High HbF Levels Research Articles

Micro RNA Profiling to Identify Regulator of Fetal Hemoglobin Expression in Sickle Cell Disease

Elevated fetal hemoglobin (HbF) expression ameliorates the clinical severity of sickle cell disease (SCD) by inhibiting hemoglobin S polymerization. Differences in HbF levels are attributed to inherited DNA genetic variations that regulate γ-globin transcription; however the role of microRNA (miRNA) genes in HbF regulation has not been investigated using clinical samples. miRNAs are small non-protein-coding RNA molecules that negatively regulate gene expression through inhibition of mRNA translation. Our goal is to identify miRNA genes with altered expression in sickle cell patients with elevated HbF levels, to elucidate mechanisms of γ-globin gene regulation. After IRB approval, peripheral blood was collected from SCD patients (not on hydroxyurea therapy), followed in the pediatric and adult Sickle Cell Clinics at Georgia Regents University. HbF levels measured by high performance liquid chromatography and complete blood and reticulocyte counts were obtained. Twelve blood samples, six each from SCD subjects with high HbF (19.9±2.1%) or low HbF (4.4±0.9%) levels were analyzed. We observed more severe anemia and higher reticulocyte counts in the low HbF group. After Histopaque separation, red blood cells were processed on a MACS column with anti-CD71 antibody to isolate reticulocytes, followed by total RNA extraction using Trizol. RNA (750ng) was hybridized to a genome-wide miRCURY LNA microRNA Array (Exiqon) containing 1,921 human probes. The microarray raw data were collected on an Agilent G2565BA Microarray Scanner System and normalized by Model-Based Background Correction and Principle Component Analysis. Characterization of miRNA profiles for low HbF compared to high HbF groups identified 327 differentially expressed genes including multiple miR-144 isoforms. We subsequently explored the function of miR-144 because it targets Nrf2 which mediates drug-induced HbF expression and Nrf2 has an antioxidant protective effect in SCD. Therefore, we conducted supervised learning of the normalized microarray data based on miR-144 expression. Interestingly, in the low HbF group we observed two subphenotypes: 1) associated with 8-fold increased miR-144 expression (3 subjects) and 2) associated with no change in miR-144 level (3 subjects) when compared to the high HbF group suggesting a role of miR-144 in HbF regulation. In the supervised learning analysis, there were 62 up-regulated and 33 down-regulated miRNAs (>2-fold; p<0.05) in the first subphenotype. We hypothesized that miRNAs up-regulated in the low HbF group might silence known γ-globin trans-activators. By TargetScan and Miranda analysis 7 miRNAs were predicted to target γ-globin including miR-96 a known negative regulator. There were 4 miRNAs predicted to target Nrf2 and 12 miRNAs that target other transcription factors such as KLF1, KLF4, BCL11A, and GATA2.To define a functional role of miR-144 we conducted studies in adult CD34+ stem cells grown in a two-phase culture system containing Stem Cell Factor (50ng/mL), Interleukin-3 (10ng/mL) and Erythropoietin (4IU/mL). At day 8 in culture, miR-144 mimic, miR-144 antagomir (inhibitor) or scrambled control (100nM, 200nM, and 300nM) were transfected using a Nucleofector System. After 72 hr incubation, RT-qPCR was conducted to measure γ-globin and Nrf2 mRNA levels. miR-144 mimic or antagomir at 100-300nM concentrations had no significant effect on γ-globin mRNA levels. By contrast, flow cytometry analysis using a FITC-anti-γ-globin antibody in erythroid cells treated with miR-144 mimic, produced a 30-70% decrease in HbF positive cells (p<0.05). On the contrary, we observed a 1.8-fold increase in HbF positive cells mediated by 300nM miR-144 antagomir. Evidence that miR-144 targets Nrf2 was established when antagomir treatment increased Nrf2 expression 1.4-fold (p<0.05). Final studies using day 8 erythroid progenitors treated with Nrf2 siRNA demonstrated a 40% decrease in γ-globin mRNA supporting a role of Nrf2 on γ-gene expression. In summary, the miRNA profiles associated with HbF expression in SCD patients combined with functional studies in human primary erythroid progenitors, support a role for miR-144 in γ-globin regulation. These findings will be expanded to a pre-clinical SCD mouse model to develop miR-144 as a potential therapeutic option. DisclosuresNo relevant conflicts of interest to declare.

RN-1, a Potent and Selective LSD1 Inhibitor, Induces High Levels of Fetal Hemoglobin (HbF) in Anemic Baboons (P. anubis)

