Fetal hemoglobin in sickle cell anemia

Abstract Fetal hemoglobin (HbF) can blunt the pathophysiology, temper the clinical course, and offer prospects for curative therapy of sickle cell disease. This review focuses on (1) HbF quantitative trait loci and the geography of β-globin gene haplotypes, especially those found in the Middle East; (2) how HbF might differentially impact the pathophysiology and many subphenotypes of sickle cell disease; (3) clinical implications of person-to-person variation in the distribution of HbF among HbF-containing erythrocytes; and (4) reactivation of HbF gene expression using both pharmacologic and cell-based therapeutic approaches. A confluence of detailed understanding of the molecular basis of HbF gene expression, coupled with the ability to precisely target by genomic editing most areas of the genome, is producing important preliminary therapeutic results that could provide new options for cell-based therapeutics with curative intent.

Download Full-text

Genome editing using CRISPR-Cas9 to create the HPFH genotype in HSPCs: An approach for treating sickle cell disease and β-thalassemia

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1612075113 ◽

2016 ◽

Vol 113 (38) ◽

pp. 10661-10665 ◽

Cited By ~ 79

Author(s):

Lin Ye ◽

Jiaming Wang ◽

Yuting Tan ◽

Ashley I. Beyer ◽

Fei Xie ◽

...

Keyword(s):

Sickle Cell Disease ◽

Genome Editing ◽

Sickle Cell ◽

Globin Gene ◽

Autologous Transplantation ◽

Clinical Manifestations ◽

Fetal Hemoglobin ◽

Compound Heterozygous ◽

Cell Disease ◽

Hematopoietic Stem

Hereditary persistence of fetal hemoglobin (HPFH) is a condition in some individuals who have a high level of fetal hemoglobin throughout life. Individuals with compound heterozygous β-thalassemia or sickle cell disease (SCD) and HPFH have milder clinical manifestations. Using RNA-guided clustered regularly interspaced short palindromic repeats-associated Cas9 (CRISPR-Cas9) genome-editing technology, we deleted, in normal hematopoietic stem and progenitor cells (HSPCs), 13 kb of the β-globin locus to mimic the naturally occurring Sicilian HPFH mutation. The efficiency of targeting deletion reached 31% in cells with the delivery of both upstream and downstream breakpoint guide RNA (gRNA)-guided Staphylococcus aureus Cas9 nuclease (SaCas9). The erythroid colonies differentiated from HSPCs with HPFH deletion showed significantly higher γ-globin gene expression compared with the colonies without deletion. By T7 endonuclease 1 assay, we did not detect any off-target effects in the colonies with deletion. We propose that this strategy of using nonhomologous end joining (NHEJ) to modify the genome may provide an efficient approach toward the development of a safe autologous transplantation for patients with homozygous β-thalassemia and SCD.

Download Full-text

Differences in Fetal Hemoglobin Induction in Sickle Cell Disease and beta-Thalassemia.

Blood ◽

10.1182/blood.v108.11.555.555 ◽

2006 ◽

Vol 108 (11) ◽

pp. 555-555 ◽

Cited By ~ 2

Author(s):

Hassana Fathallah ◽

Ali Taher ◽

Ali Bazarbachi ◽

George F. Atweh

Keyword(s):

Gene Expression ◽

Sickle Cell Disease ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Mrna Levels ◽

