Alpha Hemoglobin Stabilizing Protein (AHSP) Optimizes Hemoglobin A Synthesis by Maintaining a Pool of Viable Alpha Globin Subunits.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 650-650
Author(s):  
Xiang Yu ◽  
Yi Kong ◽  
Louis C. Dore ◽  
Anne M. Katein ◽  
John K. Choi ◽  
...  
Keyword(s):  
In Vitro Translation ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Fully Depleted ◽  
Alpha Thalassemia ◽  
Gene Ablation ◽  
Mutant Strains ◽  
Alpha Globin

Abstract AHSP binds alpha hemoglobin (Hb) to maintain its structure and limit its prooxidant activities. In addition, AHSP binds and stabilizes apo-alpha globin, which lacks heme. Previously, we demonstrated that Ahsp−/− mice exhibit hemolytic anemia with Hb precipitation in erythroid cells. Through interbreeding of mutant strains, we also showed that loss of AHSP exacerbates beta thalassemia. Together, these studies indicate that AHSP participates in Hb homeostasis and may act to neutralize potentially toxic excess alpha globin that is known to accumulate in normal erythroid precursors, and to a greater extent, in beta thalassemic ones. However, additional functions for AHSP may exist. In particular, AHSP-alpha globin complexes may also promote HbA synthesis. To test this, we depleted the pool of excess alpha globin in Ahsp−/− mice by interbreeding with alpha thalassemic ones. Compared to mice with either mutation alone, compound mutants missing both AHSP and one alpha globin allele (genotype Ahsp−/− //alpha globin*α/αα) exhibited more severe erythroid defects, including worsened anemia, hypochromia, Hb instability and ineffective erythropoiesis. Pulse-labeling of double-mutant reticulocytes showed that alpha to beta globin synthetic ratios were unaffected by loss of AHSP, but precipitation of both alpha and beta nascent chains into cell membranes was strongly enhanced. These data indicate that AHSP is important for erythropoiesis and Hb production even when alpha globin is not produced in excess. In vitro translation studies using wheat germ extracts showed that recombinant AHSP present during the synthesis of alpha globin improved its ability to become incorporated into HbA. Moreover, AHSP conferred protease resistance to nascent alpha globin, suggesting enhanced folding into the native state. Further supporting this interpretation, circular dichroism studies showed that AHSP accelerated refolding of purified denatured alpha Hb. Finally, through pulse labeling of reticulocytes followed by isoelectric focusing of soluble cytosolic fractions, we identified transient pools of free alpha Hb and AHSP-alpha Hb in vivo and showed that Ahsp gene ablation fully depleted both pools. Together, our studies indicate that AHSP acts as a molecular chaperone to promote alpha globin folding and stability prior to its incorporation into HbA. In addition, it is possible that alterations in AHSP gene function or expression could modulate alpha thalassemia severity in patients.

scholarly journals The inactive beta globin gene on a gamma delta beta thalassemia chromosome has a normal structure and functions normally in vitro

Blood ◽  
1988 ◽  
Vol 71 (3) ◽  
pp. 766-770
Author(s):  
PT Curtin ◽  
YW Kan
Keyword(s):  
Globin Gene ◽  
Large Deletion ◽  
Beta Thalassemia ◽  
Ribonuclease Protection Assay ◽  
Beta Globin ◽  
Gamma Delta ◽  
Beta Globin Gene ◽  
Thalassemia Minor

