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Alpha hemoglobin stabilizing protein: Its causal relationship with the severity of beta thalassemia
Blood Cells, Molecules and Diseases 55 (2015) 104–107
Contents lists available at ScienceDirect
Blood Cells, Molecules and Diseases journal homepage: www.elsevier.com/locate/bcmd
Alpha hemoglobin stabilizing protein: Its causal relationship with the severity of beta thalassemia Chandan S. Sagar a, Rakesh Kumar a, Dharmesh C. Sharma b, Purnima Kishor a,⁎ a b
School of Studies in Biochemistry, Jiwaji University, Gwalior, India Blood Bank, Jaya Arogya Hospital, Gajra Raja Medical College, Gwalior, India
a r t i c l e
i n f o
Article history: Submitted 17 August 2014 Revised 11 May 2015 Accepted 11 May 2015 Available online 12 May 2015 Keywords: Beta thalassemia AHSP HbF DNA damage Nucleated RBC Alpha chain toxicity
a b s t r a c t 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. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Excess alpha hemoglobin is cytotoxic and is largely responsible for the pathophysiology of beta thalassemia. Alpha-hemoglobin-stabilizing protein (AHSP), the erythroid specific molecular chaperone for free alpha globins, stabilizes and prevents the precipitation of alpha chains and might be important in β-thalassemic erythropoiesis characterized by an imbalance of globin chain synthesis [1]. In humans, AHSP expression levels vary among different individuals because of polymorphisms in regulatory regions and perhaps additional determinants that are not linked to the AHSP gene [2,3]. There is uncertainty regarding the direct relationship between AHSP expression levels and beta thalassemia phenotype. Some studies indicate that AHSP expression levels inversely correlate with the severity of thalassemia [2] while in others, disease severity was not found to correlate with the gene haplotypes [4,5]. The loss of AHSP function in mice model resulted in abnormal erythrocyte morphology, which shows cellular damage due to increased ROS and intracellular inclusion bodies. This results in an increased destruction in erythroid precursor cells and a reduced lifespan of erythrocytes [6]. These phenotypes could be the result of the loss of AHSP, causing nascent α-globin to be structurally unstable, making it incompatible for HbA formation [7]. Lim et al. [7] have reported significant ⁎ Corresponding author. E-mail address: [email protected] (P. Kishor).
http://dx.doi.org/10.1016/j.bcmd.2015.05.005 1079-9796/© 2015 Elsevier Inc. All rights reserved.
correlation between AHSP expression and mean cell hemoglobin, HbF %, α-globin, β-globin and excess α-globin expression and conclude that AHSP could be a secondary compensatory mechanism in red blood cells to counterbalance the excess α-globin chains in HbE/β-thalassemia individuals. In the present study the level of AHSP in beta thalassemia patients as compared to non-thalassemic controls has been determined and its relation with DNA damage, ineffective erythropoiesis and HbF has been demonstrated in order to provide an insight into the causal relationship between the level of AHSP and the severity of beta thalassemia. 2. Methods 2.1. Subjects Subjects included 30 regularly transfused beta thalassemia patients of the Gwalior Chambal region, registered at the blood bank of Jaya Arogya Hospital, Gajra Raja Medical College, Gwalior. The age of the patients ranges from 2–19 years, the average age being 10 years. Five patients were β° heterozygotes and the rest were homozygous for the severe β + IVS-1-5 (GNC) [HGVS nomenclature c.92+5GNC] mutation of the beta globin gene. Blood drawn for cross matching, prior to transfusion, was taken for analysis. Written informed consent was taken from the parents/guardians of all the patients prior to the analysis. Blood samples of non-thalassemic adult volunteers served as controls.
C.S. Sagar et al. / Blood Cells, Molecules and Diseases 55 (2015) 104–107
a
105
b
Fig. 1. a) Fold expression of AHSP in % cases. b) Western blot: Lane 1_ AHSP in non-thalassemic control, lanes 2_5 AHSP in beta thalassemia patients.
2.2. Determination of HbF HbF was determined by cation exchange high performance liquid chromatography by the HbA2/HbF/HbA1c dual program of the D-10 HPLC system of Bio-Rad, Laboratories.
