Sequence-Specific Conversion of βA- to βS-Globin by Small Fragment Homologous Replacement In Hematopoietic Stem/Progenitor Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3754-3754
Author(s):  
Alireza Abdolmohammadi ◽  
Rosalie Maurisse ◽  
Babek Bedayat ◽  
David DeSemir ◽  
Damian Laber ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Dna Sequences ◽  
Conflicts Of Interest ◽  
Globin Gene ◽  
Specific Gene ◽  
Small Fragment ◽  
Wild Type ◽  
Hematopoietic Stem

Abstract Abstract 3754 Introduction: An ultimate goal of gene therapy is the development of effective strategies to correct mutant genomic sequences in pathologic cells. To that end, studies have been undertaken to evaluate the therapeutic potential of an oligo/polynucleotide-based sequence-specific gene modification strategy, small fragment homologous replacement (SFHR) in the correction of the mutation giving rise to sickle cell anemia. Small DNA fragments (SDFs) comprising the sickle cell anemia mutation (an A>T transversion in codon 6) and flanking DNA sequences in the human b-globin gene were introduced into Hematopoietic Stem/Progenitor Cells (HSPCs). The studies presented indicated modification at the level of DNA, RNA, and protein when cells were differentiated into erythrocytes. Methods: In this study, SFHR was used to convert A>T in codon 6 of the b-globin gene in CD34+/CD38-/Lin- HSPCs isolated from full term umbilical cord blood as a proof of principle. HSPCs were transfected with a defined number of a 559-bp SDF using the Amaxa electroporation (nucleofection) system. After growing the transfected cells in stem cell media containing EPO for different time intervals up to 32 days, RNA was extracted and DNase I-treated before further analysis. Erythrocytes were also analyzed using antibodies that differentiate between wild-type hemoglobin A (HBA) and sickle cell hemoglobin S (HBS). Results: RFLP analysis of a 430-bp PCR product generated from mRNA-derived cDNA with the DdeI enzyme indicated conversion of bA- to bS-globin. Sequencing of the 430-bp amplicon showed the A > T conversion. Analysis of the transfected wild-type HSPC-derived erythrocytes with HBA and HBS specific antibodies demonstrated the presence of a subpopulation of cells expressing HBS. These data are consistent with previous studies showing both conversion of bS- to bA-globin in SC1 cells and bA- to bS-globin in HSPCs after electroporation and microinjection of SDF, respectively. Conclusion: These studies represent a critical next step in developing SFHR as a therapy for sickle cell disease, in that they demonstrate long-term SFHR-mediated modification of b-globin following mass transfection by electroporation of HSPCs. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Elimination of the fibrinogen integrin αMβ2-binding motif improves renal pathology in mice with sickle cell anemia

2019 ◽  
Vol 3 (9) ◽  
pp. 1519-1532 ◽  
Author(s):  
Md Nasimuzzaman ◽  
Paritha I. Arumugam ◽  
Eric S. Mullins ◽  
Jeanne M. James ◽  
Katherine VandenHeuvel ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Globin Gene ◽  
White Blood Cells ◽  
Coagulation Factor ◽  
Vascular Occlusion ◽  
Organ Damage ◽  
Binding Motif ◽  
Mutant Form ◽  
Hematopoietic Stem

Abstract Sickle cell anemia (SCA) is caused by a point mutation in the β-globin gene that leads to devastating downstream consequences including chronic hemolytic anemia, episodic vascular occlusion, and cumulative organ damage resulting in death. SCA patients show coagulation activation and inflammation even in the absence of vascular occlusion. The coagulation factor fibrinogen is not only central to hemostasis but also plays important roles in pathologic inflammatory processes, in part by engaging neutrophils/macrophages through the αMβ2 integrin receptor. To determine whether fibrin(ogen)-mediated inflammation is a driver of SCA-associated pathologies, hematopoietic stem cells from Berkeley sickle mice were transplanted into homozygous Fibγ390-396A mice that express normal levels of a mutant form of fibrin(ogen) that does not engage αMβ2. Fibγ390-396A mice with SCA displayed an impressive reduction of reactive oxygen species (ROS) in white blood cells (WBCs), decreased circulating inflammatory cytokines/chemokines, and significantly improved SCA-associated glomerular pathology highlighted by reduced glomerulosclerosis, inflammatory cell infiltration, ischemic lesions, mesangial thickening, mesangial hypercellularity, and glomerular enlargement. In addition, Fibγ390-396A mice with SCA had improved glomerular protective responses and podocyte/mesangial transcriptional signatures that resulted in reduced albuminuria. Interestingly, the fibrinogen γ390-396A mutation had a negligible effect on cardiac, lung, and liver functions and pathologies in the context of SCA over a year-long observation period. Taken together, our data support that fibrinogen significantly contributes to WBC-driven inflammation and ROS production, which is a key driver of SCA-associated glomerulopathy, and may represent a novel therapeutic target against irreversible kidney damage in SCA.

