High HbF Research Articles

A Study of Novel Beta Globin Mutation Associated with Dominant Beta-Thalassemia, Aberrant Erythroid Maturation and Differentiation Compounded with Hereditary Pyropoikilocytosis

Background: Heterozygosity for a beta-thalassemia mutation is clinically insignificant; however, microcytosis and mild anemia are oftenpresent. Rare mutations in the proximal half of the coding sequence for the beta globin gene (HBB) have been shown to cause an apparent autosomal dominant beta thalassemia that is associated with more severe anemia. These mutations often produce a frame shift in HBB mRNA; however, detection of the mutant transcript and/or protein has been controversial. In genotypically identical relatives, a milder phenotype can sometimes be observed, while others have severe anemia. It has been suggested that these phenotypic differences may be mediated by variations in destruction of mutant HBB transcripts by nonsense mediated decay .Materials: We studied a Dominican man with severe microcytic hypochromic anemia without iron deficiency, suggestive of thalassemia, and his family. We performed a detailed erythrocyte evaluation, including morphologic and hemoglobinopathy evaluations, globin sequencing, hemoglobin (Hb) stability evaluation, and band-3 testing for red blood cell (RBC) membrane abnormality, and analyzed DNA for mutations of genes encoding for RBC membrane proteins. We also performed in vitro expansion of erythroid progenitors, examined erythroid differentiation and proliferation, and searched for mutant HBB transcripts.Results: The propositus was found to have a novel beta-thalassemia mutation, HBB282delT encoding a Cys93fs (frame shift). He also had Heinz bodies and was positive for an unstable Hb (by isopropanol test), but had no electrophoretically detectable mutant Hb, and had absolute reticulocytopenia (Table). Peripheral blood had a typical hereditary pyropoikilocytosis (HPP) morphology (Figure 1), confirmed by band-3 testing. Family studies (Table) showed that the situation was more complex. His asymptomatic wife and one son had sickle cell trait (Table), and two other children inherited both the HBB282delT mutation and a Hb S mutation and were positive for band-3 deficiency testing. Their RBCs had typical HPP morphology, along with sickle cells (Figure 2), and had high HbF levels (Table). Reticulocytes were not available for detection of mutant transcripts by RNA analysis; we therefore performed in vitro expansion of erythroid progenitors in a 3-week liquid erythroid culture model (Gaikwad, et al, Exp Hemat, 2007). We detected mutant HBB282delT transcripts leading to a frameshift in exon 2, codon 93, where 9 new codons downstream from the mutation were encoded. The mutant transcript was present in equal amounts with the HBB transcript from the wild allele. Analysis of erythroid proliferation and differentiation revealed a marked decrease in proliferation (as determined by number of glycophorin-positive cells) and decreased differentiation (as evaluated by temporal changes of glycophorin and transferrin receptor positivity and intensity) in the propositus and two children with HBB 282delT. Hemoglobinization of their maturing erythroid progenitors was not discernible in 14-day cultures, in contrast to his wife, the asymptomatic son and the controls who had readily discernible hemoglobinized (red) progenitors. By day 21 of culture, he and the 2 affected children had only faintly hemoglobinized cells that appeared pink rather than red, and the erythroid progenitors of the propositus had minimal differentiation. None of the affected individuals had a SPTA1 exon 2 mutation, the most common missense mutations leading to the HPP phenotype.Discussion: We describe a novel HBB mutation associated with severe anemia, reticulocytopenia, and impaired erythropoiesis with delayed erythroid maturation and impaired hemoglobinization. The presented data suggest that the mutant protein is unstable and presumably toxic to erythroid progenitors. The mutant protein is not identifiable by routine hemoglobin studies and its presence will be evaluated by studies of precipitated hemoglobin (Heinz bodies and hemoglobin precipitates after isopropanol exposure). Effects of the mutant protein on maturation and differentiation can be assessed by RNAseq of erythroid progenitors compared to normal controls of the same stage of erythroid differentiation. The molecular basis of HPP is being evaluated by DNA sequencing of targeted genes encoding RBC membrane proteins and comparing their potential functional relevance to the HPP phenotype. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Ameliorating Effects of Very High Hemoglobin F Levels and Glucose-6-Phosphate Dehydrogenase Deficiency in Sickle Cell Patients Along with Associated Clinical Manifestations: A Report from Al Ahsa, Easter Province, Saudi Arabia