Increased levels of fetal hemoglobin are associated with decreased symptoms and increased life span in patients with sickle cell disease (SCD). Hydroxyurea, the only drug currently approved for SCD, is not effective in a large fraction of patients and therefore new agents are currently needed. Recent evidence has shown that LSD1, an enzyme that removes monomethyl and dimethyl residues from the lys4 residue of histone H3, is a repressor of γ-globin expression. Tranylcypromine (TCP), an LSD1 inhibitor, was shown to increase γ-globin expression in human βYAC transgenic mice (Shi et al Nat Med 19:291, 2013). Because the arrangement and developmental stage-specific expression pattern of the β-like globin genes is highly conserved between man and other simian primates, the use of an simian primate animal model such as the baboon (P.anubis) is the best predictor of the activity of HbF-inducing agents in man. In this investigation we compared the effect of TCP and the more potent and selective LSD1 inhibitor, RN-1, on HbF expression in anemic baboons (P. anubis). In vitro assays have shown that the LSD1 IC50 of RN-1 is at least 1000 fold less than TCP. Animals were phlebotomized for 14d prior to drug treatment to attain an Hct=20 to induce reticulocytosis and establish baseline HbF levels and were maintained at this Hct by periodic phlebotomies during the course of the experiment. In four baboons treated with varying doses of TCP (2-6mg/kg/; 10-20d; sc) low levels of HbF (4.9-7.9% HbF) were induced that were only slightly higher than those observed at the pretreatment baseline (2.2-4.1% HbF). In contrast, treatment with varying doses of RN-1 (2.5-0.125 mg/kg/d; 5-10d) induced high levels of HbF, F reticulocytes, and F cells in 5 of 6 animals (see Table). At high doses of drug the ratio of 5’Iγ/3’Vγ synthesis was >2 demonstrating a near-complete reversion to the pattern of fetal stage expression. Peak levels of F reticulocytes and γ-globin synthesis were observed 8d and HbF and F cells 11d after the first day of drug administration. Increased γ-globin mRNA levels (γ/γ+β) in reticulocytes measured by RT-PCR showed that increased HbF levels were not due to translational effects. Bisulfite sequence analysis showed that levels of DNA methylation of the γ-globin promoter were similar in pre- and post-treatment BM erythroid precursors from two animals. Flow cytometry analysis using anti-α4-integrin and anti-baboon RBC antibodies showed that RN-1 treatment altered terminal BM erythroid differentiation by increasing the proportion of less differentiated precursors, however no changes in MCHC or total hemoglobin synthesis (α/γ+β) were observed. RN-1 treatment was associated with decreased ANC (290-870 X 103/μl nadir) and increased platelets (1092-1445 X 103/μl peak) and monocytes that were likely caused by effects on hematopoietic differentiation. The ANC nadir and peak platelet and monocyte counts were observed between d14-19 and resolved with 2-3 days. Similar changes in ANC and platelets, although not in monocytes, are observed following treatment with decitabine and can be controlled by modification of dose and schedule of administration (Lavelle et al Blood 119:1240, 2012). We conclude that RN-1, a more potent LSD1 inhibitor than TCP, is a powerful HbF-inducing drug with activity similar to decitabine and predict that LSD1 inhibitors may be useful drugs for the treatment of sickle cell disease.TableHbF, F-cells, and F-retics in Baboons Treated with RN-1AnimalRN-1 Dose (mg/kg/d)HbF (%)Globin synthesis (γ/γ+β)F-cells (%)F-retics (%)PrePostPrePostPrePostPrePost85482.5 (4d)5.827.3ND0.7828.559.234.692.385490.5 (5d)2.429.50.060.6819.160.833.997.580000.25 (5d)4.120.60.150.4920.347.045.780.785480.20 (5d)3.720.50.040.5236.954.836.989.280010.125 (5d)1.84.8ND0.0613.521.822.931.785490.125 (10d)3.6160.040.2217.642.721.070.0 DisclosuresNo relevant conflicts of interest to declare.

The Role of OMICS Research in Understanding Phenotype Variation in Thalassaemia: The THALAMOSS Project

The β-thalassaemias are a group of severe and rare anaemias with monogenic inheritance, a complex systemic phenotype and several treatment-related complications, caused by more than 300 mutations of the β-globin gene. Novel therapeutic protocols, most of which are based on still experimental treatments, show great promise but significant variability of success between patients. These strategies include chemical/molecular induction of the endogenous β-like γ-globin gene or the restoration of clinically relevant β-globin levels by gene therapy. A small number of modifiers with significant impact on disease penetrance, severity and efficacy of treatments are known, but most remain elusive. Improvements of existing treatment regimens and optimization and application of novel treatments will critically depend on the characterization of additional disease modifiers and the stratification of patients for customized treatment regimens. This requires extensive analyses based on “OMICS”, an English-language neologism which refer to different but connected fields in molecular biology and biochemistry, such as genomics, transcriptomics, exomics, proteomics, metabolomics. The major objective of OMICS is a collective characterization of pools of biological molecules (gene sequences, transcripts, proteins and protein domains) controlling biological structures, functions and dynamics, including several involved in pathological conditions. One of the most interesting observations of genomics in β-thalassaemias is the association between genomic sequences and high fetal haemoglobin (HbF) levels, in consideration of the fact that high HbF levels are usually associated with milder forms of β-thalassaemia. Related to this issue, is the possibility to predict response to different therapeutic protocols on the basis of genomic analyses. For instance, three major loci (Xmn1-HBG2 single nucleotide polymorphism, HBS1L-MYB intergenic region on chromosome 6q, and BCL11A) contribute to high HbF production. Pharmacogenomic analysis of the effects of hydroxyurea (HU) on HbF production in a collection of β-thalassemia and sickle cell disease (SCD) patients allowed the identification of genomic signatures associated with high HbF. Therefore, it can hypothesized that genomic studies might predict the response of patients to treatments based on hydroxyurea, which is at present the most used HbF inducer in pharmacological therapy of β-thalassaemia. Transcriptomic/proteomic studies allowed to identify the zinc finger transcription factor B-cell lymphoma/leukemia 11A (BCL11A) as the major repressor of HbF expression. The field of research on g-globin gene repressors (including BCL11A) is of top interest, since several approaches can lead to pharmacologically-mediated inhibition of the expression of g-globin gene repressors, leading to gglobin gene activation. Among these strategies, we underline direct targeting of the transcription factors by aptamers or decoy molecules, as well as inhibition of the mRNA coding g-globin gene repressors with shRNAs, antisense molecules, peptide nucleic acids (PNAs) and microRNAs. In this respect, the THALAMOSS FP7 Project (THALAssaemia MOdular Stratification System for personalized therapy of β-thalassemia, www.thalamoss.eu) aims develop a universal sets of markers and techniques for stratification of β-thalassaemia patients into treatment subgroups for (a) onset and frequency of blood transfusions, (b) choice of iron chelation, (c) induction of fetal hemoglobin, (d) prospective efficacy of gene-therapy. The impact of THALAMOSS is the provision of novel biomarkers for distinct treatment subgroups in β-thalassaemia (500–1000 samples from participating medical centres), identified by combined genomics, proteomics, transcriptomics and tissue culture assays, the development of new or improved products for the cell isolation, characterization and treatment of β-thalassaemia patients and the establishment of routine techniques for detection of these markers and stratification of patients into treatment groups. Translation of these activities into the product portfolio and R&amp;D methodology of participating SMEs will be a major boost for them as well as for the field. THALAMOSS tools and technologies will (a) facilitate identification of novel diagnostic tests, drugs and treatments specific to patient subgroups and (b) guide conventional and novel therapeutic approaches for β-thalassaemia, including personalized medical treatments.

HbF in HbE/β-thalassemia: A clinical and laboratory correlation

IntroductionFetal hemoglobin (HbF) is the predominant hemoglobin in red cells during fetal life. Just after birth, the level of HbF decreases gradually to <1%, and is replaced mainly by adult hemoglobin (HbA) (∼97%). However, higher HbF levels could be associated with HbE/β-thalassemia, a complex thalassemia intermedia with a diverse clinical severity ranging from mild-to-severe anemia. This study investigates the correlation of HbF level with the clinical and laboratory data of HbE/β-thalassemia individuals.MethodsPeripheral blood samples from 30 HbE/β-thalassemia subjects were subjected to a full blood count, genomic as well as quantitative real-time polymerase chain reaction gene expression studies. Statistical analyses were performed using SPSS 17.0.ResultsHbF levels were influenced by age, mean cell volume (MCV), mean cell hemoglobin (MCH), HbA, β-globin, and α/β-globin expressions.DiscussionHbF production is affected by the α/β-globin chain imbalance due to the lack of β-globin gene expression as well as inversely correlates to the amount of functional hemoglobin available in the cells.

