Haemoglobinopathy survey in an Eti-Turk village.

Blood specimens were obtained from 281 inhabitants of an Eti-Turk village with a population of about 500. Starch gel (pH 8.6) and agar gel (pH 6.45) electrophoresis were performed in 279 of the specimens. Hb S was present in 105 partially interrelated persons (37.36%), three of whom had sickle-cell anaemia. Hb E was detected in 5 persons (1.79%), one of whom was a double heterozygote for Hb S and Hb E. One Hb S+alpha-thalassaemia and 7 Hb S with elevated Hb A'2 combinations were found. The beta-thalassaemia gene prevalence was 0.0377. Hb A2 was found in 4 persons (1.42%), and Hb F was slightly increased in 37 (22.3%) persons with a normal haemoglobin picture. Erythrocyte G-6-PD deficiency was 10% among males.     

Enhanced inhibition of polymerization of sickle cell hemoglobin in the presence of recombinant mutants of human fetal hemoglobin with substitutions at position 43 in the gamma-chain

Four recombinant mutants of human fetal hemoglobin [Hb F (alpha2gamma2)] with amino acid substitutions at the position 43 of the gamma-chain, rHb (gammaD43L), rHb (gammaD43E), rHb (gammaD43W), and rHb (gammaD43R), have been expressed in our Escherichia coli expression system and used to investigate their inhibitory effect on the polymerization of deoxygenated sickle cell hemoglobin (Hb S). Oxygen-binding studies show that rHb (gammaD43E), rHb (gammaD43W), and rHb (gammaD43R) exhibit higher oxygen affinity than human normal adult hemoglobin (Hb A), Hb F, or rHb (gammaD43L), and all four rHbs are cooperative in binding O2. Proton nuclear magnetic resonance (NMR) studies of these four rHbs indicate that the quaternary and tertiary structures around the heme pockets are similar to those of Hb F in both deoxy (T) and liganded (R) states. Solution light-scattering experiments indicate that these mutants remain mostly tetrameric in the liganded (R) state. In equimolar mixtures of Hb S and each of the four rHb mutants (gammaD43L, gammaD43E, gammaD43R, and gammaD43W), the solubility (Csat) of each of the pairs of Hbs is higher than that of a similar mixture of Hb S and Hb A, as measured by dextran-Csat experiments. Furthermore, the Csat values for Hb S/rHb (gammaD43L), Hb S/rHb (gammaD43E), and Hb S/rHb (gammaD43R) mixtures are substantially higher than that for Hb S/Hb F. The results suggest that these three mutants of Hb F are more effective than Hb F in inhibiting the polymerization of deoxy-Hb S in equimolar mixtures.     

A biochemical and biophysical characterization of recombinant mutants of fetal hemoglobin and their interaction with sickle cell hemoglobin.

Three recombinant mutants of human fetal hemoglobin (Hb F) have been constructed to determine what effects specific amino acid residues in the gamma chain have on the biophysical and biochemical properties of the native protein molecule. Target residues in these recombinant fetal hemoglobins were replaced with the corresponding amino acids in the beta chain of human normal adult hemoglobin (Hb A). The recombinant mutants of Hb F included rHb F (gamma 112Thr --> Cys), rHb F (gamma 130Trp --> Tyr), and rHb F (gamma 112Thr --> Cys/gamma 130Trp --> Tyr). Specifically, the importance of gamma 112Thr and gamma 130Trp to the stability of Hb F against alkaline denaturation and in the interaction with sickle cell hemoglobin (Hb S) was investigated. Contrary to expectations, these rHbs were found to be as stable against alkaline denaturation as Hb F, suggesting that the amino acid residues mentioned above are not responsible for the stability of Hb F against the alkaline denaturation as compared to that of Hb A. Sub-zero isoelectric focusing (IEF) was employed to investigate the extent of hybrid formation in equilibrium mixtures of Hb S with these hemoglobins and with several other hemoglobins in the carbon monoxy form. Equimolar mixtures of Hb A and Hb S and of Hb A(2) and Hb S indicate that 48-49% of the Hb exists as the hybrid tetramer, which is in agreement with the expected binomial distribution. Similar mixtures of Hb F and Hb S contain only 44% hybrid tetramer. The results for two of our recombinant mutants of Hb F were identical to the results for mixtures of Hb F and Hb S, while the other mutant, rHb F (gamma 130Trp --> Tyr), produced 42% hybrid tetramer. The sub-zero IEF technique discussed here is more convenient than room-temperature IEF techniques, which require Hb mixtures in the deoxy state. These recombinant mutants of Hb F were further characterized by equilibrium oxygen binding studies, which indicated no significant differences from Hb F. While these mutants of Hb F did not have tetramer-dimer dissociation properties significantly altered from those of Hb F, future mutants of Hb F may yet prove useful to the development of a gene therapy for the treatment of patients with sickle cell anemia.     