▪Introduction: Sickle cell disease (SCD) is an autosomal inherited structural disorder of hemoglobin which leads to vaso-occlusive, hematological and infectious crises, including stroke as one of the common complications. Eastern province of Saudi Arabia is known to have the highest prevalence of sickle cell anemia in the world, though resulting disease is mild. The aim of this study is to determine the characteristics of sickle cell patients in Al Asha, Eastern province of Saudi Arabia in order to determine factors ameliorating this disease in the area.Material and Methods:A total of 73 patients were included in the study. Data was collected by a questioner, which was composed of: Identity variables (MRN), social demography (age, gender), clinical manifestations (presence of co-morbidities), and laboratory results of hemoglobin (Hb) electrophoresis of sickle cell patients. Data has been analyzed by SPSS version 22.Results:Male to female ratio was 34:39. Moreover, SCD was more prevalent in females than males.Furthermore, SCD individuals less than 20 years reported as the highest prevalence among all age groups. Hemoglobin electrophoresis pattern in sickle cell patients were found to be: Hb A2 (2.26%) ± 1.005, Hb F (21.45%) ± 9.33, Hb S (76.27%)±8.73; Hb A1 was not detected. G6PD deficiency, thalassemias, aplastic anemia and hemolytic anemia with found in 11(15.1%), 4 (5.5%), 4 (5.5%) and 5 (6.8%) of patients. One of the world highest levels of HbF were detected in our SCD patients. Patients with high HbF levels and/or having G6PD deficiency had the milder form of SCD.Discussion & Conclusions:In this study, we found world highest Hb F levels in comparison to international normal range of Hb F in SCD patients which is unique to Saudi population. The study reflects that patients with SCD have vaso-occlusive crisis as the most common clinical manifestation followed by bone pain, chest pain, asthma and splenomegaly. We found an interesting case of a patient with β-Thalassemia trait and G6PD deficiency in addition to SCD. Almost 13.7% of all SCD patients had cholesteatoma. Although there are conflicting reports about effect of G6PD deficiency in SCD, we found ameliorating effect of G6PD which may account for mild form of SCD in Al Ahsa region. Further studies are required to investigate the factors responsible for ameliorating effects of different factors in SCD patients from this region which may open up new windows to clinically manage this disease in Saudi Arabia. [Display omitted] DisclosuresSaglio:Pfizer: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Roche: Consultancy, Honoraria.

New Insights into F-Cells, F-Reticulocytes and the Role of Fetal Hemoglobin in Protecting Sickle Red Cells from Hemolysis upon Mechanical Stress

Sickle cell disease (SCD) is an autosomal hereditary recessive disorder caused by a single point mutation in the first exon of the β-globin gene generating abnormal hemoglobin (HbS). Hypoxia drives HbS polymerization and fiber formation that are responsible for red blood cell (RBC) sickling. In SCD patients expression of fetal hemoglobin (HbF) is higher than in healthy individuals and varies considerably. Although clinical studies have shown that HbF levels were inversely correlated with disease severity, studies addressing the relationship between the expression level of HbF at the cellular level, i.e. in F-cells, and the impact on RBC morphology and survival are scarce.In this study, we investigated the distribution and the expression level of HbF at the cellular level in a large cohort of SCD patients (SS or Sβ°) using high throughput imaging techniques, functional sickling assays and a novel microfluidic device.We determined the percentage of HbF-expressing cells (F+RBCs) in 46 patient blood samples and found that it was highly variable (mean: 44% +/- 24%) and correlated with the percentage of HbF measured by HPLC (R2 = 0.86). To estimate the content of HbF in F+RBCs at the single cell level we developed a method based on imaging flow cytometry and defined three different populations according to the expression level of HbF: low, medium and high F+RBCs (Figure 1). We determined the percentage of each subpopulation in 30 SCD blood samples and found that the low F+RBCs were the most prevalent, with a median of 17.85% of total circulating RBCs. The high F+RBCs were the least abundant, with a median of 0.63%, while medium F+RBCs accounted for 4.03% of RBCs.Next, we investigated the relationship between F+RBCs and irreversibly sickled cells (ISCs) using imaging flow cytometry. The %ISCs was significantly higher in the low than in the medium subpopulation and no ISCs were found in the high F+RBC subpopulation. Applying Percoll density fractionation we found that F+RBCs were predominant in the low dense (LD) fraction and poorly present in the high dense (HD) fraction; this was associated with low and high %ISCs in the LD and HD fractions, respectively.To investigate the potential role of HbF in the survival of circulating sickle RBCs we determined the percentage of F-cells within the reticulocyte and mature RBC populations using flow cytometry. Analyzing 46 blood samples, we found that the percentage of F+ reticulocytes (%F+Retics) was significantly lower than the percentage of F+ mature RBCs (%F+RBCs) within the same blood sample of all patients, indicating selective advantage of the F+Retics during maturation. We hypothesized that HbF might exert a protective effect by preserving the deformability of RBCs, protecting them from mechanical destruction. To investigate our hypothesis, we designed a microfluidic biochip to evaluate the resistance of F+RBCs undergoing repetitive mechanical stress, flowing through 5 x 5 μm channels (Figure 2). We measured the %F+RBCs in cell suspensions before (input) and after (output) flowing in the chip. We observed a significant increase of %F+RBC population in the output suspensions, indicating that F+RBCs withstand mechanical stress better than non-F-cells. Moreover, we found that high but not low F+RBCs were protected against lysis, indicating that a minimal threshold is needed for HbF to exert its protective role. In line with this finding, we measured the HbF/HbS ratio in the plasma (free hemoglobin) and in RBCs of SCD patients and found that it was much lower in the plasma, providing direct evidence that intravascular hemolysis targets RBCs with no or low HbF.Finally, there was no correlation between the percentage of reticulocytes and the %HbF or the %F+RBCs. However, a negative correlation was found between the percentage of reticulocytes and the %F+Retics, indicating that this latter is a more relevant marker of hemolysis and medullar regeneration than %HbF or %F+RBCs.Our study gives new insights into the role of the mechanical dimension as a critical contributing factor to anemia in SCD. It reveals a cellular mechanism through which HbF exerts its protective role in vivo, showing evidence that high HbF levels protect RBCs from increased density and from hemolysis upon mechanical stress. Our findings pave the way to future clinical studies in which the relationship between HbF expression at the early reticulocyte stage and clinical manifestations could be finely addressed. [Display omitted] DisclosuresBrousse:Add Medica: Membership on an entity's Board of Directors or advisory committees.

