scholarly journals Sodium butyrate enhances fetal globin gene expression in erythroid progenitors of patients with Hb SS and beta thalassemia

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 454-459 ◽  
Author(s):  
SP Perrine ◽  
BA Miller ◽  
DV Faller ◽  
RA Cohen ◽  
EP Vichinsky ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Sodium Butyrate ◽  
Clinical Symptoms ◽  
Therapeutic Potential ◽  
Globin Gene ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Erythroid Progenitors ◽  
Gamma Globin

Abstract Increasing the expression of the gamma globin genes is considered a useful therapeutic approach to the beta globin diseases. Because butyrate and alpha-amino-n-butyric acid (ABA) augment gamma globin expression in normal neonatal and adult erythroid progenitors, we investigated the effects of sodium butyrate and ABA on erythroid progenitors of patients with beta thalassemia and sickle cell anemia who might benefit from such an effect. Both substances increased fetal hemoglobin (Hb F) expression in Bfu-e from 7% to 30% above levels found in control cultures from the same subjects with sickle cell anemia. The fraction of cultured erythroblasts producing Hb F increased more than 20% with sodium butyrate treatment in 70% of cultures. In most cultures, this produced greater than 20% total Hb F and greater than 70% F cells, levels which have been considered beneficial in ameliorating clinical symptoms. Alpha: non-alpha (alpha-non-alpha) imbalance was decreased by 36% in erythroid progenitors of patients with beta thalassemia cultured in the presence of butyrate compared with control cultures from the same subjects. These data suggest that sodium butyrate may have therapeutic potential for increasing gamma globin expression in the beta globin diseases.

scholarly journals Sodium butyrate enhances fetal globin gene expression in erythroid progenitors of patients with Hb SS and beta thalassemia

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 454-459 ◽  
Author(s):  
SP Perrine ◽  
BA Miller ◽  
DV Faller ◽  
RA Cohen ◽  
EP Vichinsky ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Sodium Butyrate ◽  
Clinical Symptoms ◽  
Therapeutic Potential ◽  
Globin Gene ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Erythroid Progenitors ◽  
Gamma Globin

Increasing the expression of the gamma globin genes is considered a useful therapeutic approach to the beta globin diseases. Because butyrate and alpha-amino-n-butyric acid (ABA) augment gamma globin expression in normal neonatal and adult erythroid progenitors, we investigated the effects of sodium butyrate and ABA on erythroid progenitors of patients with beta thalassemia and sickle cell anemia who might benefit from such an effect. Both substances increased fetal hemoglobin (Hb F) expression in Bfu-e from 7% to 30% above levels found in control cultures from the same subjects with sickle cell anemia. The fraction of cultured erythroblasts producing Hb F increased more than 20% with sodium butyrate treatment in 70% of cultures. In most cultures, this produced greater than 20% total Hb F and greater than 70% F cells, levels which have been considered beneficial in ameliorating clinical symptoms. Alpha: non-alpha (alpha-non-alpha) imbalance was decreased by 36% in erythroid progenitors of patients with beta thalassemia cultured in the presence of butyrate compared with control cultures from the same subjects. These data suggest that sodium butyrate may have therapeutic potential for increasing gamma globin expression in the beta globin diseases.

Influence of Globin Gene Modifiers on Baseline and Hydroxyurea-Induced Fetal Hemoglobin Levels in Children with Sickle Cell Anemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3171-3171
Author(s):  
Russell E. Ware ◽  
Barry Eggleston ◽  
Tatiana Abramova ◽  
Sherri A. Zimmerman ◽  
Alice Lail ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Globin Genes ◽  
Beta Globin ◽  
Gene Number ◽  
Gamma Globin ◽  
Alpha Globin ◽  
Hydroxyurea Therapy

