High HbF Levels Research Articles

Structural and Functional Insights on an Uncharacterized Aγ-Globin-Gene Polymorphism Present in Four β0-Thalassemia Families with High Fetal Hemoglobin Levels

Several DNA polymorphisms have been associated with high production of fetal hemoglobin (HbF), although the molecular basis is not completely understood. In order to identify and characterize novel HbF-associated elements, we focused on five probands and their four families (from Egypt, Iraq and Iran) with thalassemia major (either β(0)-IVSII-1 or β(0)-IVSI-1) and unusual HbF elevation (>98 %), congenital or acquired after rejection of bone marrow transplantation, suggesting an anticipated favorable genetic background to high HbF expression. Patient recruitment, genomic DNA sequencing, western blotting, electrophoretic mobility shift assays, surface plasmon resonance (SPR) biospecific interaction analysis, bioinformatics analyses based on docking experiments. A polymorphism of the Aγ-globin gene is here studied in four families with β(0)-thalassemia (β(0)-IVSII-1 and β(0)-IVSI-1) and expressing unusual high HbF levels, congenital or acquired after rejection of bone marrow transplantation. This (G→A) polymorphism is present at position +25 of the Aγ-globin genes, corresponding to a 5'-UTR region of the Aγ-globin mRNA and, when present, is physically linked in chromosomes 11 of all the familiar members studied to the XmnI polymorphism and to the β(0)-thalassemia mutations. The region corresponding to the +25(G→A) polymorphism of the Aγ-globin gene belongs to a sequence recognized by DNA-binding protein complexes, including LYAR (Ly-1 antibody reactive clone), a zinc-finger transcription factor previously proposed to be involved in down-regulation of the expression of γ-globin genes in erythroid cells. We found a novel polymorphism of the Aγ-globin gene in four families with β(0)-thalassemia and high levels of HbF expression. Additionally, we report evidence suggesting that the Aγ-globin gene +25(G→A) polymorphism decreases the efficiency of the interaction between this sequence and specific DNA binding protein complexes.

Prognostic Factors for Hospitalization of Children with Sickle Cell Anemia Treated with Hydroxyurea at Maximum Tolerated Dose

Background: Hydroxyurea (HU) is recommended to be offered to children with sickle cell anemia (SCA; HbSS and HbSβ0 thalassemia) beginning at 9 months of age (Yawn, JAMA 2014). Intensifying HU to a maximum tolerated dose (MTD) provides fetal hemoglobin (HbF) levels of >20% (Ware, Blood 2010). Yet, children treated with HU at MTD still experience hospitalizations due to SCA-related complications (Nottage, PLoS One 2013). The ability to identify children at high risk of experiencing SCA-related complications while on HU would enhance clinical management and facilitate performance of clinical trials of novel agents. Our objective was to identify risk factors associated with hospitalization and to develop a prediction model for hospitalization of children treated with HU at MTD.Methods: The Hydroxyurea Study of Long-Term Effects (HUSTLE) is a prospective observational study (NCT00305175) designed to describe the clinical effects of HU in children with SCA. We have analyzed children who initiated HU per protocol and whose dose was escalated to MTD. Laboratory and clinical data were abstracted at baseline and at 9-12 months after enrollment. The first laboratory values of each interval were considered representative of the period. The primary outcome measure was hospitalization. After a multivariate regression logistic model identified risk factors for hospitalization based on a stepwise selection strategy, a smoothed receiver-operating-characteristic (ROC) curve was generated to predict hospitalization for the composite model and for each individual risk factor using the method of maximum-likelihood fitting of univariate distriubutions. Atlhough HbF was not identified in regression analysis among risk factors, because of its known effects as a disease modifier, it was inserted into the composite model. A prediction model was then generated for the identified risk factors utilizing optimal cutoff values defined by the maximum Youden method.Results: 151 children (mean [SD] age, 8.3 [5.0] years) were analyzed. HU resulted in higher HbF levels (22.6% [9.1] versus 10.1% [7.1]; p<0.0001), lower absolute neutrophil counts (4000/mm3 [2600] versus 7000/mm3 [3800]; p<0.0001), and fewer hospitalizations (7.8% [10/128] versus 28.9% [39/128]; p<0.0001) after 9-12 months compared to baseline.Children hospitalized after 9-12 months of therapy had similar HbF levels compared to those not hospitalized (OR 0.98 [95% CI: 0.9-1.1]; p=0.5), but higher total bilirubin (BiliT) (OR 1.3 [95% CI: 1.0-1.6]; p=0.03), blood urea nitrogen (BUN) (OR 1.3 [95% CI: 1.1-1.7]; p=0.19), and lactate dehydrogenase (LDH) (OR 1.01 [95% CI: 1.002-1.010]; p=0.006) levels. A ROC curve including insertion of HbF showed an area under the curve (AUC) of 0.90 (Figure 1). Optimal cutoffs for risk factors were: BiliT 1.05 mg/dL, BUN 8.5 mg/dL, and LDH 473 units/L. The probability of hospitalization varied in relation to identified risk factors (Table 1).Table 1Predicted probability of hospitalization after 9-12 months of HU at MTD# of risk factorsBiliTHbFBUNLDHProbability of≥1.05(mg/dL)≤20 (%)≥8.5(mg/dL)≥473(units/L)Hospitalization (%)0NoNoNoNo<11YesNoNoNo<1NoYesNoNo1.3NoNoYesNo5.8NoNoNoYes192YesYesNoNo<1YesNoYesNo<1YesNoNoYes2.2NoYesYesNo10NoYesNoYes29NoNoYesYes653YesYesYesNo1.1YesYesNoYes3.8YesNoYesYes15NoYesYesYes774YesYesYesYes25LDH, lactate dehydrogenase; BiliT, total bilirubin; BUN, blood urea nitrogenDiscussion: In this pediatric cohort, HU therapy at MTD produced a robust laboratory response with most children having HbF >20% after 9-12 months. At such high levels of HbF, few children experienced a hospitalization due to its protective effects; however, we identified three laboratory parameters (LDH, BiliT, and BUN) that were predictive risk factors for children who continued to be hospitalized despite high levels of HbF. After 9-12 months of HU therapy at MTD the probability of hospitalization ranged from <1% to 65% for children with HbF of less than <20% and <1% to 77% in children with HbF of >20%. This model may help identify children at risk for complications of SCA while being treated with HU at MTD. [Display omitted] DisclosuresEstepp:Eli Lilly and Company: Research Funding; Daiichi Sankyo: Research Funding. Off Label Use: Off label use of hydroxyurea in children with sickle cell anemia.

