Thalassemic Mice Research Articles

Improvement of Mouse β-Thalassemia by Recombinant Human Erythropoietin

Abstract Homozygous beta thalassemic mice received 50 U (1,660 U/kg) of recombinant human erythropoietin (rhEpo) 5 days a week for 2 weeks. Hemoglobin increased from 9.2 +/- 0.6 g/dL to 10.5 +/- 0.4 g/dL (P = .002) and hematocrit increased from 29.2% +/- 0.9% to 34.1% +/- 1.9% (P = .0014). The beta minor/alpha globin chain synthesis ratio increased slightly but significantly between day -4 (0.75 +/- 0.07) and day 4 (0.81 +/- 0.04) (P = .01) and reached a minimum ratio (0.67 +/- 0.03) on day 15 (P = .001), being parallel to reticulocyte counts and to the incorporated trichloracetic acid (TCA)-insoluble radioactivity, therefore parallel to the erythropoietic output in thalassemic mice, as in normal mice. Erythrocyte defects were improved in beta thalassemic mice treated by rhEpo: membrane-associated alpha globin was significantly decreased (P less than .01), thiol group reactivity of ankyrin was significantly improved (P less than .05), spectrin alterations were reduced, and deformability of mouse thalassemic red blood cells was normalized. These results provide experimental criteria for modulating globin chain imbalance necessary for the therapy of human beta thalassemia intermedia, and suggest that rhEpo might be of interest to improve the red blood cell mass and reduce erythrocyte alterations in this disease.

Improvement of mouse beta-thalassemia by recombinant human erythropoietin

Homozygous beta thalassemic mice received 50 U (1,660 U/kg) of recombinant human erythropoietin (rhEpo) 5 days a week for 2 weeks. Hemoglobin increased from 9.2 +/- 0.6 g/dL to 10.5 +/- 0.4 g/dL (P = .002) and hematocrit increased from 29.2% +/- 0.9% to 34.1% +/- 1.9% (P = .0014). The beta minor/alpha globin chain synthesis ratio increased slightly but significantly between day -4 (0.75 +/- 0.07) and day 4 (0.81 +/- 0.04) (P = .01) and reached a minimum ratio (0.67 +/- 0.03) on day 15 (P = .001), being parallel to reticulocyte counts and to the incorporated trichloracetic acid (TCA)-insoluble radioactivity, therefore parallel to the erythropoietic output in thalassemic mice, as in normal mice. Erythrocyte defects were improved in beta thalassemic mice treated by rhEpo: membrane-associated alpha globin was significantly decreased (P less than .01), thiol group reactivity of ankyrin was significantly improved (P less than .05), spectrin alterations were reduced, and deformability of mouse thalassemic red blood cells was normalized. These results provide experimental criteria for modulating globin chain imbalance necessary for the therapy of human beta thalassemia intermedia, and suggest that rhEpo might be of interest to improve the red blood cell mass and reduce erythrocyte alterations in this disease.

The role of membrane skeletal-associated alpha-globin in the pathophysiology of beta-thalassemia

The beta-thalassemic mouse provides a useful model for testing hypotheses about the pathophysiology in human beta-thalassemia. The clinical picture of these mice and their red blood cell deformability characteristics are quite similar to those observed in human beta- thalassemia intermedia. The creation of transgenic mice that express human beta-globin (beta s) has provided an opportunity to study the effect of increasing the non-alpha-globin chain production on the thalassemic phenotype. A small increase in beta-globin production produces transgenic mice that are healthier, have almost normal hemoglobin values, and whose red blood cell deformability is increased. We quantified and characterized the membrane skeletal-associated globin in normal, transgenic thal/sickle, and thalassemic mice and showed that only alpha-globin was associated with the membrane skeleton in the pathologic red blood cells, and that the degree of rigidity as measured in the rheoscope correlated directly and closely with the amount of membrane skeletal-associated globin in these abnormal red blood cells.

