Alpha Hemoglobin Stabilizing Protein Expression Research Articles

Alpha-Hemoglobin Stabilizing Protein Gene Polymorphism (rs4499252 A/G) and its Association with Beta-Thalassemia Major in Iraqi Patients.

Beta thalassemia (β-thalassemia) major is a genetic disorder of hemoglobin production that results in a diminished rate of synthesis of one or more of the globin chains causing variable degrees of anemia. Alpha-hemoglobin-stabilizing protein (AHSP) is a specific alpha-globin factor that affects the severity of the disease in patients with β-thalassemia. A recent study was conducted to investigate the polymorphism in the AHSP (rs4499252) gene and its association with β-thalassemia in Iraq. Blood samples were obtained from 90 β-thalassemia patients and 60 healthy individuals as a control group in the Wasit Center for Hereditary Anemia from August 2020 to January 2021. After DNA extraction from the whole blood, to determine the genotype of the AHSP gene, the High-Resolution Melt (HRM) Real-Time PCR was used. The results showed a significant increase (P<0.05) in genotype GG (wild type) of the SNP (rs4499252) in β-thalassemia patients, compared to the control group. On the other hand, genotype AA was significantly higher (P≤0.05) in β-thalassemia patients than in the control group, while the genotype GA showed a non-significant difference (P<0.01) between β-thalassemia patients and the healthy controls. The results also showed that the AHSP expression is a biomarker of hemoglobin H disease severity, and the A allele was more frequent in β-thalassemia patients than the G allele in Iraqi patients.

Transient Improvement in Erythropoiesis Is Achieved Via the Chaperone AHSP With Early Administration of Propranolol in Burn Patients.

Burn patients experience erythropoietin resistant anemia in which early commitment and late maturation of erythroblasts are defective. The authors previously showed that propranolol (Prop) treatment restores erythroid committed progenitors, but terminal maturation remains impaired. Hemoglobinization and maturation occur during terminal erythropoiesis and these processes are aided by an erythroblast intrinsic functional protein called alpha-hemoglobin stabilizing protein (AHSP). The authors evaluated the role of AHSP in PBMC- (peripheral blood mono nuclear cell) derived erythroblasts and the implications of Prop in burn patients. Blood samples were collected at three time points from 17 patients receiving standard burn care (SBC) or Prop. Five healthy volunteers provided control plasma (CP). PBMCs were placed in biphasic cultures with 5% autologous plasma (BP) or CP. Erythroblasts were harvested during mid and late maturation stages; the percentage of AHSP+ erythroblasts, AHSP expression, and relative distribution of reticulocytes and polychromatophilic erythroblasts (PolyE) were determined by cytometry. During the second time point (7-10 days postburn), Prop cohort required 35% less transfusions. At mid maturation, PBMCs from Prop-treated patients cultured in BP had 33% more AHSP+ erythroblasts and 40% more AHSP expression compared with SBC. Furthermore, at late maturation, Prop had 50% more reticulocytes and 30% less PolyEs in CP vs BP compared with SBC (11% and 6%, respectively). AHSP is positively associated with late-stage maturation of PBMC-derived erythroblasts in the presence of CP. Albeit transiently, this is more pronounced in Prop than SBC. Early administration of propranolol in burn patients supports erythropoiesis via the chaperone AHSP.

Stage Specific Expression Pattern of Alpha-Hemoglobin-Stabilizing-Protein (AHSP) Portrayed in Erythroblast Chronology.

During erythropoiesis, the molecular chaperone alpha-hemoglobin-stabilizing protein (AHSP) sequesters free alpha-hemoglobin (αHb) and prevents precipitation of excess αHb. While AHSP is linked to hereditary anemia, the pattern of expression during specific erythroblast stages is poorly understood. We investigated gene and protein expressions of AHSP throughout progressive maturation stages of erythroblasts in biphasic cultures of blood and bone marrow samples from healthy donors. Differentiating erythroblasts were periodically subjected to flow cytometry, Amnis imaging and RT-qPCR analyses. We made parallel in vivo validations from naive murine bone marrow cells. Percentages of AHSP+ erythroblasts, protein expressions and AHSP gene expressions are negligible on culture day 6 (CFU-Es) and progressively increases from culture days 8–12 (peaks on day 12) and declines on day 14. Notably, sub-cellular location of AHSP is both in the cytoplasm and nucleus in the early erythroblasts while in the late stages of maturation AHSP is found predominantly in the nucleus, being expelled with it during enucleation. As both human bone marrow and peripheral blood mononuclear cells (PBMC) derived erythroblasts demonstrated similar expression patterns, sampling of erythroblasts from day 11 cultures could portray erythroblast chronology and provide optimum representative stage specific expression patterns. PBMCs may be suitable for comparison studies of AHSP expression in pathologic erythropoiesi