HbA2 induction: the merit of pancellularity in sickle cell disease.

HbA2 induction: the merit of pancellularity in sickle cell disease.

Impairment of Cerebrovascular Hemodynamics in Patients With Severe and Milder Forms of Sickle Cell Disease.

In patients with sickle cell disease (SCD), cerebral blood flow (CBF) is elevated to counteract anemia and maintain oxygen supply to the brain. This may exhaust the vasodilating capacity of the vessels, possibly increasing the risk of silent cerebral infarctions (SCI). To further investigate cerebrovascular hemodynamics in SCD patients, we assessed CBF, arterial transit time (ATT), cerebrovascular reactivity of CBF and ATT (CVRCBF and CVRATT) and oxygen delivery in patients with different forms of SCD and matched healthy controls. We analyzed data of 52 patients with severe SCD (HbSS and HbSβ0-thal), 20 patients with mild SCD (HbSC and HbSβ+-thal) and 10 healthy matched controls (HbAA and HbAS). Time-encoded arterial spin labeling (ASL) scans were performed before and after a vasodilatory challenge using acetazolamide (ACZ). To identify predictors of CBF and ATT after vasodilation, regression analyses were performed. Oxygen delivery was calculated and associated with hemoglobin and fetal hemoglobin (HbF) levels. At baseline, severe SCD patients showed significantly higher CBF and lower ATT compared to both the mild SCD patients and healthy controls. As CBFpostACZ was linearly related to CBFpreACZ, CVRCBF decreased with disease severity. CVRATT was also significantly affected in severe SCD patients compared to mild SCD patients and healthy controls. Considering all groups, women showed higher CBFpostACZ than men (p < 0.01) independent of baseline CBF. Subsequently, post ACZ oxygen delivery was also higher in women (p < 0.05). Baseline, but not post ACZ, GM oxygen delivery increased with HbF levels. Our data showed that baseline CBF and ATT and CVRCBF and CVRATT are most affected in severe SCD patients and to a lesser extent in patients with milder forms of SCD compared to healthy controls. Cerebrovascular vasoreactivity was mainly determined by baseline CBF, sex and HbF levels. The higher vascular reactivity observed in women could be related to their lower SCI prevalence, which remains an area of future work. Beneficial effects of HbF on oxygen delivery reflect changes in oxygen dissociation affinity from hemoglobin and were limited to baseline conditions suggesting that high HbF levels do not protect the brain upon a hemodynamic challenge, despite its positive effect on hemolysis.