The Role of Membrane Skeletal-Associated α-Globin in the Pathophysiology of β-Thalassemia

The beta-thalassemic mouse provides a useful model for testing hypotheses about the pathophysiology in human beta-thalassemia. The clinical picture of these mice and their red blood cell deformability characteristics are quite similar to those observed in human beta-thalassemia intermedia. The creation of transgenic mice that express human beta-globin (beta s) has provided an opportunity to study the effect of increasing the non-alpha-globin chain production on the thalassemic phenotype. A small increase in beta-globin production produces transgenic mice that are healthier, have almost normal hemoglobin values, and whose red blood cell deformability is increased. We quantified and characterized the membrane skeletal-associated globin in normal, transgenic thal/sickle, and thalassemic mice and showed that only alpha-globin was associated with the membrane skeleton in the pathologic red blood cells, and that the degree of rigidity as measured in the rheoscope correlated directly and closely with the amount of membrane skeletal-associated globin in these abnormal red blood cells.

Iron Homeostasis in β-Thalassemic Mice

Abstract To explore the pathogenesis of nontransfusional iron overload in iron- loading anemia, we examined features of external iron exchange, internal iron kinetics, and tissue iron burden in adult mice with inherited gene-deletion beta-thalassemia. Mice homozygous for beta- thalassemia display moderate anemia, reticulocytosis, and shortened red cell survival, whereas heterozygous carriers appear hematologically normal. Quantitative iron determination revealed that iron content and concentration in liver, spleen, and kidney, but not heart, were far higher (P less than .01) in 15-to 35-week old homozygous thalassemic mice than in age-matched normal and heterozygous controls; of these tissues, iron content increased with age only in kidneys (P = .01) of homozygous affected mice. Although plasma iron levels were only minimally elevated in homozygotes, plasma iron turnover was threefold greater (P less than .001) than that seen in heterozygote controls. Nevertheless hyperabsorption of enteric radioiron, discernible among homozygous thalassemic mice as late as 6 to 8 weeks after birth, was not observed in older mice, additionally, thalassemic and control mice at 18 to 34 weeks showed comparable iron excretion after intravenous radioiron. We conclude that adult mice with beta-thalassemia regain balanced external iron exchange, despite substantial tissue iron excess and accelerated internal iron transit.

Iron homeostasis in beta-thalassemic mice

To explore the pathogenesis of nontransfusional iron overload in iron-loading anemia, we examined features of external iron exchange, internal iron kinetics, and tissue iron burden in adult mice with inherited gene-deletion beta-thalassemia. Mice homozygous for beta-thalassemia display moderate anemia, reticulocytosis, and shortened red cell survival, whereas heterozygous carriers appear hematologically normal. Quantitative iron determination revealed that iron content and concentration in liver, spleen, and kidney, but not heart, were far higher (P less than .01) in 15-to 35-week old homozygous thalassemic mice than in age-matched normal and heterozygous controls; of these tissues, iron content increased with age only in kidneys (P = .01) of homozygous affected mice. Although plasma iron levels were only minimally elevated in homozygotes, plasma iron turnover was threefold greater (P less than .001) than that seen in heterozygote controls. Nevertheless hyperabsorption of enteric radioiron, discernible among homozygous thalassemic mice as late as 6 to 8 weeks after birth, was not observed in older mice, additionally, thalassemic and control mice at 18 to 34 weeks showed comparable iron excretion after intravenous radioiron. We conclude that adult mice with beta-thalassemia regain balanced external iron exchange, despite substantial tissue iron excess and accelerated internal iron transit.

Compensatory increase in levels of beta minor globin in murine beta-thalassemia is under translational control.