48 Burn Injury Impedes Late Erythropoiesis via Decreased Alpha Hemoglobin Stabilizing Protein and siglec1

Abstract Introduction Transfusions are of no benefit to burn patients with chronic anemia. We are elucidating the mechanisms of erythropoietin resistant anemia in burn injury using a mouse model in addition to a contrived system with human PBMCs. We have found that Alpha Hemoglobin Stabilizing Protein (AHSP), an erythroblast specific protein essential to effective hemoglobinization, is decreased in PBMC derived erythroblasts (EB) from burn patients (male + female). Hemoglobinization begins at the polychromatic EB (PolyE) stage and intensifies at the orthochromatic EB (OrthoE) stage, culminating in ejection of the nucleus and the cell becoming a reticulocyte (Reti). EB needs the support of a central island macrophage (EBI MØ) until they progress to maturation. We have seen in bone marrow from mice (male) following burn injury that Siglec1, an essential adhesion molecule is decreased in EBI MØ. It is these MØ that provide iron for the assembly of the hemoglobin unit in late erythropoiesis. EB maturation defects seen in burn injury could be due to the intrinsic changes in EB phenotype or the phenotype of supporting EBI MØ or a combination of the two. Our goal was to investigate in the animal model using male and female mice, how myeloid/erythroid intrinsic changes after burn impact the progression of late erythropoiesis from an EB and EBI MØ perspectives. Methods With IACUC approval, 12 male and female mice were anesthetized and subjected to sham burn or scald burn (15% TBSA) upon dorsal immersion in a 100oC water bath for 8 seconds. Animals were sacrificed on post burn day 7 (PBD 7). Total bone marrow cells (TBM) were harvested from bilateral femurs. Cells were characterized using CD71, Ter119, and Syto16 antibodies. EBI MØ were identified using a combination of F4/80, ERHR3, Vcam1, Ly6G and Siglec 1 antibodies. Geometric mean of fluorescence intensity (MFI) and cell counts were obtained by flow cytometric analysis and presented as x103 per million TBM cells. Peripheral blood was harvested via cardiac puncture and hemoglobin concentrations (g/dL) were obtained using a hematology analyzer. Results Among the erythroblasts, EEBs were 2.4 fold higher with a 2.4 fold lower AHSP expression in PBD 7 mice compared to sham as shown in left, top and bottom figures. Similarly, more EEBs were associated with EBIMØ with a lower Siglec1 expression as shown in right, top and bottom figures. Blood Hbg concentrations were lower in burn (sham=15.5±0.7 vs PBD 7= 13.1±0.5; p&amp;lt; 0.01). Conclusions The combined influence of Siglec1 and AHSP expression interferes with effective hemoglobinization impeding late maturation of erythroblasts following burn injury. Whether there is a synergistic action between the two deficiencies is being studied. Applicability of Research to Practice Underpinning the mechanism of erythropoiesis will lead to better options to ineffective transfusions in burn patients with anemia.

T4 Mechanistic Insights on Late Stage Erythropoiesis in Burn Patients Treated with Propranolol

