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The erythopoietin (Epo) is a model of proteins expressed upon hypoxia, regulated at the transcriptional and post transcriptional levels. The Hypoxia Induced Factor (HIF-I) activates the transcription of genes which exhibit the Hypoxia Regulatory Element (HRE). In addition the mRNA is stabilized upon hypoxia, increasing the hormone synthesis. The Epo gene is a convenient reporter gene to develop gene delivery systems and the in vivo regulation of the transgene. The ß thalassemia and acquired chronic anemias may benefit from the Epo gene transfer and expression, if it becomes safe, tunable and inexpensive. [ABSTRACT FROM AUTHOR]

Central to the pathophysiology of sickle cell disease are the vaso-occlusive events that lead to tissue damages and life-threatening complications. Lungs are particularly vulnerable to vaso-occlusion because of their specific vasculature. We developed a mouse model of hypoxia/reoxygenation lung injury closely mimicking the lung pathology of patients with sickle cell disease. This model involves the exposure of transgenic sickle cell (SAD) mice to hypoxia (8% oxygen) for 4, 10, and 46 hours followed by 2 hours of reoxygenation. Gene expression profiling of SAD lung tissue pointed to the specific induction of genes involved in the response to ischemic stress and microcirculation remodeling: Hspcb, Hsp86-1, Nfe2l2, Ace, and Fgf7. Hypoxia/reoxygenation also induced a marked increase in bronchoalveolar (BAL) total leukocyte and neutrophil counts, BAL total protein content, and BAL tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6), IL-1alpha, and macrophage inflammatory protein 2 (MIP-2) levels, all indicators of enhanced inflammatory response as compared with control mice. Nitric oxide (NO) was administered to SAD mice. NO (40 ppm) inhalation protected SAD mice from the histopathologic lesions of ischemic/reperfusion lung injury with corresponding normalization and/or modulation of tissue gene expression profiles. Inhaled NO (1) significantly reduced the increase in BAL total protein content, BAL total leukocyte, and neutrophil counts; (2) modulated BAL cytokine network; and (3) did not affect hemoglobin and methemoglobin levels. The present study provides evidences for the beneficial effects of inhaled NO in pulmonary injury induced by hypoxia/reoxygenation in a mouse model of sickle cell disease (SCD) and opens new avenues in drug design based on tissue gene expression profiling.

High doses of recombinant human erythropoietin (rhEpo) are required for the treatment of chronic anemia. Thus, it is clear that therapy for chronic anemia would greatly benefit from an erythropoietin derivative with increased erythropoietic activity rather than the native endogenous hormone. In this report, the activity of a human Epo-Epo dimer protein, obtained by recombinant technology, is described and compared with its Epo monomer counterpart produced under identical conditions. Although monomer Epo and dimer Epo-Epo had similar pharmacokinetics in normal mice, the increase in hematocrit value was greater with the dimer than with the monomer. Moreover, in clonogenic assays using CD34(+) human hematopoietic cells, the human dimer induced a 3- to 4-fold-greater proliferation of erythroid cells than the monomer. Controlled secretion of dimeric erythropoietin was achieved in beta-thalassemic mice by in vivo intramuscular electrotransfer of a mouse Epo-Epo plasmid containing the tetO element and of a plasmid encoding the tetracycline controlled transactivator tTA. Administration of tetracycline completely inhibited the expression of the mEpo dimer. On tetracycline withdrawal, expression of the Epo-Epo dimer resumed, thereby resulting in a large and sustained hematocrit increase in beta-thalassemic mice. No immunologic response against the dimer was apparent in mice because the duration of the hematocrit increase was similar to that observed with the monomeric form of mouse erythropoietin. (Blood. 2001;97:3776-3782)