Globin Genes ◽

Thalassemia Intermedia ◽

Cell Disease ◽

Globin Mrna ◽

Globin Gene Expression

Abstract A number of therapeutic agents including hydroxyurea, butyrate and decitabine have shown considerable promise in the treatment of sickle cell disease (SCD). However, the same agents have shown less clinical activity in β-thalassemia. As a first step towards understanding the molecular basis of the different clinical responses to these agents, we have studied the mechanisms of induction of fetal hemoglobin (HbF) by butyrate in BFU-E derived cells from 5 patients with SCD and 9 patients with β-thalassemia intermedia. Exposure to butyrate resulted in a dose-dependent augmentation of γ-globin mRNA levels in erythroid cells from patients with SCD. In contrast, induction of γ-globin expression in erythroid cells from patients with β-thalassemia intermedia was only seen at a high concentration of butyrate. The increase in γ-globin mRNA levels in patients with SCD and β-thalassemia intermedia was associated with opening of the DNA structure as manifested by decreased DNA methylation at the γ-globin promoters. Interestingly, butyrate exposure had markedly different effects on the expression of the β- and α-globin genes in the two categories of patients. Butyrate decreased the level of β-globin mRNA in 4 out of 5 patients with SCD (P = 0.04), while in β-thalassemia the levels of β-globin mRNA did not change in 7 patients and decreased in 2 patients after butyrate exposure (P = 0.12). Thus in patients with SCD, the effects of the induction of the γ-globin gene on the γ/(β+γ) mRNA ratios were further enhanced by the butyrate-mediated decreased expression of the β-globin gene. As a result, γ/(β+γ) mRNA ratios increased in all patients with SCD, with a mean increase of 31% (P = 0.002). In contrast, butyrate increased γ/(β+γ) mRNA ratios only in 4 out of 9 patients with β-thalassemia, with a more modest mean increase of 12% (P = 0.004). Interestingly, the decreased β-globin expression in patients with SCD was associated with closing of the DNA configuration as manifested by hypermethylation of DNA at the promoter of the β-globin gene while methylation of the same promoter did not change following butyrate exposure in patients with β-thalassemia intermedia. More surprisingly, the expression of the α-globin genes increased following butyrate exposure in 4 out of 9 patients with β-thalassemia, while the levels of α-globin mRNA decreased in 4 out of 5 patients with SCD. As a result, the favorable effects of the butyrate-induced increase in γ-globin gene expression on the α: non-α mRNA imbalance in patients with β-thalassemia intermedia were partly neutralized by the corresponding increase in α-globin gene expression. These differences may explain, at least in part, the more favorable effects of inducers of HbF in SCD than in β-thalassemia. Further studies are necessary to fully understand the molecular bases of the different responses to agents that induce HbF in patients with these disorders.

Download Full-text

Analysis of Fetal Hemoglobin Expression within Humanized Sickle Cell Disease Mice Overexpressing the TR2/4 Transgene.

Blood ◽

10.1182/blood.v116.21.1619.1619 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1619-1619 ◽

Cited By ~ 1

Author(s):

Andrew Campbell ◽

Osamu Tanabe ◽

Rebekah Urbonya ◽

Andrea Mathias ◽

Lihong Shi ◽

...

Keyword(s):