Impact of Hydroxyurea on Sickle Cell Patients Managed in a Community Medical Center

Background: Sickle Cell Disease (SCD), including the subtypes of HbSS(SS) or Hb S beta zero thalassemia(SB0) are characterized by frequent vasoocclusive pain crises (VOC) requiring numerous visits to emergency departments and admissions to the hospital. A NHLBI expert panel recommends that patients(pts) with SCD receive hydroxyurea(H) therapy to improve clinical outcomes. H, approved by the FDA in 1998 for treatment of clinically severe SCA is the only disease modifying agent for sickle cell disease. Its primary mechanism of action is induction of fetal hemoglobin (HbF) which in turn causes inhibition of sickling. As a part of our adult sickle cell comprehensive care program, aggressive education and counseling has been utilized to ensure that eligible adults with SCD are treated with H. The clinical benefits of this program were tracked for its impact on blood parameters and utilization of health care services.Methods: Pts with SS/SB0 over the age of 18 years attending a comprehensive SCD program were tracked for use of H, clinic and ED visits, hospitalizations and transfusions over a period of 12 months. Lab parameters monitored included WBC, Hemoglobin(Hb), MCV , platelets(plts), Hb F level. Dose of H in the steady state is reported in mg/kg . Education about the benefits and side effects was provided utilizing a newly created video, one on one counselling and educational group programs. As part of the yearly visits, H education and compliance were reinforced. Pts were excluded if attempting pregnancy, currently pregnant or breast feeding.Results: We identified 169 pts of which 135 were evaluable with complete data. 89/135 (66%) were on H and 46/135 (34%) not on H. The major barrier to not taking H was patient refusal. Pts on H were divided into two groups, Hb F < 15%(n=54) and Hb F >15% (n=35); 66/89 (75%) were SS , 22/89(25%) SB0. As expected, MCV and HB were higher in pts on H and WBC and plts were lower in pts on H and correlated with higher HbF levels (See Table 1).Table 1Not on H(n=46)Pts on H with HbF<15% ( n=54)Pts on H with HbF >15%( n=35)WBC(K/uL)11.39.68.8HB(g/dl)8.48.49.1MCV(fL)90.7102113.3Platelets(K/uL)367320308HbF (%)5.57.618.8H dose (mg/kg)N/A22.618.6The number of ED visits was significantly lower on H. Admissions trended lower on H but were not statistically different (see Table 2).Table 2Pts on HPts not on HP valueER visits (mean)1.363.150.0206Hospital Admissions (mean)3.053.410.6261Clinic Visits (mean)6.105.890.7382Pts on H had similar ED, clinic visits and admissions with HbF levels of <15% and >15%.The number of pts transfused were as follows: pts not on H 25/46(54%); on H 40/89(45%); and pts with HbF<15% -28/54(52%), while HbF>15%- 12/35(34%).Conclusion: Aggressive education in a community comprehensive care clinic has allowed for a majority of eligible patients with SS/SB0 to utilize H. Use of H has significantly reduced ED visits suggesting uncomplicated VOC can be reduced utilizing H in a community setting .Transfusions were reduced in patients on H especially those with HbF >15%. Mean H dosing did not correlate with higher HbF levels, which raises the question of compliance in some pts. Continued education, monitoring of prescriptions filled and possible higher doses of H may be beneficial in reducing hospitalizations. DisclosuresNo relevant conflicts of interest to declare.

Investigation of Two Combination HbF Induction Regimens, RN-1 and Hydroxyurea Versus RN-1 and Decitabine, in a Humanized Sickle Cell Mouse Model

Sickle cell disease (SCD) results from the substitution of glutamic acid for valine at the sixth amino acid of β-globin protein, leading to the formation of the abnormal hemoglobin S (HbS). Following deoxygenation in red blood cells (RBCs), HbS forms polymers causing the RBCs to become deformed and adherent leading to vaso-occlusive events resulting in splenic infarction, kidney failure, stroke, painful crises, chronic anemia, inflammation and decreased survival. Hydroxyurea, the only drug currently FDA approved for SCD, is not effective in some patients and, therefore, new combination regimens are needed. RN-1, a new drug targeting the histone demethylase LSD-1, is a more effective inducer of γ-globin expression in sickle cell mice and HbF in baboons (P. anubis). Decitabine(DAC), a DNMT1 inhibitor, is well known to induce high levels of HbF in non-human primates and SCD patients and is currently in clinical trials. We investigated the effect of two combination regimens: RN-1 in combination with hydroxyurea vs. RN-1 in combination with DAC. The knock-in humanized sickle cell mice (B6;129-Hbatm1(HBA)Tow Hbbtm2(HBG1,HBB*)Tow/J) were treated with 1) DMSO (vehicle control), 2) RN-1 (5mg/kg), 3) DAC(0.25mg/kg) and 4) RN-1(5mg/kg) + DAC(0.5mg/kg) for 10 days. Analysis of F-retics and γ-globin mRNA performed on d10 showed F-retics were increased two fold (p=0.01) in mice treated with RN-1(5mg/kg) or DAC(0.25mg/kg) compared to DMSO-treated. γ-globin mRNA levels (γ/γ+β) were increased three fold in RN-1(5mg/kg) or DAC(0.25mg/kg) compared to DMSO. The combination of RN-1(5mg/kg) and DAC(0.25mg/kg) did increase F-retics and mRNA levels compared to mice treated with RN-1 alone(p=0.02). Also, the RN-1 and DAC combination led to neutropenia and thrombocytopenia. We next examined the effect of RN-1(5mg/kg) and HU (100mg/kg) in combination. Knock-in humanized sickle cell mice (B6;129-Hbatm1(HBA)Tow Hbbtm2(HBG1,HBB)Tow/J) were treated with 1) DMSO (vehicle) 2) RN-1(5mg/kg) and 3) RN-1(5mg/kg) + HU (100mg/kg). While RN-1 in combination with HU did not significantly increase F-cells or F-retics, there was a significant decrease in spleen weight [(DMSO: 1.17±0..06g; RN1:0.71± 0.1g; RN1+HU: 0.46±0.05g)(p=0.004:p=0.02)] and percentage of sickle cells in a peripheral blood smear.[(DMSO: 8.4 ±1.3%; RN-1:4.1± 0.2%; RN1+HU: 1.57±0.6%)(p=0.006:p-0.014)]. We conclude that further studies to determine the effect of RN-1 in combination with HU or DAC in the sickle cell mouse model and cultures from sickle cell CD34+ cells are needed to evaluate its potential use in the treatment of sickle cell disease. These results suggest that RN-1 in combination with hydroxyurea may be a useful therapeutic regimen. DisclosuresNo relevant conflicts of interest to declare.

