Unequal Synthesis of Complementary Globin Chains of Human Fetal Hemoglobin by the Effect of L-O-Methylthreonine

High fetal hemoglobin (HbF) levels inhibit the polymerization of sickle hemoglobin (HbS) and reduce the complications of sickle cell disease. Pharmacologic agents that can reverse the switch from γ- to β-chain synthesis — γ-globin chains characterize HbF, and sickle β-globin chains are present in HbS — or selectively increase the proportion of adult erythroid precursors that maintain the ability to produce HbF are therapeutically useful. Hydroxyurea promotes HbF production by perturbing the maturation of erythroid precursors. This treatment increases the total hemoglobin concentration, reduces the vaso-occlusive complications of pain and acute chest syndrome, and attenuates mortality in adults. It is a promising beginning for pharmacologic therapy of sickle cell disease. Still, its effects are inconsistent, trials in infants and children are ongoing, and its ultimate value — and peril — when started early in life are still unknown.

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Effective erythropoiesis and HbF reactivation induced by kit ligand in β-thalassemia

Blood ◽

10.1182/blood-2007-06-097550 ◽

2008 ◽

Vol 111 (1) ◽

pp. 421-429 ◽

Cited By ~ 11

Author(s):

Marco Gabbianelli ◽

Ornella Morsilli ◽

Adriana Massa ◽

Luca Pasquini ◽

Paolo Cianciulli ◽

...

Keyword(s):

Fetal Hemoglobin ◽

Thalassemia Major ◽

Globin Genes ◽

Erythroid Progenitors ◽

Ineffective Erythropoiesis ◽

Kit Ligand ◽

Globin Chains ◽

In Vitro Response ◽

Globin Synthesis

In human β-thalassemia, the imbalance between α- and non–α-globin chains causes ineffective erythropoiesis, hemolysis, and anemia: this condition is effectively treated by an enhanced level of fetal hemoglobin (HbF). In spite of extensive studies on pharmacologic induction of HbF synthesis, clinical trials based on HbF reactivation in β-thalassemia produced inconsistent results. Here, we investigated the in vitro response of β-thalassemic erythroid progenitors to kit ligand (KL) in terms of HbF reactivation, stimulation of effective erythropoiesis, and inhibition of apoptosis. In unilineage erythroid cultures of 20 patients with intermedia or major β-thalassemia, addition of KL, alone or combined with dexamethasone (Dex), remarkably stimulated cell proliferation (3-4 logs more than control cultures), while decreasing the percentage of apoptotic and dyserythropoietic cells (<5%). More important, in both thalassemic groups, addition of KL or KL plus Dex induced a marked increase of γ-globin synthesis, thus reaching HbF levels 3-fold higher than in con-trol cultures (eg, from 27% to 75% or 81%, respectively, in β-thalassemia major). These studies indicate that in β-thalassemia, KL, alone or combined with Dex, induces an expansion of effective erythropoiesis and the reactivation of γ-globin genes up to fetal levels and may hence be considered as a potential therapeutic agent for this disease.

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Absence of messenger RNA and gene DNA for β-globin chains in hereditary persistence of fetal hemoglobin

Cell ◽

10.1016/0092-8674(76)90161-6 ◽

1976 ◽

Vol 7 (3) ◽

pp. 323-329 ◽

Cited By ~ 50

Author(s):

B.G. Forget ◽

D.G. Hillman ◽

H. Lazarus ◽

E.F. Barell ◽

E.J. Benz ◽

...

Keyword(s):

Messenger Rna ◽

Fetal Hemoglobin ◽

Globin Chains ◽

Hereditary Persistence

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LC-MS Analysis of Anti-Sickling Compounds in Cord Blood Derived RBCs Demonstrates Modification of Fetal Hemoglobin and Globin Chain Binding Preferences

Blood ◽

10.1182/blood-2018-99-116319 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1074-1074

Author(s):

Benjamin Vieira ◽

Vu P. Hong ◽

Kunal Desai ◽

Martin K. Safo ◽

David R. Light

Keyword(s):