Effect of FS (alpha 2 gamma beta s) hybrid hemoglobin on Hb S nucleation and aggregation

Asymmetrical cross-linked FS (alpha 2 gamma beta s) hybrid hemoglobin (Hb FS-fumarate) was prepared by reacting mixtures of hemoglobins F and S with double-headed aspirin, bis(3,5-dibromosalicyl) fumarate. When the molar ratio of hemoglobin to the cross-linking agent was 1 to 2 in a 1:1 FS mixture, the relative ratio of the products, cross-linked hemoglobins F (Hb F-fumarate), FS (HB FS-fumarate), and S (Hb S-fumarate), was 1.0:2.6:2.0, in contrast to a 1:2:1 ratio of cross-linked hemoglobins A, AS, and S in a 1:1 AS mixture. These results suggest that the fumaryl group reacts differently with Hb F, Hb FS and Hb S, and that the difference could be attributed to the difference in the structure in the vicinity of the EF6 Lys of non alpha-chains. The oxygen-binding properties of Hb F-fumarate, Hb FS-fumarate, and Hb S-fumarate were similar, except that the n-value of Hb F-fumarate was slightly lower than n-values of Hb S-fumarate and Hb FS-fumarate. Kinetic studies on aggregation showed that the addition of Hb FS-fumarate to unmodified Hb S did not affect the delay time prior to aggregation, but did increase the total turbidity. Electrophoretic and densitometric scanning analysis of the aggregate phase of this mixture showed the fraction of Hb FS-fumarate to be 19%. Hb F-fumarate's effect on the delay time is concentration-dependent; the greater the concentration of Hb F-fumarate, the longer the delay time. The turbidity after aggregation of the mixture of Hb S and Hb F-fumarate was much less than that of Hb S and Hb FS-fumarate. However, the fraction of Hb F-fumarate in the aggregate phase was 19%, which is similar to that of Hb FS-fumarate. These data suggest that Hb F and FS hybrid hemoglobin cannot participate in nuclei formation, but can participate in aggregation after sufficient amounts of nuclei are formed from Hb S, and that increased levels of Hb F do not have an inhibitory effect on the formation of nuclei but on the growth of aggregates.     

Single amino acid substitution in important hemoglobinopathies does not disturb molecular function and biological process

Hemoglobin is an important protein found in the red cells of many animals. In humans, the hemoglobin is mainly distributed in the red blood cell. Single amino acid substitution is the main pathogenesis of most hemoglobin disorders. Here, the author used a new gene ontology technology to predict the molecular function and biological process of four important hemoglobin disorders with single substitution. The four studied important abnormal hemoglobins (Hb) with single substitution included Hb S, Hb E, Hb C, and Hb J-Baltimore. Using the GoFigure server, the molecular function and biological process in normal and abnormal hemoglobins was predicted. Compared with normal hemoglobin, all studied abnormal hemoglobins had the same function and biological process. This indicated that the overall function of oxygen transportation is not disturbed in the studied hemoglobin disorders. Clinical findings of oxygen depletion in abnormal hemoglobin should therefore be due to the other processes rather than genomics, proteomics, and expression levels.     