Hypersegmented Neutrophil Percentage Using Automated Digital Cell Morphology: A Simple Laboratory Parameter to Monitor Hydroxyurea Therapy in Sickle Cell Disease Patients

Fetal hemoglobin (HbF) induction with hydroxyurea (HU) remains the only specific pharmacological treatment for sickle cell disease (SCD) patients. Compliance is key to achieve high HbF levels, but assessing patient adherence to long-term treatment is difficult. Since patient interviews are often unreliable, most hematologists rely on mean corpuscular volume (MCV) and HbF levels to monitor compliance, but red blood cell indices change slowly. Hypersegmented neutrophils (HN) have been for long recognized in patients taking HU, and automated digital cell morphology platforms allow routine peripheral blood smears to be conveniently photographed and stored for analysis. We have designed a protocol to determine the percentage of circulating HN and have studied how this parameter compares with others commonly used in clinical practice during HU therapy, such as HbF, MCV, and reticulocyte count.We collected blood samples from 38 patients with SCD in steady state, receiving an average dose of 20.5mg/kg/day of HU (range 10.2-33.3) at our Hematology outpatient clinic. Complete blood counts and HN percentage determinations were performed on a Sysmex XE-5000/Cellavision DM96 equipment. HbF levels were determined by HPLC (Bio-Rad). Statistical analysis was performed using GraphPad Prism 6.0 software. Standard protocol for image acquisition initially recorded 100 cells per smear and HN count was performed independently by two experienced laboratory personnel using the same image database, by counting cells with 5 or more nuclear segments. To increase precision due to the relatively low numbers of neutrophils in some samples, analysis was subsequently improved by acquiring 300 images per smear aiming to picture 100 neutrophils.Mean hemoglobin level in our population was 8.6±1.3g/dL (mean±SD), MCV 103.8±14.2fL, reticulocyte count 222,950±129,090/uL, and HbF was 13.1±7.8% (range 2.1-30.9%). HN percentage as determined with 100 images per smear displayed only borderline correlation with HbF levels (P =0.094), but acquisition of 300 images per smear yielded over 90 neutrophil images in 35/38 samples. Average HN percentage was 14,5% (range 2,0-45,0%) and correlated positively with HbF (r=0.4172, P =0.009) and MCV (r=0.4301, P =0.007). As expected, HbF also correlated with MCV (r=0.5777, P =0.0001) and reticulocyte count (r=-0.489, P =0.003). Despite our limited number of patients, ROC curve analysis showed that HN percentage had an area under curve of 0.7241 to detect patients with HbF>20% (P =0.047). Patients with more than 6 segments per neutrophil were also more likely to have higher HbF. Average daily HU dose did not correlate with HbF or any of the parameters analyzed.Lack of correlation between dose and HbF supports that medication adherence may indeed be suboptimal in the study population, but that may also be caused by individual differences in HU metabolism. While MCV still displayed the strongest correlation with HbF in our study, HN percentage performed similarly, with higher counts associated with higher HbF. Neutrophils have a much shorter half-life than erythrocytes, with an estimated half-life of hours rather than the 20 days calculated for sickle red blood cells. Therefore, digital cell morphology analysis enables clinical laboratories to determine HN counts that can change more quickly than HbF, MCV, or reticulocyte counts during HU therapy. Monitoring the number of HN may allow more timely assessment of compliance in patients starting HU or in those in need for HU in combination with sporadic blood transfusions, since neutrophil hypersegmentation should not be affected by changes in red blood cell mass. Further studies should investigate HN percentage as a potential surrogate marker of response to HU and of patient compliance.Financial support: FAPESP DisclosuresNo relevant conflicts of interest to declare.

Risk factor analysis of cerebral white matter hyperintensities in children with sickle cell disease.

Sickle cell disease (SCD) is complicated by silent cerebral infarcts, visible as white matter hyperintensities (WMHs) on magnetic resonance imaging (MRI). Both local vaso-occlusion, elicited by endothelial dysfunction, and insufficiency of cerebral blood flow (CBF) have been proposed to be involved in the aetiology. We performed an explorative study to investigate the associations between WMHs and markers of endothelial dysfunction and CBF by quantifying WMH volume on 3.0 Tesla MRI. We included 40 children with HbSS or HbSβ(0) thalassaemia, with a mean age of 12.1 ± 2.6 years. Boys demonstrated an increased risk for WMHs (odds ratio 4.5, 95% confidence interval 1.2-17.4), unrelated to glucose-6-phosphate dehydrogenase deficiency. In patients with WMHs, lower fetal haemoglobin (HbF) was associated with a larger WMH volume (regression coefficient = -0.62, R2 = 0.5, P = 0.04). Lower ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) levels were associated with lower CBF in the white matter (regression coefficient = 0.07, R2 = 0.15, P = 0.03), suggesting that endothelial dysfunction could potentially hamper CBF. The findings of our explorative study suggest that a high level of HbF may be protective for WMHs and that endothelial dysfunction may contribute to the development of WMHs by reducing CBF.

Sickle Cell Disease: Reappraisal of the Role of Foetal Haemoglobin Levels in the Frequency of Vaso-Occlusive Crisis.

