Elevated Fetal Hemoglobin Research Articles

Comparison of gene expression profiles between human erythroid cells derived from fetal liver and adult peripheral blood.

BackgroundA key event in human development is the establishment of erythropoietic progenitors in the bone marrow, which is accompanied by a fetal-to-adult switch in hemoglobin expression. Understanding of this event could lead to medical application, notably treatment of sickle cell disease and β-thalassemia. The changes in gene expression of erythropoietic progenitor cells as they migrate from the fetal liver and colonize the bone marrow are still rather poorly understood, as primary fetal liver (FL) tissues are difficult to obtain.MethodsWe obtained human FL tissue and adult peripheral blood (AB) samples from Thai subjects. Primary CD34+ cells were cultured in vitro in a fetal bovine serum-based culture medium. After 8 days of culture, erythroid cell populations were isolated by flow cytometry. Gene expression in the FL- and AB-derived cells was studied by Affymetrix microarray and reverse-transcription quantitative PCR. The microarray data were combined with that from a previous study of human FL and AB erythroid development, and meta-analysis was performed on the combined dataset.ResultsFL erythroid cells showed enhanced proliferation and elevated fetal hemoglobin relative to AB cells. A total of 1,391 fetal up-regulated and 329 adult up-regulated genes were identified from microarray data generated in this study. Five hundred ninety-nine fetal up-regulated and 284 adult up-regulated genes with reproducible patterns between this and a previous study were identified by meta-analysis of the combined dataset, which constitute a core set of genes differentially expressed between FL and AB erythroid cells. In addition to these core genes, 826 and 48 novel genes were identified only from data generated in this study to be FL up- and AB up-regulated, respectively. The in vivo relevance for some of these novel genes was demonstrated by pathway analysis, which showed novel genes functioning in pathways known to be important in proliferation and erythropoiesis, including the mitogen-activated protein kinase (MAPK) and the phosphatidyl inositol 3 kinase (PI3K)-Akt pathways.DiscussionThe genes with upregulated expression in FL cells, which include many novel genes identified from data generated in this study, suggest that cellular proliferation pathways are more active in the fetal stage. Erythroid progenitor cells may thus undergo a reprogramming during ontogenesis in which proliferation is modulated by changes in expression of key regulators, primarily MYC, and others including insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), neuropilin and tolloid-like 2 (NETO2), branched chain amino acid transaminase 1 (BCAT1), tenascin XB (TNXB) and proto-oncogene, AP-1 transcription factor subunit (JUND). This reprogramming may thus be necessary for acquisition of the adult identity and switching of hemoglobin expression.

1031 - Reactivation of fetal hemoglobin for therapy: From GWAS to the clinic

The major hemoglobin disorders, b-thalassemia and sickle cell disease (SCD), are among the most common severe Mendelian disorders worldwide. Although the molecular bases of these diseases have been known for decades, treatment has been largely supportive, except for curative bone marrow transplantation. Classical family and genetic studies long ago established the beneficial impact of elevated fetal hemoglobin on the clinical phenotypes, and stimulated investigation into approaches for reactivation of silenced fetal hemoglobin as a therapeutic strategy. Despite elucidation of the principal regulators of erythroid gene transcription more than 2 decades ago, the basis for repression of fetal hemoglobin (HbF) during late gestation and after birth was elusive. Genome-wide association studies of HbF levels in 2007/8 revealed candidate loci, including the zinc-finger transcription factor BCL11A. Subsequently, combined surrogate genetics in cells, human genetics, and CRISPR/Cas9 saturating mutagenesis have established BCL11A as a principal repressor of HbF expression and identified discrete DNA sequences within an intragenic erythroid enhancer that are favorable for gene editing therapy. Through unbiased screening of potential BCL11A binding sequences we recently identified a novel recognition motif present in embryonic and fetal, but not adult, globin genes and showed that this site is mutated in rare individuals with classical hereditary persistence of hemoglobin, an entitly described 3 decades ago. Translation of basic findings to the clinic, through genetic or pharmacological means, is under intense investigation. Summary of the current state of these efforts will be discussed. Studies of BCL11A have greatly advanced a mechanistic understanding of the switch from fetal to adult hemoglobin, and illustrate the power of integrative genetic, biochemical, and hematologic approaches in providing opportunities for transformative management of the major hemoglobin disorders.

Correlates of Pulmonary Function in Children with Sickle Cell Disease and Elevated Fetal Hemoglobin

Objective: The current study was carried out to compare pulmonary function tests (PFTs) in pediatric Kuwaiti sickle cell disease (SCD) patients to age-matched normal controls and to investigate the association of PFTs with selected clinical and laboratory parameters. Subjects andMethods: There were 38 patients with SCD and 36 controls in the study. The patients were recruited from the Pediatric Hematology Clinics of Mubarak Al-Kabeer and Al-Amiri Hospitals, Kuwait, and were studied in steady state. The controls were healthy, non-sickle cell siblings of the patients. Forced expiratory volume in 1 s (FEV₁), forced vital capacity (FVC), total lung capacity, and other PFT parameters were obtained using a constant-volume, variable-pressure, body plethysmograph. Hemoglobin, fetal hemoglobin, serum bilirubin, and lactate dehydrogenase were determined using standard methods. Results: The mean ages of the patients and controls were 10.5 ± 3.2 and 10.5 ± 3.5 years, respectively. The FEV₁% predicted of 84.1 ± 15.4% among the patients was significantly lower than the 92.1 ± 11.8% in the controls (p = 0.003). The FVC% predicted was also significantly lower (p = 0.022) in the patients than in the controls, although the values were generally within the normal range. There was no association of FEV₁ with pain phenotype, acute chest syndrome (ACS), or blood transfusions. Also, there was no significant correlation with reticulocytes, bilirubin, or lactate dehydrogenase. Conclusions: In this study, changes in PFT, especially FEV₁, developed early in the SCD patients. There was no demonstrable association with frequent vaso-occlusive crisis, ACS, and other variables. Hence, there is a need for follow-up studies with serial PFTs to identify vulnerable patients, who might need intervention to prevent early mortality.