Glycosylated minor components of human adult hemoglobin. Purification, identification, and partial structural analysis

Human hemolysate contains several minor components designated Hb A1a, Hb A1b, Hb A1c, which are post-translational modifications of the major hemoglobin component A0. Individuals with diabetes mellitus have elevated levels of Hb A1c, a hemoglobin modified with a glucose moiety at the NH2 terminus of each beta chain. A new chromatographic technique using Bio-Rex 70 is described which not only allows complete separation of Hb A1a from Hb A1b but also resolution of Hb A1a into two components, designated Hb A1a1 and Hb A1a2. Carbohydrate determinations with the thiobarbituric acid procedure revealed that Hb A1a1, Hb A1a2, and Hb A1b as well as Hb A1c were glycosylated. Total phosphate analysis revealed 2.06 and 1.01 mol of phosphorus/alphabeta dimer for Hb A1a1 and Hb A1a2 respectively; Hb A1b and Hb A1c contained no detectable phosphate. Hemoglobin incubated with D-[14C]glucose-6-P co-chromatographs precisely with Hb A1a2, strongly suggesting that Hb A1a2 is glucose-6-P hemoglobin. Levels of Hb A1a1 and Hb A1a2 are normal in individuals with diabetes mellitus. Furthermore, diabetic red cells contain normal levels of glucose-6-P. Therefore, glucose-6-P hemoglobin does not serve as a significant precursor to Hb A1c. Instead Hb A1c is formed by the direct reaction of hemoglobin with glucose. This suggests that hemoglobin can serve as a model system for nonenzymatic glycosylation of protein.     

pH-dependent Soret difference spectra of the deoxy and carbonmonoxy forms of human hemoglobin and its derivatives.

The transition from deoxy to oxystructure of hemoglobin A (Hb) is accompanied by the breaking of the salt bridges formed by C-terminal residues in deoxy-Hb. This, in turn, changes the state of the heme. The switch between these different allosteric forms can be followed by changes in the optical absorbance spectra (Perutz, M. F., Ladner, J. E., Simon, S. R., and Ho, C. (1974), Biochemistry 13, 2163). Using difference spectroscopy in the soret region, pH-dependent spectral changes of Hb and its derivatives (carbamylated at both the alpha-NH2 groups, alpha2cbeta2c; N-ethylsuccinimide hemoglobin, NES-Hb) in their deoxy and carbonmonoxy forms were measured. From these measurements, the pK values of histidine-146beta and valine-1alpha in deoxy-Hb were determined to be 8.6 +/- 0.2 and 7.7 +/- 0.1, respectively. In carbonmonoxy-Hb a pK value of 6.3 +/- 0.1 was found.     

Genetic hemoglobin abnormalities in 2363 Cuban newborns

Summary A total of 2363 Cuban newborns were screened for genetic hemoglobin abnormalities; 2187 (92.56%) had a normal electrophoretic pattern. Of the 176 samples with abnormal electrophoretic patterns, 102 (4.32%) had hemoglobins A, F plus Bart's; 54 (2.29%) had hemoglobins A, F and S; 3 (0.13%) had hemoglobins A, F, S plus Bart's; 14(0.59%) had hemoglobins A, F and C; 1 (0.04%) had hemoglobins A, F, C and Bart's. The frequency of Hb Bart's was 4.46% in AA phenotype, 5.25% in AS, and 6.67% in AC. Two newborns were found to have rare variants. A close correlation was found between the observed and expected phenotypes, which indicates the accuracy of the diagnostic methods used. The results of all hemoglobin abnormalities were entered on the infants' hospital records. In addition, these families received genetic counseling.     