Abstract Fetal hemoglobin (HbF) is recognized as a major determinant of clinical disease severity in children and adults with sickle cell anemia (SCA). Patients with elevated HbF levels have a milder disease course, and many current therapeutic protocols for SCA include pharmacological induction of HbF. However, baseline and treatment HbF levels vary widely due to presumed genetic and environmental factors. Recognized globin gene modifiers of HbF include the beta globin haplotype and a potential contribution from concomitant alpha thalassemia. To characterize more fully the influence of globin gene modifiers on both baseline and treatment HbF levels, we retrospectively determined the beta globin haplotype (Benin, CAR, Senegal, Cameroon, or Arab-Indian) by selective gamma globin gene nucleotide sequencing and the alpha globin gene number (2, 3, or 4) by PCR for 67 African-American children with SCA receiving hydroxyurea therapy at stable maximal tolerated dose (MTD). The four beta globin haplotypes and frequencies identified in our cohort of children include Benin (0.61), CAR (0.17), Senegal (0.12), and Cameroon (0.10). The number of alpha globin genes and frequencies identified were 4 genes (0.72), 3 genes (0.25) and 2 genes (0.03). Baseline and MTD HbF levels were analyzed according to each variable. The average baseline HbF value for the entire cohort of children was 7.7 ± 4.4% (median 7.6%, range 1.3 – 19.3%), while the average treatment HbF value was 23.9 ± 7.2 % (median 22.9%, range 10.2 – 40.7%). All 67 children increased their HbF in response to hydroxyurea therapy (median 16.7%, range 5.0 – 28.8%). There was a modest but statistically significant correlation between the baseline and treatment HbF (r=0.66, p<.0001). The estimated effect of one unit change in baseline HbF on treatment HbF was 1.11 (95% CI of 0.78, 1.43). When baseline %HbF was analyzed according to the beta globin haplotype, the overall ANOVA had a p-value of 0.02, indicating a statistically significant influence. Further analysis confirmed associations previously identified in adults with SCA, i.e. children with at least one copy of the CAR haplotype had a lower baseline HbF (5.9% vs 8.4%, p=.05), while those with at least one copy of the Senegal haplotype had a higher baseline HbF (11.1% vs 6.7%, p<.001). When hydroxyurea MTD (treatment) HbF values were analyzed according to beta globin haplotype while adjusting for baseline HbF, however, the effect of beta globin haplotype was not statistically significant (p=.13). Analyses of HbF according to alpha globin gene number revealed no statistically significant effects on either baseline or treatment HbF values. Taken together, these data support the hypothesis that beta globin haplotype significant influences baseline HbF values for children with SCA, but has no significant effects on hydroxyurea MTD HbF values. Accordingly, children with SCA should be offered hydroxyurea based solely on clinical indications, without consideration of baseline HbF or beta globin haplotype. Even children with low baseline HbF values or the CAR beta globin haplotype can respond to hydroxyurea therapy with an elevated %HbF. Future studies designed to identify genetic modifiers of treatment HbF values should focus on sequence polymorphisms in non-globin genes that have trans-acting effects on gamma globin gene expression.

New Concepts in Genome Regulation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. SCI-47-SCI-47
Author(s):  
Ann Dean ◽  
Jongjoo Lee ◽  
Ryan Dale ◽  
Ivan Krivega
Keyword(s):  
Sickle Cell Disease ◽  
Long Range ◽  
Control Region ◽  
Sickle Cell ◽  
Globin Gene ◽  
Locus Control Region ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Cell Disease ◽  
Gamma Globin

Abstract Manipulating gene regulation to favor gamma-globin transcription over beta-globin transcription has been a goal of research in erythropoiesis for decades because of its relevance to amelioration of the pathophysiology of sickle cell disease and beta-thalassemia. A fundamental unanswered question in biology is how the unique pattern of gene expression, the transcriptome, of the many different individual mammalian cell types arises from the same genome blueprint and changes during development and differentiation. There is a growing appreciation that genome organization and the folding of chromosomes is a key determinant of gene transcription. Within this framework, enhancers function to increase the transcription of target genes over long linear distances. To accomplish this, enhancers engage in close physical contact with target promoters through chromosome folding, or looping. These long range interactions are orchestrated by cell type specific proteins and protein complexes that bind to enhancers and promoters and stabilize their interaction with each other. We have been studying LDB1, a member of an erythroid protein complex containing GATA1, TAL1 and LMO2. The LDB1complex activates erythroid genes through occupancy of virtually all erythroid enhancers. LDB1engages in homo- and heterotypic interactions with proteins occupying the promoters of erythroid genes to bring them into proximity with their enhancers. We find that enhancer long range looping activity can be redirected. Both targeting of the beta-globin locus control region to the gamma-globin gene in adult erythroid cells by the tethering of LDB1 or epigenetic unmasking of a silenced gamma-globin gene lead to increased locus control region (LCR)/gamma-globin contact frequency and reduced LCR/beta-globin contact. The outcome of these manipulations is robust, pan-cellular gamma-globin transcription activation with a concomitant reduction in beta-globin transcription. These examples suggest that chromosome looping can be considered a therapeutic target for gene activation or gene silencing to ameliorate genetic diseases such as sickle cell disease and beta-thalassemia. Disclosures No relevant conflicts of interest to declare.