Recent progress in understanding and manipulating haemoglobin switching for the haemoglobinopathies

The major β-haemoglobinopathies, sickle cell disease and β-thalassaemia, represent the most common monogenic disorders worldwide and a steadily increasing global disease burden. Allogeneic haematopoietic stem cell transplantation, the only curative therapy, is only applied to a small minority of patients. Common clinical management strategies act mainly downstream of the root causes of disease. The observation that elevated fetal haemoglobin expression ameliorates these disorders has motivated longstanding investigations into the mechanisms of haemoglobin switching. Landmark studies over the last decade have led to the identification of two potent transcriptional repressors of γ-globin, BCL11A and ZBTB7A. These regulators act with additional trans-acting epigenetic repressive complexes, lineage-defining factors and developmental programs to silence fetal haemoglobin by working on cis-acting sequences at the globin gene loci. Rapidly advancing genetic technology is enabling researchers to probe deeply the interplay between the molecular players required for γ-globin (HBG1/HBG2) silencing. Gene therapies may enable permanent cures with autologous modified haematopoietic stem cells that generate persistent fetal haemoglobin expression. Ultimately rational small molecule pharmacotherapies to reactivate HbF could extend benefits widely to patients.

Whole transcriptome analysis of human erythropoietic cells during ontogenesis suggests a role of VEGFA gene as modulator of fetal hemoglobin and pharmacogenomic biomarker of treatment response to hydroxyurea in \u03b2-type hemoglobinopathy patients

BackgroundHuman erythropoiesis is characterized by distinct gene expression profiles at various developmental stages. Previous studies suggest that fetal-to-adult hemoglobin switch is regulated by a complex mechanism, in which many key players still remain unknown. Here, we report our findings from whole transcriptome analysis of erythroid cells, isolated from erythroid tissues at various developmental stages in an effort to identify distinct molecular signatures of each erythroid tissue.ResultsFrom our in-depth data analysis, pathway analysis, and text mining, we opted to focus on the VEGFA gene, given its gene expression characteristics. Selected VEGFA genomic variants, identified through linkage disequilibrium analysis, were explored further for their association with elevated fetal hemoglobin levels in β-type hemoglobinopathy patients. Our downstream analysis of non-transfusion-dependent β-thalassemia patients, β-thalassemia major patients, compound heterozygous sickle cell disease/β-thalassemia patients receiving hydroxyurea as fetal hemoglobin augmentation treatment, and non-thalassemic individuals indicated that VEGFA genomic variants were associated with disease severity in β-thalassemia patients and hydroxyurea treatment efficacy in SCD/β-thalassemia compound heterozygous patients.ConclusionsOur findings suggest that VEGFA may act as a modifier gene of human globin gene expression and, at the same time, serve as a genomic biomarker in β-type hemoglobinopathy disease severity and hydroxyurea treatment efficacy.

Comparative study of sickle cell anemia and hemoglobin SC disease: clinical characterization, laboratory biomarkers and genetic profiles

BackgroundIn this study, we evaluate the association of different clinical profiles, laboratory and genetic biomarkers in patients with sickle cell anemia (SCA) and hemoglobin SC disease (HbSC) in attempt to characterize the sickle cell disease (SCD) genotypes.MethodsWe conducted a cross-sectional study from 2013 to 2014 in 200 SCD individuals (141 with SCA; 59 with HbSC) and analyzed demographic data to characterize the study population. In addition, we determined the association of hematological, biochemical and genetic markers including the βS-globin gene haplotypes and the 3.7 Kb deletion of α-thalassemia (−α3.7Kb-thal), as well as the occurrence of clinical events in both SCD genotypes.ResultsLaboratory parameters showed a hemolytic profile associated with endothelial dysfunction in SCA individuals; however, the HbSC genotype was more associated with increased blood viscosity and inflammatory conditions. The BEN haplotype was the most frequently observed and was associated with elevated fetal hemoglobin (HbF) and low S hemoglobin (HbS). The -α3.7Kb-thal prevalence was 0.09 (9%), and it was associated with elevated hemoglobin and hematocrit concentrations. Clinical events were more frequent in SCA patients.ConclusionsOur data emphasize the differences between SCA and HbSC patients based on laboratory parameters and the clinical and genetic profile of both genotypes.

Existence of HbF Enhancer Haplotypes at HBS1L-MYB Intergenic Region in Transfusion-Dependent Saudi β-Thalassemia Patients.

