ALBERT

Description: Introduction Patients with Wiskott-Aldrich syndrome (WAS) including X-linked thrombocytopenia (XLT) have microthrombocytopenia, and hemorrhage is a major problem. Current management options in WAS/XLT patients include splenectomy, human stem cell transplant (HSCT) and gene therapy. In this study, we asked whether eltrombopag, a thrombopoietin mimetic, would increase platelet counts, improve platelet function, and/or reduce bleeding in WAS/XLT patients. Methods In 9 WAS/XLT patients and 8 age-matched healthy control subjects, flow cytometry was used to assess platelet function by surface expression of activated GPIIb-IIIa (reported by PAC1) and P-selectin in whole blood after stimulation with low and high concentrations of ADP or thrombin receptor activating peptide (TRAP), and by annexin V binding (a measure of surface phosphatidylserine) in platelet-rich plasma after stimulation with convulxin. Eltrombopag was administered to 5 WAS and 3 XLT patients (50 mg in 2 adults, and 1 mg/kg in 6 children up to 75 mg/day) with a goal platelet count ≥50k. Results High concentration ADP- or TRAP-induced PAC1 mean fluorescence intensity (MFI) was significantly reduced in WAS/XLT patients compared to healthy controls (Figure). Platelet surface P-selectin MFI in response to TRAP was also significantly reduced. In contrast, annexin V binding to platelets was not different between WAS/XLT and controls. As expected, platelet size of WAS/XLT patients was smaller than controls. WAS protein (which is deficient in WAS/XLT), is important for cytoskeletal movement and could therefore be involved in trafficking of surface proteins. However, surface expression of activated GPIIb-IIIa and P-selectin were no longer different in WAS/XLT patients vs. controls when corrected for size by platelet surface CD41 MFI. In 3 WAS/XLT patients whose platelet count improved on eltrombopag, platelet function did not improve. The table summarizes the results of eltrombopag treatment in 5 responders (2 WAS, 3 XLT patients) and 3 non-responders (3 WAS patients). Comparison of baseline, peak and change in immature platelet fraction in 5 WAS/XLT responders to eltrombopag vs. 7 pediatric chronic immune thrombocytopenia (ITP) patients responding to eltrombopag showed a significant decrease in all three measures, suggesting that platelet production in WAS/XLT patients is more difficult to increase than in ITP patients. Long term eltrombopag use in WAS/XLT patients showed no tachyphylaxis, transaminitis or induction of malignancy. Conclusions 1) Baseline platelet function in WAS/XLT is reduced compared to healthy age-matched controls, as measured by agonist-induced platelet surface activated GPIIb-IIIa and P-selectin. 2) This reduction is proportional to the reduced platelet size in WAS/XLT compared to controls. 3) In contrast, annexin V binding (a measure of platelet procoagulant activity) showed no differences between WAS/XLT and controls. 4) Eltrombopag has beneficial effects on the thrombocytopenia and bleeding, but not platelet function, in the majority of WAS/XLT patients. 5) This eltrombopag-induced reduction in bleeding is presumably primarily the result of the increased platelet count, but it was also observed in 2 eltrombopag “non-responders” (i.e. patients whose platelet counts did not increase after eltrombopag). 6) The production of new platelets with eltrombopag is less in WAS/XLT than in ITP. Disclosures: Off Label Use: Eltrombopag was given to WAS/XLT patients for treatment of thrombocytopenia. Michelson:Sysmex: Honoraria. Bussel:GlaxoSmithKline: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Amgen: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Cangene: Research Funding; Genzyme: Research Funding; IgG of America: Research Funding; Immunomedics: Research Funding; Ligand: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Eisai: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Shionogi: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Sysmex: Research Funding; Symphogen: Membership on an entity’s Board of Directors or advisory committees.