(goat antimouse IgG h + 1, NOVUS, 1:2000) for 3 h. The blot was again washed and then incubated with the substrate (TMB, Sigma). The image was captured and analyzed using the alpha imager software in the gel documentation system. 3. Results and discussion
2.3. Nucleated RBCCs Whole blood was stained with Giemsa stain and observed at 100× under the microscope and nucleated RBC counts were determined per 100 RBCs. 2.4. Quantitation of AHSP Relative quantitation of AHSP of beta thalassemia patients as compared to non-thalassemic controls was done by Western blotting [8]. 2.4.1. Sample preparation RBCs were separated by layering whole blood on Hi Sep (Hi Media), lysed, mixed with sample buffer (10% w/v SDS, 10 m M dithiothreitol, 20% v/v glycerol, 0.2 M Tris HCl, pH 6.8, 0.05% bromophenol blue), boiled in a water bath at 60 °C and loaded. Samples were normalized to the micrograms of protein loaded. Protein was determined by the Lowry's method [9]. 2.4.2. Electrophoresis SDS-PAGE was performed on a 15% gel [10], using Tris–glycine running buffer (25 m M tris, 200 m M glycine, 0.1% SDS, p H 8.3). 30 μg protein was loaded in each well. Protein molecular weight marker (PUREGENE, Genetix) was run simultaneously. 2.4.3. Western blotting The membrane (Immobilon-PSQ Membrane, PVDF, 0.2 μm from millipore) was cut to appropriate size and dipped in methanol for 1 min, soaked in towbin buffer (25 m M Tris–HCl, 192 m M Glycine, 0.1% w/v, 20% methanol) for 15 min, and sandwiched between blotting paper towels, also soaked in towbin buffer, and placed in the blotting apparatus (semidry, from BIOTECH). Blotting was done at 55 V for 15 min. The membrane was stained with Ponceau to confirm protein transfer and then destained. The gel was stained with Coomassie blue to confirm complete transfer. The blot was then washed with 1× TBST buffer (60.55 M Tris Cl buffer, 29.2 M NaCl, 0.1% Tween 20) and dipped in 5% BSA to block non-specific sites and washed again. It was then dipped in the primary antibody (mouse polyclonal ERAF/AHSP antibody, NOVUS, diluted 1:500) for 15 h, followed by washing with TBST buffer, and then incubation in the HRP conjugated-secondary antibody
Relative quantitation of AHSP in thalassemia major patients, as compared to controls, revealed downregulation in 64% of the patients, upregulation in 16% and was comparable to controls in 20% of the patients (Fig. 1a & b) The expression was not seen to correlate with the age of the patients, or their sex, which supports earlier data [2]. Data suggest that the factors that apparently influence the expression of AHSP are the number of nucleated RBCs, DNA damage in RBC precursors and HbF levels. 3.1. Nucleated RBCs and AHSP level Beta thalassemia is associated with an increase in alpha-beta globin chain ratio. The free-iron species released from the unpaired alpha chains induces the formation of oxygen radicals that cause cellular damage resulting in hemolysis and ineffective erythropoiesis [11]. This ineffective erythropoiesis causes the release of nucleated RBCs in the circulation [12]. In the present study, nucleated RBCs have been seen in the peripheral blood samples of patients expressing AHSP at par with, or higher than non-thalassemic controls (Fig. 2). The pattern of AHSP expression has been found to be similar to that of hemoglobin, with the highest
Fig. 2. % nucleated RBCs and fold expression of AHSP in the subjects.