We have previously described an English family with gamma delta beta- thalassemia in which a large deletion stops 25 kilobases (kb) upstream from the beta-globin gene locus, and yet the beta-globin gene is inactive in vivo. Affected family members had a beta-thalassemia minor phenotype with a normal hemoglobin A2 level. Gene mapping showed that these subjects were heterozygous for a chromosome bearing a large deletion that began in the G gamma-globin gene, extended through the epsilon-globin gene, and continued upstream for at least 75 kb. The A gamma-, delta-, and beta-globin gene loci on this chromosome were intact. To examine the possibility that an additional defect was present in the beta-globin gene, we cloned, sequenced, and examined the expression of the beta-globin gene from the affected chromosome. No mutation was found in the beta-globin gene sequence from 990 base-pairs 5′ to the cap site to 350 basepairs 3′ to the polyadenylation signal. The gene was subcloned into an expression vector and introduced into HeLa cells. Analysis of RNA derived from these cells, using a ribonuclease protection assay, revealed qualitatively and quantitatively normal transcription. Thus a structurally and functionally normal beta-globin gene is inactive in the presence of a large deletion more than 25 kb upstream. The loss of beta-globin gene function may be due to disturbance of chromatin conformation caused by the deletion or may be the result of loss of upstream sequences that are necessary for beta-globin gene expression in vivo.

scholarly journals Globin-chain specificity of oxidation-induced changes in red blood cell membrane properties

Blood ◽  
1992 ◽  
Vol 79 (6) ◽  
pp. 1586-1592 ◽  
Author(s):  
SL Schrier ◽  
N Mohandas
Keyword(s):  
Membrane Damage ◽  
Membrane Skeleton ◽  
Material Behavior ◽  
Membrane Properties ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Globin Chain ◽  
Globin Chains ◽  
Alpha Globin

Abstract We have previously shown that excess unpaired alpha- and beta-globin chains in severe alpha- and beta-thalassemia interacting with the membrane skeleton induce different changes in membrane properties of red blood cells (RBCs) in these two phenotypes. We suggest that these differences in membrane material behavior may reflect the specificity of the membrane damage induced by alpha- and beta-globin chains. To further explore this hypothesis, we sought in vitro models that induce similar membrane alterations in normal RBCs. We found that treatment of normal RBCs with phenylhydrazine produced rigid and mechanically unstable membranes in conjunction with selective association of oxidized alpha-globin chains with the membrane skeleton, features characteristic of RBCs in severe beta-thalassemia. Methylhydrazine, in contrast, induced selective association of oxidized beta-globin chains with the membrane skeleton and produced rigid but hyperstable membranes, features that mimicked those of RBCs in severe alpha- thalassemia. These findings suggest that consequences of oxidation induced by globin chains are quite specific in that those agents that cause alpha-globin chain accumulation at the membrane produce rigid but mechanically unstable membranes, whereas membrane accumulation of beta- globin chains results in rigid but mechanically stable membranes. These in vitro experiments lend further support to the hypothesis that membrane-associated alpha- and beta-chains induce oxidative damage to highly specific different skeletal components and that the specificity of this skeletal damage accounts for the differences in material membrane properties of these oxidatively attacked RBCs and perhaps of alpha- and beta-thalassemic RBCs as well.

scholarly journals Globin-chain specificity of oxidation-induced changes in red blood cell membrane properties

Blood ◽  
1992 ◽  
Vol 79 (6) ◽  
pp. 1586-1592 ◽  
Author(s):  
SL Schrier ◽  
N Mohandas
Keyword(s):  
Membrane Damage ◽  
Membrane Skeleton ◽  
Material Behavior ◽  
Membrane Properties ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Globin Chain ◽  
Globin Chains ◽  
Alpha Globin