DNA polymorphisms at BCL11A, HBS1L-MYB and Xmn1-HBG2 site loci associated with fetal hemoglobin levels in sickle cell anemia patients from Northern Brazil.

Increased levels of fetal hemoglobin (HbF, α2γ2) may reduce sickle cell anemia severity due to its ability to inhibit HbS polymerization and also reduce the mean corpuscular HbS concentration. We have investigated the influence of three known major loci on the HbF trait (HBG2, rs748214; BCL11A, rs4671393; and HBS1L-MYB, rs28384513, rs489544 and rs9399137) and HbF levels in SCA patients from the State of Pará, Northern Brazil. Our results showed that high levels of HbF were primarily influenced by alleles of BCL11A (rs4671393) and HMIP (rs4895441) loci, and to a lesser extent by rs748214 Gγ-globin (HBG2) gene promoter. The SNPs rs4671393 and rs4895441 explained 10% and 9.2%, respectively, of the variation in HbF levels, while 4.1% of trait variation was explained by rs748214. The results can be considered as in accordance with the pattern of ancestry displayed by the SCA patients: 39.6% European, 29.6% African and 30.8% Native American, and reinforce the suggestion that studies of association between genetic modifiers and clinical and laboratory manifestations in Brazil must be controlled by ancestry.

Pregnancy in beta-thalassemia intermedia: 20-year experience of a Greek thalassemia center.

Progress in the management of patients with thalassemia intermedia (TI) enabled increasing rates of pregnancies among TI women worldwide. Nevertheless, information regarding TI pregnancy management and outcome is quite limited in the literature. The aim of this study was to report our experience regarding the maternal and fetal outcome of TI patients, as well as to depict the complexity of the disease and the need for multidisciplinary and personalized management as shown by the description of two interesting pregnancy cases. We analyzed our data recorded from 60 pregnancies in 34 women over a 20-yr period. Forty-nine patients achieved full-term pregnancies (mean maternal age ± SD: 27.4 ± 6.5 yr) within 37 ± 3 gestation weeks. Their mean hemoglobin value was 8.33 ± 1.22 g/dL; 26.5% of patients were not transfused at all or they had been transfused only once during gestation. There were 11 abortions (18.3%). The spontaneous abortions (5/11) were related to high HbF levels. Six patients had more than two normal deliveries. Nineteen newborns (38.8%), which weighed 2-3 kg, required hospitalization to an intensive neonatal care unit for 1-3 wk. One patient presented with life-threatening complications (hemolytic anemia, thrombocytopenia, and enlargement of spleen) and another with spastic paraparesis due to extramedullary paravertebral masses. Although several complications can occur during a pregnancy in TI women, the careful and frequent monitoring by both hamatologists and obstetricians can lead to successful deliveries.

Long Term Safety and Efficacy Of Low Fixed Dose Hydroxyurea In Pediatric Patients With Sickle Cell Anemia: A Single Center Study From Central India

IntroductionHydroxyurea (HU) therapy ameliorates the morbidity associated with Sickle cell anemia (SCA). HU is typically escalated to maximum tolerated doses (MTD) for treatment of SCA patients. Monitoring HU for toxicity poses potential challenges in low resource settings. We have previously reported the safety and efficacy of fixed low dose HU at 10mg/kg body weight ( Jain et al 2013). We studied the long term safety and efficacy of low dose HU in pediatric patients with SCA. MethodsLong term observational follow up of 40 young Indian SCA patients was carried out for a period of 10 years in a single tertiary care center in central India. The number of clinical events including vasocclusive crises, acute chest syndrome, stroke, sequestration crises, avascular bone necrosis in the entire cohort was assessed prior to starting HU and subsequently every 3 months. Laboratory parameters including hemoglobin levels, Hb F levels, mean corpuscular volume, mean corpuscular hemoglobin concentration were also followed up over the same time period. ResultsThe total number of patients in this study was 40 (17 females & 23 males). Mean age was 12.95 years ± 9.62. All patients had HbSS. There was an increase in mean hemoglobin, HbF, MCV and MCH over time in the patient population ( Table 1). There was a decrease in the mean number of hospitalizations, vasocclusive crises, acute chest syndromes, stroke and severe anemia ( Table 2). HU was well tolerated with minimal hematological or other toxicity (Table3). Patients who experienced transient hematological, liver or renal toxicity were able to resume HU at the same dose. HU was permanently discontinued in 2 patients who had manifestations of HIV/AIDS. HU was temporarily interrupted in one patient who was receiving treatment for tuberculosis and in one patient for the duration of a normal pregnancy. There was one death attributable to severe septicemia without evidence of neutropenia after 2 years follow up. There were 3 subjects who were lost to follow up, one after 2 years follow up and remaining 2 after 5 years of follow up. Of note, patients had a high HbF level at baseline. High HbF levels at baseline, the variable relationship of HbF to disease severity and amelioration of disease in Indian patients with high HbF has been previously reported ( Jain et al 2012, Patel et al 2012, Italia et al 2009).Table 1Mean value ± S.D of Hematological parameters for patients on long term HU(n= no.of patients)Lab parametersBaseline (n=40)6 months(n=40)24 months(n=38)60 months(n=36)120 months(n=36)Hb F18.44 ± 10.5321.35 ± 9.6321.08 ± 8.5621.92 ± 8.9522.15 ± 8.74Hb (gm %)8.94 ± 1.829.29 ± 1.639.83 ± 1.749.74 ± 1.619.76 ± 1.53MCV (fl)89.27 ± 10.9892.89 ± 10.4993.31 ± 11.3393.39 ± 10.3993.36 ± 10.41MCH29.65 ± 4.4631.11 ± 4.1831.16 ± 4.3030.88 ± 3.8431.31 ± 4.15Table 2Mean number of clinical events per year ± S.D in the entire cohortClinical parametersBaseline (n=40)1 year(n=40)5 years(n=36)10 years(n=36)Hospitalizations18.34 ± 17.332.82 ± 1.252.27 ± 2.062.72 ± 2.37Vasocclusive crises13.07 ± 18.531.45 ± 0.871.65 ± 1.581.82 ± 1.81Severe anemia4.35 ± 4.310.65 ± 0.720.58 ± 0.840.62 ± 0.83Stroke0.075 ± 0.26000Sequestration crises0.075 ± 0.26000Chest syndrome0.075 ± 0.260.025 ± 0.1580.025 ± 0.1580Avascular Necrosis0.075 ± 0.260.025 ± 0.15800Table 3Toxicity Associated with HU During Study Period of 10 yearsToxicityNo. of episodesNo. of patientsANC < 500x109/L00ANC 500-1500 x109/L64Thrombocytopenia (<80 x109/L )32ALT (> 2 times upper limit of normal)85Creatinine (> upper limit of normal)22Reticulocytopenia (Absolute Reticulocyte Count < 80 x1012/L)11Hyperpigmentation33 ConclusionsLow fixed dose HU is well tolerated and is efficacious in reducing incidence of VOC, hospitalizations, severe anemia, stroke and acute chest syndrome. Low fixed dose HU may be advantageous in low resource settings because of lower toxicity and consequently less need for monitoring. There is a need for multi-center randomized comparisons of low fixed dose HU with conventional escalation of HU dose to MTD. Disclosures:Krishnamurti:GlycoMimetics, Inc.: Research Funding.