Latent Cardiogenic Potential in Endocardium and Hemogenic Endothelium Revealed in the Absence of Scl/tal1

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2362-2362
Author(s):  
Amelie Montel-Hagen ◽  
Ben Van Handel ◽  
Roberto Ferrari ◽  
Rajkumar Sasidharan ◽  
Tonis Org ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Lineage Tracing ◽  
Bhlh Transcription Factor ◽  
Wild Type ◽  
Transcriptional Program ◽  
Hemogenic Endothelium ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Abstract Abstract 2362 The endothelium in embryonic and extraembryonic hematopoietic tissues has the capacity to generate hematopoietic stem and progenitor cells (HS/PC). However, it is unknown how this unique endothelium is specified. Microarray analysis of endothelial cells from hematopoietic tissues of embryos deficient for the bHLH transcription factor Scl/tal1 revealed that Scl establishes a robust hematopoietic transcriptional program in the endothelium. Surprisingly, lack of Scl also induced an unexpected fate switching of the prospective hemogenic endothelium to the cardiac lineage. Scl deficient embryos displayed a dramatic upregulation of cardiac transcription factors and structural proteins within the yolk sac vasculature, resulting in the generation of spontaneously beating cardiomyocytes. Ectopic cardiac potential in Scl deficient embryos arose from endothelial-derived CD31+Pdgfrα+ cardiogenic progenitor cells (CPCs), which were present in all sites of HS/PC generation. Analysis of Runx1-deficient embryos revealed, that although Runx1 acts downstream of Scl during the emergence of definitive HS/PCs, it is not required for the suppression of the cardiac fate in the endothelium. The only wild type tissue that contained CD31+Pdgfrα+ putative CPCs was the heart, and this population was greatly expanded in Scl deficient embryos. Strikingly, endocardium in Scl−/− hearts also activated a robust cardiomyogenic transcriptional program and generated Troponin T+ cardiomyocytes both in vivo and in vitro. Although CD31+Pdgfrα+ CPCs from wild type hearts did not generate readily beating cells in culture, they produced cells expressing endothelial, smooth muscle and cardiomyocyte specific genes, implying multipotentiality of this novel CPC population. Furthermore, CD31+Pdgfrα+ CPCs were greatly reduced in Isl1−/− hearts, which fail to generate functional, multipotential CPCs. Lineage tracing using VE-cadherin Cre Rosa-YFP mouse strain demonstrated that, in addition to generating HS/PCs in hematopoietic tissues, endothelial cells are also the cell of origin for CD31+Pdgfrα+ CPCs in the heart. Together, these data suggest a broader role for embryonic endothelium as a potential source of tissue-specific stem and progenitor cells and implicate Scl/tal1 as an important regulator of endothelial fate choice. Disclosures: No 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

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4066-4066
Author(s):  
Paola Sebastiani ◽  
John J. Farrell ◽  
Shuai Wang ◽  
Heather L Edward ◽  
Heather M. Shappell ◽  
...  
Keyword(s):  
African Americans ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Transcription Initiation ◽  
Conflicts Of Interest ◽  
Globin Gene ◽  
Initiation Site ◽  
Skewed Distribution ◽  
African Origin ◽  
Hbf Level

Abstract 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. Disclosures No relevant conflicts of interest to declare.