Background and Objectives. β-Thalassemia and sickle cell disease are genetic disorders characterized by reduced and abnormal β-globin chain production, respectively. The elevation of fetal hemoglobin (HbF) can ameliorate the severity of these disorders. In sickle cell disease patients, the HbF level elevation is associated with three quantitative trait loci (QTLs), BCL11A, HBG2 promoter, and HBS1L-MYB intergenic region. This study elucidates the existence of the variants in these three QTLs to determine their association with HbF levels of transfusion-dependent Saudi β-thalassemia patients. Materials and Methods. A total of 174 transfusion-dependent β-thalassemia patients and 164 healthy controls from Eastern Province of Saudi Arabia were genotyped for fourteen single nucleotide polymorphisms (SNPs) from the three QTL regions using TaqMan assay on real-time PCR. Results. Genotype analysis revealed that six alleles of HBS1L-MYB QTL (rs9376090C p = 0.0009, rs9399137C p = 0.008, rs4895441G p = 0.004, rs9389269C p = 0.008, rs9402686A p = 0.008, and rs9494142C p = 0.002) were predominantly associated with β-thalassemia. In addition, haplotype analysis revealed that haplotypes of HBS1L-MYB (GCCGCAC p = 0.022) and HBG2 (GTT p = 0.009) were also predominantly associated with β-thalassemia. Furthermore, the HBS1L-MYB region also exhibited association with the high HbF cohort. Conclusion. The stimulation of HbF gene expression may provide alternative therapies for the amelioration of the disease severity of β-thalassemia.

Metformin Induces FOXO3-Dependent Fetal Hemoglobin Production in Primary Erythroid Cells

Elevated fetal hemoglobin (HbF, α2γ2) ameliorates the pathophysiology of sickle cell disease (SCD) by diluting HbS and inhibiting its polymerization in patient red blood cells. Hydroxyurea (HU) the only approved drug for SCD acts in part by raising HbF levels. However, up to 50% of adult SCD patients treated with hydroxyurea, do not have a clinically meaningful response, illustrating the need for new improved agents. We identified FOXO3, a potentially "druggable" transcription factor, as a candidate HbF inducer through analysis of rare variants from whole exome sequence data from SCD patients. Specifically, numerous rare variants that potentially impair FOXO3 activity and/or expression were associated with reduced HbF levels. FOXO3 was previously shown to facilitate erythroid precursor survival, maturation and resistance to oxidative stress.To investigate our findings from genomic studies further, we manipulated FOXO3 expression in normal human CD34+ hematopoietic stem and progenitor cells (HSPCs) induced to undergo erythroid differentiation and examined the effects on γ-globin RNA levels and HbF production. Transfection of CD34+ HSPCs with lentivirus expressing FOXO3 shRNA that reduced levels of the corresponding RNA by 80% reduced γ-globin (HBG1 and HBG2) mRNA by 60% compared to control cultures expressing scrambled shRNA (n= 3 separate experiments, p < 0.0005). A second non overlapping FOXO3 shRNA reduced the corresponding mRNA by 50% and reduced γ-globin by 31% (n=3 separate experiments, p < 0.0001). Reduction of FOXO3 mRNA was accompanied by delay in differentiation (11% GPA+ cells with 80%FOXO3 kd by day 14 compared to 72% GPA+ cells in the scramble kd).Metformin, an FDA-approved drug used for type 2 diabetes, is known to increase FOXO3 expression and therefore, we tested its effects on HbF expression in CD34+ HSPC-derived erythroblasts by treating with 30, 50 and 100 µM drug, beginning at day 5 of culture. At day 14 of culture, 59% late basophilic erythroblasts were present in control cultures, as evidenced by band3 and α4 integrin expression. In contrast, maturation was accelerated in metformin treated cultures, with 70% late basophilic erythroblasts, consistent with previous studies. Remarkably, metformin treatment also caused a dose dependent increase in HbF-immunostained cells ("F-cells", Figure 1) and in % HbF, as measured by HPLC (Figure 2). HPLC analysis at d14 showed that %HbF (HbF/(HbF + HbA) rose from 10.3% at baseline, to 19.8% and 30% at 50 and 100 µM metformin (n= 3, p=0.04 and 0.005). For dosage comparison patients on standard metformin therapy for diabetes typically exhibit plasma drug levels of 70 µM. When cultures were treated with metformin (100µM) and HU (30µM), %HbF was increased to 45% compared to 26% and 30% in cultures treated with HU or metformin respectively as single agents (n=3, p=<0.0001, p=0.0016, and p=0.005).Thus, HU and metformin show additive effects, on HbF induction, suggesting that combination therapy in patients may be more effective than standard therapy of HU alone. In addition, metformin may be a superior agent than HU as single therapy because it does not cause myelosuppression, and therefore requires minimal laboratory monitoring, and does not arrest erythropoiesis, making it a potentially effective agent for quantitative beta hemoglobinopathies. Taken together, our results indicate that FOXO3 is a positive regulator of γ-globin expression and a potential therapeutic target for HbF induction. Given our current results and the excellent safety profile of metformin, a pilot clinical trial of the drug (1500-2500 mg/day) alone or combined with hydroxyurea at stable maximum tolerated dose, in patients aged 16-40 years with sickle cell anemia or non-transfusion dependent beta thalassemia has been approved by the Baylor College of Medicine IRB. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Re-Creating Hereditary Persistence of Fetal Hemoglobin (HPFH) to Treat Sickle Cell Disease (SCD) and β-Thalassemia

