Blood Cells, Molecules, and Diseases 39 (2007) 184 – 188 www.elsevier.com/locate/ybcmd
Co-inheritance of α- and β-thalassaemia in mice ameliorates thalassaemic phenotype Hsiao Phin Joanna Voon ⁎, Hady Wardan, Jim Vadolas Cell and Gene Therapy Research Group, The Murdoch Childrens Research Institute, The University of Melbourne, Royal Children’s Hospital, Flemington Road, Parkville 3052, Melbourne, Australia Submitted 12 December 2006 Available online 9 May 2007 (Communicated by Sir D. Weatherall, F.R.S., 24 January 2007)
Abstract β-Thalassaemia is an inherited disease caused by defective synthesis of the β-globin chain of haemoglobin, leading to an imbalance in globin chains. Excess α-globin chains precipitate in erythroid progenitor cells resulting in cell death, ineffective erythropoiesis and severe anaemia. Decreased α-globin synthesis leads to milder symptoms, exemplified by individuals who co-inherit α-thalassaemia and β-thalassaemia. In this study, we set out to investigate whether co-inheritance of α- and β-thalassaemia in mice leads to reduced anaemia. Heterozygous murine β-globin knockout (KO) mice (β+/−) which display severe anaemia were mated with heterozygous α-globin KO mice (α++/−−). The resulting progeny were genotyped and classed as wild-type WT (α++/++;β+/+), heterozygous α-KO (α++/−−;β+/+), heterozygous β-KO (α++/++;β+/−) or double heterozygous (DH) α-KO/β-KO (α++/−−;β+/−). Mice were bled and full blood examinations (FBE) performed. FBE results for heterozygous β-KO mice (β+/−) showed marked reductions in haemoglobin and haematocrit levels and significant increases in red cell distribution widths and reticulocyte counts compared to WT mice. In contrast, FBE results for DH α-KO/β-KO mice showed near normal red blood cell indices. These results indicate that reduction of α-globin expression leads to correction of the globin chain imbalance in β-thalassaemic mice and therefore an improved phenotype. The analysis of DH α-KO/β-KO mice leads to the following conclusions: (1) co-inheritance of α- and β-thalassaemia in mice improves the thalassaemic phenotype, identical to the situation in humans; (2) the heterozygous murine β-globin KO mouse model is a suitable in vivo model to test for therapeutic knockdown of α-globin. © 2007 Elsevier Inc. All rights reserved. Keywords: α- and β-thalassaemia; Co-inheritance; Blood; Mouse model
Introduction Thalassaemia is an inherited haemoglobinopathy characterised by defective synthesis of either the α-chain or the β-chain of haemoglobin. In β-thalassaemia, reduced β-globin synthesis results in accumulation of free α-globin chains. Deprived of normal β-globin binding partners, excess α-globin aggregates to form toxic, insoluble precipitates in red blood cells and their precursors. The resulting inclusion bodies cause mechanical damage to membrane structures and trigger premature apoptosis in erythroid progenitor cells, leading to ineffective erythropoiesis [1–3]. The subsequent anaemia stimulates further erythropoiesis, resulting in proliferation and expansion of the bone marrow [4,5]. ⁎ Corresponding author. Fax: 613 8341 6212. E-mail address:
[email protected] (H.P.J. Voon). 1079-9796/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2007.01.006
In cases of severe thalassaemia, erythropoietic stimulation is sufficiently potent to drive extramedullary haematopoiesis in the spleen [3]. In addition, there is also evidence that the spleen harbours a significant pool of abnormal red blood cells with imbalanced globin chain ratios [6], all of which are factors that contribute to gross enlargement of the spleen. Individuals who co-inherit α- and β-thalassaemia have impaired synthesis of both chains with neither produced in excess. Without excess α-globin, there is reduced membrane disruption and therefore reduced cellular damage in erythroid progenitor cells and these individuals display milder clinical symptoms [1,3,7–9]. Therefore, a possible strategy in the treatment of β-thalassaemia could involve targeted reduction of α-chains to mimic co-inheritance of α- and β-thalassaemia. To pursue this therapeutic goal, the strategy of reducing αglobin needs to be proven effective in an animal model.