Foetal haemoglobin has been implicated in the modulation of sickle cell crisis. Its level is generally inversely proportional to the severity of sickle cell disease (SCD) for a given sickle cell phenotypes. The main aim of therapy for vaso-occlusive crisis (VOC), which is the hallmark of SCD, is to reduce the chances of sickling through the prevention of polymerization of HbS. One way of preventing this polymerization is by increasing foetal haemoglobin levels. To determine the relationship between HbF levels and the frequency of crisis in SCD patients in Ghana. A longitudinal retrospective survey covering a period of 30 months was carried out on adult SCD patients at the Sickle Cell Clinic of the Korle-Bu Teaching Hospital. Eighty-three adults aged 15 to 65 years made up of 40 males and 43 femalea were studied. Analysis of variance (ANOVA) gave significant results in Hb and HbF levels. Higher HbF levels were positively related to less frequent crisis and were significantly high in SCD patients than in controls. HbF effects on the clinical manifestations on SCD were variable. Threshold values of HbF play a role in reducing the frequency of vaso-occlusive crisis in SCD patients and this finding contributes to the body of available literature on SCD severity. However our work does not give the apparent threshold level of helpful HBF Level in SCD.

Thalassemia Modular Stratification System for Personalized Therapy of Beta-Thalassemia (THALAMOSS)

Thalassemia Modular Stratification System for Personalized Therapy of Beta-Thalassemia (THALAMOSS)

AB045. Molecular markers for disease severity in beta thalassemia/Hb E disease

Thalassemia is a hereditary disease affecting hemoglobin synthesis, characterized by microcytic hypochromic anemia. Homozygote or compound heterozygote patients usually manifested as thalassemia major which require regular treatment. There are five functional genes arranged in the order 5' e-Gγ-Aγ-ψβ-δ-β 3' that are activated during development. Expression of the individual genes within the β-globin cluster is controlled by the complex interactions between local regulatory sequence (promoter regions) within each gene and the β-locus control region (β-LCR), located 6-18 kb upstream of the e-globin gene. Beta thalassemia (β-thal) is a very heterogeneous disorder due to variations in inactivation mechanism of the β-genes. Point mutations and small deletions or insertions in the nucleotide sequences are the main molecular defects responsible for most β-thalassemia. In spite of seemingly identical genotypes, severity of β-thal patients can vary greatly. This heterogeneity in the clinical severity may occur from the nature of β-globin gene mutation, α-thalassemia (α-thal) gene interaction and difference in the amount of Hb F production that is partly associated with a specific β-globin haplotype. Co-inheritance of α-thal may ameliorate the severity of β-thal disease in those cases with mild β-thal genotypes. However, many patients who are β°/β+ thal or β°-thal/Hb E do not have a detectable α-thal haplotype but still have a mild clinical symptom suggests that there are other additional factors responsible for the mildness of the disease. Inheritance of a β-thal chromosome with the Xmn I+ haplotype at the position -158 of the Gγ-globin gene was found to be associated with increased Hb F production and milder anemia in patients with thalassemia intermedia and Xmn I +/+ haplotype is necessary to produce a significant clinical effect. Homozygosity for the Xmn I + haplotype, +/+, was also found in the mild cases of β-thal/Hb E. However, there is no severity difference among homozygous β-thal patients with Xmn I +/+, −/+ or −/−. The GWAS study of the whole genome with more than 6000,000 SNPs of 1,100 β-thal/Hb E patients with mild and severe diseases revealed SNPs in three independent genes that show significant association with the disease severity. The strongest SNPs associated with the disease severity located in three regions; the β-globin gene cluster on chromosome 11, the HBS1L-MYB intergenic region on chromosome 6q23 and the BCL11A gene on chromosome 2p15. Further analysis of Hb F level showed that Hb F level was significantly higher in mild patients than moderate and severe patients (%Hb F; mild =42.6±11.5, moderate =35.7±11.1, severe =32.4±12.1; P<0.001). The association of Hb F level and frequency of Hb F-QTLs was studied in 520 cases. All individual SNPs on Hb F-QTLs are associated with Hb F (P value <10−5). Thirteen tagging SNPs were selected from three Hb F-QTLs. Of the common haplotypes CA haplotypes of BCL11A; CG haplotypes of HMIP; TTCTGTTAA and TTCTGTTAG haplotypes of β-globin gene cluster showed association with high HbF level. Our data indicated that several genetic loci act in concert to influence Hb F levels and disease severity of β-thal/HbE patients. Understanding the genetic modifier in β-thalassemia is important for the management of β-thalassemia patients from PND to prognosis and decision for difficult treatment such as stem cell transplantation. Moreover, this may lead to future alternative treatment of β-thalassemia patients as well.

A rapid paper-based test for quantifying sickle hemoglobin in blood samples from patients with sickle cell disease.

Quantification of sickle hemoglobin (HbS) in patients with sickle cell disease (SCD) undergoing hydroxyurea or chronic transfusion therapy is essential to monitoring the effectiveness of these therapies. The clinical monitoring of %HbS using conventional laboratory methods is limited by high per-test costs and long turnaround times usually associated with these methods. Here we demonstrate a simple, rapid, inexpensive paper-based assay capable of quantifying %HbS in blood samples from patients with SCD. A 20 μL droplet of whole blood and hemoglobin solubility buffer was deposited on chromatography paper. The relative color intensities of regions of the resulting blood stain, determined by automated image analysis, are used to estimate %HbS. We compared the paper-based assay with hemoglobin electrophoresis (comparison method) using blood samples from 88 subjects. The test shows high correlation (R(2) = 0.86) and strong agreement (standard deviation of difference = 7%HbS) with conventional Hb electrophoresis measurement of %HbS, and closely approximates clinically predicted change in %HbS with transfusion therapy (mean difference 2.6%HbS, n = 5). The paper-based assay can be completed in less than 35 min and has a per-test cost less than $0.25. The assay is accurate across a wide range of HbS levels (10-97%) and hemoglobin concentrations (5.6-12.9 g/dL) and is unaffected by high levels of HbF (up to 80.6%). This study demonstrates the feasibility of the paper-based %HbS assay. The paper-based test could improve clinical care for SCD, particularly in resource-limited settings, by enabling more rapid and less expensive %HbS monitoring.

Genetic association of fetal-hemoglobin levels in individuals with sickle cell disease in Tanzania maps to conserved regulatory elements within the MYB core enhancer.