Burn patients suffer from Epo resistant anemia in which the Epo-independent stages of early commitment and late maturation of erythroblasts are defective. We have previously shown in mice and patients, that propranolol (prop) treatment after burn injury restored erythroid committed progenitors, while terminal maturation was still impaired. During terminal erythropoiesis, hemoglobinization is aided by alpha hemoglobin stabilizing protein (AHSP), which is an erythroblast intrinsic functional protein predominantly expressed in nucleated erythroblasts. Effective hemoglobinization results in maturation of polychromatic erythroblasts (PolyE) to reticulocytes. Here we demonstrate in PBMC derived ex vivo erythroblast cultures, AHSP is positively associated with late stage maturation in the presence of allogeneic control plasma (CP); and more so in combination with prop than with standard burn care (SBC) in burn patients. After IRB approval, blood samples were collected from 6 burn patients (age=39±16 yeas; %TBSA=32±7) at three time points on post burn days 2–4, 7–10 and 27–33. Control plasma was obtained from healthy volunteers. Fifteen PBMC samples were placed in growth factor cocktail (SCF, GMCSF, IL-3, Cyclosporine) for 0–5 days to nurture erythroid progenitors. On day 5, each sample was split into two and supplemented with either autologous burn plasma (BP) or CP along with SCF, EPO, and holotransferrin from days 5–15. We harvested cells on culture days 9, 11 and 15; subjected them to FACS and AMNIS to determine the expression (mean fluorescence intensity) and percentage of AHSP+ erythroblasts on days 9 and 11 and the percentage of Reticulocytes (Sytox- CD235a+ CD71+/- Syto16-) and PolyE (Sytox- CD235a+ CD71+/- Syto16+ SSChigh) on day 15. See figure. Overall, CP supplementation in PBMC cultures produced more reticulocytes with a proportional decrease in PolyE, indicating enhanced late stage maturation. Nonetheless, the decrease in PolyE and increase in reticulocytes are more striking in prop treated patients compared to SBC. This is consistent with about 20% increase in AHSP+ cells and AHSP expression in developing erythroblasts in prop compared to SBC. Together, CP imparts a positive influence on reticulocyte maturation by augmenting AHSP expression in PBMC derived erythroblasts. Plasma resuscitation in addition to prop treatment is a promising alternate strategy to reduce transfusions in burn patients.

Role of Alpha Hemoglobin Stabilizing Protein Expression in Beta Thalassemia Patients

AbstractBackground: Beta (b)-Thalassemia is an autosomal reces-sive inherited disease causing variable degrees of anemia. The molecular defects are owing to point mutations or small deletions leading to diminution or absence of b-globin chain synthesis. The unmatched a-Hb is destructive to itself and other cellular components, resulting in ineffective erythropoi-esis and hemolysis. Alpha-Hemoglobin Stabilizing Protein (AHSP) is a scavenger protein of erythroid that fastly and reversibly attach the a subunit of hemoglobin molecule preventing its precipitation and subsequent hemolysis.Aim of Work: Is to assess AHSP gene expression in thalassemia major and intermedia and its relation to clinical features, laboratory investigations and to determine its role on the severity of thalassemia.Subjects and Methods: This case-control study is consisted of 120 studied subjects categorized into 40 thalassemia major patients, 40 thalassemia intermedia patients and 40 age healthy individuals as a control group. All subjects underwent the following; history, clinical picture, complete blood picture and Hb electrophoresis, serum ferritin and measurement of AHSP, alpha globin and beta globin genes expression by real time PCR.Results: AHSP gene in thal. intermedia group was higher than both thal. major (p<0.001). Beside thal. intermedia group had higher a globin/AHSP ratio versus thal. major group (p=0.011). b globin/AHSP ratio was not significant between both thal. groups. AHSP in thal.major group correlated with a globin and b globin expression and negatively correlated with organomegally, ferritin and no. of blood transfusion. Whereas in thal. intermedia group, was positively correlated with Hb, HCT, MCV, MCH, a globin gene expression, b globin gene expression and b globin/a globin genes ratio.Conclusion: AHSP expression was higher in thalassemia intermedia group than thalassemia major group. Consequently, AHSP can be considered genetic modifier in b thalassemia.

The effect of histone deacetylase inhibitors on AHSP expression.

Alpha-hemoglobin stabilizing protein (AHSP) is a molecular chaperone that can reduce the damage caused by excess free α-globin to erythroid cells in patients with impaired β-globin chain synthesis. We assessed the effect of sodium phenylbutyrate and sodium valproate, two histone deacetylase inhibitors (HDIs) that are being studied for the treatment of hemoglobinopathies, on the expression of AHSP, BCL11A (all isoforms), γ-globin genes (HBG1/2), and some related transcription factors including GATA1, NFE2, EKLF, KLF4, and STAT3. For this purpose, the K562 cell line was cultured for 2, 4, and 6 days in the presence and absence of sodium phenylbutyrate and sodium valproate. Relative real-time qRT-PCR analysis of mRNA levels was performed to determine the effects of the two compounds on gene expression. Expression of all target mRNAs increased significantly (p < 0.05), except for the expression of BCL11A, which was down-regulated (p < 0.05) in the cells treated with both compounds relative to the levels measured for untreated cells. The findings indicated that sodium valproate had a more considerable effect than sodium phenylbutyrate (p < 0.0005) on BCL11A repression and the up-regulation of other studied genes. γ-Globin and AHSP gene expression continuously increased during the culture period in the treated cells, with the highest gene expression observed for 1 mM sodium valproate after 6 days. Both compounds repressed the expression of BCL11A (-XL, -L, -S) and up-regulated GATA1, NFE2, EKLF, KLF4, STAT3, AHSP, and γ-globin genes expression. Moreover, sodium valproate showed a stronger effect on repressing BCL11A and escalating the expression of other target genes. The findings of this in vitro experiment could be considered in selecting drugs for clinical use in patients with β-hemoglobinopathies.