Cord Blood ◽

Binding Affinity ◽

Oxygen Affinity ◽

Fetal Hemoglobin ◽

Oxygen Binding ◽

Globin Chain ◽

Employment Equity ◽

Globin Chains ◽

N Terminus ◽

Equity Ownership

Abstract Sickle cell disease (SCD) is a genetic hemoglobinopathy driven largely by a single codon mutation of the β-globin gene resulting in polymerization of hemoglobin S (HbS). Anti-sickling approaches that involve increasing the oxygen affinity of HbS to treat SCD are under development and offer the potential to directly prevent HbS polymerization and its downstream pathophysiology. Two such compounds, 5-hydroxymethylfurfural (5HMF) and voxelotor (GBT440) have entered clinical trials for SCD with promising results and exert their therapeutic effects by modifying the N-terminus of HbS α-globin chains to form a reversible Schiff base. Formation of this N-terminal adduct stabilizes the oxygen-bound R-state (in the R2 conformation) that increases the oxygen affinity of the altered HbS and delaying the polymerization of HbS. In addition, genetic and small molecule therapies designed to increase fetal hemoglobin (HbF) expression hold great potential for the treatment of SCD. Increasing the percentage of HbF in RBCs significantly slows sickling kinetics without affecting oxygen delivery. Combination approaches of high-O2-Hb modification with HbF inducing therapies clinically could result in increased efficacy in the treatment of SCD, but the impact of hemoglobin modifiers on fetal hemoglobin has not been reported. Our present studies investigated the effects of 5HMF and voxelotor in HbF-rich umbilical cord blood derived RBCs. HbF-rich (60-90%) RBCs were isolated from cord blood and incubated with commercially available 5HMF and voxelotor synthesized in-house. The effect of these compounds on hemoglobin oxygen affinity was determined by measuring the p50 of the oxygen saturation curve in whole cells. Sites of modification were determined directly by incubating compounds with the purified RBC lysate, stabilizing the N-terminal adduct by reduction to the amine, and analysis of the resulting modification by LC-MS. Similar to the reported p50 shifts with normal adult hemoglobin (HbA) and HbS, 5HMF and voxelotor increased the oxygen-binding affinity of HbF with an EC50 of 7.9 mM and 560 mM respectively. 1 mM voxelotor lowered cord RBC p50 to 4 mmHg in vitro. LC-MS analysis showed that 5HMF exclusively modified the N-terminus of the α-globin chain, with no modification of b-globin and g-globin chains. Unexpectedly, the α-globin, β-globin and γ-globin chains were all modified by voxelotor following incubation with cord blood. Voxelotor was also shown to modify both α-globin and the β-globin or βS-globin chains on purified HbA or HbS, respectively. These data contrast with published crystallography data demonstrating that voxelotor selectively modifies a single α-globin chain in CO-ligated HbS (Oksenberg et al 2016). Although anti-sickling aromatic aldehydes have similar effects on the oxygen binding affinity of HbA, HbS and HbF, they can vary in their selectivity for modification of the α-globin and beta-like chains of HbF, HbA, and HbS (Abraham et al. 1995). To further investigate our data with voxelotor and increase our understanding of this class of molecules, other hemoglobin modifying aldehyde molecules such as 5-formylsalicyclic acid (5FSA), tucaresol and velaresol (BW12C) will be examined. Disclosures Vieira: Bioverativ a Sanofi Company: Employment. Hong:Bioverativ a Sanofi Company: Employment, Equity Ownership. Desai:Bioverativ a Sanofi Company: Employment, Equity Ownership. Safo:Bioverativ a Sanofi Company: Consultancy; Virginia Commonwealth University: Employment. Light:Bioverativ a Sanofi Company: Employment, Equity Ownership.

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Fetal hemoglobin synthesis in erythroid cultures in hereditary persistence of fetal hemoglobin and beta o-thalassemia

Blood ◽

10.1182/blood.v63.6.1278.bloodjournal6361278 ◽

1984 ◽

Vol 63 (6) ◽

pp. 1278-1284 ◽

Cited By ~ 1

Author(s):

RS Weinberg ◽

SE Antonarakis ◽

HH Jr Kazazian ◽

GJ Dover ◽

SH Orkin ◽

...