We have previously shown that excess unpaired alpha- and beta-globin chains in severe alpha- and beta-thalassemia interacting with the membrane skeleton induce different changes in membrane properties of red blood cells (RBCs) in these two phenotypes. We suggest that these differences in membrane material behavior may reflect the specificity of the membrane damage induced by alpha- and beta-globin chains. To further explore this hypothesis, we sought in vitro models that induce similar membrane alterations in normal RBCs. We found that treatment of normal RBCs with phenylhydrazine produced rigid and mechanically unstable membranes in conjunction with selective association of oxidized alpha-globin chains with the membrane skeleton, features characteristic of RBCs in severe beta-thalassemia. Methylhydrazine, in contrast, induced selective association of oxidized beta-globin chains with the membrane skeleton and produced rigid but hyperstable membranes, features that mimicked those of RBCs in severe alpha- thalassemia. These findings suggest that consequences of oxidation induced by globin chains are quite specific in that those agents that cause alpha-globin chain accumulation at the membrane produce rigid but mechanically unstable membranes, whereas membrane accumulation of beta- globin chains results in rigid but mechanically stable membranes. These in vitro experiments lend further support to the hypothesis that membrane-associated alpha- and beta-chains induce oxidative damage to highly specific different skeletal components and that the specificity of this skeletal damage accounts for the differences in material membrane properties of these oxidatively attacked RBCs and perhaps of alpha- and beta-thalassemic RBCs as well.

scholarly journals Investigating Zeta Globin Gene Expression to Develop a Potential Therapy for Alpha Thalassemia Major

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 3-4
Author(s):  
Georgia L. Gregory ◽  
Beeke Wienert ◽  
Marisa Schwab ◽  
Billie Rachael Lianoglou ◽  
Roger P. Hollis ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Globin Gene ◽  
Thalassemia Major ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Wild Type ◽  
Advisory Committees ◽  
Alpha Thalassemia ◽  
Alpha Globin ◽  
Zeta Globin Gene

Introduction: Alpha globin mutations are very common worldwide, and the severity of resulting anemia depends on the number and type of mutated alleles. While the 4 gene mutation (alpha thalassemia major, ATM) was previously deemed fatal except in rare cases, emerging evidence indicates that survival to birth and good postnatal outcomes are possible with in utero transfusions. We hypothesized that the embryonic zeta globin gene, which is expressed early in gestation prior to alpha globin, may compensate for the lack of alpha globin and that induction of zeta globin after it has naturally been silenced may become a new therapy for patients with ATM. Methods: We evaluated mutations in the UCSF international registry of patients with ATM to understand factors related to patient survival with and without in utero transfusions. We then engineered Human Umbilical Cord Derived Erythroid Progenitor Cells (HUDEP-2 cells) carrying the common SEA alpha globin deletion, in which zeta globin expression is preserved (H-SEA), as well as those on which the zeta globin genes were deleted (HBZ-/-) using CRISPR-Cas9. We evaluated the expression of alpha and zeta globins using qPCR, Western blot, and flow cytometry. We generated lentiviral vectors expressing zeta globin under the control of beta-globin promoters to examine changes in both zeta and alpha globin in a dynamic way. Results: None of the registry patients who survived to birth spontaneously (n=11) had a mutation that involves a concomitant deletion in zeta globin (such as the -FIL, -THAI, or -MEDII mutation), while alpha globin mutations extending into the zeta globin gene were found in 14 of 37 (38%) patients who were diagnosed prenatally, suggesting that the presence of zeta globin may play a role in the ability to survive to birth without fetal therapy. Interestingly, we found that H-SEA clones express higher levels of zeta globin than WT cells, as illustrated by quantitative real-time PCR (Fig 1A), Western blot (Fig 1B) and flow cytometry (Fig 1C). These cells also developed beta globin dimers due to excess unpaired beta-globin chains, as demonstrated by Western blotting with and without reducing agents, indicating that they are an appropriate cell model for ATM. We next generated HUDEP-2 clones lacking zeta globin (HBZ KO) and transduced these clones with lentiviral vectors expressing high levels of zeta globin (lenti-zeta) (Fig 1D). Western blotting revealed that increasing the levels of zeta globin in these cells resulted in decreased expression of alpha globin, suggesting reciprocal control between these genes (Fig 1E). Most importantly, we saw a reduction in toxic beta-globin dimers in H-SEA cells expressing high levels of zeta-globin after transduction with lenti-zeta, suggesting that zeta globin could functionally replace the missing alpha-globin (Fig 1 F,G). To understand transcriptomic differences in H-SEA cells that may result in increased zeta globin expression, we performed bulk RNA sequencing of wild type and H-SEA clones. We confirmed that zeta expression is significantly upregulated in H-SEA compared to wild type (log2 fold change of 4.25, p=2.24E-38). Pathway analysis of differentially expressed genes is ongoing. Conclusions: Our international patient registry suggests that expression of zeta globin may play a role in the spontaneous survival to birth in a subset of patients. Zeta globin expression is increased in the setting of H-SEA cells in vitro, and restoration of zeta expression by lentivirus results in a reduction of toxic beta globin dimers in these ATM cells. Furthermore, expressing zeta globin at high levels in H-WT cells decreased alpha globin expression, suggesting a reciprocal regulation of these two genes. This concept is similar to the relationship between fetal gamma and adult beta globins which has been exploited for therapeutic editing approaches in patients with beta-thalassemia. At this point, the natural repressor of zeta globin is not yet known, but our data suggests that a strategy of upregulating zeta globin could potentially be developed to mimic the ongoing trials of using the BCL11A repressor to induce gamma globin in patients with beta thalassemia and sickle cell disease. Disclosures Wienert: Integral Medicines: Current Employment. Kohn:Allogene Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Patents & Royalties, Research Funding. MacKenzie:Acrigen: Membership on an entity's Board of Directors or advisory committees; Ultragenyx: Research Funding.