A molecular study on the role of alpha-hemoglobin-stabilizing protein in hemoglobin H disease.

The clinical course of hemoglobin H (HbH) disease is remarkably variable. It is not completely clear how genetic and environmental factors interplay to modify clinical severity in affected individuals. Previous studies suggested that altered structure or function of alpha-hemoglobin-stabilizing protein (AHSP) could modify the clinical phenotypes of thalassemias. The present study attempted to explore the potential role of AHSP in the pathophysiology of HbH disease in 95 Chinese and Thai/Sino-Thai patients with deletional and non-deletional form of this disease. We identified six polymorphic sites in AHSP which were subgrouped into major haplotype clades. No association between AHSP genotypes or haplotypes and clinical phenotypes was observed. Instead, multiple linear regression analysis indicated that expression of AHSP correlated negatively with age (P<0.001) and hemoglobin (P=0.007), but positively with reticulocyte count (P=0.003) and severity score (P=0.003). Subgroup analysis showed that AHSP expression was higher in the non-deletional form than in the deletional form (P<0.001). Moreover, specific types of non-deletional HbH disease with production of mutant alpha-globin chains that do not bind to AHSP (Hb Constant Spring and Hb Pakse) showed the highest AHSP expression. The present findings demonstrate that AHSP expression is a biomarker of HbH disease severity and infer an important role of AHSP in modulating the pathophysiology of this disease. Pharmacological or genetic means to alter AHSP expression may be a novel approach for amelioration of disease severity in HbH disease.

Study of alpha hemoglobin stabilizing protein expression in patients with β thalassemia and sickle cell anemia and its impact on clinical severity

The α hemoglobin stabilizing protein (AHSP) binds α-Hb and prevents its precipitation limiting free α-Hb toxicities. Our aim was to study AHSP expression in β thalassemia syndromes in relation to their clinical severity and to compare it with its level in sickle cell anemia.We compared patients with β-thalassemia (n=37) (β-thalassemia major (BTM) (n=19) and β-thalassemia intermedia (BTI) (n=18)) with 12 patients with sickle cell anemia as regards clinical severity, age at presentation, transfusion dependency, mean pre-transfusion hemoglobin level, use of hydroxyurea and AHSP expression by real time quantitative PCR.Median (and IQR) AHSP expression was significantly higher in patients with sickle cell anemia 2275 (3898) compared to thalassemia 283 (718), P=0.001, with no significant difference between BTM and BTI (P=0.346). It was also significantly higher in non-transfusion dependent patients with β thalassemia (NTDT) compared to transfusion dependent ones (P=0.019), and in patients on hydroxyurea therapy (P<0.001). However, there was no significant difference in its level according to clinical severity score (P=0.946) or splenectomy status (P=0.145). ConclusionAHSP expression was higher in patients with sickle cell anemia versus thalassemia, with no significant difference between BTM and BTI. Expression was higher in patients with NTDT and on hydroxyurea therapy.

Alpha hemoglobin stabilizing protein: Its causal relationship with the severity of beta thalassemia.

Thalassemia major is characterized by anemia, iron overload and cellular damage. The severity of symptoms correlates with the alpha/non-alpha globin imbalance and is proportional to the magnitude of alpha chain excess. Alpha hemoglobin stabilizing protein (AHSP), the erythroid specific alpha globin chaperone, stabilizes free alpha chains, and prevents the formation of reactive oxygen radicals. Though AHSP expression has been linked to the severity of beta thalassemia, its role as a probable genetic modifier of disease severity, has still not been unequivocally established. In the present study, the level of the chaperone has been seen to vary in regularly transfused beta thalassemia patients, being underexpressed in 64% of cases, upregulated in 16% and comparable to controls in 20% of the cases. This discrepancy may be attributed to the degree of DNA damage, % HbF, and the number of nucleated RBCs in the peripheral blood of these patients. Results reveal that a decrease in the free alpha chain pool, and hence the repertoire of unbound iron, due to elevated HbF and/or the presence of nucleated RBCs in the peripheral blood results in the upregulation of the AHSP gene.