BackgroundCommon genetic variants residing near upstream regulatory elements for MYB, the gene encoding transcription factor cMYB, promote the persistence of fetal hemoglobin (HbF) into adulthood. While they have no consequences in healthy individuals, high HbF levels have major clinical benefits in patients with sickle cell disease (SCD) or β thalassemia. Here, we present our detailed investigation of HBS1L-MYB intergenic polymorphism block 2 (HMIP-2), the central component of the complex quantitative-trait locus upstream of MYB, in 1,022 individuals with SCD in Tanzania.MethodsWe have looked at 1022 individuals with HbSS or HbS/β0 in Tanzania. In order to achieve a detailed analysis of HMIP-2, we performed targeted genotyping for a total of 10 SNPs and extracted additional 528 SNPs information from a genome wide scan involving the same population. Using MACH, we utilized the existing YRI data from 1000 genomes to impute 54 SNPs situated within HIMP-2.ResultsSeven HbF-increasing, low-frequency variants (β > 0.3, p < 10−5, f ≤ 0.05) were located in two partially-independent sub-loci, HMIP-2A and HMIP-2B. The spectrum of haplotypes carrying such alleles was diverse when compared to European and West African reference populations: we detected one such haplotype at sub-locus HMIP-2A, two at HMIP-2B, and a fourth including high-HbF alleles at both sub-loci (‘Eurasian’ haplotype clade). In the region of HMIP-2A a putative functional variant (a 3-bp indel) has been described previously, but no such candidate causative variant exists at HMIP-2B. Extending our dataset through imputation with 1000 Genomes, whole-genome-sequence data, we have mapped peak association at HMIP-2B to an 11-kb region around rs9494145 and rs9483788, flanked by two conserved regulatory elements for MYB.ConclusionsStudies in populations from the African continent provide distinct opportunities for mapping disease-modifying genetic loci, especially for conditions that are highly prevalent there, such as SCD. Population-genetic characteristics of our cohort, such as ethnic diversity and the predominance of shorter, African-type haplotypes, can add to the power of such studies.

High Foetal Haemoglobin in Sickle Cell Disease: Not so Protective?

High Foetal Haemoglobin in Sickle Cell Disease: Not so Protective?

Impact of fetal hemoglobin on micronutrients in sickle cell anemia

Background / Objective: The presence of persistent high fetal hemoglobin (HbF) in sickle cells disease (SCD) patients may be a modulator of clinical and biochemical features. This study seeks to test the hypothesis that high level of HbF may regulate the levels of calcium, magnesium, zinc, and copper in SCD patients in a steady clinical state. Materials and Methods: Serum calcium, magnesium, zinc, and copper were assayed in 100 SCD patients in steady clinical state and 50 control subjects using the colorimetric method while blood HbF was determined by alkaline denaturation method. Results: Twenty-five percent of the study group had high (>5%) HbF, while 75% had low (<4.9%) HbF levels. HbF (P < 0.001), serum copper (P < 0.001), and calcium (P = 0.002) were significantly higher in SCD patients compared with controls, while zinc and magnesium were significantly lower (P < 0.001) in SCD patient compared with controls. Serum calcium (P = 0.01) and copper (P = 0.118) were lower in SCD patients with high (≥5%) HbF while magnesium and zinc were significantly higher (P < 0.001) in SCD patients with high HbF compare with those with low (≤4.9). HbF correlated negatively with calcium (r = −0.25, P = 0.011) and copper (r = -0.11, P = 0.287) while magnesium (r = 0.60, P = 0.001) and zinc (r = 0.57, P < 0.001) correlated positively on HbF levels. Conclusion: HbF levels may have modulated the levels of these elements in SCD patients. It is suggested that HbF may be estimated along with hemoglobin electrophoresis in diagnosis, clinical management, and predicting clinical course of SCD patients. Nutritional studies may be routinely conducted in this group of patients for better management.

Amelioration of murine sickle cell disease by nonablative conditioning and γ-globin gene-corrected bone marrow cells

Patients with severe sickle cell disease (SCD) are candidates for gene therapy using autologous hematopoietic stem cells (HSCs), but concomitant multi-organ disease may contraindicate pretransplant conditioning with full myeloablation. We tested whether nonmyeloablative conditioning, a regimen used successfully for allogeneic bone marrow transplantation of adult SCD patients, allows engraftment of γ-globin gene-corrected cells to a therapeutic level in the Berkeley mouse model of SCD. Animals transplanted according to this regimen averaged 35% engraftment of transduced hematopoietic stem cells with an average vector copy < 2.0. Fetal hemoglobin (HbF) levels ranged from 20 to 44% of total hemoglobin and approximately two-thirds of circulating red blood cells expressed HbF detected by immunofluorescence (F-cells). Gene therapy treatment of SCD mice ameliorated anemia, reduced hyperleukocytosis, improved renal function, and reduced iron accumulation in liver, spleen, and kidneys. Thus, modest levels of chimerism with donor cells expressing high levels of HbF from an insulated γ-globin lentiviral vector can improve the pathology of SCD in mice, thereby illustrating a potentially safe and effective strategy for gene therapy in humans.