106
C.S. Sagar et al. / Blood Cells, Molecules and Diseases 55 (2015) 104–107
a
b
Fig. 3. a) Box plots exhibiting HbF % and DNA damage (tail moment of comets). b) Correlation between DNA damage and fold expression of AHSP.
expression coinciding with that of maximum hemoglobinization [13]. AHSP is an alpha globin chaperone and thus, as Hb synthesis declines, it is no longer needed and is degraded [14]. While hemoglobin synthesis begins in the proerythroblast phase, and persists till the reticulocyte stage, HBA expression is higher during the intermediate phase, comprising predominantly of polychromatic and orthochromatic erythroblasts, than the late phase comprising of more mature precursors [13]. It has also been demonstrated by immunohistochemistry that AHSP stains only nucleated erythroid precursors and not mature enucleate RBCs [14]. A reduction in AHSP expression has been seen to occur when the cells lose their nuclear function and hemoglobin synthesis is reduced [15]. Studies revealing maximum AHSP expression in the polychromatic and orthochromatic stages as compared to that in later stages of erythropoiesis [15], and the persistence of nucleated RBCs in the patients in the present study, could account for the prolonged synthesis of the chaperone and hence a high AHSP pool in these patients.
All the patients of this study group presented with sub-normal HbA, and 48% presented with elevated HbF which lessens tissue hypoxia as well as the unpaired alpha chain pool. Since high HbF, a consequence of enhanced gamma globin expression, relies on the genetic make-up of cells, the release of AHSP producing erythroid precursors in the circulation combats alpha chain toxicity in patients with HbF in the normal range. Low DNA damage reflects lesser production of ROS as a result of Fenton's reaction and allows AHSP producing nucleated erythroid
3.2. DNA damage and AHSP level Varying degrees of DNA damage in developing RBCs of these subjects have already been demonstrated [16]. The present study shows a weak negative correlation, with an r = −0.3109, between DNA damage and the fold change in AHSP levels (Fig. 3b). Low AHSP, as compared to non-thalassemic controls, in 64% of patients could be attributed to DNA damage in developing RBCs, thus inhibiting erythropoiesis, and allowing fewer RBCs to enter the stage of peak AHSP expression (Fig. 3a). 3.3. HbF and AHSP level Lim et al. [7] suggested that AHSP is a secondary compensatory mechanism, in addition to the HbF response, to the α/β globin chain imbalance in HbE/beta thalassemia patients. HPLC analysis in this group of patients revealed elevated HbF, ranging from 5–47%, in 48% of the patients (Fig. 4). A weak positive correlation with r = 0.3709 was seen between HbF and AHSP. Earlier reports have revealed that human AHSP expression can be affected by the iron status, via an iron sensor system, and that iron insufficiency upregulates, and its abundance downregulates, the gene [3]. Since excessive unpaired alpha chains release heme and iron which initiate self amplifying redox reactions [17], high AHSP in patients with raised HbF may be attributed to the partial compensation of insufficient beta chains by gamma globin chains, resulting in a relatively small free alpha globin pool and hence free iron, possibly adequate to prolong the expression of AHSP.
Fig. 4. Chromatogram showing 47.1% HbF in a patient.
C.S. Sagar et al. / Blood Cells, Molecules and Diseases 55 (2015) 104–107
precursors to persist in the circulation. It is speculated, that maintenance of low free iron pool could minimize DNA damage, prolong the survival of AHSP producing erythroid precursors, and prevent the downregulation of AHSP, thus, preventing the accumulation of unpaired alpha chains in β-thal patients with HbF in the normal range. Acknowledgment The authors are indebted to all the patients' guardians for their cooperation in allowing the use of blood samples of their ward. The authors thank the University Grants Commission (F.No. 36-122/2008 (SR)), New Delhi, India and the Indian Council of Medical Research (ICMR) (2010-02970) for providing funds for the work. Rakesh Kumar received a Senior research fellowship from the ICMR. Thanks are due to all the staff of the blood bank at Jaya Arogya Hospital for regularly providing blood samples of the patients. References [1] L. De Franceschi, M. Bertoldi, A. Matte, S.S. Franco, A. Pantaleo, E. Ferru, F. Turrini, Oxidative stress and -thalassemic erythroid cells behind the molecular defect, Oxidative Med. Cell. Longev. 2013 (2013) (10 pages Article ID 985210). [2] M.I. Lai, J. Jiang, N.A. Silver, et al., AHSP is a quantitative trait gene that modifies the phenotype of beta-thalassaemia, Br. J. Haematol. 133 (2006) 675–682. [3] C.O. Dos Santos, L.C. Dore, E. Valentine, et al., An iron responsive element-like stem-loop regulates α-hemoglobin-stabilizing protein Mrna, J. Biol. Chem. 283 (40) (2008) 26956–26964. [4] V. Viprakasit, V.S. Tanphaichitr, W. Chinchang, et al., Evaluation of alpha hemoglobin stabilizing protein (AHSP) as a genetic modifier in patients with beta thalassemia, Blood 103 (2004) 3296–3299.