Epigenetic effects toward new insights as potential therapeutic target in B-thalassemia

BackgroundFetal hemoglobin (HbF) induction has shown promise for the treatment of β-hemoglobinopathies. HbF induction in β-thalassemia could overcome ineffective hematopoiesis and thus terminate transfusion dependency for formerly transfusion dependant patients. Several miRNAs have been found to reactivate γ-globin expression and increase HbF. In this study, we aimed to investigate the expression of 4 miRNAs (miR-15a, miR-16-1, miR-96, and miR-486-3p) in high HbF thalassemia patients and correlate their levels with the patients’ HbF levels then, in order to predict the exact role of the studied miRNAs in hematopoiesis, a bioinformatic analysis was carried out. We went through this bioinformatic analysis to determine the network of genes regulated by miRNAs and further investigate the interaction between all of them through their involvement in hematopoiesis. In this study, the differential expression was measured by qRT-PCR for 40 patients with high HbF and compared to 20 healthy controls. Bioinformatics was conducted involving functional annotation and pathway enrichment analyses. ResultsThe studied microRNAs were significantly deregulated in thalassemia patients in correlation with HbF. Functional annotation and pathway enrichment analyses revealed a major role of miR-486-3p and miR-15a in HbF induction. ConclusionMiR-486-3p and miR-15a are crucial for HbF induction. Further validating studies are needed.

Epigenetic inactivation of ERF reactivates γ-globin expression in β-thalassemia

The fetal-to-adult hemoglobin switch is regulated in a developmental stage-specific manner and reactivation of fetal hemoglobin (HbF) has therapeutic implications for treatment of β-thalassemia and sickle cell anemia, two major global health problems. Although significant progress has been made in our understanding of the molecular mechanism of the fetal-to-adult hemoglobin switch, the mechanism of epigenetic regulation of HbF silencing remains to be fully defined. Here, we performed whole-genome bisulfite sequencing and RNA sequencing analysis of the bone marrow-derived GYPA+ erythroid cells from β-thalassemia-affected individuals with widely varying levels of HbF groups (HbF ≥ 95th percentile or HbF ≤ 5th percentile) to screen epigenetic modulators of HbF and phenotypic diversity of β-thalassemia. We identified an ETS2 repressor factor encoded by ERF, whose promoter hypermethylation and mRNA downregulation are associated with high HbF levels in β-thalassemia. We further observed that hypermethylation of the ERF promoter mediated by enrichment of DNMT3A leads to demethylation of γ-globin genes and attenuation of binding of ERF on the HBG promoter and eventually re-activation of HbF in β-thalassemia. We demonstrated that ERF depletion markedly increased HbF production in human CD34+ erythroid progenitor cells, HUDEP-2 cell lines, and transplanted NCG-Kit-V831M mice. ERF represses γ-globin expression by directly binding to two consensus motifs regulating γ-globin gene expression. Importantly, ERF depletion did not affect maturation of erythroid cells. Identification of alterations in DNA methylation of ERF as a modulator of HbF synthesis opens up therapeutic targets for β-hemoglobinopathies.

The rare hemoglobin variant Hb Mizuho: report of a Swiss family and literature review

Hb Mizuho is a very rare unstable hemoglobin; here, we describe the clinical history of three Swiss family members with Hb Mizuho together with a systematic review of the previously six published cases. The clinical history of the adult woman we report here is unique since this is the first Hb Mizuho presenting with Moyamoya complications and the first case reported with long-term erythrocyte exchange. The literature review showed that Hb Mizuho was mainly reported as a de novo mutation, with the exception of children descended from known cases. All published patients with this unstable hemoglobin showed severe hemolytic anemia with the exception of one; all were regularly transfused. Patients with higher HbF levels might require fewer transfusions. All patients underwent splenectomy at a median age of 4 years and had variable clinical improvement; some achieved complete resolution of transfusion dependency after splenectomy. Iron overload in Hb Mizuho patients seems to be mainly attributed to transfusions and has less to do with ineffective erythropoiesis. Diagnosis might be challenging; a normal hemoglobin electrophoresis should not rule out the diagnosis of unstable hemoglobin in patients with otherwise unexplained hemolytic anemia. This series shows the enormous utility of using molecular techniques for diagnosis.

Incidental Detection of Hemoglobin Variants During Evaluation of HbA1c.