Objective: A new intramuscular DNA electrotransfer method for erythropoietin (EPO) expression was evaluated in the natural mouse model of human beta-thalassemia (Hbb-thal1) in terms of its ability to reverse the anemia and improve the thalassemic features of erythrocytes., Materials and Methods: Intramuscular injection of small amounts of a plasmid encoding mouse EPO, immediately followed by controlled electric pulses, was used., Results: This procedure induced very high hematocrit levels in beta-thalassemic mice compared to nonelectrotransferred mice. The hematocrit increase was dose dependent, still increased 4 months after injection of plasmid DNA, and associated with a high transgenic EPO blood level in all mice (up to 2500 mU/mL of plasma). EPO gene electrotransfer not only led to a long-lasting and dose-dependent increase in the hematocrit but also to a 100% increase in the lifespan of erythrocytes of thalassemic mice. This was related to a nearly complete reestablishment of alpha/beta globin chain balance, as demonstrated by a marked decrease in unpaired alpha globin chain. Eight months after the first electrotransfer of pCMV-mEPO plasmid, reinjection of the same construct raised the hematocrit to a level close to that observed following the first electrotransfer., Conclusion: This is the first description of the use of plasmid DNA to achieve long-term improvement in a mouse model of a human genetic disorder.

Erythropoiesis is positively regulated by stem cell factor, interleukin 3, and erythropoietin, which synergize to allow the production of hemoglobinized red blood cells from erythroid progenitors. In contrast, interferon gamma, tumor necrosis factor alpha, and transforming growth factor B(1), (TGF-beta(1)) are powerful inhibitors of erythropoiesis. Interferon gamma and alpha act principally by inducing apoptosis. The aim of this study was to elucidate the mechanisms by which TGF-beta(1) inhibits erythropoiesis. We used an in vitro serum-free system of human red blood cell production. From a virtually pure population of CD36(+) erythroid progenitors, stem cell factor, interleukin 3, and erythropoietin allowed massive proliferation (x300) and promoted terminal red blood cell differentiation. We show here that TGF-beta(1) (2 ng/mL) inhibited the growth of CD36(+) cells by 15-fold. TGF-beta(1) markedly accelerated and increased erythroid differentiation as assessed by hemoglobin and glycophorin expression. Furthermore, May-Grünwald-Giemsa staining and ultrastructural analysis revealed that TGF-beta(1) induced full differentiation toward normal enucleated red cells even in the absence of macrophages. This acceleration of erythroid differentiation did not modify the pattern of hemoglobin chains expression from adult or fetal erythroid progenitors. Analysis of apoptosis, cell cycle and Ki-67 expression showed that TGF-beta(1) inhibited cell proliferation by decreasing the cycle of immature erythroid cells and accelerating maturation toward orthochromatic normoblasts that are not in cycle. We showed that TGF-beta(1) is a paradoxical inhibitor of erythropoiesis that acts by blocking proliferation and accelerating differentiation of erythroid progenitors.

We have examined the effect of hydroxyurea (HU), clotrimazole (CLT), magnesium oxide (Mg), and combined CLT+Mg therapies on the erythrocyte characteristics and their response to chronic hypoxia in a transgenic sickle mouse (SAD) model. SAD mice were treated for 21 days with 1 of the following regimens (administered by gavage): control (n = 6), HU (200 mg/d; n = 6), CLT (80 mg/kg/d, n = 5), Mg (1,000 mg/kg/d, n = 5), and CLT+Mg (80 and 1,000 mg/kg/d, respectively, n = 6). Nine normal mice were also treated as controls (n = 3), HU (n = 3), and CLT+Mg (n = 3). Treatment with HU induced a significant increase in mean corpuscular volume and cell K content and a decrease in density in SAD mice. Treatment with the CLT and Mg, either alone or in combination, also increased cell K and reduced density in SAD mice. After 21 days of treatment, the animals were exposed to hypoxia (48 hours at 8% O(2)) maintaining the same treatment. In the SAD mice, hypoxia induced significant cell dehydration. These hypoxia-induced changes were blunted in either HU- or Mg-treated SAD mice and were completely abolished by either CLT or CLT+Mg treatment, suggesting a major role for the Gardos channel in hypoxia-induced dehydration in vivo.