Keyword(s):

Globin Gene ◽

Fetal Hemoglobin ◽

Beta Globin ◽

Globin Chains ◽

Hereditary Persistence ◽

Enzyme Pattern ◽

Globin Synthesis ◽

The Individual ◽

Globin Gene Expression

To determine whether hemoglobin regulation is normal in diseases affecting beta-globin gene expression, globin synthesis was examined in members of a family of a patient with hereditary persistence of fetal hemoglobin/beta o-thalassemia (HPFH/beta o-thal). The HPFH defect is the Ghanian type II, with a deletion from psi beta 1 to at least 20 kb 3′ to beta. The beta o-thal gene has the haplotype II restriction enzyme pattern and has the beta 39 nonsense mutation. Erythroid colonies from blood BFU-E were radiolabeled, and globin chains were separated by gel electrophoresis. Colonies from the beta o-thal heterozygote had non-alpha/alpha ratios more balanced than in the reticulocytes. Gamma synthesis was 11% of non-alpha, which is higher than in reticulocytes, but within the range seen in normal adult colonies. Both HPFH heterozygotes produced 20%-30% gamma in erythroid colonies as well as reticulocytes, although non-alpha/alpha was more balanced in the colonies. The HPFH/beta o-thal patient produced 100% gamma in reticulocytes and in colonies. G gamma and gamma-synthetic proportions were not correlated at the individual colony level in the heterozygotes, suggesting that they had “adult” and not “fetal” progenitor cells. The Hb expression of these adult progenitors is presumably modulated normally in vivo in beta o-thal, but the normal decrease in HbF production does not occur in gene deletion HPFH.

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A murine model for the switch from fetal to adult hemoglobin during ontogeny

Blood ◽

10.1182/blood.v56.6.1100.bloodjournal5661100 ◽

1980 ◽

Vol 56 (6) ◽

pp. 1100-1105 ◽

Cited By ~ 1

Author(s):

BP Alter ◽

SC Goff

Keyword(s):

Murine Model ◽

Fetal Hemoglobin ◽

Globin Chains ◽

Adult Mice ◽

Adult Hemoglobin ◽

Globin Synthesis ◽

Murine Erythroleukemia ◽

Triton X ◽

Total Beta

In murine erythroleukemia cells, the minor/major hemoglobin (Hb) ratio depends on the cell line and the inducing agent. To determine whether mouse minor hemoglobin is a “fetal” hemoglobin in vivo, globin chain composition and synthesis rates were determined in DBA/2 mice of various ages ranging from 14-day embryos to > 6-mo adults. Globin chains were separated by electrophoresis on polyacrylamide gels containing urea and Triton X-100. This method separates the embryonic (x,y,z) and the adult (alpha, beta ma, beta mi) globin chains. Fourteen day embryos had only 5%-10% adult globins but approximately 30% of the adult beta chains were beta mi. The % beta mi decreased with age and reached 20% in adult mice. Biosynthetic studies led to more pronounced differences: beta mi synthesis was 45% of total beta chain production in 14-day embryos and declined to 22% in adults. Thus beta minor/total beta globin synthesis declines during mouse ontogeny. This resembles qualitatively the human switch from fetal to adult hemoglobin and provides a murine model for studies of hemoglobin regulation.

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Introducing Negatively Charged Residues on the Surface of Fetal Hemoglobin Improves Yields in Escherichia coli

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.721794 ◽

2021 ◽

Vol 9 ◽

Author(s):

Karin Kettisen ◽

Leif Bülow

Keyword(s):