A Twins Heritability Study on Alpha Hemoglobin Stabilizing Protein (AHSP) Expression Variability

Cytotoxic precipitation of free α-globin monomers and its production of reactive oxygen species cause red cell membrane damage that leads to anemia and eventually ineffective erythropoiesis in β-thalassemia. Alpha hemoglobin stabilizing protein (AHSP) was found to bind only to free α-globin monomers creating a stable and inert complex which remains soluble in the cytoplasm thus preventing harmful precipitations. Alpha hemoglobin stabilizing protein was shown to bind nascent α-globin monomers with transient strength before transferring α-globin to β-globin to form hemoglobin tetramer. A classical twin study would be beneficial to investigate the role of genetics and environment in the variation of alpha hemoglobin stabilizing protein expression as this knowledge will enable us to determine further investigations with regards to therapeutic interventions if alpha hemoglobin stabilizing protein is to be a therapeutic agent for β-thalassemia. This study investigates the heritability influence of alpha hemoglobin stabilizing protein expression and factors that may contribute to this. Results indicated that a major proportion of alpha hemoglobin stabilizing protein expression was influenced by genetic heritability (46%) with cis-acting factors accounting for 19% and trans-acting factors at 27%.

Transgenic Human α-Hemoglobin Stabilizing Protein Could Partially Relieve βIVS-2-654-Thalassemia Syndrome in Model Mice

Beta-thalassemia is an anemia caused by a relative excess of alpha-hemoglobin (alphaHb) due to absent or reduced beta-hemoglobin (betaHb) synthesis. In this study, we explore whether the introduction of alpha-hemoglobin stabilizing protein (AHSP), a chaperone protein for proper folding and stabilization of free alphaHb in red blood cells, thus aiding hemoglobin A (HbA) assembly, could relieve the pathogenic state of red blood cells in beta-thalassemia. For that, a human ahsp vector was constructed to generate transgenic human ahsp mice in a model of beta(IVS-2-654)-thalassemia by microinjecting the vector into fertilized eggs, resulting in the production of double heterozygous mice (h-ahsp(+)/beta(IVS-2-654+)). Real-time quantitative RT-PCR and Western blot analysis confirmed AHSP expression in three h-ahsp(+)/beta(IVS-2-654+) mice. Hematologic determination showed an improvement in the red blood cell indices of these h-ahsp(+)/beta(IVS-2-654+) mice. The red blood cell count and hemoglobin level were elevated to various extents as compared with their diseased siblings. A dramatic reduction in anisocytosis in the peripheral blood of h-ahsp(+)/beta(IVS-2-654+) mice was observed (16.2 +/- 4.6 vs. 30.0 +/- 5.2%). Few erythroid precursors appeared in the liver sinusoids of h-ahsp(+)/beta(IVS-2-654+) mice. Splenomegaly with extramedullary hematopoiesis was also ameliorated. Significantly, serum iron concentration was remarkably reduced as compared with that of h-ahsp(-)/beta(IVS-2-654+) mice (43.2 +/- 14.9 vs. 82.4 +/- 12.9 microM), and iron deposition in the liver was decreased in h-ahsp(+)/beta(IVS-2-654+) mice. All these results suggested amelioration of the anemia phenotype in h-ahsp(+)/beta(IVS-2-654+) mice after introduction of the ahsp gene. We therefore propose that an ahsp transgene could provide an adjuvant method for gene therapy of beta-thalassemia.

Reduction of AHSP synthesis in hemin-induced K562 cells and EPO-induced CD34 + cells leads to α-globin precipitation, impairment of normal hemoglobin production, and increased cell death

alpha-Hemoglobin stabilizing protein (AHSP) binds alpha-hemoglobin (Hb), avoiding its precipitation and its pro-oxidant activity. In the presence of betaHb, the alphaHb-AHSP complex is dismembered and betaHb displaces AHSP to generate the quaternary structure of Hb. The relationship between Hb formation and alterations in AHSP expression, which may affect human erythropoiesis, has not yet been described in human cells. Hence, in this study, we examined the effects of AHSP knockdown in hemin-induced K562 and erythropoietin-induced CD34(+) cells with particular reference to cellular aspects and gene expression. Short-hairpin RNA expression vectors aimed at the AHSP mRNA target sequence were cloned and transfected into K562 and CD34(+) cells. K562 and CD34(+) cells were stimulated to erythroid differentiation. Cells were examined in terms of gene expression using quantitative real-time polymerase chain reaction; reactive oxygen species (ROS) production, apoptosis, and Hb production through flow cytometry assays; and immunofluorescence assays for globin chains. RNA interference-mediated knockdown of AHSP expression resulted in considerable alphaHb precipitation, as well as in a significant decrease in HbF formation. AHSP-knockdown cells demonstrated an increased ROS production and increased rate of apoptosis. These findings strengthen the hypothesis that AHSP stabilizes the alphaHb chain, avoiding its precipitation and its ability to generate ROS, which implicate in cell death. Moreover, data indicate that AHSP may be highly significant for human hemoglobin formation and suggest that AHSP is a key chaperone protein during human erythropoiesis.