beta thalassemia (beta thal) in DBA/2J mice is a consequence of the spontaneous and complete deletion of the beta major globin gene. Homozygous beta thal mice have clinical and biological features similar to those observed in human beta thal intermedia. Erythrocytes in human beta thal are characterized by a relative cell dehydration and reduced K+ content. The role of this erythrocyte dehydration in the reduced erythrocyte survival, which typifies the disease, has not previously been evaluated. We examined for 1 month the effects on the anemia and the erythrocyte characteristics of beta thal mice of daily treatment with either clotrimazole (CLT), an inhibitor of red blood cell (RBC) dehydration via the Gardos channel, or human recombinant erythropoietin (r-HuEPO), or hydroxyurea (HU). The use of either r-HuEPO or HU induced a significant increase in hemoglobin (Hb), hematocrit (Hct), erythrocyte K+ and a decrease in percent reticulocytes, suggesting improved erythrocyte survival. CLT alone decreased only mean corpuscular hemoglobin concentration (MCHC) and cell density and increased cell K+. Thus, though the Gardos channel plays a major role in cell dehydration of murine beta thal erythrocyte survival. Combination therapy with r-HuEPO plus HU produced no incremental benefit beyond those of single drug therapy. However, addition of CLT to r-HuEPO, to HU, or to combined r-HuEPO plus HU led to statistically significant increase in Hb, Hct, and erythrocyte K+ compared with any of the regimens without CLT. These results suggest that CLT not only inhibits erythrocyte dehydration, but also potentiates the erythropoietic and cellular survival responses to r-HuEPO and HU.

Repeated injections of large doses of erythropoietin (Epo) have been shown to be of benefit in the treatment of murine and human beta-thalassemia. To determine whether Epo gene therapy could replace this treatment for long-term periods, lethally irradiated beta-thalassemic (Hbbd3th haplotype) and normal DBA/2J (Hbbd haplotype) mice were grafted with syngeneic bone marrow cells infected with a retroviral vector carrying the Epo cDNA. In normal mice, dysregulated Epo production induced elevated serum Epo levels (176 +/- 68 mU/mL), high hematocrit levels (73% +/- 8%), and elevated beta-minor globin chain synthesis. In contrast, in thalassemic mice, moderate increases in the hematocrit levels (from 33% +/- 1% to 43% +/- 9%), associated with limited increases in the initially elevated Epo levels (from 83 +/- 22 to 190 +/- 230 mU/mL), were recorded 2 months after transplantation. In mice in which the hematocrit increased most, from 33% +/- 1% before transplantation to 49% +/- 10%, the retroviral Epo gene expression induced a striking improvement of the beta-thalassemic syndrome. These mice exhibited normal or near-normal beta/alpha-globin chain synthesis ratios, induced by the activation of the beta-minor chain. This led to the elimination of the high amounts of unpaired alpha chains in erythrocytes and finally reduced the reticulocyte count despite the permanent Epo stimulation. These results show that efficient Epo gene expression corrects the erythrocyte phenotype of the mouse beta-thalassemic syndrome. However, the incidence of lethal polycythemia or of transient improvements indicates that the present strategy is only the first step toward such indirect gene therapy.

The present report provides evidence that hydroxyurea (HU) improves the beta thalassaemic phenotype in mice receiving 200 mg/kg/d for 30 d. The haematocrit rose from 29 +/- 3% at day 0 to 37 +/- 4% at day 30 (P < 0.05), despite myelosuppression and decreased reticulocyte counts. The beta minor/alpha ratio of globin chain synthesis increased from 0.78 at day 0 to 0.97 at day 30 (P < 0.001). Membrane defects improved: the proportion of bound alpha chains decreased, the proportion of spectrin and ankyrin increased and red cell deformability also increased.