Fetal Hemoglobin ◽

Globin Chains ◽

Γ Subunit ◽

Alternative Protein ◽

Free Heme ◽

Negative Surface ◽

Negative Surface Charge ◽

The Impact ◽

Enhanced Yield ◽

Culture Protocol

Fetal hemoglobin (HbF) has been developed into an important alternative protein for oxygen therapeutics. Such applications require extensive amounts of proteins, which only can be achieved via recombinant means. However, the expression of vertebrate hemoglobins in heterologous hosts is far from trivial. There are several issues that need to be dealt with. These include, among others, the solubility of the globin chains, equimolar expression of the globin chains, and access to high levels of free heme. In this study, we examined the impact of introducing negative charges on the surface of HbF. Three different HbF mutants were examined, carrying four additional negative charges on the α-subunit (rHbFα4), two additional negative charges on the γ-subunit (rHbFγ2) or a combination of these (rHbFα4/γ2). The increase in negative surface charge in these HbF mutants required the development of an alternate initial capture step in the downstream purification procedures. For the rHbFα4 mutant, we achieved a significantly enhanced yield of purified HbF with no apparent adverse effects on Hb functionality. However, the presence of non-functional Hb portions in the rHbFγ2 and rHbFα4/γ2 samples reduced the yields significantly for those mutants and indicated an imbalanced expression/association of globin chains. Furthermore, the autoxidation studies indicated that the rHbFγ2 and rHbFα4/γ2 mutants also were less oxidatively stable than rHbFα4 and wt rHbF. The study further verified the need for an improved flask culture protocol by optimizing cultivation parameters to enable yield-improving qualities of surface-located mutations.

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Fetal hemoglobin synthesis in erythroid cultures in hereditary persistence of fetal hemoglobin and beta o-thalassemia

Blood ◽

10.1182/blood.v63.6.1278.1278 ◽

1984 ◽

Vol 63 (6) ◽

pp. 1278-1284 ◽

Cited By ~ 12

Author(s):

RS Weinberg ◽

SE Antonarakis ◽

HH Jr Kazazian ◽

GJ Dover ◽

SH Orkin ◽

...

Keyword(s):

Globin Gene ◽

Fetal Hemoglobin ◽

Beta Globin ◽

Globin Chains ◽

Hereditary Persistence ◽

Enzyme Pattern ◽

Globin Synthesis ◽

The Individual ◽

Globin Gene Expression

Abstract To determine whether hemoglobin regulation is normal in diseases affecting beta-globin gene expression, globin synthesis was examined in members of a family of a patient with hereditary persistence of fetal hemoglobin/beta o-thalassemia (HPFH/beta o-thal). The HPFH defect is the Ghanian type II, with a deletion from psi beta 1 to at least 20 kb 3′ to beta. The beta o-thal gene has the haplotype II restriction enzyme pattern and has the beta 39 nonsense mutation. Erythroid colonies from blood BFU-E were radiolabeled, and globin chains were separated by gel electrophoresis. Colonies from the beta o-thal heterozygote had non-alpha/alpha ratios more balanced than in the reticulocytes. Gamma synthesis was 11% of non-alpha, which is higher than in reticulocytes, but within the range seen in normal adult colonies. Both HPFH heterozygotes produced 20%-30% gamma in erythroid colonies as well as reticulocytes, although non-alpha/alpha was more balanced in the colonies. The HPFH/beta o-thal patient produced 100% gamma in reticulocytes and in colonies. G gamma and gamma-synthetic proportions were not correlated at the individual colony level in the heterozygotes, suggesting that they had “adult” and not “fetal” progenitor cells. The Hb expression of these adult progenitors is presumably modulated normally in vivo in beta o-thal, but the normal decrease in HbF production does not occur in gene deletion HPFH.

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Molecular Analysis of Alpha Hemoglobin Stabilizing Protein (AHSP) in Caucasian Patients with different Beta-Thalassemia Phenotypes.

Blood ◽

10.1182/blood.v104.11.3770.3770 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3770-3770 ◽

Cited By ~ 4

Author(s):

M. Domenica Cappellini ◽

Chiara Refaldi ◽

Daniela Bignamini ◽

Laura Zanaboni ◽

Gemino Fiorelli

Keyword(s):

High Performance ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Clinical Severity ◽