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[5] B. Wang, Y. Fang, X. Guo, Z. Ren, J. Zhang, Transgenic human alpha-hemoglobin stabilizing protein could partially relieve beta IVS-2-654-thalassemia syndrome in model mice, Hum. Gene Ther. 21 (2) (2010) 149–156. [6] Y. Kong, S. Zhou, A.J. Kihm, A.M. Katein, X. Yu, D.A. Gell, J.P. Mackay, K. Adachi, L. Foster-Brown, C.S. Louden, A.J. Gow, M.J. Weiss, Loss of α-hemoglobin-stabilizing protein impairs erythropoiesis and exacerbates β-thalassemia, J. Clin. Investig. 114 (2004) 1457–1466. [7] W.F. Lim, M. Logeswaran, E. George, J. Sathar, L.K. Teh, G.G. Gan, M.I. Lai, αHaemoglobin stabilising protein expression is influenced by mean cell haemoglobin and HbF levels in HbE/β-thalassaemia individuals, Blood Cell Mol. Dis. 48 (2012) 17–21. [8] M. Emanuelli, M. Cecati, D. Sartini, P. Stortoni, A. Corradetti, R. Stefano, T.A. Giannubilo, A.L. Tranquilli, Placental Alpha Hemoglobin Stabilizing Protein (AHSP) and recurrent miscarriage, Cell Stress Chaperones 14 (2) (2008) 93–197. [9] O.H. Lowry, N.J. Rosenbrough, R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193 (1) (1951) 265–275. [10] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685. [11] E. Fibach, E.A. Rachmilewitz, The role of oxidative stress in hemolytic anemia, Curr. Mol. Med. 8 (7) (2008) 609–619. [12] C. Antonio, G. Renzo, O. Raffaella, Beta-thalassemia, in: R.A. Pagon, M.P. Adam, H.H. Ardinger, T.D. Bird, C.R. Dolan, C.-T. Fong, R.J.H. Smith, K. Stephens (Eds.),GeneReviews® [Internet], 2013. [13] M.E. Favero, F.F. Costa, Alpha-hemoglobin-stabilizing protein: an erythroid molecular chaperone, Biochem. Res. Int. 2011 (2011) 373859. [14] P.W. Raess, M.E. Paessler, A. Bagg, M.J. Weiss, J.K. Choi, α-Hemoglobin-stabilizing protein is a sensitive and specific marker of erythroid precursors, Am. J. Surg. Pathol. 36 (10) (2012) 1538–1547. [15] C.O. Dos Santos, A.S.S. Duarte, S.T. Olalla Saad, F.F. Costa, Expression of α-hemoglobin stabilizing protein gene during human erythropoiesis, Exp. Hematol. 32 (2) (2004) 157–162. [16] C.S. Sagar, R. Kumar, D.C. Sharma, P. Kishor, DNA damage: beta zero vs beta plus thalassemia, Ann. Hum. Biol. Early online 1–4, http://dx.doi.org/10.3109/ 03014460.2014.990921. [17] F. Waseem, K.A. Khemomal, R. Sajid, Antioxidant status in beta thalassemia major: a single-center study, Indian J. Pathol. Microbiol. 54 (2011) 761–763.