While red cells from individuals with beta thalassemias are characterized by evidence of elevated in vivo oxidation, it has not been possible to directly examine the relationship between excess alpha-hemoglobin chains and the observed oxidant damage. To investigate the oxidative effects of unpaired alpha-hemoglobin chains, purified alpha-hemoglobin chains were entrapped within normal erythrocytes. These "model" beta-thalassemic cells generated significantly (P < 0.001) greater amounts of methemoglobin and intracellular hydrogen peroxide than did control cells. This resulted in significant time-dependent decreases in the protein concentrations and reduced thiol content of spectrin and ankyrin. These abnormalities correlated with the rate of alpha-hemoglobin chain autoxidation and appearance of membrane-bound globin. In addition, alpha-hemoglobin chain loading resulted in a direct decrease (38.5%) in catalase activity. In the absence of exogenous oxidants, membrane peroxidation and vitamin E levels were unaltered. However, when challenged with an external oxidant, lipid peroxidation and vitamin E oxidation were significantly (P < 0.001) enhanced in the alpha-hemoglobin chain-loaded cells. Membrane bound heme and iron were also significantly elevated (P < 0.001) in the alpha-hemoglobin chain-loaded cells and lipid peroxidation could be partially inhibited by entrapment of an iron chelator. In contrast, chemical inhibition of cellular catalase activity enhanced the detrimental effects of entrapped alpha-hemoglobin chains. In summary, entrapment of purified alpha-hemoglobin chains within normal erythrocytes significantly enhanced cellular oxidant stress and resulted in pathological changes characteristic of thalassemic cells in vivo. This model provides a means by which the pathophysiological effects of excess alpha-hemoglobin chains can be examined.

The alpha- and beta-thalassaemias are characterized by decreased erythrocyte deformability. To determine what effects excess alpha- and beta-haemoglobin (globin) chains have on cellular and membrane deformability, purified haem-containing alpha- and beta-chains were entrapped within normal erythrocytes. Entrapment of purified alpha-chains in normal erythrocytes resulted in a significant decrease in cellular and membrane deformability similar to that observed in beta-thalassaemia intermedia. The decreased deformability was correlated with alpha-chain membrane deposition, an alteration in membrane proteins and a decrease in membrane reactive thiol groups. These changes in membrane and cellular deformability were time dependent and closely correlated with membrane alpha-chain deposition. The membrane changes and the loss of membrane deformability appeared to account for the loss of cellular deformability in the alpha-chain loaded cells. While both beta-chain loaded and Hb H erythrocytes demonstrated a significant loss of cellular deformability, this loss was less pronounced than in the alpha-chain loaded and beta-thalassaemic cells and may arise from either the increased intracellular viscosity of the beta-chain loaded cells or to the smaller amount of membrane bound globin. In summary, these studies demonstrate that alteration of cellular and membrane deformability occurs very rapidly and as a direct consequence of the autoxidation and membrane binding of the unpaired globin chains.

In order to obtain a transgenic mouse model of sickle cell disease, we have synthesized a novel human beta-globin gene, beta SAD, designed to increase the polymerization of the transgenic human hemoglobin S (Hb S) in vivo. beta SAD (beta S-Antilles-D Punjab) includes the beta 6Val substitution of the beta S chain, as well as two other mutations, Antilles (beta 23Ile) and D Punjab (beta 121Gln) each of which promotes the polymerization of Hb S in human. The beta SAD gene and the human alpha 2-globin gene, each linked to the beta-globin locus control region (LCR) were co-introduced into the mouse germ line. In one of the five transgenic lines obtained, SAD-1, red blood cells contained 19% human Hb SAD (alpha 2 human 1 beta 2SAD) and mouse-human hybrids in addition to mouse hemoglobin. Adult SAD-1 transgenic mice were not anemic but had some abnormal features of erythrocytes and slightly enlarged spleens. Their erythrocytes displayed sickling upon deoxygenation in vitro. SAD-1 neonates were anemic and many did not survive. In order to generate adult mice with a more severe sickle cell syndrome, crosses between the SAD progeny and homozygous for beta-thalassemic mice were performed. Hemoglobin SAD was increased to 26% in beta-thal/SAD-1 mice which exhibited: (i) abnormal erythrocytes with regard to shape and density; (ii) an enlarged spleen and a high reticulocyte count indicating an increased erythropoiesis; (iii) mortality upon hypoxia; (iv) polymerization of hemolysate similar to that obtained in human homozygous sickle cell disease; and (v) anemia and mortality during development.