scholarly journals Oral sodium phenylbutyrate therapy in homozygous beta thalassemia: a clinical trial

Blood ◽  
1995 ◽  
Vol 85 (1) ◽  
pp. 43-49 ◽  
Author(s):  
AF Collins ◽  
HA Pearson ◽  
P Giardina ◽  
KT McDonagh ◽  
SW Brusilow ◽  
...  
Keyword(s):  
Fetal Hemoglobin ◽  
Cell Number ◽  
Response To Treatment ◽  
Beta Thalassemia ◽  
Thalassemia Patient ◽  
Alpha Globin ◽  
Pill Counts ◽  
Sodium Phenylbutyrate

Butyrate analogues have been shown to increase fetal hemoglobin (HbF) production in vitro and in vivo. Sodium phenylbutyrate (SPB), an oral agent used to treat individuals with urea-cycle disorders, has been shown to increase HbF in nonanemic individuals and in individuals with sickle cell disease. We have treated eleven patients with homozygous beta thalassemia (three transfusion dependent) and one sickle-beta- thalassemia patient with 20 g/d (forty 500-mg tablets) of SPB for 41 to 460 days. All patients showed an increase in the percent of F reticulocytes associated with treatment, but only four patients responded by increasing their Hb levels by greater than 1 g/dL (mean increase, 2.1 g/dL; range, 1.2 to 2.8 g/dL). None of the transfusion- dependent thalassemia subjects responded. Increase in Hb was associated with an increase in red blood cell number (mean increase, 0.62 x 10(12)/L), and mean corpuscular volume (mean increase, 6 fL). Changes in percent HbF, absolute HbF levels, or alpha- to non-alpha-globin ratios as measured by levels of mRNA and globin protein in peripheral blood did not correlate with response to treatment. Response to treatment was not associated with the type of beta-globin mutation, but baseline erythropoietin levels of greater than 120 mU/mL was seen in all responders and only two of eight nonresponders to SPB. Compliance with treatment was greater than 90% as measured by pill counts. Side effects of the drug included weight gain and/or edema caused by increase salt load in 2/12, transient epigastric discomfort in 7/12, and abnormal body odor in 3/12 subjects. Two splenectomized patients who were not on prophylactic antibiotics developed sepsis while on treatment. We conclude that SPB increases Hb in some patients with thalassemia, but the precise mechanism of action is unknown.

scholarly journals The beta-globin gene on the Chinese delta beta-thalassemia chromosome carries a promoter mutation