Contents lists available at ScienceDirect
Blood Cells, Molecules and Diseases journal homepage: www.elsevier.com/locate/bcmd
Alpha hemoglobin stabilizing protein: Its causal relationship with the severity of beta thalassemia Chandan S. Sagar a, Rakesh Kumar a, Dharmesh C. Sharma b, Purnima Kishor a,⁎ a b
School of Studies in Biochemistry, Jiwaji University, Gwalior, India Blood Bank, Jaya Arogya Hospital, Gajra Raja Medical College, Gwalior, India
a r t i c l e
i n f o
Article history: Submitted 17 August 2014 Revised 11 May 2015 Accepted 11 May 2015 Available online 12 May 2015 Keywords: Beta thalassemia AHSP HbF DNA damage Nucleated RBC Alpha chain toxicity
a b s t r a c t 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. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Excess alpha hemoglobin is cytotoxic and is largely responsible for the pathophysiology of beta thalassemia. Alpha-hemoglobin-stabilizing protein (AHSP), the erythroid specific molecular chaperone for free alpha globins, stabilizes and prevents the precipitation of alpha chains and might be important in β-thalassemic erythropoiesis characterized by an imbalance of globin chain synthesis [1]. In humans, AHSP expression levels vary among different individuals because of polymorphisms in regulatory regions and perhaps additional determinants that are not linked to the AHSP gene [2,3]. There is uncertainty regarding the direct relationship between AHSP expression levels and beta thalassemia phenotype. Some studies indicate that AHSP expression levels inversely correlate with the severity of thalassemia [2] while in others, disease severity was not found to correlate with the gene haplotypes [4,5]. The loss of AHSP function in mice model resulted in abnormal erythrocyte morphology, which shows cellular damage due to increased ROS and intracellular inclusion bodies. This results in an increased destruction in erythroid precursor cells and a reduced lifespan of erythrocytes [6]. These phenotypes could be the result of the loss of AHSP, causing nascent α-globin to be structurally unstable, making it incompatible for HbA formation [7]. Lim et al. [7] have reported significant ⁎ Corresponding author. E-mail address: [email protected] (P. Kishor).
http://dx.doi.org/10.1016/j.bcmd.2015.05.005 1079-9796/© 2015 Elsevier Inc. All rights reserved.
correlation between AHSP expression and mean cell hemoglobin, HbF %, α-globin, β-globin and excess α-globin expression and conclude that AHSP could be a secondary compensatory mechanism in red blood cells to counterbalance the excess α-globin chains in HbE/β-thalassemia individuals. In the present study the level of AHSP in beta thalassemia patients as compared to non-thalassemic controls has been determined and its relation with DNA damage, ineffective erythropoiesis and HbF has been demonstrated in order to provide an insight into the causal relationship between the level of AHSP and the severity of beta thalassemia. 2. Methods 2.1. Subjects Subjects included 30 regularly transfused beta thalassemia patients of the Gwalior Chambal region, registered at the blood bank of Jaya Arogya Hospital, Gajra Raja Medical College, Gwalior. The age of the patients ranges from 2–19 years, the average age being 10 years. Five patients were β° heterozygotes and the rest were homozygous for the severe β + IVS-1-5 (GNC) [HGVS nomenclature c.92+5GNC] mutation of the beta globin gene. Blood drawn for cross matching, prior to transfusion, was taken for analysis. Written informed consent was taken from the parents/guardians of all the patients prior to the analysis. Blood samples of non-thalassemic adult volunteers served as controls.
C.S. Sagar et al. / Blood Cells, Molecules and Diseases 55 (2015) 104–107
a
105
b
Fig. 1. a) Fold expression of AHSP in % cases. b) Western blot: Lane 1_ AHSP in non-thalassemic control, lanes 2_5 AHSP in beta thalassemia patients.
2.2. Determination of HbF HbF was determined by cation exchange high performance liquid chromatography by the HbA2/HbF/HbA1c dual program of the D-10 HPLC system of Bio-Rad, Laboratories.