A viable autosomal recessive mutation (named fch, or ferrochelatase deficiency) causing jaundice and anemia in mice arose in a mutagenesis experiment using ethylnitrosourea. Homozygotes (fch/fch) display a hemolytic anemia, photosensitivity, cholestasis, and severe hepatic dysfunction. Protoporphyrin is found at high concentration in erythrocytes, serum, and liver. Ferrochelatase activity in various tissues is 2.7-6.3% of normal. Heterozygotes (+/fch) are not anemic and have normal liver function; they are not sensitive to light exposure; ferrochelatase activity is 45-65% of normal. Southern blot analysis using a ferrochelatase cDNA probe reveals no gross deletion of the ferrochelatase gene. This is the first spontaneous form of erythropoietic protoporphyria in the house mouse. Despite the presence in the mouse of clinical and biochemical features infrequent in the human, this mutation may represent a model for the human disease, especially in its severe form.

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 fate of alpha-hemoglobin chains and the cause of membrane protein defects in thalassemic erythrocytes have been studied in: (1) human beta-thalassemia syndromes, (2) mouse beta-thalassemia, and (3) normal human erythrocytes loaded with purified alpha-hemoglobin chains. The similarity and differences observed in these three systems underline the importance of insoluble alpha chains and the direct relationship between the amount of these chains and the membrane protein defects. Indeed, in addition to the alpha/non-alpha ratio of globin chain synthesis, the proteolysis and instability of alpha chains are major factors in modulating the cellular defects.

Very recently a high expression of human hemoglobin S, which causes sickle cell disease, has been obtained in transgenic mice. We have constructed a modified beta S gene, beta SAD which carries two additional mutations in order to induce polymerization of transgenic hemoglobin when diluted by endogenous mouse Hb. The transgenic SAD mice are not anemic but exhibit a low percentage of irreversible sickle cells. Sickling is induced by deoxygenation of erythrocytes in vitro. In addition, the anemia of neonates and the low incidence of SAD animals in the progeny suggest a deleterious effect of SAD Hb during development. Finally, hypoxia induces a high mortality in SAD adults suggesting the induction of vaso-occlusive events.

The G gamma and A gamma content of Hb F produced in cultures of BFU-Es from the blood of normal fetuses, neonates, and adults was determined. The results show that erythroid progenitors produce A gamma and G gamma chains in a ratio characteristic of their ontogenic stage. The analysis of the G gamma/A gamma ratio in culture of BRU-Es from thalassemic patients showed a marked heterogeneity, resembling that observed in freshly drawn cells. These results afford evidence that the type of gamma chain produced is programmed at the level of early erythroid progenitors.

In the present work we have developed an affinity chromatography system, using haptoglobin bound covalently to Sepharose 4B, to purify hemoglobin from soluble non-heme proteins. Sepharose-haptoglobin specifically binds hemoglobin. It exhibits the same characteristics in its interactions with hemoglobin and alpha or beta hemoglobin chains as does haptoglobin in solution. Globin chains can be eluted from the Sepharose-haptoglobin after removal of the heme. This method has allowed accurate measurements of globin-chain synthesis in blood and bone marrow samples and in culture of early erythroid precursors.