Hereditary persistence of fetal hemoglobin, a naturally occurring condition that substantially ameliorates disease in SCD and β-thalassemia, is associated with genetic variation at the β-globin locus. Our strategy is to use the CRISPR-Cas9 system to re-create specific genetic variants associated with HPFH in CD34+ hematopoietic stem and progenitor cells (HSPCs) and demonstrate their causal relationship to elevated fetal hemoglobin (HbF) levels as a potential therapeutic strategy to treat SCD and β-thalassemia.The CRISPR-Cas9 system has been adapted to achieve site-directed DNA cleavage by guide RNAs (gRNAs) and the Cas9 endonuclease. We have identified highly potent single gRNAs (sgRNAs) that can target regions within the β-globin locus associated with HPFH. The sgRNAs, when combined and delivered as dual sgRNAs, resulted in deletions that mimic naturally occurring deletions associated with HPFH. We have also optimized transfection dose and ratio of these sgRNAs with either Cas9 mRNA or Cas9 protein into primary human CD34+ HSPCs from mobilized peripheral blood of healthy donors and can achieve greater than 85% transfection rate with minimal cell loss (85-95% viability). We found that sgRNAs delivered with Cas9 protein resulted in better cell viability than when delivered with Cas9 mRNA especially at higher doses, while attaining the same rates of editing efficiency. Lastly, erythroid differentiation of CD34+ HSPCs demonstrated significant increase in γ-globin gene expression level by qRT-PCR as well as HbF protein levels, measured by FACS and LC/MS.In conclusion, we have optimized sgRNA and Cas9 transfection into primary human CD34+ HSPCs. We have identified sgRNAs that are highly effective in targeting the β-globin locus and employed forward genetics to re-create genetic variants associated with HPFH in HPSCs. We have demonstrated the causal relationship of different HPFH genetic variants to elevation of HbF, and obtained comparative data on upregulation of HbF in erythroid cells differentiated from edited CD34+ HSPCs. Our findings provide a viable therapeutic strategy using CRISPR-Cas9 for the treatment of β-hemoglobinopathies. DisclosuresLin:CRISPR Therapeutics: Employment. Paik:CRISPR Therapeutics: Employment. Mishra:CRISPR Therapeutics: Employment. Chou:CRISPR Therapeutics: Employment. Zhang:CRISPR Therapeutics: Employment, Equity Ownership. Tomkinson:CRISPR Therapeutics: Employment. Pettiglio:CRISPR Therapeutics: Employment. Sanginario:CRISPR Therapeutics: Employment. Woo:CRISPR Therapeutics: Employment. Lee:CRISPR Therapeutics: Employment. Allen:CRISPR Therapeutics: Employment. Cradick:CRISPR Therapeutics: Employment. Chakraborty:CRISPR Therapeutics: Employment. Porteus:CRISPR Therapeutics: Consultancy, Equity Ownership. Mavilio:Adverum Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees; CRISPR Therapeutics: Consultancy, Research Funding. Cowan:CRISPR Therapeutics: Employment, Equity Ownership. Novak:CRISPR Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Lundberg:CRISPR Therapeutics: Employment, Equity Ownership.

Correlation of SIN3A genomic variants with β-hemoglobinopathies disease severity and hydroxyurea treatment efficacy.

Hemoglobinopathies, particularly β-thalassemia and sickle cell disease, are characterized by great phenotypic variability in terms of disease severity, while notable differences have been observed in hydroxyurea treatment efficacy. In both cases, the observed phenotypic diversity is mostly dependent on the elevated fetal hemoglobin levels, resulting from the persistent fetal globin gene expression in the adult erythroid stage orchestrated by intricate mechanisms that still remain only partly understood. We have previously shown that several protein factors act as modifiers of fetal hemoglobin production, exerting their effect via different pathways. Here, we explored whether SIN3A could act as a modifier of fetal globin gene expression, as it interacts with KLF10, a known modifier of fetal hemoglobin production. We show that SIN3A genomic variants are associated both with β-thalassemia disease severity (rs11072544) as well as hydroxyurea treatment response (rs7166737) in β-hemoglobinopathies patients. Our findings further underline that fetal hemoglobin production is the result of a complex interplay in which several human globin gene cluster variants interact with protein factors encoded by modifier genes to produce the observed clinical outcome.

A case of undetectable glycosylated hemoglobin

Cation-exchange high-performance liquid chromatography separates hemoglobin species based on charge differences. Since the glycosylated hemoglobin [HbA1c] test is based on normal hemoglobin, hemoglobinopathies can affect the test by altering the normal process of glycation of HbA to A1C, producing an abnormal peak on chromatogram or making the erythrocytes more prone to hemolysis, thereby decreasing the time for glycosylation to occur and producing a falsely low A1C result. A 51-year-old male presented with complaint of generalized weakness. HbA1c test done to rule out abnormal glucose tolerance revealed undetectable levels. He had low hemoglobin, microcytic red cell indices, 86.2 % of hemoglobin E (corrected A2), and minor elevation of fetal hemoglobin. This case is significant for the incidental discovery of HbE homozygosity in an apparently healthy and active person, through incidental finding of undetectable levels of HbA1c.