HemoglobinA1c (HbA1c) is used to diagnose Diabetes mellitus and monitor glycemic control over the previous eight to twelve weeks in diabetic patients. Detection of HbA1c by cation exchange-high performance liquid chromatography (CE-HPLC) gives a chromatogram by which abnormal hemoglobin variants are also picked up. Some of these may interfere with HbA1c values affecting clinical management. Due to increased inter-state migration as well as medical tourism, there is a high possibility of finding various hemoglobin variants in any part of India. We did a prospective analysis over 1.5years, of the hemoglobin variants detected during all the HbA1c runs. The HbA1c was tested on Bio-Rad D10 dual HbA2/F/A1c platform, which uses the CE-HPLC method. Every chromatogram was carefully studied to look for unknown peaks. The samples showing unknown peaks >6% were re-run in extended HbA2/F mode to categorize the hemoglobin variants. We had 9595 HbA1c samples, of which 70 cases showed a variant window. There were 40 males and 30 females, age ranging from 28 to 76years. The different hemoglobin variants detected were HbD, HbE and HbS in heterozygous state, high HbF (with a differential diagnosis of HPFH heterozygous and delta-beta thalassemia heterozygous), HbE homozygous, HbQ heterozygous and HbJ heterozygous. We conclude that in the process of monitoring glycemic control using HbA1c, we can also pick-up hemoglobin variants. Hence, it is essential to review HbA1c graphs, so that the diagnosis of hemoglobin variants is not missed and the HbA1c reported is reliable.

DNA methylation patterns of \u03b2-globin cluster in \u03b2-thalassemia patients

BackgroundReactivation of fetal hemoglobin (HbF, α2γ2) holds a therapeutic target for β-thalassemia and sickle cell disease. Although many HbF regulators have been identified, the methylation patterns in β-globin cluster driving the fetal-to-adult hemoglobin switch remains to be determined.ResultsHere, we evaluated DNA methylation patterns of the β-globin cluster from peripheral bloods of 105 β0/β0 thalassemia patients and 44 normal controls. We also recruited 15 bone marrows and 4 cord blood samples for further evaluation. We identified that the CpG sites in the locus control region (LCR) DNase I hypersensitive site 4 and 3 (HS4-3) regions, and γ- and β-globin promoters displayed hypomethylation in β0/β0-thalassemia patients, especially for the patients with high HbF level, as compared with normal controls. Furthermore, hypomethylations in most of CpG sites of the HS4-3 core regions were also observed in bone marrows (BM) of β0/β0-patients compared with normal controls; and methylation level of γ-globin promoter -50 and + 17 CpG sites showed lower methylation level in patients with high HbF level compared with those with low HbF level and a negative correlation with HbF level among β0-thalassemia patients. Finally, γ-globin promoter + 17 and + 50 CpG sites also displayed significant hypomethylation in cord blood (CB) tissues compared with BM tissues from normal controls.ConclusionsOur findings revealed methylation patterns in β-globin cluster associated with β0 thalassemia disease and γ-globin expression, contributed to understand the epigenetic modification in β0 thalassemia patients and provided candidate targets for the therapies of β-hemoglobinopathies.

Fetal Hemoglobin in Sickle Hemoglobinopathies: High HbF Genotypes and Phenotypes.

Fetal hemoglobin (HbF) usually consists of 4 to 10% of total hemoglobin in adults of African descent with sickle cell anemia. Rarely, their HbF levels reach more than 30%. High HbF levels are sometimes a result of β-globin gene deletions or point mutations in the promoters of the HbF genes. Collectively, the phenotype caused by these mutations is called hereditary persistence of fetal hemoglobin, or HPFH. The pancellularity of HbF associated with these mutations inhibits sickle hemoglobin polymerization in most sickle erythrocytes so that these patients usually have inconsequential hemolysis and few, if any, vasoocclusive complications. Unusually high HbF can also be associated with variants of the major repressors of the HbF genes, BCL11A and MYB. Perhaps most often, we lack an explanation for very high HbF levels in sickle cell anemia.

MicroRNA expression patterns in HbE/β-thalassemia patients: The passwords to unlock fetal hemoglobin expression in β-hemoglobinopathies

Hemoglobin E (HbE)/β-thalassemia is a form of β-hemoglobinopathy that is well-known for its clinical heterogeneity. Individuals suffering from this condition are often found to exhibit increased fetal hemoglobin (HbF) levels – a factor that may contribute to their reduced blood transfusion requirements. This study hypothesized that the high HbF levels in HbE/β-thalassemia individuals may be guided by microRNAs and explored their involvement in the disease pathophysiology. The miRNA expression profile of hematopoietic progenitor cells in HbE/β-thalassemia patients was investigated and compared with that of healthy controls. Using miRNA PCR array experiments, eight miRNAs (hsa-miR-146a-5p, hsa-miR-146b-5p, hsa-miR-148b-3p, hsa-miR-155-5p, hsa-miR-192-5p, hsa-miR-335-5p, hsa-miR-7-5p, hsa-miR-98-5p) were identified to be significantly up-regulated whereas four miRNAs (hsa-let-7a-5p, hsa-miR-320a, hsa-let-7b-5p, hsa-miR-92a-3p) were significantly down-regulated. Target analysis found them to be associated with several biological processes and molecular functions including MAPK and HIF-1 signaling pathways – the pathways known to be associated with HbF upregulation. Results of dysregulated miRNAs further indicated that miR-17/92 cluster might be of critical importance in HbF regulation. The findings of our study thus identify key miRNAs that can be extrinsically manipulated to elevate HbF levels in β-hemoglobinopathies.