Description: The αIIbβ3 integrin receptor mediates platelet aggregation, and individuals without functional αIIbβ3 manifest the mucocutaneous bleeding disorder Glanzmann thrombasthenia. Since platelets also participate in the pathophysiology of thrombosis, anti-αIIbβ3 agents have been successful in reducing mortality after percutaneous coronary interventions and from platelet-mediated coronary thrombosis. Accordingly, much research has focused on the structure, activation and signaling of the mature receptor on the cell surface. There is much to be gained medically and scientifically by understanding the intracellular processes by which integrins are assembled and expressed. Some forms of Glanzmann thrombasthenia result from mutations that disrupt normal integrin folding and assembly, causing the integrin subunits to be retained in the ER and degraded. These cases indicate the existence of a quality control mechanism for αIIbβ3 by which misfolded subunits are recognized, retained and degraded. Previous studies in HEK293 cells and murine megakaryocytes have identified the calnexin cycle as part of this quality control mechanism. We have expanded upon these findings and studied their functional implications by determining the effect of siRNA-induced knock down of proteins from six families of post-translational processing proteins on αIIbβ3 surface expression on human megakaryocyte-lineage cells derived from umbilical cord blood (UCB): lectins, glucosyltransferases, mannosidases, HSP90s, HSP70s, BiP, protein disulfide isomerases, and tetraspanins. We report that siRNA against two proteins in the calnexin cycle decreased surface expression of natively-folded αIIbβ3 on UCB-derived megakaryocytes as measured by binding of 10E5, an αIIbβ3 complex-dependent mAb. siRNA against UDP-glucose ceramide glucosyltransferase-like 1(UGGT, a glucosyltransferase) decreased binding by 36 +/− 9%, and siRNA against EDEM1 (a mannosidase) decreased binding by 15 +/− 14%, both p 〈 0.05. Additionally, siRNA against CD9 increased binding by 17 +/− 8%, p 〈 0.05, suggesting that CD9 may be a negative regulator of αIIbβ3 surface expression. UGGT is a glucosyltransferase protein and folding sensor of the calnexin cycle that recognizes and binds to glycoproteins that are in nearly-native folding conformations, but does not bind to fully folded glycoproteins. Glycoproteins that are glucosylated by UGGT may be bound by calnexin and retained in the ER, increasing their time for folding. Fully folded proteins cannot be reglucosylated by UGGT and escape the calnexin cycle, exiting the ER. EDEM1 is a mannosidase that interacts with calnexin to accept terminally misfolded proteins and direct them to degradation. Together, these data suggest a model in which the level of expression of native-folded αIIbβ3 on the cell surface is regulated in the ER by the calnexin cycle.

Description: Introduction Maximizing proplatelet formation from cultured megakaryocytes will be a critical step in ex-vivo production of platelets for transfusion. However, mechanisms triggering proplatelet formation are not completely identified. Apoptosis factors are suggested to play an important role in initiating the process of platelet release. However, their role is controversial. Megakaryocytes might employ the apoptotic machinery to facilitate platelet production through the intrinsic pathway and caspase 3/9 activation, and pro-survival factors, such as Bcl-xL, are necessary to enable megakaryocyte survival during proplatelet formation. However, removing the pro-apoptotic intrinsic pathway factors Bak and Bax did not limit proplatelet formation. To shed some light on this controversy we performed experiments in which we increased megakaryocyte proplatelet formation by inhibiting two vital cytoskeletal proteins, actin and myosin-II. We found that such an inhibition of actin or myosin-II late in megakaryocytopoiesis correlated with activation of the apoptosis pathways. We analyzed the effect of actin or myosin-II inhibition on the extrinsic and intrinsic apoptosis pathways and whether the increase in proplatelet formation due to actin or myosin inhibition was dependent on apoptosis. Methods Human cord blood