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Various thalassaemic mouse models are available for use [10– 12], with the simplest being the heterozygous β-globin knockout (β-KO) mouse model [13]. These heterozygous βKO mice display distinct haematological abnormalities consistent with β-thalassaemia [13–15]. A heterozygous αglobin knockout (α-KO) counterpart also exists with a much milder thalassaemic phenotype [14,16,17]. In this study, thalassaemic heterozygous β-KO mice were crossed to heterozygous α-KO mice to produce double heterozygous (DH) α-KO/β-KO mice, analogous to co-inherited α-/βthalassaemia in humans. The resulting double heterozygous progeny display a markedly improved phenotype compared to thalassaemic β-KO littermates. Materials and methods Genotyping Established heterozygous β-KO mouse models [13] and heterozygous α-KO mouse models [16] were obtained from the Jackson Labs and crossed to produce double heterozygous α-KO/ β-KO offspring. Resulting progeny were genotyped using DNA obtained from tail biopsies using previously described multiplex PCR reactions to determine α-globin [17] and β-globin [15] genotypes. In brief, α-globin genotypes were determined using a multiplex PCR with primers specific for the neomycin KO cassette and primers specific for α-globin with the following PCR conditions: 94 °C (5 min), followed by 30 cycles of 94 °C (30 s), 60 °C (30 s), 72 °C (1 min) and a final elongation phase of 10 min at 72 °C. β-Globin genotypes were determined using a separate multiplex PCR with primers specific for the HPRT KO cassette and primers specific for β-globin with the following PCR conditions: 94 °C (5 min), followed by 33 cycles of 94 °C (30 s), 55 °C (30 s), 72 °C (2 min) and a final elongation phase of 10 min at 72 °C. Mice were classed as wild-type (WT) (α ++/++ ;β +/+ ), heterozygous α-KO (α ++/−− ;β +/+ ), heterozygous β-KO (α ++/++ ;β +/− ) or double heterozygous α-KO/β-KO (α ++/−− ;β +/− ).
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Full Blood Examination Blood samples were collected from mice with different genotypic backgrounds by retro orbital eye bleeding into EDTA tubes. Full blood examination was performed using an automated Roche Cobas Helios haematological analyser at the Walter and Eliza Hall Institute, Melbourne. Flow Cytometry Mice were culled and spleens were removed and weighed. Cells were extracted from bone marrow and spleen by flushing with phosphate buffered saline (PBS) containing 2% fetal calf serum. Red cells were lysed with Tris ammonium chloride at 37 °C for 5 min then washed with PBS prior to antibody labeling. A total of 2 × 105 cells were labeled on ice for 20 min with 0.2 μg of FITC-conjugated anti-mouse CD71 (transferrin receptor) and 0.2 μg of PE-conjugated anti-mouse TER119 (erythroid marker) (BD Pharmingen, California). Samples were washed three times with PBS containing 2% fetal calf serum then analysed on an LSR II flow cytometer (Becton Dickinson) using FACS Diva software (Becton Dickinson).
Results Heterozygous β-KO mice displayed classic thalassaemic traits clearly detectable by full blood examinations (Fig. 1). These included reduced haemoglobin (Hb) and haematocrit (HCT) levels (40% and 33% reduction, respectively) combined with significant increases in reticulocyte numbers (8.3-fold increase) and increased red cell distribution widths (RDW) (2.6-fold increase) compared to WT mice. In contrast to the heterozygous β-KO mice (α++/++;β+/−), double heterozygous αKO/β-KO mice (α++/−−;β+/−) displayed a substantially improved phenotype, reflecting the situation of co-inherited α- and βthalassaemia in humans. The DH α-KO/β-KO mice displayed
Fig. 1. Full blood examinations of WT (α++/++;β+/+), α-KO (α++/−−;β+/+), β-KO (α++/++;β+/−) and DH α-KO/β-KO (α++/−−;β+/−) mice. Heterozygous β-KO mice display significantly decreased levels of Hb and HCT as well as increased RDWs and reticulocyte counts compared to WT mice. All parameters are restored to near WT levels in the DH α-KO/β-KO mice. All values are expressed as mean average ± SD.