(goat antimouse IgG h + 1, NOVUS, 1:2000) for 3 h. The blot was again washed and then incubated with the substrate (TMB, Sigma). The image was captured and analyzed using the alpha imager software in the gel documentation system. 3. Results and discussion
2.3. Nucleated RBCCs Whole blood was stained with Giemsa stain and observed at 100× under the microscope and nucleated RBC counts were determined per 100 RBCs. 2.4. Quantitation of AHSP Relative quantitation of AHSP of beta thalassemia patients as compared to non-thalassemic controls was done by Western blotting [8]. 2.4.1. Sample preparation RBCs were separated by layering whole blood on Hi Sep (Hi Media), lysed, mixed with sample buffer (10% w/v SDS, 10 m M dithiothreitol, 20% v/v glycerol, 0.2 M Tris HCl, pH 6.8, 0.05% bromophenol blue), boiled in a water bath at 60 °C and loaded. Samples were normalized to the micrograms of protein loaded. Protein was determined by the Lowry's method [9]. 2.4.2. Electrophoresis SDS-PAGE was performed on a 15% gel [10], using Tris–glycine running buffer (25 m M tris, 200 m M glycine, 0.1% SDS, p H 8.3). 30 μg protein was loaded in each well. Protein molecular weight marker (PUREGENE, Genetix) was run simultaneously. 2.4.3. Western blotting The membrane (Immobilon-PSQ Membrane, PVDF, 0.2 μm from millipore) was cut to appropriate size and dipped in methanol for 1 min, soaked in towbin buffer (25 m M Tris–HCl, 192 m M Glycine, 0.1% w/v, 20% methanol) for 15 min, and sandwiched between blotting paper towels, also soaked in towbin buffer, and placed in the blotting apparatus (semidry, from BIOTECH). Blotting was done at 55 V for 15 min. The membrane was stained with Ponceau to confirm protein transfer and then destained. The gel was stained with Coomassie blue to confirm complete transfer. The blot was then washed with 1× TBST buffer (60.55 M Tris Cl buffer, 29.2 M NaCl, 0.1% Tween 20) and dipped in 5% BSA to block non-specific sites and washed again. It was then dipped in the primary antibody (mouse polyclonal ERAF/AHSP antibody, NOVUS, diluted 1:500) for 15 h, followed by washing with TBST buffer, and then incubation in the HRP conjugated-secondary antibody
Relative quantitation of AHSP in thalassemia major patients, as compared to controls, revealed downregulation in 64% of the patients, upregulation in 16% and was comparable to controls in 20% of the patients (Fig. 1a & b) The expression was not seen to correlate with the age of the patients, or their sex, which supports earlier data [2]. Data suggest that the factors that apparently influence the expression of AHSP are the number of nucleated RBCs, DNA damage in RBC precursors and HbF levels. 3.1. Nucleated RBCs and AHSP level Beta thalassemia is associated with an increase in alpha-beta globin chain ratio. The free-iron species released from the unpaired alpha chains induces the formation of oxygen radicals that cause cellular damage resulting in hemolysis and ineffective erythropoiesis [11]. This ineffective erythropoiesis causes the release of nucleated RBCs in the circulation [12]. In the present study, nucleated RBCs have been seen in the peripheral blood samples of patients expressing AHSP at par with, or higher than non-thalassemic controls (Fig. 2). The pattern of AHSP expression has been found to be similar to that of hemoglobin, with the highest
Fig. 2. % nucleated RBCs and fold expression of AHSP in the subjects.
106
C.S. Sagar et al. / Blood Cells, Molecules and Diseases 55 (2015) 104–107
a
b
Fig. 3. a) Box plots exhibiting HbF % and DNA damage (tail moment of comets). b) Correlation between DNA damage and fold expression of AHSP.
expression coinciding with that of maximum hemoglobinization [13]. AHSP is an alpha globin chaperone and thus, as Hb synthesis declines, it is no longer needed and is degraded [14]. While hemoglobin synthesis begins in the proerythroblast phase, and persists till the reticulocyte stage, HBA expression is higher during the intermediate phase, comprising predominantly of polychromatic and orthochromatic erythroblasts, than the late phase comprising of more mature precursors [13]. It has also been demonstrated by immunohistochemistry that AHSP stains only nucleated erythroid precursors and not mature enucleate RBCs [14]. A reduction in AHSP expression has been seen to occur when the cells lose their nuclear function and hemoglobin synthesis is reduced [15]. Studies revealing maximum AHSP expression in the polychromatic and orthochromatic stages as compared to that in later stages of erythropoiesis [15], and the persistence of nucleated RBCs in the patients in the present study, could account for the prolonged synthesis of the chaperone and hence a high AHSP pool in these patients.