Hemoglobin Tarrant: alpha126(H9) Asp leads to Asn. A new hemoglobin variant in the alpha1beta1 contact region showing high oxygen affinity and reduced cooperativity.

Hemoglobin (Hb) Tarrant was detected by its electrophoretic mobility on cellulose acetate (pH 8.4) and citrate agar (pH 6.2). On cellulose acetate it moved as a band between hemoglobins F and S, and on citrate agar as a band at hemoglobin S. The test for solubility in 2 M phosphate buffer with Na2S2O4 was negative. The new variant has a substitution of asparagine for aspartic acid in position 126 of the alpha-chain, one of the sites involved in the alpha1beta1 contact. Furthermore, in deoxyhemoglobin aspartic acid 126 of each alpha chain also forms a non-covalent electrostatic salt bridge with arginine 141 of the corresponding alpha chain (Perutz, M. F. and Ten Eyck, L. F. (1972) Cold Spring Harbor Symp. Quant. Biol. 36, 295-310 and Perutz, M. F. (1970) Nature 228, 726-739). As a consequence of this substitution in hemoglobin Tarrant, the deoxy conformation or T state is destabilized because these two bridges cannot be formed. This condition is reflected in high oxygen affinity and low cooperativity.     

Effect of hemoglobin solution on compensation to anemia in the erythrocyte-free primate

Hemoglobin solutions are undergoing clinical trials as erythrocyte substitutes. Some of these solutions have higher O2 affinities compared with normal erythrocyte hemoglobin. Also, they appear to interact with endothelial-derived smooth muscle relaxation. The purpose of this study was to evaluate the nature and limits of compensation to acute normovolemic anemia in the erythrocyte-free primate maintained with a hemoglobin solution. The experimental group consisted of six anesthetized paralyzed adult baboons (Papio anubis) that were exchange transfused (ET) with a pyridoxylated polymerized hemoglobin solution [hemoglobin concentration [( Hb]) = 14 g/dl, O2 half-saturation pressure of hemoglobin (P50) = 19.6 Torr] until a hematocrit less than 1% was achieved. They underwent a second ET with Dextran-70 until [Hb] = 1 g/dl. A control group (n = 6) underwent an ET with Dextran-70 until [Hb] = 1 g/dl. Both groups maintained O2 consumption (VO2) until [Hb] = 3 g/dl. Both groups were stable until [Hb] less than 1 g/dl, and both groups increased their cardiac output. The relation between VO2 and O2 delivery was similar for both groups. In vivo P50 and mixed venous O2 tension were significantly lower in the experimental group. The nature and limits of compensation to diminished O2 delivery due to anemia were similar in the two groups.     

Hemoglobin New Mexico: beta 100 (G2) Pro----Arg. A variant hemoglobin associated with erythrocytosis

Hemoglobin New Mexico beta 100 Pro----Arg was found in a 4-year-old black male and represents a new mutation. The propositus is also heterozygous for Hb S. The variant shows high oxygen affinity, reduced cooperatively, and a lowered alkaline Bohr effect. Addition of allosteric effectors leads to improved cooperativity and a Bohr effect that is similar to that of Hb A. The high percentage of the variant (53.5%) and its increased oxygen affinity result in erythrocytosis in this patient. The hemoglobin level and packed cell volume values are elevated. In spite of these factors the patient appears healthy and shows no discomfort. The altered oxygen-linked properties of this variant can be related to the fact that the substituted residue contributes to the alpha 2 beta 1/alpha 1 beta 2 subunit interface, an area that is critical not only to the allosteric transitions between the oxy and deoxy states but also to stabilizing the hemoglobin tetrameer.     

Oxidation-reduction potentials of human fetal hemoglobin and gamma chains. Effects of blocking sulfhydryl groups.