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corrected anaemia with Hb and HCT conspicuously elevated from β-KO levels to values approaching normal. Clear improvements in RDW and reticulocyte numbers were also observed with both parameters restored to within WT range. Increased reticulocyte numbers can be attributed to enhanced erythropoietic stimulation due to anaemia [18]. Enhanced erythropoiesis in the β-KO mice was confirmed by labeling cells extracted from bone marrow with an FITC-conjugated anti-CD71 and a PE-conjugated anti-TER119 (Fig. 2A). As haematopoietic progenitor cells generally express mid to high levels of both CD71 and TER119, it was possible to quantify the progenitor population by labeling for these markers. In WT mice, ∼ 21% of extracted bone marrow cells were double positive for CD71 and TER119 and could be classed as early to mid haematopoietic progenitor cells. In thalassaemic heterozygous β-KO mice, the progenitor cell population roughly doubled to ∼ 53% confirming a state of enhanced erythropoiesis consistent with increased reticulocyte numbers seen in FBE
profiles. Haematopoietic progenitor cell numbers were close to normal in the DH α-KO/β-KO mice (∼ 28%), reflecting corrected erythropoiesis in these mice with balanced globin synthesis. Enhanced erythropoiesis in anaemic heterozygous β-KO mice also results in the spleen being utilised as a site of secondary erythropoiesis [3,19]. Antibody labeling of splenocytes with anti-CD71 and anti-TER119 confirmed secondary erythropoiesis in heterozygous β-KO mice. A large population of double positive haematopoietic cells was detected in spleens of anaemic β-KO mice (∼ 40% of total cell population), while only minimal numbers were present in WT spleens (∼ 3%). The spleens of DH α-KO/β-KO mice resembled WT spleens and contained only a limited population of haematopoietic cells (∼ 3.5%) (Fig. 2B). In addition, spleens of thalassaemic heterozygous β-KO mice are often grossly enlarged due to a combination of increased erythropoiesis and a tendency for the spleen to sequester and
Fig. 2. Representative flow cytometry profiles of cells extracted from (A) bone marrow and (B) spleens of (i) WT, (ii) α-KO, (iii) β-KO and (iv) DH α-KO/β-KO mice. (v) Average % haematopoietic cells in bone marrow and spleen. All values are expressed as mean average ± SD. Cells were double stained with FITC-conjugated CD71 (transferrin receptor) antibody and PE-conjugated TER119 antibody which binds a murine erythroid marker. Haematopoietic progenitor cells are defined as CD71+ and TER119+. β-KO mice have abnormally large populations of erythroid progenitor cells in both bone marrow and spleen whereas DH α-KO/β-KO mice with reduced anaemia have reduced erythropoietic stimulus and therefore display a normal haematopoietic profile.
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Fig. 3. Analysis of spleen weights. (A) Average spleen weights (mean ± SD) and (B) representative spleen sizes of WT, α-KO, β-KO and DH α-KO/β-KO mice.