derived CD34+ cells were isolated and cultured with thrombopoietin (TPO) and stem cells factor (SCF) for 11 days. Megakaryocytes were isolated on day 8 and replated in culture medium containing only TPO. The day 8 megakaryocytes were treated with an actin inhibitor (AI, Latrunculin-A) 10µM or myosin-II inhibitor (MI, Blebbistatin) 20µM. On day 11 of culture, cell ploidy was analyzed by flow cytometry using propidium Iodide. Proplatelets were counted using inverted light microscopy and their structure characterized by fluorescence microscopy using a β-1 tubulin-specific antibody. Western blots were performed for Bcl-xL, Bak, and Bax. Mitochondrial outer membrane permeabilization (MOMP) and phosphatidylserine (PS) externalization (Annexin-V binding) were measured using flow cytometry. Caspases 3 and 7 were analyzed using a luminescence assay (CaspaseGlo3/7). In some experiments apoptosis was inhibited on day 8 with a pan-caspase inhibitor ZVAD-fmk (20µM). All results were compared to untreated control megakaryocytes. Results Treatment with either actin or myosin-II inhibitor increased the level of polyploidization in megakaryocytes (both p=0.03). There was no additive effect in final ploidy with combination of reagents. Proplatelet extension by megakaryocytes treated with AI or MI was higher than untreated control cells (p=0.02, p=0.05) and proplatelet structure was normal as assessed by fluorescence microscopy. Bcl-xL, Bak and Bax protein levels were not different from controls after AI or MI treatment. MOMP and PS externalization were increased in both AI (p=0.04) and MI (p=0.04) treated megakaryocytes compared to untreated controls. Caspase 3/7 activities were also increased in treated megakaryocytes. The addition of a pan-caspase inhibitor prevented the release of proplatelets in both AI and MI treated megakaryocytes. Discussion Inhibition of actin or myosin-II led to increased levels of polyploidization and proplatelet formation in cultured megakaryocytes. This increased proplatelet formation was simultaneous with increased final apoptosis activation. Apoptosis analysis showed no difference in protein levels of the intrinsic pathway pro-apoptotic (Bak and Bax) or pro-survival (Bcl-xL) factors. However, both flow cytometry and caspase 3/7 assays showed that the final apoptosis process was activated in cells treated with AI or MI. Thus the apoptosis activation was mediated through a pathway independent of the intrinsic pathway factors Bcl-xL, Bak and Bax. Proplatelet formation in response to AI or MI was inhibited by the pan-caspase inhibitor, suggesting that the effects of AI and MI on proplatelet formation are caspase dependent. These results suggest that inhibition of actin or myosin-II late in megakaryocytopoiesis increases proplatelet formation through activation of apoptosis. Our findings suggest that proplatelet formation is dependent on the activation of apoptosis but may not be mediated through the intrinsic pathway. Determining the pathway leading to final apoptosis and proplatelet formation deserves further investigation. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2111 INTRODUCTION: The integrin αIIbβ3 mediates platelet aggregation by binding to fibrinogen and other plasma proteins. Inherited defects of αIIbβ3 result in the mucocutaneous bleeding disorder, Glanzmann thrombasthenia (GT). However, pharmacological inhibition of αIIbβ3 can reduce mortality after myocardial infarction and percutaneous stent placement. Thus the degree to which αIIbβ3 functions is critical. One element influencing the degree of αIIbβ3 function is its level of surface expression on the platelet. The post-translational mechanisms controlling surface expression remain unclear, but their stringency is evident in certain GT patients whose full length αIIbβ3 complexes are completely retained and degraded intracellularly. A clearer understanding of the post-translational processes controlling αIIbβ3 surface expression can provide insight into a pathway that is a potential target for novel αIIbβ3-targeted therapies. Toward that end we previously reported the interaction of αIIb with DNAJC10, an hsp40 type chaperone protein, which was identified by a