Imaging Flow Cytometry and Microfluidic Flow Assays Demonstrate Heterocellular Aggregation Of Immature Sickle Erythrocytes To Neutrophils Via Mac-1/VLA-4 Interactions

BackgroundVaso-occlusion (VO) is a major acute complication of sickle cell disease (SCD). Sickle mouse models indicate that the recruitment of leukocytes to the endothelium may participate in the initiation/propagation of VO, consequently interacting with circulating erythrocytes and other cell types to impair blood flow. We investigated, in vitro, whether neutrophils (Neut) circulate in heterocellular aggregates with red blood cells (RBC) in sickle cell anemia (SCA) patients, and whether Neut from SCA individuals interact with RBC once tethered to vessel wall components. MethodsPeripheral blood was obtained from healthy control individuals (CON) and SCA patients in steady state, off (SCA) and on hydroxyurea (SCAHU; 15-30mg/kg/day). After Ficoll-sedimentation of granulocytes and incubation with antibodies against RBC/Neut surface proteins (RBC: CD235a, CD71; Neut: CD66b, CD11a, CD11b), neutrophil-red blood cell aggregates (Neut-RBC agg) were evaluated using the Amnis® ImageStreamX MKII imaging flow cytometer. CD66b+ events (6000) were acquired at 40X and data analyzed by IDEAS 6.0. Neutrophil adhesion and heterotypic interactions with RBC were also monitored using a Cellix VenafluxTM microfluidics platform, employing 800-μm-channel biochips lined with activated human umbilical vein endothelial cells (HUVECs), or E-selectin (4µg/mL). ResultsCirculating Neut-RBC agg (CD66+CD235a+ events) were significantly increased in SCA, compared to CON individuals (Mean ±SEM % of Neut aggregated with RBC; SCA, 10.9 ±1.6%; SCAHU, 11.5 ±2.6%; CON, 5.4 ±1.7%; n≥7; p<0.05, SCA, comp. to CON) (See Figure). Neut-RBC agg were composed mainly of CD235+CD71+ reticulocytes, as opposed to mature RBC (CD235+CD71-) (Mean ±SEM; SCA, 6.9 ±1.6%; SCAHU, 7.2 ±1.9%; CON, 1.8 ±1.3% Neut aggregated to reticulocytes; n≥7; p<0.01). Sizes of aggregates were variable, with each neutrophil binding to 1 or 2 or more RBCs. Fetal hemoglobin (HbF) levels in SCA patients inversely correlated with the percentage of Neut-RBC agg (rS=-0.55; p<0.05; n=17). While the surface expressions of the neutrophil β2 integrins, LFA-1 and Mac-1, were not significantly different between CON, SCA and SCAHU patients, Mac-1 (expressed on Neut), and VLA-4 (expressed on reticulocytes)-blocking antibodies and a peptide inhibitor of ICAM-4 (expressed on mature RBC), significantly decreased Neut-RBC agg found in SCA samples, compared to the respective isotype negative controls (INC) (Mean ±SEM; INC, 7.75 ±1.7%; anti-Mac-1, 4.9 ±1.5%, p<0.01; anti-VLA-4, 5.67 ±1.5%, p<0.05; anti-ICAM-4, 5.63 ±1.5%, p<0.05; n≥13). Using a microfluidic platform to monitor interactions of RBC with Neut adhered to vessel wall components, perfused SCA RBC were found to interact significantly more with autologous Neut previously adhered to activated HUVECs, compared to RBC from SCAHU and CON individuals (Mean ±SEM of RBC adhered/Neut: 2.5 ±0.5; 1.0 ±0.3; 1.0 ±0.3, respectively; n≥09; p=0.01). Furthermore, the duration of interactions of RBC with autologous Neut pre-adhered to recombinant E-selectin was significantly higher for SCA RBC, compared to CON RBC and RBC from patients on HU (Mean ±SEM duration of interactions: SCA, 1.57 ±0.10s; CON, 1.17 ±0.04s; SCAHU, 1.19 ±0.04s; n≥05; p<0.01 SCA, comp. to CON). ConclusionA significant proportion of neutrophils of SCA patients circulate in the form of neut-RBC agg, particularly involving reticulocytes; higher HbF levels were associated with decreased Neut-RBC agg formation. Furthermore, Neut from SCA individuals, when adhered to activated endothelial cells or endothelial ligands, interact more readily with SCA RBC; additionally, HU therapy is associated with a reduction in these heterotypic interactions. Antibodies or pharmacological approaches that reduce the formation of Neut-RBC agg by targeting Mac-1/VLA-4 interactions may hold potential for clinical benefits in SCD. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Targeting BCL11A and KLF1 For The Treatment Of Sickle Cell Disease and β-Thalassemia In Vitro using Antisense Oligonucleotides