Further Evidence That HO-1 Regulates SDF-1 Expression in the Bone Marrow Microenvironment and That HO-1-Deficient Mice Show a Defect in the SDF-1–CXCR4 Retention Axis of Hematopoietic Stem/Progenitor Cells in Bone Marrow and Thus Are Easy Mobilizers - Studies in HO-1 Mutant Mice, Irradiation Chimeras, and the Effect of in Vivo Pharmacological Inhibition of HO-1

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 344-344
Author(s):  
Marcin Wysoczynski ◽  
Janina Ratajczak ◽  
Gregg Rokosh ◽  
Roberto Bolli ◽  
Mariusz Z Ratajczak
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Crucial Role ◽  
Conflicts Of Interest ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Cell Counts ◽  
Deficient Mice

Abstract Abstract 344 Background: Stromal derived factor-1 (SDF-1), which binds to the CXCR4 receptor expressed on the surface of hematopoietic stem/progenitor cells (HSPCs), plays an important role in the retention of HSPCs in BM niches. Heme oxygenase (HO-1) is a stress-responsive enzyme that catalyzes the degradation of heme and plays an important function in various physiological and pathophysiological states associated with cellular stress, such as ischemic/reperfusion injury, atherosclerosis, and cancer. Interestingly, it has also been reported that HO-1 regulates the expression of SDF-1 in myocardium (J Mol Cell Cardiol. 2008;45:44–55). Aim of study: Since SDF-1 plays a crucial role in retention and survival of HSPCs in BM, we become interested in whether HO-1 is expressed by BM stromal cells and whether deficiency of HO-1 affects normal hematopoiesis and retention of HSPCs in BM. Experimental approach: To address this issue, we employed several complementary strategies to investigate HO-1–/–, HO-1+/–, and wild type (wt) mouse littermates for i) the expression level of SDF-1 in BM, ii) the number of clonogenic progenitors from major hematopoietic lineages in BM, iii) peripheral blood (PB) cell counts, iv) the chemotactic responsiveness of HSPCs to an SDF-1 gradient as well as to other chemoattractants, including sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), and extracellular nucleotiodes (ATP, UTP), iv) the adhesiveness of clonogenic progenitors to immobilized SDF-1 and stroma, v) the number of circulating HSPCs in PB, and vi) the degree of mobilization in response to granulocyte-colony stimulating factor (G-CSF) or AMD3100, assessed by enumerating the number of CD34–SKL cells and clonogeneic progenitors (CFU-GM) circulating in PB. We also exposed mice to the small HO-1 molecular inhibitor tin protoporphyrin IX (SnPP) and studied the effect of this treatment on G-CSF- or AMD3100-induced mobilization of HSPCs. Finally, to prove an environmental HSPC retention defect in HO-1-deficient mice, we created radiation chimeras, wild type mice transplanted with HO-1-deficient BM cells, and, vice versa, HO-1-deficient mice reconstituted with wild type BM cells. Results: Our data indicate that under normal, steady-state conditions, HO-1–/– and HO+/– mice have normal PB cell counts and numbers of circulating CFU-GM, while a lack of HO-1 leads to an increase in the number of erythroid (BFU-E) and megakaryocytic (CFU-GM) progenitors in BM. However, while BMMNCs from HO-1–/– have normal expression of the SDF-1-binding receptor, CXCR4, we observed that the mRNA level for SDF-1 in BM-derived fibroblasts was ∼4 times lower. This corresponded with the observation in vitro that HSPCs from HO-1–/– animals respond more robustly to an SDF-1 gradient, and HO-1–/– animals mobilized a higher number of CD34–SKL cells and CFU-GM progenitors into PB in response to G-CSF and AMD3100. Both G-CSF and AMD3100 mobilization were also significantly enhanced in normal wild type mice after in vivo administration of HO-1 inhibitor. Finally, mobilization studies in irradiation chimeras confirmed the crucial role of the microenvironmental SDF-1-based retention mechanism of HSPCs in BM niches. Conclusions: Our data demonstrate for the first time that HO-1 plays an important and underappreciated role in modulating the SDF-1 level in the BM microenvironment and thus plays a role in retention of HSPCs in BM niches. Furthermore, our recent data showing a mobilization effect by a small non-toxic molecular inhibitor of HO-1 (SnPP), suggest that blockage of HO-1 could be a promising strategy to facilitate mobilization of HSPCs. Further studies are also needed to evaluate the role of HO-1 in homing of HSPCs after transplantation to BM stem cell niches. Disclosures: No relevant conflicts of interest to declare.

