ALBERT

Description: The Ldb1/GATA-1/TAL1/LMO2 complex mediates long-range interaction between the β-globin locus control region (LCR) and gene in adult mouse erythroid cells, but whether this complex mediates chromatin interactions at other developmental stages or in human cells is unknown. We investigated NLI (Ldb1 homolog) complex occupancy and chromatin conformation of the β-globin locus in human erythroid cells. In addition to the LCR, we found robust NLI complex occupancy at a site downstream of the Aγ-globin gene within sequences of BGL3, an intergenic RNA transcript. In cells primarily transcribing β-globin, BGL3 is not transcribed and BGL3 sequences are occupied by NLI core complex members, together with corepressor ETO2 and by γ-globin repressor BCL11A. The LCR and β-globin gene establish proximity in these cells. In contrast, when γ-globin transcription is reactivated in these cells, ETO2 participation in the NLI complex at BGL3 is diminished, as is BCL11A occupancy, and both BGL3 and γ-globin are transcribed. In these cells, proximity between the BGL3/γ-globin region and the LCR is established. We conclude that alternative NLI complexes mediate γ-globin transcription or silencing through long-range LCR interactions involving an intergenic site of noncoding RNA transcription and that ETO2 is critical to this process.

Description: All sickle cell anemia patients (HbSS, SCA) have the same genetic mutation, but the clinical phenotype is highly variable and difficult to predict prior to the onset of complications. Transcranial Doppler (TCD) is the only validated predictive indicator of severe SCA currently available, identifying SCA patients age 2 years or older with the greatest stroke risk. Preliminary studies have identified that elevated absolute reticulocyte count (ARC) between 2 and 6 months of age is associated with an increased risk of hospitalization for SCA complications, stroke and death.1,2 To determine if early reticulocyte levels in SCA patients are useful in classifying children at highest risk of developing an abnormal (abTCD) or conditional (cdTCD) TCD, 121 consecutive patients with SCA who had TCD screening were identified after IRB review granted a waiver of consent. Retrospective chart review was then performed to collect the steady state ARC between 2 and 6 months of age; patients were excluded if this value was not available in the medical record. Steady state was defined as a sample collected at least 30 days from an acute illness and at least 60 days since the patient received a blood transfusion. ARC and hematologic data were analyzed using Cox regression analysis to determine the relationship between ARC levels and time to cdTCD/abTCD. TCDs were considered normal (nlTCD) if the Time Average Mean Maximum Velocity (TAMMV) in the middle cerebral artery or distal internal carotid artery was

Description: Abstract 2666 Acute erythroid stress causes elevated levels of fetal hemoglobin (HbF) in primates. However, relationships between the chronic erythroid stress in children with Sickle Cell Disease (HbSS) and HbF production are less well understood. For this study, peripheral blood from children with HbSS was analyzed within 72 hours of collection and storage at 4°C. The level of erythroid stress was determined by absolute reticulocyte counts (ARC) as well as reticulocyte flow cytometry phenotyping and sorting using CD71 (HiCD71, LoCD71 and NegCD71) and CD36. Immature reticulocytes were identified by HiCD71 expression and included the CD36+ population. Sorted populations from at least three subjects were utilized to assess fetal globin expression during reticulocyte maturation according to quantitative polymerase chain reaction (qPCR), and high pressure liquid chromatography (HPLC). While the number of gamma-globin mRNA copies/cell decreased with reticulocyte maturation, the ratio of gamma/gamma+beta mRNA remained stable. HbF protein levels in the HiCD71 and LoCD71 populations were equivalent for each patient with average values of 4.3±4.2% HiCD71 vs. 4.4±4.0% LoCD71, p=0.96. By comparison, HbF levels in the NegCD71 were increased in all subjects (14.2±9.5% NegCD71, p