Sickle cell anemia (SCD) is a hereditary blood disorder in which red blood cells (RBC) become sickle-shaped and block blood vessels, leading to painful vaso-occlusive episodes. Sickling occurs because of a point-mutation in the β-globin gene of hemoglobin. Fetal hemoglobin (HbF, α2γ2) is the main oxygen transport protein with greater oxygen binding affinity in the fetus during the last months of embryonic development and the first few months of life after birth. HbF inhibits sickling by interfering with the polymerization of hemoglobin S. Higher HbF levels in SCD correlate with better survival and because HbF production can be reactivated pharmacologically in adults, it can be used for the treatment of SCD as well as β-thalassemia. In β-thalassemia, there is reduced or absent synthesis of the β-globin gene, causing ineffective erythropoiesis.B-cell lymphoma/leukemia 11A (BCL11A) is a transcription factor in the zinc-finger protein family and is expressed in B cells and erythroid cells. BCL11A represses fetal hemoglobin expression by binding to the GGCCCGG motif in the β-globin promoter region. Erythroid Kruppel-like factor (KLF1) is an erythroid-specific transcription factor that regulates β-globin expression through direct interaction with its promoter and indirectly regulates γ-globin expression through the regulation of BCL11A. By reducing the expression of BCL11A and KLF1, we can promote production of HbF through the upregulation of γ-globin expression.To demonstrate upregulation of γ-globin mRNA expression in vitro, we used MEL-h-b-BAC line#7 cells, a murine erythroleukemic cell line harboring the entire human beta globin locus and expressing mouse BCL11A and KLF1 (Tim Townes, Univ. of Alabama at Birmingham). Antisense oligonucleotides (ASOs) targeting mouse BCL11A or mouse KLF1 were added to the cells in a dose-dependent manner. Seven days later, with free uptake of the ASOs into the cells, we observed dose-dependent reduction of mouse BCL11A mRNA (IC50 = 0.7 μM) and mouse KLF1 mRNA (IC50 = 3 μM). Consequently, we observed a 300 +/- 8% upregulation of human γ-globin mRNA expression after achieving ∼90% reduction in BCL11A mRNA expression after ASO treatment compared to the untreated control cells. Similarly, KLF1 ASO treatment caused a 500 +/- 58% up regulation of human γ-globin mRNA expression after achieving ∼80% mRNA reduction in KLF expression. These data indicate that targeting BCL11A and/or KLF1 with ASO treatment can cause an increase in γ-globin expression that is necessary for the upregulation of fetal hemoglobin and may be used for the treatment of sickle-cell anemia and β-thalassemia. Disclosures:Peralta: Isis Pharmaceuticals, Inc.: Employment. Low: Isis Pharmaceuticals, Inc.: Employment. Kim: Isis Pharmaceuticals, Inc.: Employment. Murray: Isis Pharmaceuticals, Inc.: Employment. Guo: Isis Pharmaceuticals, Inc.: Employment. Freier: Isis Pharmaceuticals, Inc.: Employment. Townes: University of Alabama at Birmingham: Employment. Hung: Isis Pharmaceuticals, Inc.: Employment.

Fetal Hemoglobin In Sickle Cell Anemia: A Glass Half Full?

HbF modulates the phenotype of sickle cell anemia by inhibiting deoxyHbS polymerization. HbF is confined to erythrocytes called F-cells that can be detected by FACS when these cells contain sufficient HbF. Measuring the amount of HbF/F-cell is difficult and not clinically available. African-Americans with sickle cell anemia have 2-80% F-cells with an average HbF/F-cell of 6.4±1.6 pg. The distribution of HbF/F-cell is highly individual regardless of HbF level. People with HbS-gene deletion hereditary persistence of HbF (HPFH) have a mean HbF of 30%, and HbF is evenly distributed among their erythrocytes. Polymer is not present in these cells either experimentally or after calculating the HbS polymer fraction at 70% O2 saturation. Therefore, each cell contains about 10 pg. of HbF. DeoxyHbS polymerization is prevented at physiologic venous and capillary O2 saturations of 40-70% when HbF/F-cell is 9-12 pgs. We call this the “protective” level of HbF. F-cells need not contain “protective” levels of HbF. Some β-globin gene cluster haplotypes are associated with high HbF. Carriers of these haplotypes can have milder disease. Nevertheless, even patients with high HbF can have frequent painful episodes, acute chest syndrome and osteonecrosis. Patients with HbS-δβ thalassemia have 15 to 25% HbF but are anemic and have vasoocclusive complications, albeit less often than in sickle cell anemia. Hydroxyurea reduces the morbidity and mortality of sickle cell anemia, an effect likely to be mediated by its induction of HbF. Patients treated with hydroxyurea are better and probably live longer, but adults are anemic and rarely asymptomatic. In all these patient groups, HbF is unevenly distributed among erythrocytes. In contrast, people with HbS-HPFH are nearly asymptomatic and not anemic. The failure of HbF to modulate uniformly all complications of sickle cell disease might be related to the heterogeneous concentration of HbF in sickle erythrocytes. HbF is associated with protection from the development of certain disease subphenotypes but has limited prognostic value in individuals. In many cross-sectional studies, high HbF was associated with a reduced rate of acute painful episodes, fewer leg ulcers, less osteonecrosis, less frequent acute chest syndromes and reduced disease severity. HbF had a weak or no clear association with priapism, urine albumin excretion, stroke and silent cerebral infarction, systemic blood pressure and tricuspid regurgitant velocity. Perhaps this is because intravascular hemolysis of cells with little or no HbF causes nitric oxide scavenging, or because these complications are less dependent on HbS polymerization. No study provides information on the concentration of HbF/F-cell other than providing the relatively meaningless calculated mean value. Rather than the total number of F-cells or the concentration of HbF in the hemolysate, HbF/F-cell and the proportion of F-cells that have “protective” HbF is the most critical predictor of the likelihood of some disease subphenotypes. Hypothetical distributions of HbF-cells with different levels of HbF/F-cell can be plotted for different concentrations of HbF. With mean HbF levels of 5%, 10% and 20%, and HbF content per cell of 1.5, 3 and 6 pg., assuming a fixed mean, the variance was changed to show how the distribution of HbF per cell can greatly vary, even if the mean is constant. For example, with 20% HbF, as few as 1% and as many as 24% of cells have “protective” HbF. When HbF is lower, few or no “protected” cells can be present. Due to the heterogeneous concentrations of HbF, HbS can polymerize in some F-cells that have sub-polymer inhibiting concentrations of HbF. Inducing high levels of HbF is one approach to treating sickle cell disease. Inactivating BCL11A, a repressor of γ-globin gene expression, abrogates sickle cell disease in transgenic sickle mice. Their HbF was distributed homogeneously, and their phenotype mimicked HbS-HPFH. If it becomes possible in humans to target BCL11A or its pathway with agents that affect gene transcription, will it result in pancellular HbF? Broadening the distribution of HbF amongst sickle erythrocytes with drugs like hydroxyurea that effect the kinetics of erythropoiesis, coupled with an agent whose primary mechanism of action is to increase the transcription of the γ-globin genes, might be the most fruitful approach to HbF induction therapy and more efficacious than single agent treatment. Disclosures: No relevant conflicts of interest to declare.