Mechanistic Insights into Forced Chromatin Looping Mediated Activation of Fetal Globin Gene Expression

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 331-331
Author(s):  
Caroline Bartman ◽  
Gerd A. Blobel ◽  
Jeremy Grevet ◽  
Chris C.S. Hsiung ◽  
Jeremy W. Rupon ◽  
...  
Keyword(s):  
Gene Expression ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Conflicts Of Interest ◽  
Globin Gene ◽  
Gene Promoters ◽  
Globin Genes ◽  
Chromatin Architecture ◽  
Chromatin Looping ◽  
Globin Promoter

Abstract The β-globin enhancer, called locus control region (LCR), is required for high level expression of all b-type globin genes. The LCR is in physical proximity with the genes it controls, with contacts shifting from embryonic (ε) to fetal (γ) and finally to adult (δ and β) globin gene promoters during development. In prior studies we showed that forced chromatin contacts between the LCR and the β-globin promoter led to transcriptional activation, suggesting that LCR-promoter looping causally underlies β-globin transcription (Deng et al. Cell 2012). In these studies, the transcription co-factor Ldb1 was tethered to the β-globin promoter using artificial zinc finger (ZF) DNA binding proteins, to trigger the promoter-LCR interaction. We subsequently showed that tethering Ldb1 to the promoters of developmentally silenced embryonic or fetal globin genes reactivated their expression in adult erythroblasts in a manner dependent on looped contacts with the LCR (Deng et al. Cell 2014). This work established a novel strategy to raise fetal globin expression in patients with sickle cell anemia. To examine mechanistically the effects of chromatin looping on gene expression we performed single molecule RNA FISH experiments to precisely measure transcription output at individual alleles before and after enforced LCR-γ-globin looping. The experiments were carried out in primary human erythroblasts, which produce elevated levels of γ-globin when exposed to culture conditions. Preliminary data suggest that the majority of transcripts emerge from the β-globin gene with a smaller fraction of transcripts coming from the γ-globin gene as expected. Among cells producing any type of globin primary transcripts, a significant fraction (25%-35%) of cells co-express γ- and β-globin. Importantly, γ- and β-globin are frequently transcribed from the same allele. Forced juxtaposition of the LCR and the γ-globin promoter increases the number of alleles expressing only γ-globin while reducing the number of alleles expressing only the β-globin gene. This result is consistent with the γ- and β-globin genes competing for LCR activity, and emphasizes the usefulness of this approach in the context of sickle cell anemia in which not only elevated levels of fetal hemoglobin but also reduction of the mutant hemoglobin are desirable. Surprisingly, however, the proportion of alleles co-expressing γ-globin and β-globin remains largely constant. We are testing whether co-expression from the same allele is LCR independent. Finally, our studies suggest that LCR-promoter contacts increase the probability of transcription of a given allele. We will also present work addressing the critical question as to how alteration of chromatin architecture overcomes the action of transcriptional repressive complexes, such as Bcl11a, which normally maintain embryonic and fetal globin genes in a repressed state throughout adulthood. In sum, our studies produce a deeper understanding of the interplay of chromatin architecture and gene expression in a system that holds great potential for therapeutic application in patients with hemoglobinopathies. Disclosures No relevant conflicts of interest to declare.

scholarly journals A novel human gamma-globin gene vector for genetic correction of sickle cell anemia in a humanized sickle mouse model: critical determinants for successful correction

Blood ◽  
2009 ◽  
Vol 114 (6) ◽  
pp. 1174-1185 ◽  
Author(s):  
Ajay Perumbeti ◽  
Tomoyasu Higashimoto ◽  
Fabrizia Urbinati ◽  
Robert Franco ◽  
Herbert J. Meiselman ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Globin Gene ◽  
Regulatory Control ◽  
Critical Parameters ◽  
Minimal Amount ◽  
Reduced Intensity Conditioning ◽  
Hematopoietic Stem ◽  
F Cells ◽  
Reduced Intensity