The Grndad Registry: Contemporary Natural History Data and an Analysis of Real-World Patterns of Use and Limitations of Disease Modifying Therapy in Adults with SCD

The Grndad Registry: Contemporary Natural History Data and an Analysis of Real-World Patterns of Use and Limitations of Disease Modifying Therapy in Adults with SCD

Combinatorial Administration of Pharmacological Inhibitors Targeting DNMT1 and KDM1A Produces Synergistic and Additive Increases in F Retiulocytes and γ-Globin Expression in Normal Baboons

Sickle cell disease (SCD) is an inherited blood disorder that severely impacts the quality of life and reduces the life expectancy of approximately 100,000 patients in the U.S. and millions worldwide. Elevated levels of Fetal Hemoglobin (HbF; α2γ2) reduce the severity of symptoms and lengthen the life span of patients with sickle cell disease by inhibiting deoxy HbS polymerization. Hydroxyurea (HU), the only FDA-approved drug shown to increase HbF, is not effective in a subset of patientsand importantly, in the refractory individuals the increased HbFis heterogeneously distributed within the erythrocyte population resulting in a large fraction of erythrocytes lacking protective levels. Simian primates such as the baboon are widely acknowledged as the best animal models for testing the ability of new drugs to increase γ-globin expression because results in baboons are predictive of effects in man due to conservation of the structure and developmental stage-specific regulation of the β-like globin genes in simian primates. Our previous studies in baboons have shown that the most powerful drugs for reactivation of HbF are DNMT1 and KDM1A inhibitors targeting enzymes located within the co-repressor complexes that act to establish and maintain repressive epigenetic modifications at the γ-globin promoter to repress γ-globin expression. Treatment of baboons with either decitabine, an inhibitor of DNMT1, or RN-1, an inhibitor of KDM1A, strongly stimulates HbF. Dose-limiting effects of these drugs include neutropenia, thrombocytopenia, and/or thrombophilia. In this investigation we have explored the hypothesis that a treatment regimen comprised of both decitabine and RN-1 could achieve additive or synergistic effects on HbF while minimizing adverse hematological side effects. Three baboons (PA8695, PA8697, PA8698) were treated with two courses of different doses of RN-1 (0.25 and 0.125mg/kg/d) administered subcutaneously for 3 days to select an optimal RN-1 dose for each animal. The response to RN-1 varied between individual animals and was associated with pretreatment levels of γ-globin expression. Higher pretreatment levels of γ-globin expression were observed in PA8697 (0.08 γ/γ+β) compared to PA8695 (0.014+0.005 γ/γ+β) and PA8698 (0.004+0.0007 γ/γ+β) and the effect on γ-globin expression in PA8697 (0.24+0.5 γ/γ+β) at the 0.125mg/kg/d dose was greater than the effect of 0.25 mg/kg/d dose in PA8695 (0.17+0.05 γ/γ+β) and PA8698 (0.121+0.006 γ/γ+β). For combinatorial studies with decitabine, an RN-1 dose of 0.125mg/kg/d was chosen for PA8697 and 0.25mg/kg/d for PA8695 and 8698. Decitabine (0.3mg/kg/d; 2d), administered as a single agent subcutaneously for two days increased F retics nearly 3 fold (23.6+12.5%; p&amp;lt;0.02) compared to pretreatment levels (8.26+9.90%) but changes in γ-globin expression (0.066+0.076 γ/γ+β) compared to the pretreatment levels (0.03+0.042 γ/γ+β) were not significant. For combinatorial experiments, decitabine (0.3mg/kg/d) was administered for 2 days followed by administration of RN-1 (0.25mg/kg/d PA8695 and 8698; 0.125mg/kg/d PA8697) for 3 days. This staggered dose schedule was used to avoid any possible effects of RN-1 on cell cycle kinetics that could potentially reduce incorporation of decitabine during S phase. The decitabine/RN-1 combination produced significantly higher increases in F retics (63.0+23.6%; p&amp;lt;0.002) than either RN-1 (29.9+27.1%) or decitabine (23.6+12.5%) alone. The effect of the decitabine/RN-1 combination on F retics was greater than additive (p&amp;lt;0.01; Figure 1A) in the two low HbF animals, PA8695 and 8698, and additive for the high HbF animal PA8697 (Figure 1B). Although increased levels of γ-globin expression produced by the decitabine/RN-1 combination (0.22+0.08 γ/γ+β) were not significantly different overall from those induced by RN-1 alone (0.178+0.06 γ/γ+β), individual analysis of PA8697 revealed an additive effect (Figure 1C). Plt and ANC remained with the normal range. In conclusion, targeting separate components of the γ-globin co-repressor complex by combinatorial administration of decitabine and RN-1 generated synergistic and additive increases in F retics and γ-globin expression in the absence of hematological toxicity. Future experiments will explore different doses, alternative schedules, and the effect of longer term treatment regimens. Disclosures Saunthararajah: EpiDestiny: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties.