RN-1, a New and Potent Lsd-1 Inhibitor, Increases γ-Globin Expression In a Humanized Sickle Cell Mouse Model

Increased levels of fetal hemoglobin (HbF) decrease symptoms and increase life expectancy in Sickle Cell Disease (SCD). DNA Methyltransferase 1 (DNMT1) and Lysine Specific Demethylase-1, (LSD-1), a mono- and dimethyl-histone H3 K4 demethylating enzyme, are components of the DRED multiprotein complex, a repressor of γ-globin expression (Cui et al Mol Cell Biol 31:3298, 2011). While DNMT1 inhibitors are well known to induce high levels of HbF in non-human primates and SCD patients, it was recently shown that the LSD1 inhibitor tranylcypromine (TC) increased γ-globin expression in cultured human erythroid progenitors and human βYAC mice (Shi et al Nat Med 19:291, 2013). We tested the effect of TC in non-human primates by administering varying doses of TC (2-6mg/kg/d; 10d; sc; n=4) to anemic baboons. Small increases in HbF were observed at the highest TC dose (ΔHbF=4%; n=2) that were 10-15 fold less than in baboons treated with decitabine (DAC; 0.5mg/kg/d; 10d). To identify LSD1 inhibitors with increased potency that may be more powerful in vivo inducers of HbF, a screening assay for LSD1 activity was developed based on the recent report that TC in combination with low-dose ATRA induced differentiation of AML cells (Shenk et al Nat Med 18:605, 2012). The U9367 cell line was treated with various LSD1 inhibitors in combination with low-dose ATRA. Effects on differentiation were assessed by measurement of the differentiation marker CD11b by flow cytometry. The TC derivative RN-1 induced differentiation with a 64 fold increased potency compared to TC. Experiments were then conducted to compare the effects of TC and RN-1 on γ-globin expression in cultured baboon erythroid progenitors. Cultures (n=2) were treated with varying doses of either RN-1 or TC on d7 and d10 and globin chain expression measured by HPLC. Low level γ-globin expression (0.021, 0.048 γ/γ+β) was observed in controls. TC (5μM) increased γ-globin expression (0.32 γ/γ+β) in the absence of cytotoxicity while similar levels of γ-globin (0.24, 0.36 γ/γ+β) were induced by non-cytotoxic doses of RN-1 that were 20-70 fold lower (0.25, 0.07μM). We next compared the effects of RN-1, TC, and DAC on HbF induction in a knock-in humanized mouse model. Humanized B6;129-Hbatm1(HBA)TowHbbtm3(HBG1,HBB)Tow/J mice were treated with phenylhydrazine for 2 days followed by varying doses of DAC, TC or RN-1 for 3 days. Analysis of F cells and γ-globin mRNA performed on d8 and 10 showed that F cells were increased 2-3 fold (p<0.002) in mice treated with 10mg/kg RN-1 (9.70+1.96%) and 20mg/kg RN-1 (9.55+1.20%) compared to DMSO-treated controls (3.56+1.70%) while γ-globin mRNA levels (γ/γ+β) were increased 5-8 fold in RN-1 treated mice (p<0.002). Reticulocyte counts did not differ significantly between control and RN-1-treated mice (10mg/kg). Higher doses of RN-1 (30mg/kg) induced greater increases in γ-globin mRNA (14 fold; p<0.023) but were associated with decreased reticulocyte counts. In contrast, no changes F cells or γ-globin mRNA were observed in mice treated with TC (6mg/kg/ip; n=3 and 10mg/kg/ip; n=3). DAC treatment resulted in dose-dependent increases in F cells and γ-globin mRNA. F cells were increased 2-3 fold (p<0.01) and γ-globin mRNA (γ/γ+β) 7 fold (p<0.004) in mice treated with 0.25mg/kg DAC in the absence of significant cytotoxicity while higher doses of DAC were associated with decreased reticulocyte counts. Therefore, in this model, doses of either RN-1 (10mg/kg) or DAC (0.25mg/kg) that are non-toxic induce similar increases in F cells and γ-globin mRNA. Experiments were then performed to test the ability of RN-1 to increase F cells and γ-globin mRNA in the sickle cell mouse B6;129-Hbatm1(HBA)TowHbbtm2(HBG1,HBB*)Tow/J model. Mice were injected for four days with RN-1 (10mg/kg). F cells in RN-1 treated mice were increased 2.6 fold (p<0.0001) compared to controls while γ-globin mRNA levels (γ/γ+β) were increased 7.3 fold (p<0.004). These results demonstrate that RN-1, a recently developed LSD-1 inhibitor with increased potency and selectivity compared to TC, increases γ-globin expression in 1) cultured baboon erythroid progenitors, 2) a phenylhydrazine-treated humanized transgenic mouse model, and 3) sickle cell mice. We conclude that further studies to determine the effect of RN-1 in the non-human primate model be performed to evaluate its potential use in the treatment of sickle cell disease. Disclosures:No relevant conflicts of interest to declare.

Sickle cell disease in Saudi Arabia: the phenotype in adults with the Arab‐Indian haplotype is not benign

Sickle cell disease (SCD) in Saudi patients from the Eastern Province is associated with the Arab-Indian (AI) HBB (β-globin gene) haplotype. The phenotype of AI SCD in children was described as benign and was attributed to their high fetal haemoglobin (HbF). We conducted a hospital-based study to assess the pattern of SCD complications in adults. A total of 104 patients with average age of 27 years were enrolled. Ninety-six per cent of these patients reported history of painful crisis; 47% had at least one episode of acute chest syndrome, however, only 15% had two or more episodes; symptomatic osteonecrosis was reported in 18%; priapism in 17%; overt stroke in 6%; none had leg ulcers. The majority of patients had persistent splenomegaly and 66% had gallstones. Half of the patients co-inherited α-thalassaemia and about one-third had glucose-6-phosphate dehydrogenase deficiency. Higher HbF correlated with higher rate of splenic sequestration but not with other phenotypes. The phenotype of adult patients with AI SCD is not benign despite their relatively high HbF level. This is probably due to the continued decline in HbF level in adults and the heterocellular and variable distribution of HbF amongst F-cells.