A 3.7-kilobase pair deletion including the entire beta major globin gene results in beta-thalassemia in a murine model of the disease (Skow, L. C., Burkhart, B. A., Johnson, F. M., Popp, R. A., Popp, D. M., Goldberg, S. Z., Anderson, W. F., Barnett, L. B., and Lewis, S. E. (1983) Cell 34, 1043-1052). There is a compensatory increase in synthesis of beta minor globin, resulting in a beta minor/alpha globin ratio of 0.75 in the homozygous thalassemic mouse, as compared to 0.2 in the normal homozygous diffuse mouse. The results presented here demonstrate that the increase in beta minor globin synthesis occurs at translation rather than at transcription. RNase T1 analysis of reticulocyte mRNA reveals that the beta/alpha ratio of globin mRNA is 0.3, significantly lower than the globin synthetic ratio of 0.7. However, the beta/alpha ratio of mRNA on polysomes is higher than unassociated mRNA, demonstrating that beta minor mRNA is preferentially translated. Elevated synthesis of beta minor globin is maintained during in vitro translation in thalassemic reticulocyte lysate. In this system, partial inhibition of translational elongation by cycloheximide decreases the beta minor/alpha globin synthetic ratio, whereas partial inhibition of initiation by hemin deficiency increases the beta minor/alpha synthetic ratio. This suggests that beta minor mRNA competes with alpha-mRNA for a limiting mRNA binding factor at initiation of translation.

Independent expression of the two mouse adult ?-globin genes

Dot blot hybridization was used to determine the relative amounts of the beta-major and beta-minor globin RNAs present in reticulocytes of mice at 14.5 and 17.5 days of gestation, newborns, and adults of the Hbab/Hbab;Hbbs2/Hbbs2 globin genotype. RNAs isolated from embryonic yolk sac, fetal liver, and adult reticulocytes were hybridized with the following labeled DNA probes: alpha-1, beta-minor specific, and beta-major specific. The level of beta-sminor RNA in reticulocytes at 14.5 and 17.5 days of gestation is nearly the same as in induced reticulocytes of adult mice. In contrast, the level of beta-s2major RNA in reticulocytes at 14.5 days of gestation is 0.23 X and at 17.5 days of gestation is 0.66 X the amount found in reticulocytes of adult mice. These results correlate well with earlier observations that the beta-sminor globin gene approaches its normal adult level of expression by 14.5 days of gestation, whereas the beta-s2major globin gene expression increases between 14.5 days of gestation and 6 days postnatally. They indicate that the differential expression of beta-sminor and beta-s2major globins during development is regulated at the level of transcription. Expression of the beta-minor globin gene in reticulocytes of adult normal mice is not maximal, however, because the levels of the beta-minor globin and its RNA are increased further in reticulocytes of thalassemic mice.

Spontaneous iron overload in alpha-thalassemic mice

Because clinical disorders of spontaneous iron overload have no experimental counterpart, we studied iron distribution (atomic absorption analysis) and intestinal absorption (59Fe) in mice with hereditary alpha-thalassemia. Mice heterozygous for a radiation-induced alpha-Hb gene deletion exhibit a mild hemolytic anemia, like the human condition, with microcytosis, reticulocytosis, splenomegaly, and chemical evidence of defective alpha-chain synthesis. Quantitative iron determination showed that total iron content in spleen, liver, and kidney, but not heart or lung, of adult alpha-thalassemic mice was greater (P less than .05) than that in unaffected littermates. Iron concentration was also increased in liver (P less than .001), spleen (P = .025), kidney (P = .058), and heart (P = .010); in general, the greater the iron concentration in liver, the greater that in spleen (r = .39, P = .009), kidney (r = .70, P less than .001), and heart (r = .46, P less than .001). In mice examined 8 months postoperatively, splenectomy, as compared to sham operation, significantly raised iron content in extrasplenic tissues, but did not affect total body iron. At 10-11 weeks of age, but no longer at 12-14 weeks, thalassemic mice showed higher rates of iron absorption than age-matched controls. Thus, alpha-thalassemic mice display an early occurring iron absorption defect, leading to a modest, sustained, nonprogressive iron overload, and thereby represent a valuable model for exploring disorders of iron homeostasis.