The oxidation-reduction equilibrium of the gamma chains of human fetal hemoglobin (Hb F) has been studied and compared with that of the alpha and beta chains of human adult hemoglobin (Hb A). The effects of the sulfhydryl (--SH) reagents, iodoacetate, iodoacetamide, and p-mercuribenzoate (PMB), on the three kinds of chains and on Hb F have been compared. The midpoint potentials (E-m) of all three sorts of chains are lower than those of tetrameric hemoglobin A or F. The E-m values of alpha chains are the lowest, E-m = 0.049 volt at 6 degrees, and are unaffected by pH change or by PMB treatment, at least from pH 6 to 8. The E-m values of beta-SH chains are higher; E-m = 0.102 volt at pH 7, decreasing to 0.050 volt at pH 8, both at 6 degrees. These results agree with those of Banerjee and Cassoly ((1969) J. Mol. Biol. 42, 337-349). They reported no effect of PMB on beta chains, but we find that 2 eq of PMB/chain raise E--M to 0.139 volt at pH 7 at 6 degrees, chiefly as the result of reaction at beta-93, not at beta-112. Carboxymethylation at beta-93 has an insignificant effect compared with that of PMB. The oxidation-reduction potential of gamma chains is similar to that of beta chains. E-m = 0.098 volt at pH 7 at 6 degrees, decreasing to 0.064 at pH 8 and 0.010 at pH 9. The effects of --SH reagents, reacting at position gamma-93 (the only --SH group present in gamma chains), are essentially the same as those seen with beta chains. The oxidation-reduction potential of Hb F is almost identical with that of Hb A, except for being 0.008 volt lower at pH 6 at 6 degrees. This agrees with the results reported by Flohe and Uehleke ((1966) Life Sci. 5, 1041-1045). PMB or iodoacetamide treatment lowers E-m by 0.02 to 0.03 volt, depending on the pH, from 6 to 9, in much the same way as previously reported for Hb A(Brunori, M., Taylor, J.F., Antonini, E., Wyman, J., and Rossi-Fanelli, A. (1967) J. Biol. Chem. 242, 2295-2300). The "residual oxidation Bohr effect" noted in Hb F can be attributed to the oxidation Bohr effect of the gamma chains. The apparent pK of the heme-linked water molecule was found at 25 degrees to be, for Hb F, 8.1; for gamma-SH chains, 7.85; for gamma-PMB chains, 8.35; and for gamma chains treated with iodoacetate, 7.80. Sedimentation coefficients, s-20, w, at a protein concentration of 5 mg/ml, were found to be, for fetal hemoglobin 4.09, for iodoacetamide-treated fetal hemoglobin 4.04, for PMB-treated fetal hemoglobin 3.41, for fetal gamma-SH chains 4.25, and for fetal gamma-PMB chains 3.08.     

Isolation and functional characterization of hemoglobin Casper: beta106(G8) Leu replaced by Pro.

Hemoglobin Casper (beta106Leu replaced by Pro) can be separated from hemoglobin (Hb) A by isoelectric focusing on polyacrylamide gel. This abnormal hemoglobin was estimated to be 30% of teh total by both isoelectric focusing and heat lability kinetics. Its oxygen equilibrium curves indicate a high oxygen affinity, low degree of subunit interaction, and a decreased Bohr effect. Mixtures of Hb Casper and Hb A appear to bind oxygen as if no hybrid molecules exist.     

Involvement of the distal histidine in the low affinity exhibited by Hb Chico (Lys beta 66----Thr) and its isolated beta chains.