South-Italy -thalassemia: a novel deletion not removing the -globin silencing element and with 3' breakpoint in a hsRTVL-H element, associated with -thalassemia and high levels of HbF

Hereditary persistence of fetal hemoglobin (HbF) in adulthood may be due to: mutations in the γ-globin genes promoter, mutations in the transcriptional factors involved in their regulation, polymorphic sequences or large deletions in the β-globin cluster.[1][1] Recently, Sankaran clarified the

Association between baseline fetal hemoglobin levels and incidence of severe vaso‐occlusive pain episodes in children with sickle cell anemia

The ameliorating effect of high fetal hemoglobin (HbF) levels on the incidence of pain episodes in sickle cell anemia (SCA) is well-known; however, in children this relationship is less clearly established. We hypothesized that higher HbF levels in children with SCA are associated with fewer severe pain episodes. A meta-analysis of data from the Silent Infarct Transfusion Trial (n = 456) and the Cooperative Study of Sickle Cell Disease (n = 764), demonstrated that baseline HbF levels were associated with the incidence of severe pain, commonly defined across studies as an event requiring hospitalization (P-value = 0.02).

PP006. Gene expression profiling of first trimester placentas from pregnancies at high risk of developing preeclampsia

Preeclampsia (PE) is an important cause of fetal and maternal morbidity and mortality. It is considered a two-stage disease, the first stage characterized by a defect placentation and the second stage by maternal manifestations. Details of the patho-physiology behind the transition from stage one to stage two remain unclear. Was to study first trimester placental gene expression in patients identified as high risk for PE by either Doppler ultrasound or the biochemical markers cell free fetal hemoglobin (HbF) and alpha-1-microglobulin (A1M). Placental samples were obtained from seven women at highrisk of PE as determined by Doppler ultrasound of the uterine arteries and eight women with normal uterine artery resistance who for other reasons terminated their pregnancies surgically. Maternal serum samples were analyzed for HbF and A1M. The patients were risk stratified according to two risk classifications: (I) High vs. low uterine artery resistance and (II) High HbF and A1M vs. low HbF and A1M. Total RNA from the placentas was used for whole genome microarray. The results were analyzed by bioinformatics and genes of interest confirmed with qPCR. A total of 453 and 332 significantly altered genes were identified in the two study groups. Bioinformatics revealed 12 genes of interest in study group I and 7 genes of interest in study group II. Genes related to vascular tonus regulation and inflammatory response were identified in study group I suggesting that a lack of tonus regulation and increased inflammation might contribute to the high uterine artery resistance seen in this group. Genes related to regulation of hematopoiesis was found in group II suggesting dysfunctional hematopoiesis as a factor explaining the high levels of cell-free HbF seen.

Fetal Haemoglobin and β-globin Gene Cluster Haplotypes among Sickle Cell Patients in Chhattisgarh.

Foetal Haemoglobin (HbF) is the best-known genetic modulator of sickle cell anaemia, which varies dramatically in concentration in the blood of these patients. The patients with SCA display a remarkable variability in the disease severity. High HbF levels and the β-globin gene cluster haplotypes influence the clinical presentation of sickle cell disease. To identify the genetic modifiers which influence the disease severity, we conducted a β-globin haplotype analysis in the sickle cell disease patients of Chhattisgarh. The foetal haemoglobin and the β-globin gene haplotypes of the sickle cell trait and the sickle cell disease patients from Chhattisgarh were investigated. A total of 100 sickle cell patients (SS), 50 sickle cell trait patients (AS) and 50 healthy control individuals were included in the present study. The distribution of the β-globin gene haplotype was done by the PCR-RFLP method. PCR-RFLP showed that the homozygous Arab-Indian haplotype (65%) was the most frequent one, followed by the heterozygous Arab-Indian haplotype (11%) in the sickle cell patients (SS), while the AS patients had a higher frequency of the heterozygous Arab-Indian haplotype (38%) in comparison to homozygous one (32%). Four atypical haplotypes, 3 Benin and 1 Cameroon were also observed, although they were in lower frequencies. In the present study, the HbF levels were higher in the AS and the SS patients, with one or two Arab-Indian haplotypes as compared to the other haplotypes. The presence of the Arab-Indian haplotype as the predominant haplotype might be suggestive of a gene flow to/from Saudi-Arabia or India and it was associated with higher HbF levels and a milder disease severity.

Identification of a New Functional HDAC Complex Composed by HDAC5, GATA1 and EKLF in Human Erythroid Cells