proteomic analysis of αIIb-interacting proteins. Continuing that investigation, we have explored the function of DNAJC10 in αIIbβ3 biogenesis pathway. METHODS: Megakaryocytes were derived from human umbilical cord blood (UCB) by enrichment of UCB leukocytes for CD34+ progenitor cells followed by culture in serum-free medium with thrombopoietin and stem cell factor. Under our conditions the UCB leukocytes differentiate into a population of large cells of which 〉 90% express αIIbβ3, 〉 80% express GPIb, and about 50% express α2β1. Interaction of αIIb and β3 with DNAJC10 was tested by co-immunoprecipitation with mAbs specific for αIIb (CA3, M148, B1B5), b3 (7H2), or the αIIbβ3 complex (10E5). Megakaryocytes were treated with a proteasome inhibitor (MG-132) to increase the capture of αIIb (and potential interacting proteins) that had been routed to the proteasome for degradation. DNAJC10, αIIb, and β3 localization within megakaryocytes and platelets was observed with immunofluorescence, and compared to localization of the endoplasmic reticulum (ER) and Golgi. The effect of siRNA-mediated knockdown of DNAJC10 on αIIbβ3 expression was assessed by flow cytometry and immunofluorescence. RNA depletion was confirmed by quantitative RT-PCR. RESULTS: DNAJC10 co-immunoprecipitated with αIIb-specific (CA3, M148, B1B5), β3-specific (7H2), and complex-specific (10E5) mAbs, indicating that it associated with αIIbβ3 in megakaryocytes. An antibody (B1B5) that recognized pro-αIIb and pro-αIIbβ3 co-immunoprecipitated much more DNAJC10 than antibodies preferentially recognizing mature αIIb and αIIbβ3. The amount of DNAJC10 immunoprecipitated was markedly increased after cells were treated with a proteasome inhibitor. siRNA against DNAJC10 resulted in significantly elevation of αIIbβ3 surface expression on megakaryocytes as judged by flow cytometry. Immunofluorescence with confocal microscopy surprisingly revealed that DNAJC10, nominally an endoplasmic reticulum protein, did not colocalize with ER markers, but was distributed in foci throughout the megakaryocytes, proplatelet processes, platelet buds, and mature peripheral blood platelets. CONCLUSIONS: DNAJC10 engages the precursor forms of αIIb and αIIbβ3 at a point upstream of degradation and may be an intermediate step in targeting these to the proteasome. Since siRNA mediated knockdown of DNAJC10 resulted in increased αIIbβ3 surface expression, this chaperone may be a negative regulator of αIIbβ3 surface expression. Disclosures: No relevant conflicts of interest to declare.

Description: Red blood cell alloimmunization can be a life-threatening complication for patients with sickle cell disease (SCD) receiving therapeutic transfusions. However, it remains unknown why only some (20-60%) SCD patients develop alloantibodies whereas others do not. Because of ongoing hemolysis in SCD, we have investigated the effects of toxic hemin on T cell responses of patients with SCD on chronic transfusion therapy. We found impaired monocyte control of proinflammatory T cells in response to hemin in alloimmunized SCD patients, partly due to defective levels of monocyte heme-oxygenase-1 (HO-1), an anti-inflammatory heme degrading enzyme that catabolizes heme into iron, biliverdin and carbon monoxide (CO). Monocytes are precursors of dendritic cells (DCs), which represent the antigen presenting cells most able to initiate and regulate immune responses. However, little is known about the functional properties of DCs in SCD patients. We therefore hypothesized that DCs of alloimmunized SCD patients would also have impaired response to hemin. Monocyte-derived DCs (moDCs) from healthy donor controls (n=11) and a cohort of SCD patients (aged 15-30 on chronic transfusion therapy every 3-4 weeks for at least two years using C,E,K phenotyped-matched, leukodepleted units) grouped either as "non-alloimmunized" (no history of antibody production, n=6), versus "alloimmunized" (with a history of having produced at least one alloantibody, n=7) were matured in the absence or presence of hemin (5uM and 20uM) with TLR agonists: LPS/IFNg (TLR4 agonist+STAT1 activation) or R848 (TLR7/8 agonist). Hemin-exposed mature and immature (no TLR agonist) moDCs were then assayed for HO-1 expression, cytokine production, co-stimulation, and T cell priming. Interestingly, upon hemin exposure, immature moDCs from healthy donors and non-alloimmunized patients upregulated more HO-1 (1056±206 fold; mean fluorescent intensity (MFI): 12283±1818 at 20uM hemin) than alloimmunized patients (fold increase 494±49; MFI: 7422±959, p