Mechanisms Regulating Increased Embryonic βh1 Globin Expression in Adult Nan anemic Mice

Sickle Cell Disease affects 90-100,000 in the US including 1/500 African-Americans born each year. Elevation of fetal hemoglobin (HbF) by co-inheritance of positive genetic modifiers of HbF expression or hydroxyurea (HU) treatment ameliorates disease severity. Because HU can have significant side effects, novel therapies aimed at elevating postnatal HbF expression are actively being sought. Three major loci modify HbF expression. Together, they account for ~50% of the variation in HbF expression, indicating that additional modifiers exist. Previously, we described the semi-dominant inbred mouse model Nan (neonatal anemia) that carries a missense mutation (E339D) in the second zinc finger of Krüppel-like factor 1 (KLF1/formerly EKLF) causing severe anemia accompanied by a striking failure of hemoglobin switching in Nan/+ mice (homozygotes die in utero). Embryonic βh1 globin expression is upregulated in Nan E14.5 fetal liver and in adult spleen where, remarkably, it accounts for nearly 100% of β-like globin gene expression. To extend these studies, we examined potential mechanisms regulating βh1 expression in adult Nan. Nan expression of Bcl11a, a downstream target of KLF1 that plays a major, conserved role in β-like globin gene switching, is 60-80% that of both untreated and phenylhydrazine treated (PHZ) wild type (WT) controls in the spleen. In peripheral blood, Nan BCL11A protein level is >50% of WT by western blotting. Importantly, prior studies by other investigators showed that newborn Bcl11a heterozygous knockout mice expressing Bcl11a at 50% of WT levels are haplosufficient, showing no differences in β-like globin gene expression. These data indicate that upregulated βh1 expression in Nan is BCL11A independent. We next examined erythropoiesis by flow cytometry using CD44, Ter119, and forward scatter (FSC) as markers. Nucleated erythroid precursors were strikingly decreased in Nan vs. PHZ-treated and phlebotomized (PHB) spleen, unusual in an anemic mouse model. Similar results were obtained using CD71, Ter119 and FSC gating. Despite this, βh1 expression normalized to saline-injected non-anemic controls was dramatically higher in Nan (93.0 ± 13.7, AU, X ± SEM) than either PHZ (3.6 ± 0.7) or PHB (7.4 ± 0.7) mice (p < 0.0001). Thus, increased βh1 in Nan is not simply due to stress erythropoiesis with concomitantly increased erythroid precursors. To analyze βh1 expression genetically, we constructed two Nan congenic lines on two different inbred genetic backgrounds, BALB/cBy and 129/SvImJ. Marked variation in adult spleen βh1 expression levels is seen among the three Nan strains. Similarly, we analyzed βh1 expression in an outbred high resolution mapping population derived from eight inbred strains, the diversity outcross (DO). Substantial variation in expression was seen among DO individuals. These data firmly establish the existence of modifying genes exerting profound influences on βh1 expression. We established an F2 intercross between 129S1/SvImJ-Nan/+ and C57BL/6J mice to take advantage of both Nan and DO mice to identify quantitative trait loci (QTL) modifying βh1 expression. Preliminary statistical analysis of 173 phenotyped (βh1 expression by RT-PCR) and SNP-genotyped F2 Nan/+ mice using R/qtl software identified a highly significant QTL for βh1 on Chr 7 encompassing the β-globin cluster and 3 suggestive QTL (Chr 4, 5 and 14). Analysis of 261 DO mice using QTL/ReL software identified 2 significant QTL (Chr 6, 7) and 6 suggestive QTL (Chr 2, 4[2], 6, 10, 14), with three in common with the QTL identified in F2 mice. Our analyses to date identify QTL overlapping three loci known to influence β-like globin gene switching (the β globin locus, Chr 7; LSD1, Chr 4; Mi-2β, Chr 6), providing proof of principle for our strategy. More importantly, additional loci identified contain no known modifiers, indicating the influence of novel genes. In summary, elevated βh1 expression in adult Nan spleen (1) occurs independently of Bcl11a; (2) is not mediated solely by stress erythropoiesis; (3) is highly influenced by genetic background; and (4) is influenced by novel genetic regulators of β-like globin switching. DisclosuresNo relevant conflicts of interest to declare.

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.

Pharmacological Inhibition of Histone Deacetylases 1 and 2 (HDAC1/2) Induces Fetal Hemoglobin (HbF) through Activation of Gata2