Description: Abstract 2076 Persistent expression of HbF ameliorates the clinical symptoms of β-thalassemia and sickle cell disease; therefore, reactivation of the γ-globin gene in adults is of substantial interest for the clinical management of β-hemoglobinopathies. The recently identified γ-globin repressor, BCL11A, has been shown to regulate HbF expression level in adult erythroblasts. Cytokine signal transduction also increases HbF expression in cultured erythroblasts due in part to reducing the chromatin occupancy of BCL11A within the β-globin locus. Cultured CD34+ cells from at least six healthy research subjects were utilized to investigate the potential for combined effects of genetic manipulation of BCL11A with signal transduction upon globin gene regulation. The cells were transduced with BCL11A shRNA lentiviral vectors under culture conditions with and without addition of HbF-inducing cytokines. Under all culture conditions, nuclear BCL11A protein levels were reduced to nearly undetectable levels by the shRNA lentiviral vectors. The combination effects of BCL11A knockdown and HbF-inducing cytokines were quantitated by QRT-PCR. The absolute copy numbers of the eight globin genes (β-like genes; ε-, γ-, δ-, β-globins and α-like genes; ζ-, μ-, α- and θ-globins) at the proerythroblast stage of maturation were determined using standard curve methods. As previously reported, cytokine-mediated induction of HbF significantly increased the expression of γ-, ε-, and ζ-globin mRNAs while β- and δ-globins were down-regulated (p-value

Description: Introduction Benign neutropenia (BN) is an asymptomatic condition observed in individuals of African descent. Patients with BN tend to have normal myeloid maturation within the bone marrow but release fewer neutrophils into the peripheral circulation. Previous studies of BN in pediatrics have reported no evidence of increased infections but otherwise lacked a detailed clinical description. The purpose of this study is to identify individuals with BN and better characterize the clinical findings seen in patients with this condition. Methods A single institution retrospective study from 2008-2013 was conducted at an urban tertiary care children’s hospital in Washington, DC. Potential patients were initially identified by searching for ICD9 codes 288.0 (neutropenia) and 288.09 (neutropenia, not otherwise specified). Absolute neutrophil count (ANC) less than1500 was required for inclusion. Exclusion criteria included other hematologic disorder, pregnancy, low B12 or folate levels, prior cancer diagnosis, or concurrent immunosuppressant therapy. Demographic, laboratory, and clinical information on all included patients was recorded. All outpatient visits with a hematologist with a documented ANC were evaluated and recorded as encounters. Results Searching by ICD9 codes yielded 136 potential patients. 99 were excluded: 43 had antibody positive autoimmune neutropenia, 26 had transient or self-resolved neutropenia, 22 had concurrent malignancy, while 4 were receiving other immunosuppressant medications, and 4 had other known causes of neutropenia. The remaining 37 patients were included as BN, and were seen in a total of 130 encounters in the outpatient hematology clinic. Median age at evaluation was 5.3 years; 19 were males and 18 females. 22 (59.5%) patients were of African descent, 4 (10.8%) Caucasian, 3 (8.1%) Hispanic, 2 (5.4%) Asian, and 6 (16.2%) ethnicity was not documented. A summary of patient reported symptoms is in Table 1. Table 1. Key clinical findings from BN individuals Finding Patients % Reporting Upper respiratory infection 8 21.6 Rash 6 16.2 Poor weight gain or weight loss 4 10.8 Headache 3 8.1 Oral ulcers 3 8.1 Bone or joint pain 2 5.4 Cervical lymphadenopathy 2 5.4 Gingivitis 1 2.7 Dental problems 1 2.7 Night sweats or chills 1 2.7 Delayed wound healing and dyspnea were not reported during by any patients. There were 2 patients with drug allergies and 1 with a food allergy. No first degree relatives of any of the patients were noted to have a history of neutropenia. For all patients in the study there were 5 total hospitalizations: 2 for febrile neutropenia, 1 each for mastoiditis, weight loss and neutropenia, and pneumonia. None of these hospitalizations required ICU management. 5 patients had bone marrow biopsies, 3 of which were normal and the others had focal/mild hypocellularity. In terms of laboratory findings, BN individuals had a median ANC of 893 x 10^6 cells per L. Additional laboratory statistical information can be seen in Table 2. Table 2. Key laboratory results from BN individuals Result Units Minimum 25th Quartile Median 75th Quartile Maximum White blood cell cells*109/L 2.3 3.4 4.8 6.1 10.4 ANC cells*106/L 158 628 893 1255 3614 Neutrophils % 7 13 17.3 28.7 50.7 Lymphocytes % 37.4 53.7 67.4 70.7 81.1 Monocytes % 2 8.2 10.1 11.8 18 Eosinophils % 0 1.6 2.7 4 12.4 Hemoglobin gm/dL 9.4 11.3 11.8 12.7 14.1 Hematocrit % 26.4 32.7 35 36.8 42.4 Platelets cells*109/L 170 220 265 316 472 Immunoglobulin G mg/dL 445 672 1049 1351 1851 Immunoglobulin A mg/dL 29 50 106 152 203 Immunoglobulin M mg/dL 22 64 86 112 156 Discussion Compared to reported ANC values from patients with congenital neutropenias, BN patients in our sample rarely had ANCs 〈 500 and were hospitalized infrequently. The gender and racial characteristics of our study sample were atypical. BN has been previously reported to have a male predilection. A quarter of patients self-reported Caucasian, Hispanic, or Asian heritage – three backgrounds to which this clinical entity is quite atypical. Conclusion BN is often considered as a normal variant that does not confer increased risk of morbidity or mortality. Larger, prospective studies are needed to further elucidate the clinical course of this condition as well as to better understand molecular genetics. Disclosures No relevant conflicts of interest to declare.