AHSP Knockdown in Human Erythroleukemia Cell Line and Human Hematopoietic Stem Cells Results in Alpha Hemoglobin Chain Precipitation, Decreased Hemoglobin Levels and Increased Cell Death.

Alpha Hemoglobin Stabilizing Protein (AHSP) binds alpha hemoglobin chain (αHb), avoiding its precipitation and its pro-oxidant activity. In the presence of beta hemoglobin chain (βHb), the αHb-AHSP complex is dismembered and βHb displaces AHSP to generate the quaternary structure of hemoglobin. These data have been obtained in vitro and in mouse cells, but strongly suggest the importance of AHSP for normal hemoglobin synthesis in humans. To the best of our knowledge, the relationship between hemoglobin formation and alterations in AHSP expression has not yet been described in human red cells. Hence, to investigate the consequences of a reduced AHSP synthesis in human red cells, we established the RNA interference-mediated knockdown of AHSP expression in human erythroleukemia cell line (K562 cells) and human hematopoietic stem cells (CD34+ cells) induced to erythroid differentiation, and analyzed the consequent cellular and molecular aspects of AHSP knockdown in these cells. shRNA expression vectors, aimed at the AHSP mRNA target sequence, were cloned and transfected into K562 and CD34+ cells using a non-liposomal lipid reagent. Following transfection, K562 cells that stably expressed AHSP-shRNA and CD34+ cells that transiently expressed AHSP-shRNA were selected. K562 and CD34+ cells were stimulated to erythroid differentiation by hemin and erythropoietin (EPO) respectively. The cells were examined in terms of gene expression using quantitative real-time PCR; production of reactive oxygen species (ROS), apoptosis and hemoglobin production through flow cytometry assays; and immunofluorescence assays for globin chains. AHSP-shRNA hemin-induced K562 cells and AHSP-shRNA EPO-induced CD34+ cells presented 71% and 75% decreases in AHSP expression levels, respectively. The RNAi-mediated knockdown of AHSP expression resulted in a considerable αHb precipitation, as well as in a significant decrease in fetal hemoglobin formation. In addition, AHSP-knockdown cells demonstrated an increased ROS production and increased rate of apoptosis. These findings strengthen the hypothesis that AHSP stabilizes the alpha hemoglobin chain, avoiding its precipitation and its ability to generate ROS which implicate in cell death. Moreover, data indicate that AHSP may be highly significant for human hemoglobin formation and suggest that AHSP is a key chaperone protein during human erythropoiesis.

Population analysis of the alpha hemoglobin stabilizing protein (AHSP) gene identifies sequence variants that alter expression and function

Alpha-hemoglobin stabilizing protein (AHSP) is a potential modifier of beta-thalassemia by virtue of its ability to detoxify excess free alpha-globin. However, examination of patients with beta-thalassemia from a few geographic regions failed to identify obvious AHSP mutations. We extended these studies by analyzing AHSP gene sequences in 366 anonymous individuals from five different areas of the world. We detected numerous polymorphisms comprising 18 different haplotypes and two rare missense mutations. Two sequence variations produce functional effects in laboratory assays. First, a rare missense mutation in a Brazilian/Mediterranean cohort converts asparagine to isoleucine at position 75 of AHSP protein and impairs its ability to inhibit reactive oxygen species production by alpha-hemoglobin. Second, a high-frequency polymorphism in intron 1 of the AHSP gene (12391 G>A) alters an Oct-1 transcription factor binding site previously shown to be important for optimal gene expression. The 12391A polymorphism impairs Oct-1 binding and inhibits the ability of AHSP regulatory sequences to activate expression of a linked luciferase reporter. Although structural mutations predicted to alter AHSP protein function or ablate its activity are rare, the 12391 G>A SNP is common and represents a potential mechanism through which genetically determined variations in AHSP expression could influence beta-thalassemia.