Induction of HbF is an established therapeutic strategy for the treatment of sickle cell disease, and could also be effective in treating beta-thalassemia. Genetic ablation of HDAC1 or HDAC2, but not HDAC3, results in the induction of the fetal beta-like globin gene (HbG) transcript (Bradner JE, Proc Natl Acad Sci, 2010). We have previously shown that selective chemical inhibitors of HDAC1/2 elicit a dose and time dependent induction of HbG mRNA and HbF protein in cultured human CD34+ bone marrow cells undergoing erythroid differentiation (Shearstone JS, ASH Annual Meeting Abstracts, 2012). In this work, we have utilized our proof of concept molecule ACY-957, a selective inhibitor of HDAC1/2, to discover a novel role for Gata2 in the activation of HbG.To identify genes affected by HDAC1/2 inhibition, CD34+ bone marrow cells undergoing erythroid differentiation were treated with ACY-957 or vehicle, followed by mRNA expression profiling. Among the genes differentially regulated by both pharmacological inhibition and genetic ablation of HDAC1/2 were Bcl11a and Sox6, known HbG repressors, and Gata2, a potential HbG activator. Quantitative real time PCR (QRT-PCR) time course experiments confirmed that ACY-957 treatment leads to a 2-fold and 10-fold decrease in Bcl11A and Sox6, respectively, and an 8-fold increase in Gata2 mRNA. Unlike Bcl11a and Sox6, Gata2 induction by ACY-957 was highly correlated with HbG induction, suggesting a possible role for this transcription factor in the direct activation of HbG.To investigate this possibility, lentiviral infection was utilized to overexpress full length Gata2 transcript in differentiating primary erythroblasts. After 5 days of differentiation, Gata2 overexpression resulted in a 2.5-fold increase in HbG mRNA, while the level of the major adult beta-like globin chain (HbB) mRNA was unaffected. HbG mRNA remained elevated by Gata2 overexpression at day 7 of differentiation, while HbB was reduced by 1.6-fold. Gata2 overexpression appeared to have minimal effect on cell differentiation, as determined by the cell surface markers CD71 and GlycophorinA, a finding consistent with observations in ACY-957 treated cells with elevated Gata2. Furthermore, lentiviral delivery of short hairpin RNA (shRNA) targeting Gata2, attenuated HbG induction by ACY-957. These data suggest that elevated levels of Gata2 resulting from HDAC1/2 inhibition is sufficient to induce HbG at early stages of erythroid cell differentiation.To understand how HDAC1/2 inhibition drives Gata2 activation, chromatin immunoprecipitation coupled with either next generation sequencing (ChIP-seq) or QRT-PCR was performed in ACY-957 and vehicle treated cells. HDAC1 and HDAC2 were present throughout the Gata2 gene body and promoter regions, and HDAC1/2 binding levels were highly correlated, suggesting co-occupancy of these enzymes at this locus. ACY-957 treatment led to elevated histone acetylation at previously described Gata2 gene regulatory regions (Bresnick et. al. 2010, J Biol Chem). Specifically, the -1.8 kb and -2.8 kb regulatory regions showed a 6-fold increase in histone H3K9 and H2BK5 acetylation, while the +9.5 kb and -3.9 kb regions showed a 3-fold increase. The Gata2 protein showed increased binding at these regulatory regions in response to ACY-957 treatment, with a maximum increase of 3-fold at the -1.8 kb region. This finding is consistent with the known positive autoregulation of the Gata2 gene. Taken together, these data suggest that selective inhibition of HDAC1/2 leads to elevated Gata2 through acetylation-induced activation of a positive autoregulatory loop.The tight temporal correlation between Gata2 and HbG activation following HDAC1/2 inhibition argues that Gata2 may affect the beta-globin locus directly. ChIP-seq data across the 70-kb beta-globin locus demonstrated that ACY-957 treatment altered Gata2 binding only at a single region, lying within the promoter for delta globin. This region is suspected in playing a role in switching from fetal to adult globin during development, as naturally occurring deletions of this region are associated with elevated fetal hemoglobin in adults (Sankaran et. al. 2011, NEJM). Whether the change in GATA2 binding to this region is responsible for the increased expression of HbG in cells treated with HDAC1/2-selective inhibitors is under investigation. DisclosuresShearstone:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Golonzhka:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Chonkar:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Jarpe:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership.

RASA4 undergoes DNA hypermethylation in resistant juvenile myelomonocytic leukemia

Aberrant DNA methylation at specific genetic loci is a key molecular feature of juvenile myelomonocytic leukemia (JMML) with poor prognosis. Using quantitative high-resolution mass spectrometry, we identified RASA4 isoform 2, which maps to chromosome 7 and encodes a member of the GAP1 family of GTPase-activating proteins for small G proteins, as a recurrent target of isoform-specific DNA hypermethylation in JMML (51% of 125 patients analyzed). RASA4 isoform 2 promoter methylation correlated with clinical parameters predicting poor prognosis (older age, elevated fetal hemoglobin), with higher risk of relapse after hematopoietic stem cell transplantation, and with PTPN11 mutation. The level of isoform 2 methylation increased in relapsed cases after transplantation. Interestingly, most JMML cases with monosomy 7 exhibited hypermethylation on the remaining RASA4 allele. The results corroborate the significance of epigenetic modifications in the phenotype of aggressive JMML.

HBS1L-MYB intergenic variants modulate fetal hemoglobin via long-range MYB enhancers

Genetic studies have identified common variants within the intergenic region (HBS1L-MYB) between GTP-binding elongation factor HBS1L and myeloblastosis oncogene MYB on chromosome 6q that are associated with elevated fetal hemoglobin (HbF) levels and alterations of other clinically important human erythroid traits. It is unclear how these noncoding sequence variants affect multiple erythrocyte characteristics. Here, we determined that several HBS1L-MYB intergenic variants affect regulatory elements that are occupied by key erythroid transcription factors within this region. These elements interact with MYB, a critical regulator of erythroid development and HbF levels. We found that several HBS1L-MYB intergenic variants reduce transcription factor binding, affecting long-range interactions with MYB and MYB expression levels. These data provide a functional explanation for the genetic association of HBS1L-MYB intergenic polymorphisms with human erythroid traits and HbF levels. Our results further designate MYB as a target for therapeutic induction of HbF to ameliorate sickle cell and β-thalassemia disease severity.

