.5244 base pairs (bp) were sequenced in TERF1 in 47 patients. SNPs were not present in exons 1–8. The proximal promoter, exon 9 and exon 10 were sequenced in the additional 95 patients and 289 controls. One patient had a mutation resulting in a conservative amino acid change (exon 9 Ala-Val). One SNP in intron 9 (C>T) was significant in the additive (p=0.049) and in the MD model, odds ratio 1.59 (p=0.033, 95% CI 1.06-2.39, 37.3% case and 31.8% control allele frequency [AF]). Two synonymous SNPs in exon 10 did not occur in patients but each had an AF of 1.4% in controls (p=0.044 for each). The MD model for these two SNPs suggested an association (p=0.044) but the low frequencies precluded additional calculations. Haplotype analysis showed statistically significant differences between cases and controls in global tests (HaploStats p=0.03 and PHASEv2.1 p=0.01). One haplotype had borderline significance in ordinal trait analysis and comparison to the most common haplotype (p= 0.057 and 0.079, respectively, 35.6 % case and 28.9% control frequency). These data suggest that genetic variation in TERF1 may be associated with aplastic anemia.Sequence analysis of TERF2 consisted of 4515 bp. The proximal promoter, exons 1–2, 4–5 and 7–10 did not have SNPs or mutations. A mutation (Ala-Ser, exon 6) in TERF2 was seen in one patient and no controls. SNPs seen in exons 3 and 6 were also sequenced in 95 patients and 289 controls. SNPs and haplotypes in TERF2 were not associated with AA. This pilot study identified a SNP and haplotype in TERF1 that may be associated with increased risk for AA. Variation at this site in TERF1 in combination with variation at other sites in other genes of the telomere complex could be risk factors for aplastic anemia. Highly penetrant mutations in TERF1 and TERF2 were not identified. These results should be confirmed in a larger study.

Sex Hormones Up-Regulate Telomerase Activity of Normal Human Hematopoietic Cells and Restore Telomerase Activity in Carriers of Telomerase Complex Mutations.

Mutations in components of the telomerase complex (TERT and TERC) are risk factors for acquired aplastic anemia. Telomerase is a reverse transcriptase responsible for the maintenance of telomeres, repetitive nucleotide sequences capping the chromosome ends. Patients with marrow failure with TERT or TERC mutations have low telomerase activity, short telomere length in leukocytes, reduced hematopoietic function, and they do not respond adequately to immunosuppressive therapy. The constitutional aplastic anemia dyskeratosis congenita is also caused by mutations in TERC (autosomal recessive type) or in an additional component of the telomerase complex, dyskerin, encoded by the DKC1 gene (X-linked type). The majority of patients with dyskeratosis congenita and some patients with acquired aplastic anemia benefit from androgen therapy, but the mechanism by which this hormone stimulates hematopoiesis is not well understood. We investigated whether sex hormones stimulated telomerase activity in hematopoietic tissue. Peripheral blood lymphocytes from either healthy controls or mutation carriers were cultured with phytohemagglutinin and interleukin-2 for 1–7 days in the presence of methyltrienolone, estradiol, hydroxycortisone, and/or tamoxifen. Telomerase enzymatic activity was measured using a fluorescent telomere-repeating amplification protocol (TRAP), and TERT mRNA expression was assayed by real-time PCR. Androgens and estradiol up-regulated telomerase activity in lymphocytes from normal subjects in a dose-dependent fashion (controls, 124±24 total product generated [TGP] units per reaction; methyltrienolone 1 μM, 242±16 TGP units/reaction; methyltrienolone 10 μM, 757±87 TGP units/reaction; P<0.01). Increased telomerase activity correlated to higher TERT mRNA levels (P<0.05). Steroidal effects on telomerase activity were neutralized by tamoxifen, an estrogen antagonist. TERT mutation carriers had lower telomerase activity compared to controls (P<0.05), but their enzymatic function was restored to normal levels when cultured with methyltrienolone. As a control, hydrocortisone at 1 μM inhibited telomerase activity without influencing cell cycle or inducing apoptosis. We also investigated the effects of androgen on hematopoietic progenitor cells. Bone marrow CD34+ cells from normal subjects were immunomagnetically separated and cultured for eight days in StemSpan supplemented with Flt-3 ligand, stem cell factor, and interleukins 3 and 6, in the presence methyltrienolone. Up-regulation of telomerase activity was observed in CD34+ cells cultured with methyltrienolone (P<0.05), the increase correlated with greater cell numbers, a higher proportion of CD34+ cells after eight days of culture (from 10% to 15% at 10 μM methyltrienolone and to 19% at 100 μM), without changes in cell cycle or cell differentiation, based on CD45, CD71, and CD33 expression. These results indicate that sex hormones stimulate TERT expression via the estrogen receptors, leading to increased telomerase activity in hematopoietic cells and increased proliferation of hematopoietic progenitors. Androgens were able to restore telomerase activity to normal levels in TERT mutation carriers. Sex hormone effects on telomerase in normal cells may have implications for the normal aging process and in hematological malignancy (leukemic cells have high telomerase activity), and our results suggest that androgens might be useful in the treatment of patients bearing telomerase complex mutations.