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1470-1474 ◽  
Author(s):  
GF Atweh ◽  
XX Zhu ◽  
HE Brickner ◽  
CH Dowling ◽  
HH Jr Kazazian ◽  
...  
Keyword(s):  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Promoter Mutation ◽  
Beta Globin Gene ◽  
Gamma Globin ◽  
New Type

A new type of delta beta-thalassemia characterized by decreased expression of the beta-globin gene and increased expression of both G gamma and A gamma globin gene in the absence of a detectable deletion has recently been described in the Chinese population. In this study we characterize the mutant beta-globin gene from this delta beta- thalassemia chromosome. An A to G transversion is identified in the “ATA” sequence of the promoter region that leads to decreased expression of the beta-globin gene in vivo and in vitro. We also demonstrate the presence of this mutation in every individual with a high fetal hemoglobin phenotype in this family and its absence in every individual with a normal hemoglobin phenotype. This same promoter mutation has recently been detected in Chinese beta-thalassemia genes where it is present on chromosomes of the same haplotype as that of the delta beta-thalassemia chromosome we are studying. These data support the hypothesis that an as yet unidentified mutation occurred on the ancestral chromosome carrying the promoter mutation and subsequently gave rise to the delta beta-thalassemia phenotype.

scholarly journals Molecular basis for alpha-thalassemia associated with the structural mutant hemoglobin Suan-Dok (alpha 2 109leu----arg) [published erratum appears in Blood 1991 Mar 15;77(6):1404]

Blood ◽  
1990 ◽  
Vol 76 (12) ◽  
pp. 2630-2636 ◽  
Author(s):  
I Weiss ◽  
FE Cash ◽  
MB Coleman ◽  
A Pressley ◽  
JG Adams ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Globin Gene ◽  
Mrna Level ◽  
Early Time ◽  
In Vitro Translation ◽  
Globin Mrna ◽  
Time Points ◽  
Alpha Thalassemia ◽  
Alpha Globin

Abstract Hemoglobin (Hb) Suan-Dok (alpha 109Arg) is a rare alpha-globin structural mutation that is linked to an alpha-thalassemia (alpha-thal) determinant. When inherited in trans to an alpha-thal-1 mutation (-), it results in Hb H disease associated with low levels (9%) of the Suan- Dok Hb. The nature of the thalassemic defect associated with the alpha SD mutation has been investigated by structural and functional studies. Sequence analysis of the cloned Suan-Dok allele showed a missense mutation (T----G) at codon 109 in an otherwise normal alpha 2-globin gene. When the alpha 2SD-globin gene was introduced into mouse erythroleukemia cells, the steady state alpha-globin messenger RNA (mRNA) level was equivalent to the alpha A-globin gene control. Although in vitro translation of a synthetic alpha 2SD-globin mRNA generated levels of alpha globin equivalent to alpha 2A-globin mRNA at early time points, the ratio of alpha SD to alpha A globin decreased markedly at later time points. These data suggest that the thalassemic defect associated with the Suan-Dok mutation results from a significant instability of the alpha SD globin.

scholarly journals Characterization and comparison of the red blood cell membrane damage in severe human alpha- and beta-thalassemia

Blood ◽  
1992 ◽  
Vol 79 (4) ◽  
pp. 1058-1063 ◽  
Author(s):  
R Advani ◽  
S Sorenson ◽  
E Shinar ◽  
W Lande ◽  
E Rachmilewitz ◽  
...  
Keyword(s):  
Blood Cell ◽  
Red Blood Cell ◽  
Membrane Damage ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Cell Membrane Damage ◽  
Protein 4.1 ◽  
Globin Chains ◽  
Alpha Thalassemia ◽  
Alpha Globin