Characterization of a Transcript Found within the HBS1L-MYB Intergenic Region

Fetal hemoglobin (HbF, α2γ2) is the main hemoglobin in the fetus, but its levels decline after birth to <1% in adults. However, there are individuals who continue to have high levels of HbF well into adulthood. High HbF in sickle cell anemia is often associated with milder disease hence the effort to understand more about its regulation for possible therapeutic gain. Some of the variation in HbF among patients with sickle cell anemia can be accounted for by differences in three major HbF quantitative trait loci (QTL) that were found in genome-wide association studies to be associated with high HbF. However, depending on the population studied, these QTLs only account for up to 10-50% of HbF variance. Therefore, determining other factors for high HbF levels is critical in understanding the regulation of HbF expression and finding other possible therapeutic targets to induce HbF production in patients with hemoglobinopathies. Previously, we found that HbF variability is associated with a 3-bp deletion within the HBS1L-MYB intergenic region (Farrell et al, Blood 2011) and this work has been recently confirmed (Stadhouders et al, JCI, 2014). This deletion was found to be in strong linkage disequilibrium (LD) with the QTL HMIP, the HBS1L-MYB intergenic region on chr6q23. Functional analyses of the region around the 3-bp deletion using ChIP assay in K562 cells, which are heterozygous for the 3-bp deletion, found that four transcription factors that are involved in erythroid cell differentiation—GATA-2, TAL1, E47 and RUNX1—all bind in the region of the 3-bp deletion. ENCODE datasets show that RNA polymerase II and a 50 bp RNA transcript are found in this same area. Based on these observations, we set out to determine the extent of the 50-bp transcript found by ENCODE. Using cDNA from K562 cells and the 50-bp transcript as a guide, we were able to amplify by PCR a fragment greater than 1 kb, which includes the 3-bp deletion and the binding sites for the erythroid-specific transcription factors mentioned above. Sequencing of this PCR product confirmed that the transcript is transcribed from the HBS1L-MYB intergenic region. Next, 5′- and 3′-rapid amplification of cDNA ends (RACE) reactions were done to determine the full length of this transcript. Sequencing results of the RACE products show that the 3′-end of the transcript is more than 100 bp downstream from what was originally found by PCR. Results for the 5′-end are pending. By knowing the sequence of this transcript, we were able to make multiple transcript-specific primers and use them in qPCR to determine if expression of this transcript changes with induction of hemoglobin synthesis in K562 cells. Preliminary data show after the induction of HBG2 expression with butyrate, various areas of this novel transcript increased as well. Because globin synthesis in K562 cells is fixed at the fetal stage of development we also looked for expression of this transcript in iPSC-derived erythroid progenitor cells from a disease-free individual and an individual with sickle cell anemia on day 25 of differentiation, and in CD34+ cord blood-derived erythroid cells on days 7 and 14 of differentiation. qPCR showed that in these primary erythroid cells the transcript is expressed at similar levels as in K562 cells. This novel transcript within the HBS1L-MYB intergenic region seems to increase with hemoglobin induction in K562 cells, and is also found in other primary erythroid cells derived from iPSCs and CD34+ cells. This transcript is a candidate for being a long noncoding RNA (lncRNA). LncRNAs are transcripts of 200 nucleotides or greater that can be transcribed from any region of the genome. They have been shown to be involved in gene regulation and various biological processes including hematopoiesis. HMIP has a primary effect on hematopoiesis. As this fragment is transcribed from a region known to be associated with HbF variability, it will be of interest to determine its role in HbF expression. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

BCL11A enhancer Haplotypes Are Associated with the Distribution of HbF in Arab-Indian and African Haplotype Sickle Cell Anemia but Not the Different Population Levels of HbF

Fetal hemoglobin (HbF) modulates the phenotype of sickle cell anemia. In the Middle East and India the HbS gene is often on an Arab-Indian HBB haplotype that is associated with high HbF levels. HbF is “normally” distributed in this population with a mean ~20%. In African HbS haplotypes, HbF levels are much lower (mean value ~6%) with a highly skewed distribution. BCL11A is an important modulator of γ-globin gene (HBG2 and HBG1) expression and BCL11A is regulated by erythroid specific enhancers in its 2nd intron. The enhancers consist of 3 DNase hypersensitive sites (HS) +62, +58 and +55 kb from the transcription initiation site of this gene. Polymorphisms (SNPs) in these enhancers are associated with HbF. The strongest association with HbF levels in African Americans with sickle cell anemia was with rs1427407 in HS +62 and to a lesser extent, rs7606173 in HS+55. Using the results of whole genome sequencing of 14 AI haplotype patients—half with HbF <10%, half with HbF >20%—6 SNPs in the BCL11A enhancer region, rs1427407, rs7599488, rs6706648, rs6738440, rs7565301, rs7606173 and 2 indels rs3028027 and rs142027584 (CCT, CCTCT and AAAAC respectively), were detected as possibly associated with HbF level. There were no novel polymorphisms detected. We genotyped the 6 SNPs and studied their associated haplotypes in 137 Saudi (HbF18.0±7.0%) and 44 Indian patients (HbF23.0±4.8%) with the Arab-Indian HBB haplotype; 50 African Americans with diverse African haplotypes, including 4 Senegal haplotype heterozygotes, (20 with HbF 17.2±4.6% and 30 with HbF 5.0±2.5%) and imputed genotypes for these SNPs in 847 African Americans with sickle cell anemia and diverse haplotypes (HbF 6.6±5.5%). Four SNPs (rs1427407, rs6706648, rs6738440, and rs7606173) in the HS sites showed consistent associations with HbF levels in all 4 cohorts. Haplotype analysis of these 4 SNPs showed that there were 4 common and 10 rare haplotypes. The most common, GCAG, was found in ~54% of Arab-Indian haplotype carriers (HbF, ~20%) and in ~33% of African origin haplotype carriers (HbF, ~5.5%). Two haplotypes, GTAC and GTGC, were carried by ~40% of African American patients and were associated with lower levels of HbF (3.6%-4%). These same haplotypes were carried by 18% of Arab-Indian haplotype carriers and their average HbF level was 17%. These differences were significant. Haplotype TCAG was present in 20% of Arab-Indian and 25% of African haplotype cases, and carriers had on average higher HbF levels (~22% in the Arab-Indian haplotype, ~8% in African Americans). The analysis shows that: BCL11A enhancer haplotypes are differentially distributed among patients with the HbS gene on Arab-Indian or African origin haplotypes; haplotype pairs TCAG/TCAG and GTAC/GTGC are associated with the highest and lowest HbF levels in all the studied groups; the population-specific prevalence of HbF BCL11A enhancer haplotypes are likely to explain the different distributions of HbF in African origin and Arab-Indian haplotypes but do not account for the differences in average population levels of HbF or the high HbF of the Arab-Indian haplotype. Novel SNPs in BCL11A do not explain the high HbF of the Arab-Indian haplotype. Other important loci must have a predominant role in the differential expression of HbF among HbS Arab-Indian haplotype carriers. DisclosuresNo relevant conflicts of interest to declare.