Body Weight ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Beta Globin ◽

Cell Disease ◽

Hemoglobin F ◽

Gamma Globin ◽

Fold Induction

Abstract Abstract 1619 Background: Sickle Cell Disease (SCD) is a chronic debilitating hematologic condition caused by a missense mutation within the adult beta globin gene leading to significant morbidity and mortality. Increased Fetal Hemoglobin production has been shown to significantly ameliorate SCD symptoms and improve survival. A novel specific DNA-binding factor DRED (direct repeat erythroid definitive) was recently identified that regulated epsilon and gamma globin gene expression (Tanimoto et al Genes Dev 2000). Purification of DRED revealed that it harbored the nuclear orphan hormone receptors, TR2/TR4, as its DNA binding core (Tanabe et al EMBO 2002). Overexpression of TR2/TR4 Transgene within Human Beta Globin Yeast Artificial Chromosome Transgenic Mice resulted in 4-fold induction of the gamma globin mRNA levels (Tanabe et al EMBO 2007). Therefore, we wanted to determine if the overexpression of TR2/TR4 within a humanized sickle cell disease model would result in fetal hemoglobin induction. Methods: Humanized Homozygous Knock-In UAB-Sickle Cell (UAB-Hbahα/hα Hbbhβs/hβs) Mice (Wu et al Blood 2006) was mated to TR2/TR4 Overexpressing Mice (TgTR2/TR4) to generate homozygous SS-TR2/TR4 compound heterozygotes (UAB-Hba hα/hα Hbb hβs/hβs TgTR2/TR4). We generated four 2–3 month old homozygous SS-TR2/TR4 transgenic mice and compared hemoglobin F levels, complete blood cell counts and % body weight (liver, spleen, kidney) to six 2–3 month old homozygous SS mice (Hbahα/hα Hbb hβs/hβs)without the overexpressing TgTR2/TR4. Tail PCR genotyping of all sickle cell mice (with and without TgTR2/TR4) and Hemoglobin F(Hgb F) and Sickle (HgbS) levels were confirmed by HPLC Hemoglobin electrophoresis. Results: The mean Hgb F: 7.8% (n=6, sd 1.63+/−) in the homozygous SS control mice vs. 16.5% (n=4, sd 2.64+/−)in the homozygous SS-TR2/4 Mice (2 Fold higher). Hematologic profile revealed a mean Hct: 25.2 (n=6, sd 5.50 +/−) mean MCV: 75.4 (n=6, sd 10+/−) and a mean WBC: 22.6 (n= 6, sd 13.9 +/−) in the homozygous SS control mice vs. a mean Hct: 31.25(n=4, sd 6.89+/−), mean MCV: 61(n=4, sd 3.5+/−) mean WBC: 16.3(n= 4, sd 5.99+/−) in the homozygous SS-TR2/TR4 mice. Lastly, initial organ (spleen, liver, kidney) pathology evaluation revealed decreased % body weight (bw) in homozygous SS TR2/TR4 Mice vs. homozygous SS controls: 1) Spleen %bw: 4.3% vs. 3.5% TgTR2/TR4), 2) Liver % bw: 8.8% vs. 7.7% TgTR2/TR4), and 3) Kidney %bw: 1.14% vs. 1.02% TgTR2/TR4). Conclusions: Our preliminary analysis revealed that TR2/TR4 overexpression within a humanized sickle cell disease mouse model resulted in a 2-fold induction of fetal hemoglobin based on HPLC hemoglobin electrophoresis. Further, increased TR2/TR4 overexpression improved anemia and organomegaly within sickle cell disease mice. TR2/TR4 may be an attractive target for fetal hemoglobin induction for the treatment of sickle cell disease. Ongoing studies will determine if TR2/TR4 decreases organ specific disease pathology. We will also determine the cellular distribution of fetal hemoglobin in future studies. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Heme oxygenase-1 gene promoter polymorphism is associated with reduced incidence of acute chest syndrome among children with sickle cell disease

Blood ◽

10.1182/blood-2011-06-361642 ◽

2012 ◽

Vol 120 (18) ◽

pp. 3822-3828 ◽

Cited By ~ 53

Author(s):

Christopher J. Bean ◽

Sheree L. Boulet ◽

Dorothy Ellingsen ◽

Meredith E. Pyle ◽

Emily A. Barron-Casella ◽

...

Keyword(s):

Sickle Cell Disease ◽

Heme Oxygenase ◽

Sickle Cell ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Promoter Polymorphism ◽

Adverse Outcomes ◽

Acute Chest Syndrome ◽

Heme Oxygenase 1 ◽

Cell Disease

Abstract Sickle cell disease is a common hemolytic disorder with a broad range of complications, including vaso-occlusive episodes, acute chest syndrome (ACS), pain, and stroke. Heme oxygenase-1 (gene HMOX1; protein HO-1) is the inducible, rate-limiting enzyme in the catabolism of heme and might attenuate the severity of outcomes from vaso-occlusive and hemolytic crises. A (GT)n dinucleotide repeat located in the promoter region of the HMOX1 gene is highly polymorphic, with long repeat lengths linked to decreased activity and inducibility. We examined this polymorphism to test the hypothesis that short alleles are associated with a decreased risk of adverse outcomes (hospitalization for pain or ACS) among a cohort of 942 children with sickle cell disease. Allele lengths varied from 13 to 45 repeats and showed a trimodal distribution. Compared with children with longer allele lengths, children with 2 shorter alleles (4%; ≤ 25 repeats) had lower rates of hospitalization for ACS (incidence rate ratio 0.28, 95% confidence interval, 0.10-0.81), after adjusting for sex, age, asthma, percentage of fetal hemoglobin, and α-globin gene deletion. No relationship was identified between allele lengths and pain rate. We provide evidence that genetic variation in HMOX1 is associated with decreased rates of hospitalization for ACS, but not pain. This study is registered at www.clinicaltrials.gov as #NCT00072761.

Download Full-text

Fetal hemoglobin levels and beta s globin haplotypes in an Indian populations with sickle cell disease

Blood ◽

10.1182/blood.v69.6.1742.bloodjournal6961742 ◽

1987 ◽

Vol 69 (6) ◽

pp. 1742-1746

Author(s):

AE Kulozik ◽

BC Kar ◽

RK Satapathy ◽

BE Serjeant ◽

GR Serjeant ◽

...