Abstract The aim of the present work was to understand the pathophysiology of the severe human thalassemias as represented by beta-thalassemia intermedia and hemoglobin (Hb) H (alpha-thalassemia) disease. We have previously shown that the material properties of the red blood cell (RBC) and its membrane differ in severe alpha- and beta-thalassemia, and we now show that this difference is probably caused by accumulation of alpha-globin chains at the cytoskeleton in beta-thalassemia, whereas beta-globin chains are associated with the cytoskeleton in alpha- thalassemia. In both alpha- and beta-thalassemia, some of these globin chains have become oxidized as evidenced by loss of the free thiols. Furthermore, there is similar evidence of oxidation of protein 4.1 in beta-thalassemia, whereas beta-spectrin appears to be subject to oxidation in alpha-thalassemia. These observations support the idea that the association of partly oxidized globin chains with the cytoskeleton results in oxidation of adjacent skeletal proteins. The abnormality of protein 4.1 in beta-thalassemia is consistent with a prior observation, and is also in accord with the known importance of protein 4.1 in maintenance of membrane stability, a property that is abnormal in beta-thalassemic membranes.

scholarly journals The beta-globin gene on the Chinese delta beta-thalassemia chromosome carries a promoter mutation

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1470-1474 ◽  
Author(s):  
GF Atweh ◽  
XX Zhu ◽  
HE Brickner ◽  
CH Dowling ◽  
HH Jr Kazazian ◽  
...  
Keyword(s):  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Promoter Mutation ◽  
Beta Globin Gene ◽  
Gamma Globin ◽  
New Type

Abstract A new type of delta beta-thalassemia characterized by decreased expression of the beta-globin gene and increased expression of both G gamma and A gamma globin gene in the absence of a detectable deletion has recently been described in the Chinese population. In this study we characterize the mutant beta-globin gene from this delta beta- thalassemia chromosome. An A to G transversion is identified in the “ATA” sequence of the promoter region that leads to decreased expression of the beta-globin gene in vivo and in vitro. We also demonstrate the presence of this mutation in every individual with a high fetal hemoglobin phenotype in this family and its absence in every individual with a normal hemoglobin phenotype. This same promoter mutation has recently been detected in Chinese beta-thalassemia genes where it is present on chromosomes of the same haplotype as that of the delta beta-thalassemia chromosome we are studying. These data support the hypothesis that an as yet unidentified mutation occurred on the ancestral chromosome carrying the promoter mutation and subsequently gave rise to the delta beta-thalassemia phenotype.

scholarly journals Molecular basis for alpha-thalassemia associated with the structural mutant hemoglobin Suan-Dok (alpha 2 109leu----arg) [published erratum appears in Blood 1991 Mar 15;77(6):1404]

Blood ◽  
1990 ◽  
Vol 76 (12) ◽  
pp. 2630-2636 ◽  
Author(s):  
I Weiss ◽  
FE Cash ◽  
MB Coleman ◽  
A Pressley ◽  
JG Adams ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Globin Gene ◽  
Mrna Level ◽  
Early Time ◽  
In Vitro Translation ◽  
Globin Mrna ◽  
Time Points ◽  
Alpha Thalassemia ◽  
Alpha Globin

Hemoglobin (Hb) Suan-Dok (alpha 109Arg) is a rare alpha-globin structural mutation that is linked to an alpha-thalassemia (alpha-thal) determinant. When inherited in trans to an alpha-thal-1 mutation (-), it results in Hb H disease associated with low levels (9%) of the Suan- Dok Hb. The nature of the thalassemic defect associated with the alpha SD mutation has been investigated by structural and functional studies. Sequence analysis of the cloned Suan-Dok allele showed a missense mutation (T----G) at codon 109 in an otherwise normal alpha 2-globin gene. When the alpha 2SD-globin gene was introduced into mouse erythroleukemia cells, the steady state alpha-globin messenger RNA (mRNA) level was equivalent to the alpha A-globin gene control. Although in vitro translation of a synthetic alpha 2SD-globin mRNA generated levels of alpha globin equivalent to alpha 2A-globin mRNA at early time points, the ratio of alpha SD to alpha A globin decreased markedly at later time points. These data suggest that the thalassemic defect associated with the Suan-Dok mutation results from a significant instability of the alpha SD globin.

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