Abstract We show that lentiviral delivery of human γ-globin gene under β-globin regulatory control elements in hematopoietic stem cells (HSCs) results in sufficient postnatal fetal hemoglobin (HbF) expression to correct sickle cell anemia (SCA) in the Berkeley “humanized” sickle mouse. Upon de-escalating the amount of transduced HSCs in transplant recipients, using reduced-intensity conditioning and varying gene transfer efficiency and vector copy number, we assessed critical parameters needed for correction. A systematic quantification of functional and hematologic red blood cell (RBC) indices, organ pathology, and life span was used to determine the minimal amount of HbF, F cells, HbF/F-cell, and gene-modified HSCs required for correcting the sickle phenotype. We show that long-term amelioration of disease occurred (1) when HbF exceeded 10%, F cells constituted two-thirds of the circulating RBCs, and HbF/F cell was one-third of the total hemoglobin in sickle RBCs; and (2) when approximately 20% gene-modified HSCs repopulated the marrow. Moreover, we show a novel model using reduced-intensity conditioning to determine genetically corrected HSC threshold that corrects a hematopoietic disease. These studies provide a strong preclinical model for what it would take to genetically correct SCA and are a foundation for the use of this vector in a human clinical trial.

Association of G6PD −202/−376 with Lower Hemoglobin Concentration but Not Increased Hemolysis in Patients with Sickle Cell Anemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1511-1511
Author(s):  
Mehdi Nouraie ◽  
Noel S. Reading ◽  
Andrew Campbell ◽  
Caterina Minniti ◽  
Sohail R Rana ◽  
...  
Keyword(s):  
Steady State ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Conflicts Of Interest ◽  
Globin Gene ◽  
Principal Component ◽  
Hemoglobin Concentration ◽  
Hemoglobin F ◽  
Independent Effects ◽  
Definitive Erythropoiesis

Abstract Abstract 1511 Poster Board I-534 Background What determines the degree of hemolysis and of anemia in patients with hemoglobin SS is not fully known. The rate of hemolysis and severity of anemia are ameliorated by the presence of alpha thalassemia and by higher hemoglobin F percentage. Mild G6PD deficiency in the form of G6PD-202/-376 may be associated with episodic hemolysis in individuals of African descent, but past studies indicated little influence of G6PD-202/-376 on the degree of hemolysis and anemia in sickle cell disease patients (1,2). In this study we examined the roles of single and double α-globin deletions and G6PD-202/-376 on the degree of hemolysis and the hemoglobin concentration in hemoglobin SS patients. Methods Two hundred sixty two children and adolescents with hemoglobin SS were recruited at three tertiary medical centers and studied at steady state. Principal component analysis was used to develop a hemolytic component from concentrations of lactate dehydrogenase, aspartate aminotransferase and bilirubin. PCR was used to determine the presence of α-thalassemia and G6PD-202/-376. Multivariate models were employed to determine the independent effects of these genotypes on hemoglobin concentration and degree of hemolysis. Results Single a-globin deletion was associated with an estimated 0.4 g/dL increase in steady-state hemoglobin concentration and double α-globin gene deletion with a 0.8 g/dL increase (P = 0.005 for trend) due to, progressively lower degrees of hemolysis (P = 0.004). G6PD-202/-376 was associated with an estimated 0.7 g/dL decrease in the hemoglobin concentration (P = 0.003) (Figure 1a), but this observation could not be explained by increased hemolysis. Rather, the reticulocyte count was an estimated 22% lower with G6PD-202/-376 (P = 0.032) (Figure 1b). Discussion G6PD -202/-376 may be associated with lower hemoglobin concentration in sickle cell anemia and the mechanism is probably impaired erythropoiesis rather than hemolysis. A recent study (3) indicates that G6PD is needed for definitive erythropoiesis as well as for normal survival of red blood cells in the periphery. Our present findings raise the possibility that, in the setting of the markedly increased erythropoiesis of sickle cell anemia, G6PD-202/-376 may result in impairment in erythropoiesis that is discernible in the peripheral blood hemoglobin concentration. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4691-4691
Author(s):  
Martin H. Steinberg ◽  
David H.K. Chui ◽  
George J. Dover ◽  
Paola Sebastiani ◽  
Abdulrahman Alsultan
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Conflicts Of Interest ◽  
Globin Gene ◽  
Single Agent ◽  
Acute Chest Syndrome ◽  
Cell Disease ◽  
Sickle Erythrocytes ◽  
F Cells