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Sickle Cell Trait ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Globin Genes ◽

Cell Disease ◽

Hemoglobin F ◽

S Haplotype ◽

Asian Populations

To further explore the cause for variation in hemoglobin F (Hb F) levels in sickle cell disease, the beta globin restriction-fragment length polymorphism haplotypes were determined in a total of 303 (126 SS, 141 AS, 17 S beta degrees, 7 A beta, degrees and 12 AA) Indians from the state of Orissa. The beta s globin gene was found to be linked almost exclusively to a beta S haplotype ( -++-), which is also common in Saudi Arabian patients from the Eastern Province (referred to as the Asian beta s haplotype). By contrast, the majority of beta A and beta degree thalassemia globin genes are linked to haplotypes common in all European and Asian populations (+-----[+/-]; --++-++). Family studies showed that there is a genetic factor elevating Hb F levels dominantly in homozygotes (SS). This factor appears to be related to the Asian beta s globin haplotype, and a mechanism for its action is discussed. There is also a high prevalence of an independent Swiss type hereditary persistence of fetal hemoglobin (HPFH) determinant active in both the sickle cell trait and in sickle cell disease.

Download Full-text

Identifying genetic variants and pathways associated with extreme levels of fetal hemoglobin in Sickle Cell Disease in Tanzania.

10.21203/rs.2.22041/v2 ◽

2020 ◽

Author(s):

Siana Nkya ◽

Liberata Mwita ◽

Josephine Mgaya ◽

Happiness Kumburu ◽

Marco van Zwetselaar ◽

...

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Genetic Variants ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Cell Disease ◽

Synonymous Mutations ◽

Targeted Next Generation Sequencing ◽

Blood Disorder ◽

Common Genetic Variants

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

Download Full-text

Additive Effect of Butyrate and Hemin on Fetal Hemoglobin Induction in Erythroid Cells from Sickle Cell Disease and Beta-Thalassemia.

Blood ◽

10.1182/blood.v108.11.1224.1224 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1224-1224

Author(s):

Hassana Fathallah ◽

Ali Taher ◽

Ali Bazarbachi ◽

George F. Atweh

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Histone Deacetylases ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Beta Thalassemia ◽

Erythroid Cells ◽

Cell Disease ◽

Globin Mrna ◽

In Erythroid Cells

Abstract High levels of fetal hemoglobin (HbF) are known to ameliorate the pathophysiology of β-globin disorders. The objective of this study is twofold: the first is to evaluate the efficacy of hemin as an inducer of HbF in erythroid cells from patients with sickle cell disease (SCD) and β-thalassemia (β-thal); the second is to determine if the combination of butyrate and hemin can induce higher levels of expression of HbF than either agent alone. BFU-E derived cells from the peripheral blood of two patients with homozygous SCD, three patients with β-thal, one patient with sickle β-thalassemia (S/β-thal) and one normal individual (AA) were cultured in the absence (control) or presence of butyrate (B), hemin (H) or butyrate and hemin (B+H). As expected, the levels of γ-globin mRNA [expressed as % γ/(β+γ)] increased upon butyrate exposure in progenitor-derived erythroid cells from SS and S/β-thal patients, and to a lesser extent in patients with β-thal (P = 0.01). In contrast, butyrate did not increase γ-globin expression in BFU-E derived colonies from the AA individual. Moreover, hemin exposure increased the γ/(β+γ) ratio in all subjects (P = 0.02). These findings confirm that hemin can be an effective HbF inducing agent in SCD and β-thal. Although the mechanism of induction of HbF by hemin is not known, unlike butyrate, hemin is clearly not a direct inhibitor of histone deacetylases and is likely to induce HbF by a different mechanism of action. Thus, we investigated the effect of the combination of hemin and butyrate on γ-globin gene expression. Interestingly, the combination of butyrate and hemin resulted in additive increases in the γ/(β+γ) ratios in all patients compared to butyrate alone (P = 0.03) or hemin alone (P = 0.01) (Table I). Just as importantly, exposure to both drugs resulted in a decrease in the α/(β+γ) mRNA imbalance in β-thal, which is the predominant pathophysiological feature of this disorder. In conclusion, combination therapy consisting of butyrate and hemin, which are two agents with different mechanisms of action and different toxicity profiles, may provide a more effective way of inducing HbF in patients with SCD and β-thal. Table I mRNA SCD β-Thal S/β-Thal AA n 2 3 1 1 %γ/(β+γ) Control 36 42 26 7.1 B 45 50 41 6.9 H 55 55 52 15 B+H 60 61 59 13 α/(β+γ) Control 3.1 8.9 1.8 1.9 B 2.0 7.7 2.9 1.7 H 3.0 7.5 1.7 1.0 B+H 2.9 6.4 2.2 1.3