Beta Thalassemia ◽

Red Cell ◽

Globin Genes ◽

Nucleotide Polymorphisms ◽

Thai Population ◽

Globin Chains

Abstract Beta-thalassemia is a inherited hemoglobin disorder characterized by absent or reduced synthesis of the b globin chains. The pathophysiology and the severity of b-thalassemias reflect the degree of globin chain imbalance and the excess of free a globin chains that precipitate and cause oxidative damage in red cell precursors inducing their premature destruction in the bone marrow (ineffective erythropoiesis). Although the phenotype of b thalassemias can be modified by inherited factors such as different number of a globin genes or increased fetal hemoglobin production, other mechanisms appear to be involved. Recently, a protein, named alpha hemoglobin stabilizing protein (AHSP), that acts as a molecular chaperone specifically for free a globin chains, preventing their precipitation in red cell precursors, has been identified. To establish whether AHSP might have a role in modifying the clinical outcome of b thalassemias, we have analyzed the AHSP gene in 70 Caucasian b thalassaemic subjects: 26 patients with b°/b° genotype (Thalassaemia Major),24 patients with Thalassemia Intermedia (b°/b+ or b+/b+) and 20 patients with a Thalassaemia Intermedia phenotype but with only one mutation in the b globin gene, a normal a globin genotype and no other causes of anemia. In all the subjects, we have performed Denaturing High-Performance Liquid Chromatography (DHPLC) of the three exons and the direct genomic sequencing of coding and noncoding regions (~ 1.5 kb) of AHSP gene. No mutations able to modify the structure or function of AHSP have been found, however we identified eight single nucleotide polymorphisms (SNPs) spanned along the whole gene that segregate in four different aplotypes. To evaluate a possible relationship between a particular aplotype and b thalassemia severity, the allele frequency of each single aplotype in the tree groups has been established and compared to that of 33 Caucasian normal controls: no statistically significant association has been proved. Even though the loss of AHSP aggravates the b thalassaemia phenotype in mice, in Thalassemic Caucasian population the AHSP apparently doesn’t make changes in the clinical severity of b thalassemia confirming the results recently found in Thai population.

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Featured Article: Modulation of fetal hemoglobin in hereditary persistence of fetal hemoglobin deletion type-2, compared to Sicilian δβ-thalassemia, by BCL11A and SOX6-targeting microRNAs

Experimental Biology and Medicine ◽

10.1177/1535370216668052 ◽

2016 ◽

Vol 242 (3) ◽

pp. 267-274 ◽

Cited By ~ 3

Author(s):

Thais A Fornari ◽

Carolina Lanaro ◽

Dulcinéia M Albuquerque ◽

Regiane Ferreira ◽

Fernando F Costa

Keyword(s):

Cell Function ◽

Quantitative Polymerase Chain Reaction ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Regulation Of Transcription ◽

Phenotypic Differences ◽

Large Deletions ◽

Globin Chains ◽

Hereditary Persistence

Hereditary persistence of fetal hemoglobin deletion type-2 (HPFH-2) and Sicilian-δβ-thalassemia are conditions described as large deletions of the human β-like globin cluster, with absent β-globin chains and a compensatory variable increase in γ-globin. HPFH, in general, may be distinguished from DB-Thalassemia by higher fetal hemoglobin (HbF) levels, absence of anemia and hypochromic and microcytic erythrocytes. MicroRNAs (miRNAs) regulate a range of cellular processes including erythropoiesis and regulation of transcription factors such as the BCL11A and SOX6 genes, which are related to the regulation of γ-globin expression. In this report, a possible association among the overexpression of miRNAs and the expression of the γ-globin gene was analyzed in these two conditions. Forty-nine differentially expressed miRNAs were identified by microarrays in CD34+-derived erythroid cells of two subjects heterozygous for Sicilian-δβ-thalassemia, 2 for HPFH-2 and 3 for controls after 13 days of culture. Some of these miRNAs may participate in γ-globin gene regulation and red blood cell function. The BCL11A gene was found to be potentially targeted by 12 miRNAs that were up-regulated in HPFH-2 or in DB-Thal. A down-regulation of BCL11A gene expression in HPFH-2 was verified by quantitative polymerase chain reaction. These data suggest an important action for miRNA that may partially explain the phenotypic differences between HPFH-2 and Sicilian δβ-thalassemia and the increased expression of γ-globin in these conditions.

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