Overexpression Of DNMT3a and DNMT3b Is Related To Downregulation Of Mir-29a In Juvenile Myelomonocytic Leukemia (JMML)

Juvenile myelomonocytic leukemia (JMML) is a mixed myelodysplastic /myeloproliferative disorder (MDS/MPD). It occurs in infancy and young children with a progressive course leading to death within one year after diagnosis. This disease is characterized by monocytosis, leukocytosis, elevated fetal hemoglobin, hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF), a low percentage of myeloblasts in the bone marrow, and absence of the Philadelphia chromosome or the BCR/ABL fusion gene. Mutations or other abnormalities in RAS, NF1, PTPN11, and CBL have been linked to be responsible for the pathogenesis of JMML in up to 85% of cases. Treatment is very difficult in JMML, and only allogeneic stem cell transplantation (SCT) can extend survival. However, the relapse rate from allogeneic SCT is inordinately high in JMML (28-55%), with 5-year disease-free survival rates of 25-40%. JMML occurs in an age-range when genes are actively being turned on or off in children in adaption to the oxygenized environment after birth. Epigenetics plays a key role in this developmental plasticity. We previously reported hypermethylation on the promoter of PTEN in 77% of JMML patients, and decitabine, a DNA-hypomethylating reagent, significantly inhibited colony formation (CFU-GM) in JMML cells in vitro. In addition, other groups found that aberrant DNA methylation on promoters of BMP4, CALCA, CDKN2B, and RARB is significantly associated with poor prognosis in JMML. Taking together, these data suggest that epigenetic mechanisms may contribute to the pathogenesis of JMML.MicroRNAs (miRNAs) have been reported to play an important role in myeloid differentiation and activation. miRNA function is highly dependent on the cell type. Recently, we reported that miR-183 is overexpressed in JMML. Other groups have reported aberrant expression of miR-29a in acute myeloid leukemia and other cancers. Both miR-183 and miR-29a are located on chromosome 7q32 in humans, which is frequently disrupted in JMML. We hypothesized that miR-29a may be deregulated in JMML, and contribute to the aberrant epigenetic regulation in JMML.In order to test our hypothesis, we collected peripheral blood or bone marrow from 41 JMML patients and 14 normal individuals. Total RNAs were extracted from mononuclear cells (MNCs) using Trizol. We first evaluated the expression levels of miR-29a by using relative-quantitative real-time RT-PCR (qRT-PCR). We found that the expression levels (RQ) of miR-29a in patients are significantly lower than that in normal individuals (median 0.45 vs. 1.11, p< 0.001). By analysis of the expression levels of miR-29a together with previous data on miR-183 in these JMML patients and normal controls, we found that the RQ of miR-29a is inversely correlated with RQ of miR-183 (Spearman's r=-51, p<0.001). This suggests that the expression of miR-29a is segregated from that of miR-183 in MNCs, although they are located only 1.1 million base-pairs apart on chromosome 7q32. When further evaluating the mRNA expression levels of miR-29a targeting genes by using qRT-PCR, we found that RQ of DNA methyltransferases 3A and 3B (DNMT3a and DNMT3b) were significantly increased in JMML patients' samples in comparison with normal controls (median 5.16 vs. 1.00, p=0.001 for DNMT3a; median 3.45 vs. 1.08, p=0.001). Strikingly, the RQ of miR-29a was inversely correlated with the RQs of both DNMTs (Spearman's r=-0.62 for DNMT3a; Spearman's r=-0.79 for DNMT3b, p<0.001), which suggests that miR-29a plays a significant role in the regulation of DNMT 3a and DNMT3b in MNCs. In conclusion, we found that overexpression of DNMT3a and DNMT3b, the two key molecules in DNA methylation of epigenetic modification in developmental plasticity, is related to downregulation of miR-29a in JMML. In addition to our previous report on overexpression of miR-183 in JMML, this finding provides new insights into the role of miRNAs in the aberrant epigenetic regulation in JMML, and it may apply to other pediatric malignancies. Further investigation is ongoing to uncover the mechanism of down-regulated miR-29a in JMML. If the underexpression of miR-29a in JMML is due to the hypermethylation on miR-29a promoter, as reported in prostate cancer cells, demethylating agents, such as decitabine, or soybean products, isoflavone, could be potential drugs to treat JMML. Disclosures:No relevant conflicts of interest to declare.

Interpreting elevated fetal hemoglobin in pathology and health at the basic laboratory level: new and known γ‐ gene mutations associated with hereditary persistence of fetal hemoglobin

Fetal hemoglobin may be slightly or significantly elevated in post-natal life due to a number of causes. We report two novel mutations found on the promoter of the Aγ gene and summarize all common and rare determinants associated with hereditary persistence of fetal hemoglobin (HPFH) described thus far. Hematological and molecular analysis of the Aγ globin gene in two cases of HPFH. Comparison of the novel cases with all those described in the literature. We have found two novel mutations in three Italian patients with HbF values between 5.9% and 6.5% without an elevated HbA(2) and with normal hemoglobin parameters. In two probands (mother and son), a -197 C>T transition was observed, while in a single individual, a -113 A>G transition was present on the distal CCAAT box of the Aγ gene. As no other abnormalities were present in both γ-gene promoters and the changes are located on regulatory sequences, we may conclude that these mutations are responsible for the HPFH phenotype shown by the carriers. The laboratory should be able to discriminate between elevated HbF due to artifacts or to serious causes including bone marrow malignancies, aplastic anemia, and β-thalassemia major or recessive traits such as β-thalassemia minor, δβ-thalassemia, or nonpathological conditions induced by mutations or polymorphisms of the γ-gene promoters that may even be beneficial when present in patients with thalassemia major or sickle cell disease and, in particular, when these patients are treated with hydroxyurea.