All the patients of this study group presented with sub-normal HbA, and 48% presented with elevated HbF which lessens tissue hypoxia as well as the unpaired alpha chain pool. Since high HbF, a consequence of enhanced gamma globin expression, relies on the genetic make-up of cells, the release of AHSP producing erythroid precursors in the circulation combats alpha chain toxicity in patients with HbF in the normal range. Low DNA damage reflects lesser production of ROS as a result of Fenton's reaction and allows AHSP producing nucleated erythroid
3.2. DNA damage and AHSP level Varying degrees of DNA damage in developing RBCs of these subjects have already been demonstrated [16]. The present study shows a weak negative correlation, with an r = −0.3109, between DNA damage and the fold change in AHSP levels (Fig. 3b). Low AHSP, as compared to non-thalassemic controls, in 64% of patients could be attributed to DNA damage in developing RBCs, thus inhibiting erythropoiesis, and allowing fewer RBCs to enter the stage of peak AHSP expression (Fig. 3a). 3.3. HbF and AHSP level Lim et al. [7] suggested that AHSP is a secondary compensatory mechanism, in addition to the HbF response, to the α/β globin chain imbalance in HbE/beta thalassemia patients. HPLC analysis in this group of patients revealed elevated HbF, ranging from 5–47%, in 48% of the patients (Fig. 4). A weak positive correlation with r = 0.3709 was seen between HbF and AHSP. Earlier reports have revealed that human AHSP expression can be affected by the iron status, via an iron sensor system, and that iron insufficiency upregulates, and its abundance downregulates, the gene [3]. Since excessive unpaired alpha chains release heme and iron which initiate self amplifying redox reactions [17], high AHSP in patients with raised HbF may be attributed to the partial compensation of insufficient beta chains by gamma globin chains, resulting in a relatively small free alpha globin pool and hence free iron, possibly adequate to prolong the expression of AHSP.
Fig. 4. Chromatogram showing 47.1% HbF in a patient.
C.S. Sagar et al. / Blood Cells, Molecules and Diseases 55 (2015) 104–107
precursors to persist in the circulation. It is speculated, that maintenance of low free iron pool could minimize DNA damage, prolong the survival of AHSP producing erythroid precursors, and prevent the downregulation of AHSP, thus, preventing the accumulation of unpaired alpha chains in β-thal patients with HbF in the normal range. Acknowledgment The authors are indebted to all the patients' guardians for their cooperation in allowing the use of blood samples of their ward. The authors thank the University Grants Commission (F.No. 36-122/2008 (SR)), New Delhi, India and the Indian Council of Medical Research (ICMR) (2010-02970) for providing funds for the work. Rakesh Kumar received a Senior research fellowship from the ICMR. Thanks are due to all the staff of the blood bank at Jaya Arogya Hospital for regularly providing blood samples of the patients. References [1] L. De Franceschi, M. Bertoldi, A. Matte, S.S. Franco, A. Pantaleo, E. Ferru, F. Turrini, Oxidative stress and -thalassemic erythroid cells behind the molecular defect, Oxidative Med. Cell. Longev. 2013 (2013) (10 pages Article ID 985210). [2] M.I. Lai, J. Jiang, N.A. Silver, et al., AHSP is a quantitative trait gene that modifies the phenotype of beta-thalassaemia, Br. J. Haematol. 133 (2006) 675–682. [3] C.O. Dos Santos, L.C. Dore, E. Valentine, et al., An iron responsive element-like stem-loop regulates α-hemoglobin-stabilizing protein Mrna, J. Biol. Chem. 283 (40) (2008) 26956–26964. [4] V. Viprakasit, V.S. Tanphaichitr, W. Chinchang, et al., Evaluation of alpha hemoglobin stabilizing protein (AHSP) as a genetic modifier in patients with beta thalassemia, Blood 103 (2004) 3296–3299.
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