Assessment of HbF QTLs Affecting Disease Severity and Genetic Analysis in Patients Homozygous for Codon 8 (–AA) β0-Thalassemia Mutation

In β-thalassemia major, an inherited disorder caused by homozygosity or compound heterozygosity for β-thalassemia mutations, most patients are severely anemic and require chronic RBC transfusions. Elevated levels of HbF can ameliorate the disease severity and 3 major HbF quantitative trait loci (QTL) on chr 11p, 6q (the HBS1L-MYB intergenic polymorphism) and 2p (BCL11A) are associated with HbF levels in all populations studied. We studied Iraqi-American non-identical twins with markedly elevated HbF who are homozygous for codon 8 (–AA) β0-thalassemia mutation, but are clinically well and transfusion independent. MLPA analysis of the β-globin gene cluster did not show any hereditary persistence of fetal hemoglobin deletions or γ-globin gene quadruplication. Sequencing HBG2 and HBG1 promoters excluded other γ-globin gene promoter non-deletional HPFH mutations. We characterized 12 Turkish and Iraqi patients who are homozygous for codon 8 (–AA) frame-shift β-thalassemia mutation. Among this group, three patients from two Turkish families have a mild disease phenotype, six patients from four Turkish families have a severe phenotype, and three Iraqi patients from three families have a severe phenotype. To assess the contribution of 3 major HbF QTLs to disease severity, we genotyped polymorphisms in these QTL in all 14 patients (Table). Homozygosity for rs7482144 minor alleles (the -158 5′ HBG2 Xmn1 C-T SNP) in the HBB gene cluster is present in 13 of 14 patients with the exception being one patient with a severe phenotype. No distinct pattern for the BCL11A sentinel SNP rs766432 minor allele is found between mild and severely affected patients. Homozygosity for rs9399137 minor alleles marking the HBS1L-MYB intergenic region is present in two patients with a mild phenotype, while heterozygosity for minor alleles and homozygosity for major alleles are present in both mild and severe patients. Although the Iraqi-American twins first studied have the most minor alleles (5 of 6 possible minor alleles), the other three patients with mild phenotype have 4, 3 and 2 minor alleles, respectively. Strikingly, one patient with mild phenotype is only homozygous for the rs7482144 minor alleles. Patients with severe phenotypes have 2 to 4 QTL minor alleles. Neither individual QTL genotypes nor the number of QTL minor alleles can distinguish the mild phenotype and severe phenotype in patients with β-thalassemia major of Iraqi and Turkish origin suggesting that other factors contribute to the mild phenotype and high HbF levels in these patients. The entire 14.7 kb intergenic region of between HBG1 and HBD of one twin was sequenced and revealed 19 known SNPs and 4 novel SNPs. The potential roles of these SNPs in regulating γ-globin gene expression are under investigation. We are also investigating whether there are any other potential cis- or trans-acting factors that contribute to mild disease phenotype in these patients using genome-wide SNP arrays, whole genome sequencing and whole exome sequencing. By studying these unusual cases we hope to provide new understanding and insights for elevated HbF production in β-hemoglobinopathies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Targeting KLF1 for the Treatment of Sickle Cell Disease 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 in the fetus during the last months of embryonic development and the first few months of life after birth. HbF has a slightly greater oxygen binding affinity than adult hemoglobin (HbA, α2β2) and 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.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 KLF1, we can promote production of HbF through the upregulation of γ-globin expression.Since rodents don’t express γ-globin, we have employed both human and engineered mouse cell lines 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 KLF1, and treated with antisense oligonucleotides (ASOs) targeting mouse KLF1. After 7 days of free uptake with the ASOs, we observed a 6-fold increase of human γ-globin mRNA expression after achieving 65% mRNA reduction of mouse KLF1 compared to the untreated control. We were also able to demonstrate significant upregulation of human γ-globin protein expression in these cells by western blot. We have shown similar results in a human erythroleukemia cell line, K562, using ASOs targeting human KLF1. K562 cells were electroporated with the KLF1 ASOs and 4 days later, we observed a 5-fold increase of human γ-globin mRNA expression after achieving 40% mRNA reduction of human KLF1 compared to the untreated control. These data indicate that targeting mouse or human KLF1 with ASO treatment can cause an increase in human γ-globin expression in vitrothat is necessary for the upregulation of fetal hemoglobin.We have also shown that we are able to target the bone marrow in both mice and rats through subcutaneous administration of our KLF1 ASOs. In wild type mice, at a dose of 100 mpk/wk for 4 weeks, we observed KLF1 target reduction of 88% and a β-globin reduction of 58% compared to the saline control in whole bone marrow. In Sprague-Dawley rats, at a dose of 50 mpk/wk for 4 weeks, we observed KLF1 target reduction of 83% and a β-globin reduction of 77% compared to the saline control in whole bone marrow. Therefore, we are able to achieve significant β-globin mRNA reduction in the bone marrow in both mice and rats after subcutaneous administration of KLF ASOs. These data indicate that reducing KLF1 with antisense oligonucleotides is a viable option for the treatment of sickle cell anemia. DisclosuresPeralta:Isis Pharmaceuticals, Inc.: Employment. Low:Isis Pharmaceuticals, Inc.: Employment. Booten:Isis Pharmaceuticals, Inc.: Employment. Zhou:Univeristy of Alabama at Birmingham: Employment. Kim:Isis Pharmaceuticals, Inc.: Employment. Freier:Isis Pharmaceuticals, Inc.: Employment. Guo:Isis Pharmaceuticals, Inc.: Employment. Murray:Isis Pharmaceuticals, Inc.: Employment. Townes:University of Alabama at Birmingham: Employment. Hung:Isis Pharmaceuticals, Inc.: Employment.