Erythroid transcription factor EKLF/KLF1 mutation causing congenital dyserythropoietic anemia type IV in a patient of Taiwanese origin: Review of all reported cases and development of a clinical diagnostic paradigm

KLF1 is an erythroid specific transcription factor that is involved in erythroid lineage commitment, globin switching and terminal red blood cell maturation. Various mutations of KLF1 have been identified in humans, which have led to both benign and pathological phenotypes. The E325K mutation, within the second zinc finger of the KLF1 gene, has been shown to cause a new form of congenital dyserythropoietic anemia (CDA) now labeled as CDA type IV. We report the fourth documented case of this mutation, and propose a clinical diagnostic model to better identify this disease in other patients. Our patient is a Taiwanese child who presented to us at 8years of age with severe hemolytic anemia, splenomegaly, elevated fetal hemoglobin (HbF), iron overload, and dyserythropoiesis in the bone marrow. KLF1 sequence analysis revealed a G-to-A transition in one allele of exon 3, which resulted in the substitution of a glutamate 325 by a lysine. Flow cytometry analysis revealed decreased protein expression of CD44 on the red blood cells, and decreased red blood cell deformability as measured using an ektacytometer. Blood typing revealed his red blood cells to be Co(a-b-), In(b-), LW(ab-) and Lu(b+), even though DNA testing predicted that he would be Co(a+b-) and LW(a+b-). This newly discovered CDA combines features of a hemoglobinopathy, RBC membrane defect and hereditary persistence of HbF (HPFH) which are not seen in the previous types of CDA. Increased awareness of this phenotype may improve the more prompt and accurate diagnosis of these patients.

Timing of the Initiation of Hydroxyurea and Hematologic Outcomes in Patients with Sickle Cell Disease (SCD)

AbstractAbstract 1004▪▪This icon denotes a clinically relevant abstractHydroxyurea (HU) is approved for use in adults with Sickle Cell Disease (SCD) and increases the production of fetal hemoglobin (HbF). Increased HbF is associated with decreased clinical severity in adults and children with SCD, such as decreased numbers of vaso-occlusive events, transfusions, and hospitalizations. Higher HbF at initiation of HU is predictive of HbF response, but association between age of hydroxyurea initiation and HbF response has not been investigated. We hypothesize that starting hydroxyurea at an early age may improve hematological and clinical response. In order to determine if younger age at hydroxyurea initiation affects the percentage of HbF achieved with hydroxyurea, we conducted a retrospective cohort study. We identified subjects enrolled in the Duke University Medical Center Comprehensive Sickle Cell program who initiated hydroxyurea when they were less than 17.99 years of age and were prescribed hydroxyurea for at least six months. The following data were abstracted from the medical record between December 1996 and April 2011: age, hemoglobin, percentage HbF, and mean corpuscular volume (MCV) at start of HU and at maximum tolerated dose (MTD) of HU therapy. The correlation coefficient and p-values for various parameters were calculated. Seventy-three patients (41 males and 32 females) were included in the analysis. The mean age at hydroxyurea initiation was 5.5 years (1.2–14.1). The mean hydroxyurea dose at MTD was 28.6 ± 3.2 mg/kg/day. At initiation, the mean hemoglobin was 8.2 ± 1.2 g/dL, the mean MCV was 83±7.4 fl and mean HbF was 10 ± 5.7%. At MTD, the mean hemoglobin was 9.4 ± 1.1 g/dL, the mean MCV was 99 ± 11.1 fl, and the mean HbF was 21.7 ± 9.4%. As expected, at MTD, an elevated MCV was correlated with elevated fetal hemoglobin (r2= 0.19, p= 0.0001) [Table 1]. There was a statistically significant relationship between the age at HU initiation and the HbF at MTD (r2= 0.08, p= 0.015) [Figure 1] as well as the age at HU initiation and the hemoglobin at MTD (r2= 0.19, p= 0.016). The relationship between the age at starting HU and the overall change in HbF (DHbF) was not statistically significant (r2= 0.01, p= 0.41). There was not a statistically significant relationship between age at HU initiation and the MTD of HU (r2= 0.003, p= 0.61). The 6 patients started on HU at age less than 2 years (mean 1.5 ± 0.3 years) maintained a mean elevated HbF of 19.1 ± 5% at last documented follow-up with follow-up ranging from 1.4–13 year of uninterrupted hydroxyurea use. Starting hydroxyurea therapy at a younger age appears to improve HbF response as measured at MTD, although there is variability in the level of fetal hemoglobin attained. There is not an association seen with the DHbF or dose at MTD and age at hydroxyurea initiation. In summary, starting hydroxyurea at a younger age, when HbF is >20%, leads to persistence of HbF production and overall improvement in hematological efficacy. This was not simply the result of achieving MTD at a younger age before physiologic decline of HbF. [Display omitted] [Display omitted] Disclosures:Off Label Use: Hydroxyurea for complications of sickle cell disease in pediatrics. Shah:Eisai: Research Funding; Adventrx: Consultancy.