Download Full-text

Emerging Genetic Therapy for Sickle Cell Disease

Annual Review of Medicine ◽

10.1146/annurev-med-041817-125507 ◽

2019 ◽

Vol 70 (1) ◽

pp. 257-271 ◽

Cited By ~ 34

Author(s):

Stuart H. Orkin ◽

Daniel E. Bauer

Keyword(s):

Gene Therapy ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Gene Editing ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Current Therapy ◽

Cell Disease ◽

Genetic Manipulations ◽

Lentiviral Gene Therapy

The genetic basis of sickle cell disease (SCD) was elucidated >60 years ago, yet current therapy does not rely on this knowledge. Recent advances raise prospects for improved, and perhaps curative, treatment. First, transcription factors, BCL11A and LRF/ZBTB7A, that mediate silencing of the β-like fetal (γ-) globin gene after birth have been identified and demonstrated to act at the γ-globin promoters, precisely at recognition sequences disrupted in rare individuals with hereditary persistence of fetal hemoglobin. Second, transformative advances in gene editing and progress in lentiviral gene therapy provide diverse opportunities for genetic strategies to cure SCD. Approaches include hematopoietic gene therapy by globin gene addition, gene editing to correct the SCD mutation, and genetic manipulations to enhance fetal hemoglobin production, a potent modifier of the clinical phenotype. Clinical trials may soon identify efficacious and safe genetic approaches to the ultimate goal of cure for SCD.

Download Full-text

Effects of 5-aza-2′-deoxycytidine on fetal hemoglobin levels, red cell adhesion, and hematopoietic differentiation in patients with sickle cell disease

Blood ◽

10.1182/blood-2003-05-1738 ◽

2003 ◽

Vol 102 (12) ◽

pp. 3865-3870 ◽

Cited By ~ 179

Author(s):

Yogen Saunthararajah ◽

Cheryl A. Hillery ◽

Don Lavelle ◽

Robert Molokie ◽

Louise Dorn ◽

...

Keyword(s):

Cell Adhesion ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Mechanism Of Action ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Red Cell ◽

Cell Disease ◽

Hemoglobin F ◽

Total Hemoglobin

Abstract Fetal hemoglobin (HbF) decreases polymerization of sickle hemoglobin (HbS) and improves outcomes in sickle cell disease (SSD). Therefore, a therapeutic goal in SSD is pharmacologic reactivation of HbF. Silencing of the γ-globin (HbF) gene is associated with DNA methylation. The cytosine analog 5-aza-2′-deoxycytidine (decitabine) hypomethylates DNA by inhibiting DNA methyltransferase. We examined if subcutaneous decitabine could increase HbF levels and improve SSD pathophysiology without cytotoxicity. Eight symptomatic SSD patients resistant or intolerant of standard treatment with hydroxyurea received decitabine 0.2 mg/kg subcutaneously 1 to 3 times per week in 2 cycles of 6-week duration. Treatment decreased neutrophils and increased mean HbF (6.5% to 20.4%, P < .0001) and mean total hemoglobin (76 to 96 g/L [7.6 to 9.6 g/dL], P < .001). Features of vaso-occlusive crisis pathophysiology such as red cell adhesion, endothelial damage, and coagulation pathway activity significantly improved. γ-Globin gene promoter methylation decreased, and platelets and the proportion of megakaryocytes and erythroid cells in the marrow increased without a decrease in marrow cellularity, consistent with a DNA hypomethylating, noncytotoxic mechanism of action. Weekly subcutaneous decitabine produces cumulative increases in HbF and total hemoglobin through a noncytotoxic mechanism of action. Chronic dosing and sustained increases in hemoglobin F and total hemoglobin levels may be possible. Further studies in SSD and thalassemia are indicated.

Download Full-text