Hemoglobin (Hb) Chico (Lys beta 66----Thr at E10) has a diminished oxygen affinity (Shih, D. T.-b., Jones, R. T., Shih, M. F.-C., Jones, M. B., Koler, R. D., and Howard, J. (1987) Hemoglobin 11, 453-464). Our studies show that its P50 is about twice that of Hb A and that its cooperativity, anion, and Bohr effects between pH 7 and 8 are normal. The Bohr effect above pH 8 is somewhat reduced, indicating a small but previously undocumented involvement of the ionic bond formed by Lys beta 66 in the alkaline Bohr effect. Since the oxygen affinity of the alpha-hemes is likely to be normal, that of the beta-hemes in the tetramer is likely to be reduced by the equivalent of 1.2 kcal/mol beta-heme in binding energy. Remarkably, both initial and final stages of oxygen binding to Hb Chico are of lowered affinity relative to Hb A under all conditions examined. The isolated beta chains also show diminished oxygen affinity. In T-state Hb A, Lys(E10 beta) forms a salt bridge with one of the heme propionates, but comparison with other hemoglobin variants shows that rupture of this bridge cannot be the cause of the low oxygen affinity. X-ray analysis of the deoxy structure has now shown that Thr beta 66 either donates a hydrogen bond to or accepts one from His beta 63 via a bridging water molecule. This introduces additional steric hindrance to ligand binding to the T-state that results in slower rates of ligand binding. We measured the O2/CO partition coefficient and the kinetics of oxygen dissociation and carbon monoxide binding and found that lowered O2 and CO affinity is also exhibited by the R-state tetramers and the isolated beta chains of Hb Chico.     

Hemoglobin Atlanta or alpha 2 beta 2 75 Leu-Pro (E19): an unstable variant found in several members of a Caucasian family.

Hemoglobin Atlanta, alpha 2 beta 2 75 Leu-Pro (E19), has been found in several members of three generations of a Caucasian family living in metropolitan Atlanta. The abnormal hemoglobin is one of the nine unstable variants in which either a leucyl or an alanyl residue is replaced by a prolyl residue. These substitutions have been observed in the B, E, F, and G helixes of the beta chain and in the H helix of the alpha-chain. Hemoglobin Atlanta heterozygotes are mildly affected by the presence of this unstable hemoglobin.     

Stability of asymmetrical hybrid hemoglobins. Determination by anaerobic high performance liquid chromatography

Asymmetrical hybrid hemoglobins formed in mixtures of Hb A and Hb S, Hb F and Hb S, Hb S and Hb York(beta 146 His----Pro), and Hb A and Hb York were separated by high performance liquid chromatography on cation and anion exchange columns under anaerobic conditions. The ratio of the hybrid hemoglobin to the total mixture was consistently lower than that theoretically expected and decreased with longer elution times. The hybrid tetramer appears to be unstable even under anaerobic conditions and dissociates into alpha beta dimers. The time course of dissociation of the hybrid hemoglobins was determined by varying the separation programs and thus separating the hybrid hemoglobin at different elution times. The rate of the dissociation of the hybrid hemoglobins studied follows first order kinetics. The lines representing the time course of dissociation of hybrid hemoglobins were extrapolated to time 0 to determine the fraction of the hybrid hemoglobin in the mixture prior to separation. The values obtained for equimolar mixtures of Hb A and Hb S and Hb York and Hb S or Hb A were in agreement with the expected theoretical value (50%). In contrast, the value obtained for hybrid hemoglobin FS was slightly less (about 40%). AY and SY hybrid hemoglobins dissociated into dimers at a considerably faster rate than did AS and FS hybrid hemoglobins, possibly because of the mutation at the beta 146-position in hybrid hemoglobins containing alpha beta Y dimers. This mutation hinders the formation of salt bridges that normally stabilize the "T" quaternary conformation. Since such hybrid hemoglobins have a partial "R" conformation even when deoxygenated, their rate of dissociation to dimers is expected to increase.     