Description: Background: As patient-centered care advances, measurement of health-related quality of life (HRQL) has become increasingly important when assessing the impact of a disease or therapy on a child. Red blood cell transfusions are an effective preventative therapy for some acute and chronic complications in children with sickle cell disease (SCD). This study aimed to determine the impact of transfusion therapy on health-related quality of life (HRQL) outcomes of children with SCD. Procedure: Children (n = 196) who participated in the Multicenter Silent Infarct Transfusion (SIT) Trial were grouped per protocol into either those who received 18 months or more of transfusion or less than 18 months of transfusion (observation). Parents/guardians of children ages 5 to 18 years completed assessments of HRQL using the Child Health Questionnaire at baseline and at the time of study exit or neurological event, if that occurred. Results: Children (43% female) had a mean age of 9.55 years (SD= 2.59) at study enrollment, and 92% were Black. There were no differences between study groups (effectively transfused vs. observation) in regards to gender, disease severity, rates of pain and acute chest syndrome, or baseline levels of HRQL. At study exit, independent samples t-tests revealed children in the effectively transfused group had significantly higher scores than the observation group for the following HRQL domains: Physical Function (M = 12.68, SE = 3.52), t (174) = 3.61, p ≤ 0.001; Bodily Pain (M = 13.16, SE = 3.74), t (174) = 3.51, p ≤ 0.001; and Change in Health (M = 0.39, SE = 0.14), t(166) = 2.71, p=0.01. Additionally, children in the effectively transfused group scored 4.98 (SE = 1.98) points higher on Physical Summary Scores than children in the observation group, t (170) = 2.52, p= 0.01. Thus, parents report that children who received at least 18 months of transfusions had better overall physical functioning, less bodily pain, and more improved overall health than children who had fewer than 18 months of transfusions. Both groups reported changes in HRQL over time. Compared to study entry, paired samples t-tests revealed children in the observation group indicated an increase of 0.42 (SE = 0.14) points for Change in Health scores [t (77) = 3.06, p ≤ 0.001], but had a decrease of 5.95 (SE = 2.07) points for Self-Esteem at study exit, t (85) = -2.87, p = 0.01. Children in the transfusion group improved by 7.22 (SE = 3.11) points in regards to pain over the course of the study, t (78) = 2.32, p = 0.02. Additionally, these children had better overall health as exhibited by their Change in Health scores (MD = 0.93, SE = 0.14), t (72) = 6.80, p = ≤ 0.001, and General Health scores (MD = 4.13, SE = 1.83), t (77) = 2.26, p = 0.03. Further improvements over time were noted for the effectively transfused group for Physical Functioning (MD = 6.58, SE = 3.09), t (78) = 2.13, p = 0.04, and Physical Summary Scores(MD = 4.89, SE = 1.82), t (73) = 2.69, p= 0.01. Although both study groups reported improvements in Change in Health scores over the course of the study (Effectively Transfused: MD = 0.93, SE = 0.14; Observation: MD = 0.42, SE = 0.14), an estimated Least Square Means analysis revealed children in the observation group did not improve as much as children in the effectively transfused group, Difference Estimate = -0.46, p= 0.02. Conclusions: This study provides the first evidence that blood transfusion improves HRQL in children with SCD. Children in the SIT trial who received at least 18 months of chronic blood transfusion therapy felt better and had better overall HRQL than those who had less than 18 months of transfusion therapy. Disclosures Casella: Mast Therapeutics, previously Adventrix, ImmunoArray: Consultancy, Honoraria, Other, Patents & Royalties, Research Funding.