Significance of heme oxygenase-1(HMOX1) gene on fetal hemoglobin induction in sickle cell anemia patients

Though the patients with sickle cell anemia (SCA) inherit same genetic mutation, they show considerable phenotypic heterogeneity. It has been observed that patients with elevated fetal hemoglobin (HbF) levels have a relatively mild clinical course. There is sparse literature on the association of higher HbF levels leading to reduction in the oxidative stress in SCA patients. Hence in this study, the significance between the HMOX1 gene polymorphisms and the HbF levels has been studied. Preliminary screening was carried out. Genotyping of 3 variants in the HMOX1 gene was performed in 90 SCA patients and 50 healthy controls by PCR–RFLP, GeneScan and direct DNA sequencing. It was observed that SCA patients with higher HbF levels, showed improved hematological indices with an inverse effect on HbS levels. The TT genotype of rs2071746 (A→T) polymorphism was found to be associated with elevated HbF levels (P: 0.012). Also, the long form (> 25 GT repeats) of rs3074372 (GT)n repeats was found to be linked with increased HbF levels. We could not find any association of rs2071749 (A→G) polymorphism with the HbF levels. As, the sickle cell anemia patients show significant oxidative stress due to hemolysis, the study of polymorphisms in the HMOX1 gene may act as a potential independent marker for elevated HbF levels.

Investigation of miR-144-3p expression levels in HbSS cases with high and normal HbF

Abstract Background Sickle cell anemia (HbSS) is a hereditary blood disease that affects millions of people worldwide. Increased the HbF levels affects the clinical course of HbSS positively. The aim of this study was to investigate the association between miR-144-3p and HbSS. Materials and methods In this study 84 cases (44 HbSS/HbS combination and 40 HbAA) were performed. The expression of miR-144-3p was determined by RT-PCR. Statistical analysis was performed by the Mann-Whitney U test (SPSS 20.00 for Windows and p&lt;0.005). Results The miR-144-3p expression levels were higher in the HbSS cases (p≤0.001). Additionally, it was determined that the expression of miR-144-3p was higher in the cases with HbF&lt;3 (p=0.043). Discussion In our study, the increase in the miR-144-3p levels in low HbF levels may be associated with the severity of the disease. Conclusion Considering these results, suppressing miR-144-3p may be considered as a new treatment option in HbSS.

Identifying genetic variants and pathways associated with extreme levels of fetal hemoglobin in sickle cell disease in Tanzania

BackgroundSickle cell disease (SCD) is a blood disorder caused by a point mutation on the beta globin gene resulting in the synthesis of abnormal hemoglobin. Fetal hemoglobin (HbF) reduces disease severity, but the levels vary from one individual to another. Most research has focused on common genetic variants which differ across populations and hence do not fully account for HbF variation.MethodsWe investigated rare and common genetic variants that influence HbF levels in 14 SCD patients to elucidate variants and pathways in SCD patients with extreme HbF levels (≥7.7% for high HbF) and (≤2.5% for low HbF) in Tanzania. We performed targeted next generation sequencing (Illumina_Miseq) covering exonic and other significant fetal hemoglobin-associated loci, including BCL11A, MYB, HOXA9, HBB, HBG1, HBG2, CHD4, KLF1, MBD3, ZBTB7A and PGLYRP1.ResultsResults revealed a range of genetic variants, including bi-allelic and multi-allelic SNPs, frameshift insertions and deletions, some of which have functional importance. Notably, there were significantly more deletions in individuals with high HbF levels (11% vs 0.9%). We identified frameshift deletions in individuals with high HbF levels and frameshift insertions in individuals with low HbF. CHD4 and MBD3 genes, interacting in the same sub-network, were identified to have a significant number of pathogenic or non-synonymous mutations in individuals with low HbF levels, suggesting an important role of epigenetic pathways in the regulation of HbF synthesis.ConclusionsThis study provides new insights in selecting essential variants and identifying potential biological pathways associated with extreme HbF levels in SCD interrogating multiple genomic variants associated with HbF in SCD.