SCD-Biochip: A Functional Assay for Red Cell Adhesion in Sickle Cell Disease

Vaso-occlusion, a hallmark of sickle cell disease (SCD), is caused by abnormal adhesion of blood cells to the endothelium. Analyses of RBC-endothelial interactions are technically challenging, which has limited the widespread use of these evaluations clinically or as a research tool. Here, we present a microfluidic device (SCD-Biochip) that allows serial quantitative and qualitative examination of RBC adhesion and deformability to endothelium associated protein-immobilized microchannels (fibronectin, FN, and laminin, LN), in a closed preprocessing-free system (Fig. 1A).15 µL of surplus blood, after clinical lab tests had been completed, was taken from subjects with HbSS (transfused, non-transfused, and with high or low levels of HbF), HbSC, HbSBeta-thalassemia and from control subjects (HbAA). Samples were injected, without processing, on to FN- and LN-immobilized microchannels. This was followed by wash steps at or above the physiological flow velocities of post-capillary venules (0-10 mm/s). Adhered RBCs were then quantified. We observed a statistically significant difference in RBC adherence per unit area to FN (Fig. 1B), after 0.8 mm/s flow velocity (low wash), amongst RBCs from subjects with HbSS (90±84.0, n=26), with compound heterozygous HbSC or Sβthalassemia (21±6.8, n=4), or with normal hemoglobin (HbAA, 4±3, n=10, Mann-Whitney, p<0.05). Strikingly, this test was sensitive to physiological changes that were not well-reflected in routine lab tests (e.g., adherence to FN did not differ between samples from transfused or non-transfused HbSS patients (77±62, n=6, compared with 88.1±82.6, n=17, P=0.75, respectively), despite routine scheduled transfusions of HbAA-containing RBCs in the former. Samples from HbSS subjects with a low HbF were more likely to be highly adherent to FN than were samples from subjects with a high HbF (5/7 with <8% HbF compared with 1/10 ≥8% HbF, p=0.035). Increased adherence of HbSS- (707±904.8, n=7), compared with HbAA-containing RBCs (9±6, n=7), to LN at low wash was also seen. Of interest, a single sample from an HbSS subject with acute pain showed an adherence to FN and LN of 250 and 2176, respectively.Qualitatively, HbSS-containing RBCs showed decreased adherence to FN at increased flow rates; adhered RBCs decreased from 90±84 (n=25) at low flow wash to 69±52 (n=13) at 3.3 mm/s (intermediate flow) and 36±48.2 (n=11) at 41.7 mm/s (high flow). Analyses revealed deformable HbAA- and HbSS-containing RBCs, but only HbSS-containing samples also had a significant population of non-deformable RBCs. We analyzed single cell deformation and detachment in the presence of physiological fluid flow. Deformable HbAA- and HbSS-containing RBCs detached at relatively low flow rates (6.11±0.48 n=3, and 9.3±1.44 n=6, respectively), compared to non-deformable HbSS-containing RBCs (40±6.97 n=5). Deformable HbAA- or HbSS-containing RBCs displayed a single adhesion pivot point, compared to non-deformable HbSS-containing RBCs, which showed multiple adhesion sites. At highest flow (41.7mm/s), 75±17% (n=9) of adhered RBCs were non-deformable on FN. Of note, flow evaluations revealed two characteristic RBC profiles in HbSS subjects: either one in which adherent non-deformable cells predominated at all flow rates or one in which less adherent deformable cells comprised >50% of the RBC population.The SCD-Biochip evaluates, simply and with small blood sample volumes, complex biophysical properties of adhesion and deformability, which reflect clinical phenotypes, including hemoglobin composition. This adaptable technology may give important biophysical insights into disease pathophysiology when widely applied in SCD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Analysis of (δβ)0 Thalassemia and HPFH Deletions Suggest a Hierarchy of Cis-Acting Elements Regulating Fetal Hemoglobin Gene Expression.

Hereditary persistence of fetal hemoglobin (HPFH) and (δβ)0 thalassemia are caused by deletions within the β-globin gene (HBB) cluster that remove elements that affect the expression of the γ-globin genes (HBG2 and HBG1, or HBG). These deletions are of different lengths and have different 5’ and 3’ breakpoints. The phenotypes associated with heterozygous carriers of (δβ)0 thalassemia and HPFH deletions are differentiated by levels of 5-15% HbF distributed heterocellularly in the former and 15-30% HbF distributed pancellularly in the latter. We found a novel 588.6 kb deletion that removed both the 3.5 kb fragment 5’ to HBD that is deleted in Corfu β thalassemia and contains a BCL11A binding site, and the known cis-acting elements downstream of HBB. The proband with this deletion had a HbF of 5.4% (Morrison et al, Blood, 2014 abstract 3452). To study the relative importance of 5’ and 3’ regulatory elements in HBG expression we studied 209 cases culled from the literature and from our laboratory where the 3.5 kb element 5’ to HBD and enhancers 3’ to HBB were deleted and HBG remained intact. We used a backwards stepwise regression statistical analysis to determine which deleted elements had the greatest effect on HbF levels. The combination of the deletion of 3.5 kb intergenic region 5’ to HBD, the presence of the HPFH-1 “3D” enhancer juxtaposed to HBG, and the deletion of the 3’ HS1 region accounted for 66.7% of the HbF variation in heterozygotes for HPFH and (δβ)0-thalassemia deletions. The HPFH-1 “3D” enhancer juxtaposed to HBG— the main difference between HPFH-1 and 2 compared with Spanish (δβ)0-thalassemia—was associated with an increase in HbF of 20.78% (p<2e-16) after adjusting for the effects of the other 5’ and 3’ cis-acting elements. The next most significant factor was the deletion of the 3.5 kb fragment 5’ to HBD which resulted in an increase of 10.62% HbF after similar adjustments (p<2e-16); deletion of the 3’ HS1 region accounted for an increase in HbF of 5.25% (p<1.05e-5). The HPFH-3 and HPFH-6 enhancer regions each accounted for a less than 1% increase in HbF and were not significantly associated with HbF in this model. Among 194 individuals where both 5’ and some 3’ elements affecting γ-globin gene expression—excluding the “3D” enhancer—were deleted, HbF was 20±9.3%; in 13 cases where all 3’ enhancers—including the “3D” enhancer—were deleted, HbF was 6.8±3.7% (p=8.9e-07). To determine which combinations of cis-acting elements were associated with high and low HbF levels we performed a classification and regression tree (cART) analysis on HbF. The results of the regression tree (Figure) only included the deletion of the 5’ 3.5 kb fragment region, the presence of the HPFH-1 “3D” enhancer and the deletion of the 3’ HS1 region and were consistent with the results of the backwards selection model. The absence of the 5’ 3.5 kb fragment 5’ to HBD combined with the presence of the HPFH-1 “3D” enhancer was associated with the highest average HbF of 27.02%. The absence of the 3.5 kb fragment 5’ to HBD combined with the absence of the HPFH-1 “3D” enhancer was associated with the lowest average HbF of 6.82%.The 588.6 kb deletion is the largest deletion reported in the HBB cluster that leaves the γ-globin genes intact, and the second to remove both the BCL11A binding site and all known 3’ enhancer elements. By studying deletions in the HBBgene cluster we have further defined the hierarchy of cis-acting elements that modulate HbF levels in adults and suggest a paramount role of the distal “3D” enhancer. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.