Description: Therapeutic regulation of globin genes is a primary goal of translational research aimed toward hemoglobinopathies. Signal transduction was used to identify chromatin modifications and transcription factor expression patterns that are associated with globin gene regulation. Histone modification and transcriptome profiling were performed using adult primary CD34+ cells cultured with cytokine combinations that produced low versus high levels of gamma-globin mRNA and fetal hemoglobin (HbF). Embryonic, fetal, and adult globin transcript and protein expression patterns were determined for comparison. Chromatin immunoprecipitation assays revealed RNA polymerase II occupancy and histone tail modifications consistent with transcriptional activation only in the high-HbF culture condition. Transcriptome profiling studies demonstrated reproducible changes in expression of nuclear transcription factors associated with high HbF. Among the 13 genes that demonstrated differential transcript levels, 8 demonstrated nuclear protein expression levels that were significantly changed by cytokine signal transduction. Five of the 8 genes are recognized regulators of erythropoiesis or globin genes (MAFF, ID2, HHEX, SOX6, and EGR1). Thus, cytokine-mediated signal transduction in adult erythroid cells causes significant changes in the pattern of globin gene and protein expression that are associated with distinct histone modifications as well as nuclear reprogramming of erythroid transcription factors.

Description: Key Points LIN28B regulates HbF expression in erythroblasts that are cultured from umbilical cord and adult human blood. LIN28B expression manifested a more fetal-like phenotype among adult human erythroblasts.