Identification of Two New Synthetic Histone Deacetylase Inhibitors That Modulate Globin Gene Expression in Erythroid Cells from Healthy Donors and Patients with Thalassemia

We have identified two new histone deacetylase (HDAC) inhibitors (9 and 24) capable of inducing the expression of gamma-globin and/or beta-globin promoter-driven reporter genes in a synthetic model of Hb switch. Both compounds also increased, with different mechanisms, the gamma/(gamma+beta) ratio expressed in vitro by normal human erythroblasts. Compound 9 increased the levels of gamma-globin mRNA and the gamma/(gamma+beta) ratio (both by 2-fold). Compound 24 increased by 3-fold the level of gamma-globin and decreased by 2-fold that of beta-globin mRNA, increasing the gamma/(gamma+beta) ratio by 6-fold, and raising (by 50%) the cell HbF content. Both compounds raised the acetylation state of histone H4 in primary cells, an indication that their activity was mediated through HDAC inhibition. Compounds 9 and 24 were also tested as gamma/(gamma+beta) mRNA inducers in erythroblasts obtained from patients with beta(0) thalassemia. Progenitor cells from patients with beta(0) thalassemia generated in vitro morphologically normal proerythroblasts that, unlike normal cells, failed to mature in the presence of EPO and expressed low beta-globin levels but 10 times higher-than-normal levels of the alpha hemoglobin-stabilizing protein (AHSP) mRNA. Both compounds ameliorated the impaired in vitro maturation in beta(0) thalassemic erythroblasts, decreasing AHSP expression to normal levels. In the case of two patients (of five analyzed), the improved erythroblast maturation was associated with detectable increases in the gamma/(gamma+beta) mRNA ratio. The low toxicity exerted by compounds 9 and 24 in all of the assays investigated suggests that these new HDAC inhibitors should be considered for personalized therapy of selected patients with beta(0) thalassemia.

AHSP (Alpha Hemoglobin Stabilizing Protein) Gene Expression during Normal and β-Thalassemic Erythroid Differentiation.

β-thalassemia severity is stricktly related to the amount of free a-chains depending at least in part from different b-thalassemic genotype (β0 /β0, β0/β+, β+ /β+). Recently a new erythroid-specific protein, named AHSP (Alpha Hemoglobin Stabilizing Protein), able to form a stable complex with free α-hemoglobin, has been identified. This protein probably acts as a molecular chaperone to prevent the harmful aggregation and precipitation of α-globin that occurs during normal erythropoiesis and to a greater extent in different β-thalassemias. The aim of this study was to evaluate if the AHSP expression in erythroid cells depends on free α-chains amount. In order to verify this hypothesis, we studied the relative expression of AHSP and globin genes during differentiation of erythroid progenitors derived from peripheral blood of both normal subjects and β-thalassemic patients with different genotypes. Erythroid cultures were set up accordingly to the two phases liquid colture method described by Fibach. Total cytoplasmatic mRNA was extracted by the method of Chomczynsky-Sacchi and quantitative real-time PCR was performed using SYBRGreen to evaluate AHSP, α, β and γ globin genes expression. To quantify gene expression a mathematical model using calibration data (2-ΔΔct) was used and quantification of the house-keeping gene S14 was the internal control. In normal subjects AHSP expression was first detected at proerythroblast stage (on day 3 of culture), peaked at orthocromatic stage (on day 9) and declined to low levels by polychromatophilic-reticulocytes stage (day 13) (a decrease of 65% vs day 9 expression). Globin genes expression reached a steady state (a/non-a ratio value of 0.78 very near to a clinical equilibrium) on day 13. In this situation few free α-globin are detectable explaning the lower need of AHSP expression. In β-thalassemic subjects we observed a persistance of globin gene unbalance with an excess of free α-chains also in the advanced differentiation steps: interestingly the worst was the genotype, the highest was the globin ratio and higher was AHSP expression. In β+/β+ thalassemic patients AHSP expression declined on day 13 (a decrease of 15% vs day 9 expression) and globin gene ratio was 2.4, expression of a persistance of free α-chains excess. In β0 /β+ patients globin gene ratio on day 13 was 2.8 and AHSP expression increased of 2.5 times. Finally in β0/β0 patients globin gene ratio was very high with values up to 9–10; AHSP expression profiles instead were different between different subiects and related not only to globin unbalance but also to ineffective erythropoiesis rate and γ-globin chains persistance. All these findings underline an evident relation between AHSP and globin genes expression, suggesting a probable function of this protein during erythropoiesis.

AHSP Promoter Homopolymer in Beta-Thalassemia Syndromes.