Genome editing strategies for fetal hemoglobin induction in beta-hemoglobinopathies.

Genome editing to correct a defective β-globin gene or induce fetal globin (HbF) for patients with beta-hemoglobinopathies has the potential to be a curative strategy available to all. HbF reactivation has long been an area of intense interest given the HbF inhibition of sickle hemoglobin (HbS) polymerization. Patients with HbS who also have high HbF tend to have less severe or even minimal clinical manifestations. Approaches to genetically engineer high HbF include de novo generation of naturally occurring hereditary persistence of fetal hemoglobin (HPFH) mutations, editing of transcriptional HbF repressors or their binding sites and/or regulating epigenetic intermediates controlling HbF expression. Recent preclinical and early clinical trial data show encouraging results; however, long-term follow-up is lacking, and the safety and efficacy concerns of genome editing remain.

Genetic variants in the G gamma-globin promoter modulate fetal hemoglobin expression in the Colombian population.

Fetal hemoglobin (HbF) is a determining factor for the development of sickle cell anemia. High HbF levels lower the intensity of symptoms of this disease. HbF levels can vary in patients with sickle cell anemia and individuals without the disease. The purpose of this study was to identify the genetic variants in the G gamma-globin gene promoter that can modulate HbF expression in patients with sickle cell anemia and healthy individuals from Colombia. In total, 413 bp of the G gamma-globin gene promoter were sequenced in 60 patients with sickle cell anemia and 113 healthy individuals. The allelic and genotype frequencies of the identified variants were compared between individuals with low and high HbF for both patients and healthy individuals. In total, we identified 15 variants in both groups, only three of which were shared between patients and healthy individuals. In healthy individuals, sites -16 and -309 (rs112479156) exhibited differences in allele frequencies. The mutant allele of -16 lowered the production of HbF, whereas the mutant allele of -309 increased its production. These results reveal the presence of different mechanisms of HbF regulation between patients with sickle cell and healthy individuals.

The Effects of Hydroxyurea Therapy on the Six-Minute Walk Distance in Patients with Adult Sickle Cell Anemia: An Echocardiographic Study.

BackgroundThe impact of hydroxyurea (HU) medication as treatment of choice has not been evaluated in adult sickle cell anemia (SCA) patients in terms of the 6-min walk distance (6 MWD).AimThe aim of the study was evaluating the effects of HU on the 6 MWD, serum brain natriuretic peptide (NT-pro BNP) level, and pulmonary hypertension (PH) measured by tricuspid regurgitation velocity (TRV).MethodsIn this cross-sectional, prospective study, 110 patients with homozygous SCA were studied and compared with age- and gender-matched healthy controls. Every patient was investigated via pulsed and tissue Doppler echo evaluation, 6-min walk test (6 MWT), and blood level for the level of NT-pro-BNP hormone. Data were compared in patients with (n = 59; group 1, G1) and without (n = 51; group 2, G2) HU medication. Pearson correlation analysis was applied and clinical follow-up for the frequency of acute chest syndrome (ACS). Analysis of variance (ANOVA) multivariate statistical analysis was applied between groups.ResultsIn the study, 110 patients with SCA were studied and compared with 110 control patients. Patients in G1 compared with G2 had a longer 6 MWD (491 ± 64.4 m vs 428.6 ± 54.3 m, p < 0.005), higher HbF% (21 ± 2.5% vs 8 ± 1.8%, p < 0.005), and lower NT-pro-BNP level (314.1 ± 27.5 pmol/L vs 407 ± 18.9 pmol/L, p = 0.05). The mean TRV values were 2.8 ± 0.5 m/s in G1 versus 3.4 ± 0.4 m/s in G2, p < 0.005, and 1.5 ± 0.7 m/s in the control group. The high probability of PH based on a TRV > 3.4 m/s was 10.1% in G1 versus 17.6% in G2 and 3.6% in the control. There were weak positive correlations between NT-pro-BNP and TRV (r = 0.264; p = 0.005) and HbF% and 6 MWD (r = 0.452; p = 0.001). After 12 months of follow-up, frequency of acute chest syndrome (ACS) was twice as high in G2, at 32 patients, versus 16 in G1.ConclusionPatients with SCA on HU medication compared with no HU had significantly longer 6 MWD, lower level of NT-pro-BNP, higher HbF% level. After 1-year follow-up HU patients had less frequency of ACS. There were significant positive correlations between the level of NT-pro BNP level and TRV in m/s on echo.