The association between the polymorphism of the A gamma chain of human fetal haemoglobin and the DNA polymorphism at the beta gene cluster has been investigated. The A gamma 75 threonine mutation was found in association with haplotypes II and VI described by Orkin et al. (1982), which share the HindIII cleavage site in the A gamma IVS 2 sequence. The distance between the two polymorphic sites is 868 base-pairs. In contrast, haplotypes I, III, V and IX which do not possess this HindIII cleavage site were associated with the normal A gamma 75 isoleucine allele. The simple detection of the gamma gene polymorphism at the protein level can be useful for identifying DNA haplotypes, and therefore beta thalassaemic mutations.

A human leukemic cell line KU 812 was recently established and described as a basophilic cell line. In the present study we show that KU 812 and two of its clones are at least bipotent: in addition to a minor component of basophils, the majority of KU 812 cells belongs to the erythroid cell lineage with a significant percentage (about 15%) of mature hemoglobinized erythroblasts. This terminal differentiation is associated with the synchronized synthesis of the main erythroid proteins, including glycophorins, spectrin beta chain, band 3, and hemoglobin. The predominant hemoglobins are adult, fetal, and Bart's hemoglobin. Adult hemoglobin represented up to 75% of all hemoglobins in the KU 812 F clone in passages containing a high number of mature erythroblasts. Transcripts of all human globin chains were present with ten times less embryonic chain messenger RNA (mRNA) than alpha-, beta- or gamma-chain mRNA. Hemin slightly increased the total hemoglobin production of the cell line, especially gamma-globin chain synthesis, but did not modify the percentage of hemoglobinized cells. Phorbol myristate acetate (PMA) had a complex effect, inducing a proportion of KU 812 cells to adhere to the plastic culture flask. The adherent cell fraction expressed a very low level of specific erythroid proteins, but their ultrastructure was consistent with immature erythroid cells. In contrast, approximately 40% of the nonadherent cells were mature erythroid cells. Cell-sorting experiments showed that this paradoxic effect of PMA is mostly due to cell selection, the more mature cells being unable to adhere. In addition, KU 812 F was found to be sensitive to erythropoietin, which slightly increased its plating efficiency range (from 0% to 50%) in semisolid medium and enhanced hemoglobin accumulation twofold. In binding experiments using 125I erythropoietin, a single class of high-affinity Epo receptors (Kd: 250 pM) was detected by binding with a density of 205 receptors per cell. The KU 812 cell line is therefore a unique model for studying cell commitment toward different hematopoietic lineages and erythroid differentiation.

The K562 cell line was cloned by dilution and the pattern of hemoglobin (Hb) production was analyzed in the clones thus obtained. The pattern of hemoglobin synthesis was different from one clone to another. According to the Hb phenotype the clones were classified in three main groups; group I no detectable Hb; group II Hb Portland; group III Hb Portland + Hb Gower I. The addition of hemin induced a better hemoglobinization and a shift in the pattern of Hb production: clones of group I were induced to produce Hb Portland and clones of group II to synthesize Hb Gower I. A constant order in the sequential expression of the different hemoglobins was observed: no Hb leads to Hb Portland + Hb Gower I. The proportion of the different globin chains varied from one clone to another, but an inverse correlation between the synthesis of G gamma and epsilon chains was observed. The alpha/non-alpha chain ratio was unbalanced in all the clones and the addition of hemin induced only a moderate increase of the synthesis of alpha chains. Recloning of three primary clones increased the homogeneity of the hemoglobin pattern, in particular after hemin induction.

An affinity chromatography system, using haptoglobin bound covalently to Sepharose 4B, has been developed to purify human hemoglobin from soluble non-heme proteins. Agarose-haptoglobin specifically binds hemoglobin. Globin chains were eluted from the agarose-haptoglobin after removal of the heme. This method has allowed accurate measurements of globin chain synthesis in blood and bone marrow samples from adults and in culture of early erythroid precursors present in adult blood or bone marrow.