Alpha hemoglobin stabilizing protein (AHSP) is an erythroid protein that binds specifically to the alpha globin chains to form a stable complex, preventing their precipitation before the assembly of the hemoglobin tetramere. Studies have shown thatAHSP is required for normal erythropoiesis,knock-out mice has a phenotype similar to beta thalassemia,loss of AHSP worsens the severity of the disease in thalassemic mice.Therefore, altered AHSP expression or function could account for some of the variability in beta-thalassemia phenotype. At position -160bp upstream of exon 1 of the AHSP gene, two main homopolymers (T18 and T15) have been reported that may influence the AHSP expression and hence the hematological phenotype. In particular the frequence of the shorter T15 homopolymer, which is associated with a lower AHSP expression, was much higher than expected (on the basis of the allele frequency in the healthy population) in a group of 9 patients with thalassemia intermedia, due the compound heterozygosity for beta-thalassemia and the triple alpha globin gene (Mei et al, 2006). We have studied the frequency of T15 and T18 homolpolymers in a group of 20 normal subjects and in patients with different thalassemia syndromes, i.e. beta zero 39 homozygotes with thalassemia major (14 patients) and intermedia (19), compound heterozygotes for beta-thalassemia and triple alpha globin gene (13) and simple carriers for beta thalassemia with thalassemia intermedia phenotype (5). We found the following genotype distribution reported in figure1. There is no difference in the frequency of the genotypes among the groups, except in the severe beta-thalassemia carriers, where the short T15 homopolymer is more common. These findings are consistent with the reduced AHSP expression previously reported in the reticulocytes and in the erythroid cultures of these unusually severe beta thalassemia carriers. (Galanello et al, abs 1881. ASH 2003). Therefore, only in the peculiar group of beta-thalassemia carriers with the clinical features of thalassemia intermedia, the AHSP genotype appears to be a relevant contributory factor in the hematological phenotype. [Display omitted]

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

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.

Regulation of Alpha Hemoglobin Stabilizing Protein (AHSP) mRNA Stability by a 3′ UTR Iron Response Element.

AHSP is an erythroid protein that binds the alpha globin subunit of hemoglobin (Hb) to maintain its structure and limit its oxidative activity. Biochemical and genetic studies demonstrate that AHSP is essential for normal Hb homeostasis and may act via at least two mechanisms: First, as a molecular chaperone to promote the folding and stability of alpha globin prior to its incorporation into HbA (alpha2beta2). Second, to bind and detoxify excess alpha globin that accumulates in normal erythroid precursors, and to a larger extent, in beta thalassemic ones. The existence of a potential iron responsive element (IRE) in the 3′-UTR of the AHSP mRNA raises the interesting possibility that iron homeostasis impacts on alpha globin stability via AHSP. IREs form stem-loop structures that bind cytosolic iron-sensing proteins (IRPs). Typically, IRP binding to the 3′ UTR stabilizes mRNA. Association with iron releases the IRP, enhancing mRNA degradation. Computational algorithms identified IRE-like stem-loop structures in AHSP mRNA of multiple species, yet the primary sequences deviate significantly from canonical IRE consensus sequences determined by studies of classical IREs, such as Transferrin receptor and Ferritin. Several lines of evidence now show that the AHSP IRE binds IRPs to regulate mRNA stability in an iron-dependent fashion:1) in vitro gelshift competition assays demonstrate that human and mouse AHSP IREs bind tightly to IRPs.2) AHSP mRNA co-immunoprecipitates with IRPs in erythroid cells in a fashion that depends on an intact IRE.3) AHSP mRNA is destabilized by iron in both erythroid and heterologous cells; disruption of the IRE renders the mRNA constitutively unstable.To study how iron regulates AHSP expression in vivo, we treated mice with iron dextran for 10 days and then examined AHSP mRNA in Ter119+ erythroid progenitors by RT-PCR. We found that short-term iron overload reduced AHSP mRNA levels by about 50% (p > 0.05). Our findings indicate that AHSP mRNA stability is regulated by iron via an atypical 3′-UTR IRE. These findings extend the potential repertoire for functional IREs that do not conform to previously defined canonical consensus sequences. In addition, they provide a potential mechanism by which erythroid cells can regulate globin stability according to iron status. For example, induction of AHSP during iron deficiency might stabilize apo- alpha globin that could accumulate from lack of heme. Conversely, iron overload could destabilize alpha globin by reducing AHSP levels. As such, iron overload, which occurs in patients with beta thalassemia, might aggravate the disease by further elevating the levels of toxic free alpha globin.