scholarly journals Fetal hemoglobin in sickle cell anemia: relation to regulatory sequences cis to the beta-globin gene. Multicenter Study of Hydroxyurea

Blood ◽  
1996 ◽  
Vol 87 (4) ◽  
pp. 1604-1611 ◽  
Author(s):  
ZH Lu ◽  
MH Steinberg
Keyword(s):  
Gene Expression ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Regulatory Sequences ◽  
Beta Globin ◽  
Beta Globin Gene ◽  
Gamma Globin ◽  
Globin Gene Expression

Very different fetal hemoglobin levels among adult sickle cell anemia patients suggest genetic modulation of gamma-globin gene expression. In sickle cell anemia, different fetal hemoglobin levels are associated with distinct beta-globin gene haplotypes. Haplotype may be a marker for linked DNA that modulates gamma-globin gene expression. From 295 individuals with sickle cell anemia, we chose for detailed studies 53 patients who had the highest or the lowest fetal hemoglobin levels and 7 patients whose fetal hemoglobin levels were atypical of their haplotype. In these individuals, we examined portions of the beta- globin gene locus control region hypersensitive sites two and three, an (AT)x(T)y repeat 5′ to the beta-globin gene, a 4-bp deletion 5 to the A gamma T gene, promoters of both gamma-globin genes, 5′ flanking region of the G gamma-globin gene, and A gamma-globin gene IVS-II. Of the regions we studied all polymorphisms were always haplotype-linked and no additional mutations were present. This suggested that variations in these areas are uncommon mechanisms of fetal hemoglobin modulation in sickle cell anemia. Whereas unexamined cis-acting sequences may regulate gamma-globin gene transcription, trans-acting factors may play a more important role.

scholarly journals Complementary Base Editing Approaches for the Treatment of Sickle Cell Disease and Beta Thalassemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3352-3352 ◽  
Author(s):  
Ling Lin ◽  
Adrian P. Rybak ◽  
Conrad Rinaldi ◽  
Jonathan Yen ◽  
Yanfang Fu ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Cell Disease ◽  
Hematopoietic Stem ◽  
Base Editing ◽  
Gamma Globin

Sickle cell disease (SCD) and Beta thalassemia are disorders of beta globin production and function that lead to severe anemia and significant disease complications across a multitude of organ systems. Autologous transplantation of hematopoietic stem cells engineered through the upregulation of fetal hemoglobin (HbF) or correction of the beta globin gene have the potential to reduce disease burden in patients with beta hemoglobinopathies. Base editing is a recently developed technology that enables precise modification of the genome without the introduction of double strand DNA breaks. Gamma globin gene promoters were comprehensively screened with cytosine and adenine base editors (ABE) for the identification of alterations that would derepress HbF. Three regions were identified that significantly upregulated HbF, and the most effective nucleotide residue conversions are supported by natural variation seen in patients with hereditary persistence of fetal hemoglobin (HPFH). ABEs have been developed that significantly increase the level of HbF following nucleotide conversion at key regulatory motifs within the HBG1 and HBG2 promoters. CD34+ hematopoietic stem and progenitor cells (HSPC) were purified at clinical scale and edited using a process designed to preserve self-renewal capacity. Editing at two independent sites with different ABEs reached 94 percent and resulted in up to 63 percent gamma globin by UPLC. The levels of HbF observed should afford protection to the majority of SCD and Beta thalassemia patients based on clinical observations of HPFH and non-interventional therapy that links higher HbF dosage with milder disease (Ngo et al, 2011 Brit J Hem; Musallam et al, 2012 Blood). Directly correcting the Glu6Val mutation of SCD has been a recent goal of genetic therapies designed for the SCD population. Current base editing technology cannot yet convert mutations like those that result from the A-T transversion in sickle beta globin; however, ABE variants have been designed to recognize and edit the opposite stranded adenine residue of valine. This results in the conversion of valine to alanine and the production of a naturally occurring variant known as Hb G-Makassar. Beta globin with alanine at this position does not contribute to polymer formation, and patients with Hb G-Makassar present with normal hematological parameters and red blood cell morphology. SCD patient fibroblasts edited with these ABE variants achieve up to 70 percent conversion of the target adenine. CD34 cells from healthy donors were then edited with a lead ABE variant, targeting a synonymous mutation in an adjacent proline that resides within the editing window and serves as a proxy for editing the SCD mutation. The average editing frequency was 40 percent. Donor myeloid chimerism documented at these levels in the allogeneic transplant setting exceeds the 20 percent that is required for reversing the sickle phenotype (Fitzhugh et al, 2017 Blood). These next generation editing approaches provide a promising new modality for treating patients with Beta thalassemia and SCD. Disclosures No relevant conflicts of interest to declare.