Description: Background Immune Thrombocytopenia (ITP) is a bleeding disorder due to a combination of increased platelet destruction and reduced production, often secondary to anti-platelet/megakaryocyte antibodies. The presence of antibodies to glycoproteins (GP) IIb/IIIa (integrin αIIbβ3) and GPIb/IX, detected in majority of ITP patients, may correspond to different responses to treatment, i.e., anti-GPIb is associated with more severe disease, and less responsive to intravenous immunoglobulins and steroids. Thrombopoietin Receptor Agonists (TPO-RA) increase platelet production by stimulation of megakaryopoesis. Predictors of response to TPO-RA and influence of antibody profile on response are currently unknown. In our previous study we investigated Absolute Immature Platelet Fraction (A-IPF) prior to TPO-RA treatment and did not find a correlation between A-IPF, anti-GP antibodies, and platelet counts. The aims of this study were to further investigate: 1. The role of anti-GP antibodies in response to TPO-RA; 2. Effect of patients' antibodies on megakaryocyte (MK) viability, maturation, apoptosis and formation of proplatelets (in vitro); 3. The influence of patients' clinical characteristics on response to TPO-RA. Materials and Methods 91 patients with persistent or chronic ITP, were treated at Weill Medical College of Cornell University until January 2015 with TPO-RAs: 52 patients received eltrombopag, 22 romiplostim and 17 avatrombopag. Serum samples of 84 patients were analyzed for the presence of anti-GP by MAIPA assay as previously described. Patients with baseline platelet counts less than

Description: Background A pathogenic mechanism of Immune Thrombocytopenia (ITP) is insufficient platelet production by bone marrow megakaryocytes (MKs). MKs release platelets through a multistep process consisting of differentiation of MK progenitors, MK maturation, and formation of proplatelets, which in turn give rise to platelets. Many of these processes are primarily driven by thrombopoietin (TPO). However, the mechanism of proplatelet formation is not completely understood. It is controversial whether activation of apoptosis contributes to formation of proplatelets. Thrombopoiesis is generally increased in patients receiving TPO-receptor agonists (TPO-RA), therefore we hypothesized that MKs in these patients might show activation of apoptosis. This would support a role for apoptosis in thrombopoiesis in patients. To test this hypothesis, we investigated activation of apoptosis in MKs isolated from ITP patients treated with TPO-RA, and in umbilical cord blood (UCB) derived MKs cultured in the presence of TPO-RA. Materials and methods Bone marrow aspirates of 16 chronic ITP patients (8 male and 8 female), median age 45.2 years old, were analyzed. Responders had at least 2 out of 3 recent platelet counts 〉503/µl. Samples were analyzed by flow cytometry (FACS) for Mitochondrial Outer Membrane Potential (MOMP) and PhosphatidylSerine (PS) in the CD41+ (MK) population. MKs positive for both MOMP and PS were considered apoptotic. UCB CD34+ cells were cultured with Stemspan SFEM medium, 5 ng/ml Stem Cell Factor, and 50 ng/ml TPO (control). MKs were selected for assays on day 12 of culture by CD61 magnetic microbeads. Some cells were cultured with an additional 100 ng/ml of the TPO-RA Romiplostim (Romiplostim-treated) to mimic the presence of both endogenous TPO and TPO-RA in the human bone marrow. A third group of cells was cultured with 100 ng/ml TPO (2xTPO). Proplatelets were counted using inverted light microscopy on day 12 of culture in 10 random-field images. MOMP and PS externalization were measured by FACS. Caspase 3 and 7 activation, other markers of apoptosis, was measured by a Caspase 3/7 luminescence assay. Results There was no significant difference in age, gender and splenectomy