Hemoglobin Tilburg: alpha 2-beta 2 73 (E 17) Asp----Gly. A new hemoglobin with reduced oxygen affinity

A new hemoglobin variant has been found in a Dutch Caucasian girl and detected also in members of three generations of her family. This variant is characterized by the substitution of an aspartic acid at position 73 (E 17) of the beta-chain with a glycine residue. Hemoglobin Tilburg makes up to 42% of the total hemoglobin in the blood of the proposita, it is stable at the isopropanol test, and not associated with significant hematological abnormalities in heterozygous carriers. The oxygen dissociation curve of the purified variant, carried out at different pH values, shows a definite reduction of the affinity for oxygen and a normal alkaline Bohr effect. Three more hemoglobins with a single amino acid substitution at the same site have been previously described: Hb Korle-Bu (Asp----Asn), Hb Mobile (Asp----Val) and Hb Vancouver (Asp----Tyr). In all these proteins the affinity for oxygen is lowered to an extent which is variable and characteristic of each mutant. In this paper we discuss the possible mechanism responsible for the abnormal behaviour of hemoglobins substituted at beta 73.     

Crystallization and preliminary X-ray structural studies of hemoglobin A2 and hemoglobin E,isolated from the blood samples of beta-thalassemic patients

Hemoglobin A(2) (alpha(2)delta(2)), a minor (2-3%) component of circulating red blood cells, acts as an anti-sickling agent and its elevated concentration in beta-thalassemia is a useful clinical diagnostic. In beta-thalassemia major, where there is a failure of beta-chain production, HbA(2) acts as the predominant oxygen delivery mechanism. Hemoglobin E, is another common abnormal hemoglobin, caused by splice site mutation in exon 1 of beta globin gene, when combines with beta-thalassemia, causes severe microcytic anemia. The purification, crystallization, and preliminary structural studies of HbA(2) and HbE are reported here. HbA(2) and HbE are purified by cation exchange column chromatography in presence of KCN from the blood samples of individuals suffering from beta-thalassemia minor and E beta-thalassemia. X-ray diffraction data of HbA(2) and HbE were collected upto 2.1 and 1.73 A, respectively. HbA(2) crystallized in space group P2(1) with unit cell parameters a=54.33 A, b=83.73 A, c=62.87 A, and beta=99.80 degrees whereas HbE crystallized in space group P2(1)2(1)2(1) with unit cell parameters a=60.89 A, b=95.81 A, and c=99.08 A. Asymmetric unit in each case contains one Hb tetramer in R(2) state.     

Coexistence of Malaria and Thalassemia in Malaria Endemic Areas of Thailand

Hemoglobinopathy and malaria are commonly found worldwide particularly in malaria endemic areas. Thalassemia, the alteration of globin chain synthesis, has been reported to confer resistance against malaria. The prevalence of thalassemia was investigated in 101 malaria patients with Plasmodium falciparum and Plasmodium vivax along the Thai-Myanmar border to examine protective effect of thalassemia against severe malaria. Hemoglobin typing was performed using low pressure liquid chromatography (LPLC) and α-thalassemia was confirmed by multiplex PCR. Five types of thalassemia were observed in malaria patients. The 2 major types of thalassemia were Hb E (18.8%) and α-thalassemia-2 (11.9%). There was no association between thalassemia hemoglobinopathy and malaria parasitemia, an indicator of malaria disease severity. Thalassemia had no significant association with P. vivax infection, but the parasitemia in patients with coexistence of P. vivax and thalassemia was about 2-3 times lower than those with coexistence of P. falciparum and thalassemia and malaria without thalassemia. Furthermore, the parasitemia of P. vivax in patients with coexistence of Hb E showed lower value than coexistence with other types of thalassemia and malaria without coexistence. Parasitemia, hemoglobin, and hematocrit values in patients with coexistence of thalassemia other than Hb E were significantly lower than those without coexistence of thalassemia. Furthermore, parasitemia with coexistence of Hb E were 2 times lower than those with coexistence of thalassemia other than Hb E. In conclusion, the results may, at least in part, support the protective effect of thalassemia on the development of hyperparasitemia and severe anemia in malaria patients.     

Towards a transgenic mouse model of sickle cell disease: hemoglobin SAD.

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.