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

scholarly journals Haploinsufficiency of CALR Confers Hematopoietic Stem Cells (HSCs) with a Clonal Advantage over Wild-Type Cells, and, in Setting of Myeloproliferative Neoplasms, Compensates for the Functions of HSCs Impaired By the Calr Mutation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 97-97 ◽  
Author(s):  
Kotaro Shide ◽  
Takuro Kameda ◽  
Ayako Kamiunten ◽  
Masaaki Sekine ◽  
Yoshinori Ozono ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Negative Influence ◽  
Driver Mutations ◽  
Spleen Weight ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Cell Counts ◽  
Calr Mutation

Abstract CALR exon9 frameshift mutations function as driver mutations in essential thrombocythemia (ET) and primary myelofibrosis patients with non-mutated JAK2 or MPL. The mutations augment signal transducer and activator of transcription activity in the presence of MPL, induce increased cell proliferation and growth factor independence in cell lines, and cause ET-like myeloproliferative neoplasms (MPN) in mice. In tumor cells bearing the CALR mutation, mutant CALR protein expression occurs while wild-type (WT) CALR expression is decreased by half. Although the biological activity of mutant CALR has been elucidated in detail, the significance of CALR haploinsufficiency is unclear. The purpose of this study was therefore to clarify the influence of CALR haploinsufficiency on hematopoiesis in normal and CALR-mutated MPN in mice. First, we analyzed the effect of CALR haploinsufficiency on hematopoiesis using CALR heterozygous knockout (CALR-hKO) mice (Tokuhiro et al. Sci Rep. 2015). Blood cell counts, liver and spleen weight, histology, cell fraction in bone marrow (BM) and spleen, and survival of CALR-hKO mice were all equivalent to that observed in WT mice. In the analysis of progenitor cells by fluorescence-activated cell sorting and colony formation assays, no difference was observed in the amount of progenitor cells and in colony replating capacity between WT mice and CALR-hKO mice. Interestingly, in competitive serial transplantation experiments using whole BM cells in primary and secondary transplanted mice, CALR-hKO cells showed higher levels of chimerism than WT cells. Next, the effect of CALR haploinsufficiency on hematopoiesis in mutant CALR-del52 overexpressing mice was analyzed. We compared three groups of mice, WT mice, CALR-del52 transgenic (TG) mice (Shide et al. Leukemia 2017) and CALR-del52 TG/CALR-hKO double-mutant (TG-hKO) mice. Both TG mice and TG-hKO mice developed ET-like MPN. Compared to WT mice, increases in megakaryocytes, platelets and hematopoietic progenitor cells (including HSCs) were observed in these mice. However, no differences were observed between TG mice and TG-hKO mice. Finally, 4000 LSK cells sorted from WT mice, TG mice, and TG-hKO mice (B6-Ly5.2) and B6-Ly5.1 competitor cells (1 × 106 WT BM cells) were mixed and injected into lethally irradiated B6-Ly5.1 recipient mice, and the percent chimerism of donor cells was followed for 1 year after transplantation. From 12 weeks after transplantation, the chimerism of TG cells was significantly lower than that of control WT cells, suggesting that the CALR mutation has a negative influence on clonal expansion of HSCs. The recipient mice transplanted with TG LSK cells showed very mild thrombocythemia. On the other hand, chimerism in TG-hKO cells was significantly higher than that in control WT cells up to 12 weeks after transplantation. Chimerism at 20 weeks after transplantation was equivalent to that in control WT cells, and the recipient mice transplanted with TG-hKO LSK cells showed severe thrombocythemia. These findings show that CALR haploinsufficiency compensates for HSCs functioning, which has been impaired by the CALR mutation, enhancing the ability of HSCs to develop ET. Mice experiments have showed that HSCs with the JAK2V617F mutation are fragile, and it is considered that mutations in DNMT3A or TET2 often occurred prior to mutations in JAK2 to compensate for the defect in self renewal capacity. Conversely, since the CALR mutation is not typically preceded by any mutations, it is considered that the CALR mutation may not require any preceding mutations. Our results showed that, like JAK2 V617F mutants, overexpression of the CALR-del52 mutant impairs HSC functioning. Furthermore, we found that CALR haploinsufficiency restores the functions of impaired HSCs to the same extent as that found in WT HSCs. When an HSC acquires the CALR mutation, "defect" and "recovery" states thus occur simultaneously in the cell. This finding may explain why mutant CALR clones expand without needing to undergo any preceding mutation. Disclosures No relevant conflicts of interest to declare.