Description: Abstract 827 The highly-conserved Lin28 genes regulate cellular metabolism as well as the timing of developmental events and cell fates in multicellular organisms. Lin28 protein acts primarily by negatively regulating biogenesis of let-7 RNA, a microRNA family whose targets include growth-related signaling and transcription factor proteins. Published studies showed significantly increased expression of let-7 in purified adult blood reticulocytes compared to umbilical cord blood reticulocytes (1). This pattern correlates inversely with Lin28B expression. While present in the fetal liver and umbilical cord blood, Lin28B decreased to undetectable levels in adult bone marrow (2). Based upon the association of human ontogeny with hemoglobin switching, Lin28 was explored to identify novel mechanisms for hemoglobin regulation that may be useful for therapeutic application among patients with thalassemia or other hemoglobinopathies. To study the effects of Lin28B upon erythropoiesis and hemoglobin, ectopic expression of Lin28B was accomplished using retroviral transduction of human CD34+ cells cultivated ex vivo in erythropoietin-supplemented, serum-free cultures for 21 days. All experiments were performed in triplicate using cells from three separate adult volunteers. Lin28B over-expression (Lin28B-OE) was confirmed by Q-RT-PCR (control: 0.14 ± 0.37 copies/ng, Lin28B-OE: 1.8E+04 ± 353.8 copies/ng, p=0.01). Western analyses confirmed protein expression, and confocal microscopy revealed Lin28B predominantly in the cytoplasm of the transduced cells. Proliferation, maturation and morphology assays revealed that Lin28B-OE did not inhibit erythropoiesis when compared to control (empty vector) transductions. Terminal maturation with loss of CD71 from the erythroblast surface and enucleation by culture day 21 was detected in the control and Lin28B-OE samples. Expression levels of globin genes were evaluated upon Lin28B-OE by Q-RT-PCR. Lin28B-OE enhances gamma-globin mRNA expression (control: 5.14E+06 ± 2.6E+06 copies/ng, Lin28B-OE: 1.81E+07 ± 5.82E+06 copies/ng, p=0.038). Protein analysis confirmed the increased expression of gamma-globin. Fetal hemoglobin (HbF) levels were also increased in the Lin28B-OE cultures (control: 5.82 ± 4.54%, Lin28B-OE: 33.63 ± 9.38%; p=0.011). The increased HbF expression was maintained throughout differentiation including enucleated populations of culture-generated erythrocytes. Possible mechanism(s) for the increased expression of HbF caused by Lin28B-OE were investigated. Q-RT-PCR analyses demonstrated suppression of the let-7 microRNA family with greater-than 70% reductions of let-7a, let-7b, let-7c, let-7d, let-7e, let-7f-2, let-7g and let-7i. Expression patterns of several transcription factors including BCL11A, KLF1, SOX6 and GATA1 were explored. No major changes were detected with the exception of BCL11A. Lin28B-OE caused a 65% reduction in BCL11A expression (control: 3.07E+03 ± 1.5E+02 copies/ng, Lin28B-OE: 1.07E+03 ± 18 copies/ng; p=0.02). Western blot analyses of Lin28B-OE showed a consistent reduction of BCL11A protein. By comparison with Lin28B-OE, separately performed studies of BCL11A knockdown in adult CD34+ cells produced comparable increases in gamma-globin expression, but Lin28B expression in those cells was not affected. In addition to a more general role in development and metabolism, these experimental results suggest that Lin28B increases fetal hemoglobin and regulates BCL11A in human erythroblasts. Lin28B is thus identified as the first defined link between the regulation of a developmental clock and hemoglobin switching in humans. Disclosures: No relevant conflicts of interest to declare.

Description: Background Red blood cell (RBC) alloimmunization occurs at a much higher rate in patients with sickle cell disease (SCD) compared to other multiply transfused populations. Reasons for this include: altered immunologic responses, frequent transfusions during times of elevated inflammatory states, and disparate donor/recipient RBC antigens. Providing Rh and Kell matched RBCs has been shown to decrease but not eliminate RBC alloimmunization in patients with SCD. Although it has been shown in murine models that recipient inflammatory state at time of transfusion has the ability to regulate alloimmunization, direct clinical evidence for this effect is lacking in SCD patients. Methods With IRB approval, medical records of alloimmunized pediatric SCD patients were retrospectively reviewed to determine the influence of SCD-related complications often considered to be pro-inflammatory, at time of transfusion on RBC alloimmunization. The degree of antigen matching, age, and additive solution of each RBC unit were also assessed. Potential pro-inflammatory states were classified as: acute chest syndrome (ACS), acute stroke, acute febrile illness in the absence of another sickle-co morbidity, splenic sequestration, aplastic crisis, priapism, VOC with and without leukocytosis (WBC count ≥ 20k/μL) and elective surgery. Initial alloantibody detection dates were ascertained from blood bank records. Clinical events at time of transfusion were compared between transfusions resulting in a new alloantibody (AlloAb) and transfusions that did not. Univariable analysis was performed using Wilcoxon rank sum and Pearson’s Chi square test. Logistic regression modeling was used to estimate adjusted effects of stated variables on alloimmunization. Results A total of 3166 prestorage leukoreduced RBC transfusions (mean age 19.3 days) were provided to 52 SCD patients; 128 transfusions resulted in RBC alloantibodies; 3038 transfusions did not. On univariable analysis, 14.1% of transfusions during any inflammatory event resulted in a new AlloAb compared to 1.4% of transfusions in the absence of inflammation (p