status between responders (N=8) and non-responders (N=8). The percentage of MKs was similar in all samples. FACS analysis revealed a significantly higher percentage of MOMP + PS positive apoptotic MKs in responders (mean 37.8±12.56) than in non-responders (17.7±5.53), p=0.02 (Figure 1). FACS analysis revealed a higher percentage of apoptotic cells in Romiplostim cultured UCB MKs than in control MKs (mean 57% versus 44%, p=0.03), and 2xTPO MKs (40%, p=0.02). No difference was found between 2xTPO and control cells (p=0.6) Patient MKs did not grow in culture but MKs derived from UCB CD34+ cells cultured with Romiplostim were compared to control TPO MKs and 2xTPO MKs. While there was no difference in number of MKs per field between the 3 culture conditions; the number of proplatelets per MK was significantly higher in Romiplostim cultured vs. control (TPO) MKs (p

Description: Most studies of αIIbβ3 biogenesis have been conducted in transfected HEK293 and CHO cells or megakaryocyte-like cell lines, but the protein processing mechanisms in native megakaryocytes may differ. To address this issue we have studied αIIbβ3 biogenesis in human megakaryocyte-like cells derived from umbilical cord blood (UCB) and in 293 cells stably expressing αIIbβ3, and developed a mathematical model to express the kinetics. The leukocyte pool of whole UCB units was separated by Dextran and Ficoll sedimentation and enriched for CD34+ progenitor cells by negative selection using a commercial antibody panel. These cells were then cultured in the presence of 20 ng/ml thrombopoietin. By day 10 of culture approximately 90% of cells expressed αIIbβ3, 80% expressed GPIb, 45% expressed α2β1 and 55% expressed P-selectin. The cells adhered to fibrinogen and collagen, spread in a manner similar to platelets, and formed focal adhesions as judged by vinculin and phalloidin (F-actin) staining. While αIIbβ3 on the surface of 293 cells cannot undergo inside-out activation, 18% of the UCB-derived cells exhibited increased binding of PAC-1, an activation-specific monoclonal antibody, in response to TRAP, and the majority bound PAC-1 after adhesion to collagen. The ploidy of the UCB-derived cells ranged from 2 to 128. The dynamics of αIIbβ3 biosynthesis was analyzed by pulse-chase analysis with 35S-Cys/Met followed by immunoprecipitation, SDS-PAGE, and densitometry of exposed x-ray film. Using non-linear regression, curves were fitted to the observed data and a 3-compartment mathematical model (pro-αIIb, mature αIIb in complex with β3, and degraded αIIb) was used to describe the αIIb dynamics. There were important similarities and differences between the two types of cells. In both cell types, pro-αIIb decreased over time in a pattern best described by a simple exponential function, P = (a)exp(-bt), where P is the amount of pro-αIIb, a is the initial amount of pro-αIIb, and b is a rate constant. The half-lives (ln2/b) of pro-αIIb in the UCB-derived cells and the 293 cells were similar (120 and 140 min, respectively). In both cell types, mature αIIb increased over time with a pattern best described by a sigmoidal function, M = c/(1+exp(−(x−x0)/d), where M is the measured amount of mature αIIb, c is the asymptotic value of αIIb, d is the rate constant, and x0 is the time to half maximum mature αIIb. In the megakaryocyte-like cells, the time to half maximum (x0) was 120 min, while in the 293 cells it was only 55 min. The amount of pro-αIIb that has been degraded at any time point (D) can be calculated by subtracting the amounts of pro-αIIb and mature αIIb at that time point from the initial amount of pro-αIIb (D = a-P-M). In the UCB-derived cells ~ 25% of pro-αIIb is degraded without being processed to mature αIIb, while in 293 cells ~ 40% is degraded. In both cell types there is a ~90 minute lag time before the onset of net αIIb degradation. These findings suggest that while the ability to degrade αIIb may be similar between megakaryocytes and 293 cells, the folding, complex formation, and quality control mechanisms are quite different. These observations have implications for the study of integrin dynamics and may be useful in analysis of the molecular mechanisms of integrin biogenesis.