scholarly journals Analysis of hemoglobin F production in Saudi Arabian families with sickle cell anemia

Blood ◽  
1987 ◽  
Vol 70 (3) ◽  
pp. 716-720 ◽  
Author(s):  
BA Miller ◽  
M Salameh ◽  
M Ahmed ◽  
N Olivieri ◽  
G Antognetti ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Cell Disease ◽  
Erythroid Progenitors ◽  
Hemoglobin F ◽  
Gamma Globin ◽  
Hemoglobin Production

Erythrocytes and progenitor-derived erythroblasts of sickle cell anemia patients from the Eastern Province of Saudi Arabia contain increased fetal hemoglobin and G gamma globin. A distinctive DNA polymorphism haplotype in the beta globin gene cluster (++- +-), tightly coupled to a C----T substitution at position -158 5′ to the cap site of the G gamma globin gene, is strongly associated with sickle cell disease in this region. To determine whether the increased fetal hemoglobin production and/or elevated G gamma globin content are tightly linked to this haplotype, we studied 55 members of five Saudi families in which sickle cell disease is present. The results did not suggest a tight linkage of the haplotype to increased fetal hemoglobin production. On the other hand, several sickle trait family members heterozygous for the haplotype had normal fetal hemoglobin production in culture but elevated G gamma to A gamma ratios in peripheral blood. This observation suggests that in this genetic background increased expression of the G gamma globin gene may occur without a measurable increase in total fetal hemoglobin production. The family studies also clearly demonstrate that increased fetal hemoglobin production by erythroid progenitors is dependent on zygosity for the sickle gene in this population. These findings strongly suggest that other factors, such as the products of genes stimulated by hemolytic stress or other genetic determinants associated with the Saudi beta S chromosome, may interact with the -158 C----T substitution and influence gamma globin gene expression in this population.

scholarly journals Analysis of hemoglobin F production in Saudi Arabian families with sickle cell anemia

Blood ◽  
1987 ◽  
Vol 70 (3) ◽  
pp. 716-720 ◽  
Author(s):  
BA Miller ◽  
M Salameh ◽  
M Ahmed ◽  
N Olivieri ◽  
G Antognetti ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Cell Disease ◽  
Erythroid Progenitors ◽  
Hemoglobin F ◽  
Gamma Globin ◽  
Hemoglobin Production