clear damaged red blood cells [4,5]. The DH α-KO/β-KO mice, with reduced globin imbalance, have spleens indistinguishable from wild-type mice (Fig. 3). Discussion In human patients with β-thalassaemia, much of the pathology can be attributed to an imbalance in globin chain synthesis and subsequent accumulation of excess α-globin. The excess αglobin chains aggregate to form toxic insoluble precipitates known as inclusion bodies in red blood cells and their precursors, resulting in membrane damage and premature cell death. Reduction of α-globin synthesis, for example through coinheritance of α-thalassaemia, is a well-documented background modifier in ameliorating disease severity in β-thalassaemia [1–3]. In this study, we show that the thalassaemic phenotypes of heterozygous β-KO mice are similarly corrected when α-globin synthesis is reduced. Double heterozygous α-KO/β-KO mice with half the gene dosage of α-globin complementing the reduced expression from the β-globin locus, display a haematological profile that more closely resembles WT mice than heterozygous β-KO mice, an improvement which can only be attributed to reduced globin imbalance in the DH α-KO/β-KO mice. In the heterozygous β-KO mice, as in human patients, the problems caused by globin chain imbalance begin at the cellular level when excess α-globin chains associate with membrane structures, causing disruptions that result in wide variations of RDWs, an indirect indicator of membrane damage. In addition, the formation of inclusion bodies in haematopoietic progenitor cells and subsequent apoptosis results in ineffective erythropoiesis and a markedly anaemic phenotype, as evidenced by the significant reductions in Hb and HCT levels. This in turn increases erythropoietic stimulation resulting in an abnormally large number of erythroid progenitor cells in the bone marrow and
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spleen and generating an expanded number of circulating reticulocytes. Furthermore, red cells that survive to reach the peripheral circulation in spite of α-globin toxicity nevertheless have a shortened life span and either undergo apoptosis resulting in haemolysis, or else are removed from circulation by the spleen and either sequestered or destroyed [2]. The increased clearance of abnormal blood cells, combined with the extramedullary erythropoiesis that occurs in the spleen, leads to a striking enlargement of this organ in the heterozygous β-KO mice. In contrast, heterozygous β-KO mice that co-inherit a defect in α-globin synthesis have more balanced globin chain production. Double heterozygous α-KO/β-KO mice carrying only half the normal number of α-globin genes display a normal range of RDWs indicating they do not suffer significant membrane abnormalities. Consequently, erythroid progenitor cells survive to maturation and a much less anaemic phenotype is observed with Hb and HCT almost completely restored to WT levels. Erythropoietic stimulus is therefore removed resulting in a normal population of haematopoietic cells in bone marrow and spleen, indistinguishable from those found in WT mice. Finally, the reduced drive for extramedullary erythropoiesis combined with reduced clearance of damaged red blood cells results in a marked reduction in spleen size. This indicates that both red cell damage and stimulus for enhanced erythropoiesis has been removed and one of the most prominent clinical features of thalassaemia, splenomegaly, has been fully corrected in these mice. This study demonstrates that decreased α-globin synthesis in heterozygous β-KO mice result in significant phenotypic improvements, identical to the situation in human patients. Our results also illustrate that the heterozygous β-KO mouse is a suitable in vivo model to use in testing strategies for therapeutic downregulation of α-globin. Targeted reductions of α-globin, using short-interfering RNA or antisense oligonucleotides, equivalent to those in heterozygous α-globin KO mice, should produce phenotypic improvements in heterozygous β-KO mice. The definitive results obtained from this study form a crucial control for future experiments aiming to develop a therapy for β-thalassaemia involving the reduction of α-globin. Acknowledgments This work was supported by grants from the National Health and Medical Research Council (NHMRC) of Australia and the Thalassaemia Society of Victoria. References [1] E. Kanavakis, J. Traeger-Synodinos, S. Lafioniatis, C. Lazaropoulou, T. Liakopoulou, G. Paleologos, A. Metaxotou-Mavrommati, A. Stamoulakatou, I. Papassotiriou, A rare example that coinheritance of a severe form of beta-thalassemia and alpha-thalassemia interact in a “synergistic” manner to balance the phenotype of classic thalassemic syndromes, Blood Cells Mol. Diseases 32 (2004) 319–324. [2] S.L. Thein, Pathophysiology of {beta} thalassemia—a guide to molecular therapies, Hematology (Am. Soc. Hematol. Educ. Program) (2005) 31–37. [3] D. Weatherall, J. Clegg, The Thalassaemia Syndromes, Blackwell Science, London, 2001. [4] N.F. Olivieri, The beta-thalassemias, N. Engl. J. Med. 341 (1999) 99–109.
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