Description: Abstract 2154 Congenital dyserythropoietic anemias (CDA) are a rare, inherited form of blood disorders characterized by dyserythropoiesis in the bone marrow, anemia, jaundice and splenomegaly. There are three major types of CDAs, although rarer variants have been identified. We describe a patient with an unusual type of CDA, of which only four other cases have been reported. Our patient had severe hemolytic anemia, increased fetal hemoglobin and abnormal bone marrow pathology inconsistent with previously described forms of CDA. On further study he was also found to have a mutation in KLF1, the gene encoding Erythroid-Kruppel like growth factor (EKLF). Here we describe the full clinical characteristics of our patient and define the diagnostic clinical features of this new variant of CDA by comparing with one of the previously reported patients designated as ME in the table. EKLF is an erythroid specific transcription factor that is essential for b-globin expression, the switch from fetal to adult globin and definitive erythropoiesis (Siatecka, M and Bieker, J. Blood prepublished May 2011). Various mutations in KLF1 have been identified, some causing the benign In(Lu) type of Lu blood group phenotype. Recently, a missense, dominant-negative KLF1 mutation was reported, c.973G〉A, which resulted in a previously unidentified type of CDA (Singleton et al. ASH Abstract 162, 2009; Arnaud et al. Am J Hum Genet 2010,87:721-727). The G-to-A transition in exon 3 of KLF1 results in the substitution of glutamate 325 by a lysine (E325K) in the second zinc finger. The mutated area of the zinc finger was found to be essential for binding of EKLF to DNA motifs causing a profound dysregulation of globin gene expression. The mutation was found to have a dominant-negative effect on the transcriptional activity of EKLF, thus making the heterozygous patients symptomatic. Our patient, JL, is an 8 year old male Taiwanese immigrant found to have hyperbilirubinemia and anemia at birth. He is a developmentally normal child with height 10th centile, weight in the 25th centile and spleen palpable to his suprapubic area. He has chronic hemolytic anemia, with baseline hemoglobin 7–9 g/dL, MCV 83 fL and RDW 22% and reticulocyte count 16%. Peripheral blood smear shows marked anisopoikilocytosis, schistocytes, mild polychromasia and nucleated RBCs, many with double nuclei. Bone marrow aspirate revealed a hypercellular marrow with erythroid hyperplasia and dyserythropoiesis. Electron microscopy analysis of the bone marrow showed rare immature erythroid cells with marked heterochromatin. Several cells showed a peripheral double membrane of the cytoplasm and there was rare invagination of nuclear membrane with intranuclear precipitated material. JL received blood transfusions every 2 months for the first 3 years of his life while living in Taiwan. Since moving to the U.S. in 2003, he has only received 2 transfusions secondary to aplastic crisis. Osmotic fragility testing showed mildly increased increased fragility. Hemoglobin electrophoresis revealed an elevated fetal hemoglobin level of 42%. Gene analysis for alpha or beta globin mutations was negative. RBC enzyme testing revealed an ADA of 6.1 and decreased FFK. Due to the unique combination of anemia, elevated fetal hemoglobin, and bone marrow morphology suggestive of, but not fully diagnostic for CDA I, II or III, we tested his EKLF gene and identified the heterozygous E325K mutation. JL's two siblings, mother and father have normal hemoglobin levels and peripheral blood smears. They also have normal In(Lu) blood group phenotype. Since the E325K mutation is dominant-negative, his phenotypically normal family members were not tested for the mutation. Four other patients have also been identified as having an E325K mutation in their EKLF gene. The patients had severe hemolytic anemia, elevated fetal hemoglobin, and bone marrow morphology showing dyserythropoiesis. Patients' erythrocytes also had low CD44 and water channel AQP1 expression, which is known to be regulated by EKLF. One patient was described with multiple congenital anomalies: hepatomegaly, micropenis, hypospadias, enlarged fontanel and hypertelorism. Taken together, the key clinical characteristics of this rare CDA are: severe normocytic anemia, highly elevated Hb F, presence of nucleated RBCs in the peripheral blood, erythroid hyperplasia with limited dyserythropoiesis in the bone marrow, splenomegaly, and growth delay. Disclosures: No relevant conflicts of interest to declare.