Spontaneous Iron Overload in α-Thalassemic Mice

Abstract Because clinical disorders of spontaneous iron overload have no experimental counterpart, we studied iron distribution (atomic absorption analysis) and intestinal absorption (59Fe) in mice with hereditary alpha-thalassemia. Mice heterozygous for a radiation-induced alpha-Hb gene deletion exhibit a mild hemolytic anemia, like the human condition, with microcytosis, reticulocytosis, splenomegaly, and chemical evidence of defective alpha-chain synthesis. Quantitative iron determination showed that total iron content in spleen, liver, and kidney, but not heart or lung, of adult alpha-thalassemic mice was greater (P less than .05) than that in unaffected littermates. Iron concentration was also increased in liver (P less than .001), spleen (P = .025), kidney (P = .058), and heart (P = .010); in general, the greater the iron concentration in liver, the greater that in spleen (r = .39, P = .009), kidney (r = .70, P less than .001), and heart (r = .46, P less than .001). In mice examined 8 months postoperatively, splenectomy, as compared to sham operation, significantly raised iron content in extrasplenic tissues, but did not affect total body iron. At 10–11 weeks of age, but no longer at 12–14 weeks, thalassemic mice showed higher rates of iron absorption than age-matched controls. Thus, alpha-thalassemic mice display an early occurring iron absorption defect, leading to a modest, sustained, nonprogressive iron overload, and thereby represent a valuable model for exploring disorders of iron homeostasis.

Three mouse models of human thalassemia.

Three types of mice with globin gene mutations, called 352HB, 27HB, and Hbath-J, appear to be true animal models of human thalassemia. Expression of the alpha-globin genes in three stocks of mice, each one heterozygous for one of the alpha-globin mutations, was examined at the polypeptide, RNA, and DNA levels. alpha-Globin polypeptide chains, relative to beta-globin chains in heterozygous thalassemic mice, are present at approximately 80% of normal. The ratios of alpha-globin to beta-globin RNA sequences are also 75-80% of normal, exactly reflecting the alpha-globin to beta-globin chain ratios. In the case of mutant 352HB, at least one alpha-globin gene is deleted. Thalassemic mouse erythroid cells appear to compensate partially for the loss of half of their alpha-globin genes.

Effects of Alpha Thalassemia on Mouse Development

Matings were made between (1) normal mice, (2) heterozygous alpha thalassemic females and normal males, and (3) heterozygous alpha thalassemic mice to produce litters of (1) normal mice, (2) normal and heterozygous alpha thalassemic mice, and (3) normal, heterozygous, and homozygous alpha thalassemic mice, respectively, in order to determine what effects the heterozygous and homozygous forms of alpha thalassemia might have on mouse development. Genetic tests showed that the viable alpha thalassemic progeny of matings between heterozygous alpha thalassemic mice were heterozygotes which suggests that the homozygotes die. Examination of preimplantation embryos from the uterus of normal and heterozygous alpha thalassemic females at 86 h after mating with normal males showed that these embryos had reached the blastocyst stage of development. When heterozygous alpha thalassemic females were mated with heterozygous alpha thalassemic males, however, only three-fourths of the embryos had reached the early blastocyst stage of development at 86 h, while one-fourth of them (presumed homozygous alpha thalassemic embryos) were still at the morula stage of development. At 11.5 though 15.5 days of gestation, about one-fourth of the implantation sites did not contain live fetuses, which suggests that death had occurred earlier. Histologic examination of embryos at 5.5 though 8.5 days of gestation showed that the homozygous alpha thalassemic embryos implanted and developed to the late blastocyst stage, at which time they became necrotic. Microscopic examination of blood films from fetuses at 11.5 through 15.5 days of development showed that the yolk sac-derived erythrocytes of alpha thalassemic heterozygotes were morphologically normal, but the fetal liver-derived erythrocytes contained eosinophilic inclusions. Electrophoresis of blood from the fetuses showed that alpha thalassemic heterozygotes contained a unique embryonic hemoglobin (y 4 ), which is homologous to Bart's γ 4 in humans.