Abstract Erythrocytes and progenitor-derived erythroblasts of sickle cell anemia patients from the Eastern Province of Saudi Arabia contain increased fetal hemoglobin and G gamma globin. A distinctive DNA polymorphism haplotype in the beta globin gene cluster (++- +-), tightly coupled to a C----T substitution at position -158 5′ to the cap site of the G gamma globin gene, is strongly associated with sickle cell disease in this region. To determine whether the increased fetal hemoglobin production and/or elevated G gamma globin content are tightly linked to this haplotype, we studied 55 members of five Saudi families in which sickle cell disease is present. The results did not suggest a tight linkage of the haplotype to increased fetal hemoglobin production. On the other hand, several sickle trait family members heterozygous for the haplotype had normal fetal hemoglobin production in culture but elevated G gamma to A gamma ratios in peripheral blood. This observation suggests that in this genetic background increased expression of the G gamma globin gene may occur without a measurable increase in total fetal hemoglobin production. The family studies also clearly demonstrate that increased fetal hemoglobin production by erythroid progenitors is dependent on zygosity for the sickle gene in this population. These findings strongly suggest that other factors, such as the products of genes stimulated by hemolytic stress or other genetic determinants associated with the Saudi beta S chromosome, may interact with the -158 C----T substitution and influence gamma globin gene expression in this population.

scholarly journals Efficacy of ruxolitinib as inducer of fetal hemoglobin in primary erythroid cultures from sickle cell and beta-thalassemia patients

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Alice Pecoraro ◽  
Antonio Troia ◽  
Aurelio Maggio ◽  
Rosalba Di Marzo
Keyword(s):  
Mrna Expression ◽  
Sickle Cell ◽  
Cultured Cells ◽  
Globin Gene ◽  
Beta Thalassemia ◽  
Erythroid Progenitors ◽  
Globin Mrna ◽  
Gamma Globin

High levels of HbF may ameliorate the clinical course of β-thalassaemia and SCD. Hydroxyurea (HU) is the only HbF inducer approved for the treatment of patients. However not all patients respond to the treatment, for this reason it is noteworthy to identify new HbF inducers. Ruxolitinib is a JAK inhibitor that decreases the phosphorilation of STAT proteins. In particular STAT3 is a repressor of gamma-globin gene. The decrease of STAT3 phosphorilation could derepress gamma-globin gene and reactivate its trascription. In this study we evaluated the efficacy of ruxolitinib as inducer of HbF production. The analyses were performed in cultured erythroid progenitors from 16 beta-thalassemia intermedia (TI) and 4 sickle cell disease (SCD) patients. The use of quantitative RT-PCR technique allowed us to determine the increase of gamma-globin mRNA expression in human erythroid cultured cells treated with ruxolitinib. The results of our study demonstrated an increase in vitro of gamma-globin mRNA expression in almost all patients. These data suggest that ruxolitinib could be a good candidate to be used in vivo for the treatment of hemoglobinopathies.

scholarly journals Molecular analysis of Japanese delta beta-thalassemia

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1771-1776
Author(s):  
S Shiokawa ◽  
H Yamada ◽  
Y Takihara ◽  
E Matsunaga ◽  
Y Ohba ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Beta Thalassemia ◽  
Globin Genes ◽  
Beta Globin ◽  
Beta Globin Gene ◽  
Gamma Globin ◽  
Large Deletions ◽  
Deletion Junction

A DNA fragment containing the deletion junction region from a Japanese individual with homozygous delta beta-thalassemia has been cloned. A clone containing the normal DNA surrounding the 3′ breakpoint of this deletion and a clone carrying the G gamma- and A gamma-globin genes of this patient were also isolated. Sequences of the deletion junction and both gamma-globin genes were determined. A comparison of these sequences with previously determined sequences of the normal counterparts revealed that the 5′ breakpoint is located between 2,134 and 2,137 base pairs (bp) 3′ to the polyA site of the A gamma-globin gene, the 5′ breakpoint is located just downstream of the 3′ border of the fetal gamma-globin duplication unit, and no molecular defects are evident within the gamma-globin gene region. A comparison between the sequences of the normal DNA surrounding the 3′ breakpoint and the normal DNA surrounding the 5′ breakpoint shows that deletion is the result of a nonhomologous recombination event. There are A+T-rich stretches near the 5′ and 3′ breakpoints in the normal DNA, and a portion of an Aly repeat is located in the region 3′ to the 3′ breakpoint. Southern blot analysis using probes 3′ to the beta-globin gene showed that the deletion extends in the 3′ direction further than any other deletions associated with delta beta-thalassemia and hereditary persistence of fetal hemoglobin (HPFH) heretofore reported. These results are discussed in terms of the mechanism generating large deletions in mammalian cells and three models for the regulation of gamma-globin and beta-globin gene expression in humans.

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