A murine model of sickle cell disease was tested by studying the polymerization of hybrid hemoglobin tetramers between alpha mouse and human beta S or beta S Antilles chains were prepared from Hb S Antilles, which was a new sickling hemoglobin inducing a sickle cell syndrome more severe than Hb S. The hybrid molecules did not polymerize in solution, indicating that the mouse alpha chains inhibited fiber formation. Consequently, a mouse model for sickle cell disease requires the transfer and expression of both alpha and beta S or beta S Antilles genes.

The relative proportions of gamma chains of human fetal haemoglobin G gamma, A gamma 75 Ileu (A gamma I) and A gamma 75 Thr (A gamma T) were investigated in homogeneous populations of patients in Algeria exhibiting sickle cell disease and in patients in Algeria and Sardinia with beta-thalassaemia. The restriction site haplotypes within the beta gene cluster were known. The results suggest a tight genetic regulation of the G gamma/A gamma + G gamma ratio (G gamma ratio) which is associated with the G gamma Hind III site of polymorphism (p less than 0.0001). From the present results and those in the literature the high G gamma ratio is associated with the presence of 3 polymorphic restriction sites: Xmn1 5' to the G gamma gene, Hind III in the G gamma IVS II and Hinc II in the psi beta gene. Familial studies showed that the expression of the A gamma alleles is genetically determined. The wide variation of the A gamma T/A gamma T + A gamma I ratio (A gamma ratio) between families is most probably related to the various haplotypes bearing the A gamma I alleles.

The soluble pool of alpha hemoglobin chains present in blood or bone marrow cells was measured with a new affinity method using a specific probe, beta A hemoglobin chain labeled with [3H]N-ethylmaleimide. This pool of soluble alpha chains was 0.067 +/- 0.017% of hemoglobin in blood of normal adult, 0.11 +/- 0.03% in heterozygous beta thalassemia and ranged from 0.26 to 1.30% in homozygous beta thalassemia intermedia. This elevated pool of soluble alpha chains observed in human beta thalassemia intermedia decreased 33-fold from a value of 10% of total hemoglobin in bone marrow cells to 0.3% in the most dense red blood cells. The amount of insoluble alpha chains was measured by using the polyacrylamide gel electrophoresis in urea and Triton X-100. In beta thalassemia intermedia the amount of insoluble alpha chains was correlated with the decreased spectrin content of red cell membrane and was associated with a decrease in ankyrin and with other abnormalities of the electrophoretic pattern of membrane proteins. The loss and topology of the reactive thiol groups of membrane proteins was determined by using [3H]N-ethylmaleimide added to membrane ghosts prior to urea and Triton X-100 electrophoresis. Spectrin and ankyrin were the major proteins with the most important decrease of thiol groups.

Two new techniques have been devised for the prenatal diagnosis of hemoglobinopathies performed on fetal blood samples. Isoelectric focusing (IEF) of hemoglobins was compared to the classical chromatography of labelled globin chains for 51 fetal blood samples, 40 being obtained for prenatal diagnosis of hemoglobinopathies, in Paris. In all cases the two methods provided identical results. Adult hemoglobin was quantitatively evaluated. In addition blood samples obtained in other centers after abortion of 22 fetuses homozygous for beta thalassemia did not exhibited measureable amounts of Hb A by IEF. The fetal blood must be free of maternal contamination. If present maternal red blood cells can be completely eliminated by selective lysis using the 0RSKOV reaction. A chromatography of hemoglobins on Biorex 70 has been devised very recently to overcome the two limitations of IEF: First the contamination of fetal blood samples by maternal cells, and second the impossibility to evaluate Hb A when present in proportion below 1%. The chromatography of hemoglobins on Biorex 70 is performed in 75 minutes with 0.1 mg of hemoglobin present in membrane free hemolysate. The optical density recording allows to evaluate .5% of Hb A and to detect .1% of Hb A. In addition, the radioactivity profile of the chromatography can be determined. It has to be used in case of maternal contamination.