Fetal Hemoglobin In Sickle Cell Anemia: Molecular Characterization of Saudi Patients From the Eastern Province

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1627-1627
Author(s):  
Abdulrahman Alsultan ◽  
Idowu Akinsheye ◽  
Nadia Solovieff ◽  
Hazem Ghabbour ◽  
Duyen A. Ngo ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Conflicts Of Interest ◽  
Globin Gene ◽  
Snp Array ◽  
Fetal Hemoglobin ◽  
Homozygosity Mapping ◽  
Eastern Province ◽  
Saudi Patients

Abstract Abstract 1627 In the Eastern Province of Saudi Arabia, sickle cell anemia (HbSS) is associated with the Saudi Indian (SI) HBB-gene cluster haplotype, high levels of fetal hemoglobin (HbF) and milder disease, when compared with Southwestern Province HbSS patients who have lower HbF levels and different HBB haplotypes. An association between HbF and the Xmn1 restriction site in the HBG2 promoter present in both the SI and African-derived Senegal haplotypes is well known, but the causal elements of this association are unknown. Moreover, among individuals with the SI haplotype, only HbSS patients have high HbF while individuals with sickle cell trait (HbAS) or normal hemoglobin (HbAA) do not. Furthermore, HbF levels are far higher in SI haplotype patients, as shown below, compared with Senegal haplotype homozygotes. For example African patients homozygous for the Senegal haplotype had 12.3±5.3% HbF. To better understand the genetic basis for high HbF in SI haplotype HbSS cases, we compared sequences in the HBB gene cluster in patients with SI and Senegal haplotypes. We hypothesized that the causal elements that modify HbF in Saudi patients are in linkage disequilibrium (LD) with the βS globin gene in this population. Accordingly, we studied 5 Saudi families from the Eastern Province. Seven SI haplotype patients with HbSS (median age 5 yrs, range 2.5–49 yrs) were homozygous for the Xmn I site and had Hb 9.7 ± 1.6 g/dL, MCV 76.5 ± 8.3 fl and median Hb F 30.3 (range 18–41). Seventeen SI haplotype individuals had HbAS (median HbF 1.2, range 0–4.2); and 2 were normal. We first determined the genotypes of 3 known HbF QTLs, BCL11A (rs766432); HBS1L-MYB (rs7775698 and rs9399137); and OR51B5/6 (rs5006884). There were no consistent genotypes among these 7 patients to explain their universal high HbF. Next, we performed homozygosity mapping using Illumina Human610-Quad SNP array and identified runs of homozygosity (RoH) of variable length (from 160 kb to nearly 2 mb) within and surrounding the HBB cluster only in HbSS patients. RoH were absent elsewhere in the genome in HbSS. The RoH that was shared by all HbSS patients was 126.6 kb in chr11:5153026-5279647 (NCBI36/hg18) and contains SNPs from rs11036090 to rs7118113 of the Illumina Human610-Quad SNP array. This region contains: OR51B4, the complete HBB cluster, and OR51V1. Homozygosity mapping in 6 Senegal haplotype homozygotes showed a slightly larger RoH from chr11:4909490-5314457 and SNPs rs840713-rs10837822. Both the Saudi patients and Senegal homozygotes had the same homozygous genotypes for the overlapping region of chr11:5205580-5235931 ranging from rs11036364 to rs5010981.To identify potential genetic modifiers of HbF level in the region detected in the Saudi cases, we sequenced areas within or near the Corfu deletion that is known to cause HPFH, the HBD-HBG1 intergenic region, and core regions of HS- 2, 3, and 4 in the LCR. Core regions of HS-3 and HS-4 were identical to the reference sequences. In the core of HS-2, the 10TA.2CA.2TA.CG.12TA motif was present. This motif is known to be associated with the SI haplotype but not with any other haplotypes. Within the region of the Corfu deletion, many polymorphisms were identified highlighting the complexity of SI haplotype and HBB haplotypes in general. Many of these polymorphisms lead to creation or abolition of transcription factor binding sites when this was examined in silico using the TFBS search program ConSite (consite.genereg.net). Some of these putative sites bind transcription factors presumed to have regulatory roles in globin gene expression. Complete sequencing of the 126.6 kb interval with comparison to other HBB haplotypes associated with high and low HbF might focus attention on areas of interest that can be examined in functional studies. Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document