AbstractAbstract 979Histone deacetylation, the reaction that maintains chromatin in a condensed configuration preventing gene expression, is catalyzed by the histone deacetylase (HDAC) superfamily. The human HDAC family includes 18 different isoforms classified on the basis of their sequence homology to HDACs from Saccharomyces Cerevisiae into class I (HDAC1, −2, −3, and −8), IIa (HDAC4, −5, −7, and −9), IIb (HDAC6 and −10) and IV (HDAC11). Class I HDACs bind the DNA directly while class IIa HDACs shuffles other proteins between nucleus and cytoplasm. While the role of individual class I HDACs in erythropoiesis is starting to emerge, that of class IIa and b HDACs is still largely unknown. To clarify the role played by class IIa HDACs in the control of human erythropoiesis, an extensive analysis of expression, activity, and function of different classes of HDACs during the maturation of erythroblasts derived in vitro from adult blood or cord blood was performed. HDACs expression/activity.Erythroid maturation was associated with increased expression of class I HDACs (both mRNA and protein) which, in the case of HDAC1, was also associated with increased enzymatic activity and association with its NuRD partner GATA1. By contrast, reductions either in expression (HDAC4) or activity (HDAC5) of class IIa HDACs were observed with maturation. In addition, GATA1 and EKLF were consistently found associated in human erythroblasts but EKLF was not found associated with HDAC1. The extent of nuclear-cytoplasmic trafficking of class I (HDAC1 and 2) and IIa (HDAC4 and 5) and of the transcription factors EKLF and GATA1 in response to EPO was determined. HDAC2/EKLF/GATA1 and HDAC4 were found constitutively present in the nucleus and in the cytoplasm, respectively. By contrast, the nuclear concentration of HDAC1 increased while that of HDAC5 and of GATA1fl decreased upon stimulation with EPO. The last two observations suggested that HDAC5, GATA1 and EKLF might be associated in a complex. Identification of the HDAC5/EKLF/GATA1 complex.A series of IPs followed by WB experiments showed that HDAC5 was consistently associated with EKLF and GATA1 and conversely, both GATA1 (preferentially GATA1fl over GATA1s) and EKLF were consistently associated with HDAC5 (Fig 1A and not shown). Interestingly also pERK was detected in IPs with HDAC5, EKLF and GATA1 antibodies. These results indicate that in erythroid cells HDAC5 forms a complex with GATA1, EKLF and pERK. Identification of the biological activity of the HDAC5/GATA1/EKLF/pERK complex.The association between GATA1/EKLF was greater in cells generated with cord blood (which express high HbF levels) than in those derived from adult blood and their association decreased with maturation, suggesting that the complex may regulate HbF expression. To confirm this hypothesis, HDAC5/GATA1 association and γ/(γ+ β) mRNA ratios were determined in erythroid cells induced to mature in the presence of a pan-class II-specific (APHA9, ID50=20 μM for HDAC4) HDAC inhibitor (HDACi) (Fig 1B)1. Cells exposed in parallel to the class I/IIa-specific (UBHA24, ID50 =0.2 and 0.6 μM for HDAC1 and HDAC4, respectively) HDACi, were used as control. Exposure to APHA9 reduced the association between GATA1 and HDAC5 and increased γ/(γ + β) mRNA expression ratio, while this association was not affected by exposure to the class I/II HDACi which, as expected, also increased γ/(γ+ β) mRNA ratio. Conclusions.These data identify a new HDAC complex formed by HDAC5, EKLF and GATA1 that regulates γ/(γ + β) ratio. We hypothesize that the biological role of this new complex is to shuffle GATA1 and EKLF from the cytoplasm to the nucleus, making them able to engage into the NuRD and Sin3A complex respectively, and that inhibition of the activity of this complex affects γ-globin expression indirectly by limiting the amount of GATA1and EKLF available to associate with NuRD and Sin3A. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Impact of βS-Globin Haplotypes on Oxidative Stress in Patients with Sickle Cell Anemia in Steady State

In Brazil, sickle cell anemia (SCA) is one of the most common genetic disorders. The levels of fetal hemoglobin (HbF) may be influenced by the presence of genetic modifiers; among these are the β(S)-globin haplotypes, associated with the clinical heterogeneity presented by the disease. Patients with SCA have an imbalance between the production of reactive oxygen species and antioxidant capacity, generating oxidative stress. Nitric oxide (NO) is a potent vasodilator and may be involved in the mechanism of HbF induction. The aim of this study was to evaluate the impact of β(S)-globin haplotypes in oxidative stress in patients with SCA. The study included 47 patients with SCA in steady state. The molecular diagnosis of SCA and characterization of the β(S)-globin haplotype was performed by β(S) chain polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP). Concentration of HbF was determined by high-performance liquid chromatography (HPLC). Serum levels of nitrite/nitrate (NOx) and malondialdehyde were determined by spectrophotometry. The most prevalent haplotype in the study population was Bantu. The Benin/n group presented significantly higher HbF and nitrite levels as compared to the Bantu/n group. Our results confirm data reported in the literature where the Benin and Bantu haplotypes are respectively correlated to high levels and decreased HbF. In addition, haplotypes associated with high levels of HbF showed high levels of nitrite, demonstrating that as the HbF, serum levels of NOx may prove useful as a prognostic biomarker in patients with SCA.

Foetal haemoglobin and disease severity in sickle cell anaemia patients in Kampala, Uganda.

BackgroundSickle cell anaemia (SCA) is a major chronic health problem in Uganda. In patients with SCA, the level of foetal haemoglobin (HbF) has been found to be important in influencing the clinical course of the disease. Thus populations with high levels of HbF like those in Saudi Arabia have been described as having a milder clinical course with fewer complications as compared to populations with lower levels. Disease modifying drugs can increase the Hb F levels and modify the presentation of SCA.MethodsThis was a cross sectional study in which we determined foetal haemoglobin levels and examined the relationship between HbF levels and disease severity in SCA patients in Mulago Hospital, Kampala, Uganda. We consecutively enrolled 216 children aged 1 year to 18 years with SCA attending the Sickle Cell Clinic at Mulago Hospital whose guardians had given consent. The history included age at onset of initial symptoms and diagnosis, number of hospitalisations and blood transfusions and other complications of SCA (cardiovascular accidents, avascular hip necrosis and priapism). A detailed physical examination was performed to assess the current state and help describe the disease severity for each patient. Blood samples were drawn for HbF levels. HbF levels ≥10% was defined as high.ResultsOf the 216 children, (80) 37% had HbF levels ≥10%. Significant correlations were observed between HbF level and several clinical parameters independent of age including age at diagnosis (p value 0.013), number of hospitalisations (p value 0.024) and transfusions (p value 0.018) since birth.ConclusionA third of the children with SCA attending the Sickle cell clinic in Mulago Hospital have high HbF levels. Higher HbF level is associated with later onset of symptoms and presentation, and less severe disease characterised by fewer hospitalisations and blood transfusions. We suggest HbF levels should be determined at initial contact for patients with SCA to guide counselling and identify those who may need closer follow up and consideration for disease modifying drugs.

Association of Inflammatory Biomarker C-Reactive Protein, Lipid Peroxidation and Antioxidant Capacity Marker with HbF Level in Sickle Cell Disease Patients from Chattisgarh.

This study was undertaken to determine the association of inflammatory biomarker, oxidative stress and antioxidant capacity marker with fetal haemoglobin (HbF) level among sickle cell trait and sickle cell disease (SCD) patients in Chattisgarh. The study group consisted of 51 SCD (SS) patients with painful episode, 49 SCD (SS) patients with steady state, 50 sickle cell trait (AS) and 50 controls. Malondialdehyde (MDA), CRP, total antioxidant power (FARP), total thiol and HbF levels were quantified. We found a significant positive (p<0.0001) association between CRP and MDA levels and its inverse association with HbF level in SS patients. We also observed that antioxidant capacity had significantly positively (p<0.0001) associated with HbF level. The protective effect of HbF was found, because the increase in HbF levels resulted in decrease in lipid peroxidation and inflammation in SCD patients. A decrease in the HbF level and its antioxidant capacity has been associated with the pathogenesis of SCD. These finding may explain the high level of HbF is ameliorating oxidative stress and inflammation in SCD patients.