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Hb G-Coushatta [beta22(B4)Glu--Ala] is found in geographically separated ethnic groups. Commonest along the Silk Road region of China but also present in the North American Coushatta, we sought to determine whether this variant had a unicentric or multicentric origin. We examined the haplotype of the beta-globin gene cluster in two Chinese families and in five Louisiana Coushatta heterozygous for this mutation. Chinese and Louisiana Coushatta had different haplotypes associated with the identical Hb G mutation. These haplotypes were defined by the presence of a HindIII restriction site in the Agamma-globin gene and AvaII restriction site in the beta-globin gene in Chinese subjects and their absence in the Louisiana Coushatta. We found a CAC at codon beta2 (beta-globin gene framework 1 or 2) linked to the Hb G-Coushatta gene in Chinese, and a CAT (framework 3) in Louisiana Coushatta, indicating different beta-globin gene frameworks. Both the Hb G-Coushatta mutation (GAA--GCA) and the codon 2 CAC--CAT polymorphism are normal delta-globin gene sequences, suggesting the possibility of gene conversion. We conclude that Hb G-Coushatta had at least two independent origins. This could be due to separate mutations at codon beta22 in Chinese and Louisiana Coushatta, a mutation at this codon and a beta--delta conversion, or two beta--delta gene conversion events.

Hb Seal Rock was first reported in a young African-American women and her 2-year-old daughter (1). It is an extended alpha chain variant which, like Hb Constant Spring, is present in small quantity and is expressed as an alpha-thalassemia. The mutation, TAA--GAA affects codon 142 of the alpha 2 gene. In this family, the index case was a compound heterozygote for Hb Seal Rock trait and for alpha-thalassemia trait (-3.7 kb). Her hematologic expression was similar to mild Hb H disease, presumably because the Seal Rock mutation affects the alpha 2 gene that is normally responsible for approximately 70% of alpha-globin synthesis. Her daughter had only Hb Seal Rock trait, but was phenotypically alpha-thalassemia-2 trait due to the expression of the Seal Rock mutation on one of her alpha 2-globin genes, the other three alpha-globin genes being unaffected.

A four base pair deletion 5' to A gamma T-globin gene at positions -222 to -225 has been reported to reduce the expression of this gene. To evaluate the prevalence and effect of this deletion, PCR-based methods were employed. The deletion had a gene frequency of 0.06 in a sample of African-American individuals with sickle cell trait, 0.18 in adult African-Americans with normal Hb AA, and 0.36 in caucasians. Seventy cord blood samples from African-American newborns with Hb AA were evaluated by both HPLC and PCR. The frequency of the A gamma T allele was 0.13. The A gamma T-globin chain was always present in a lower proportion than the A gamma I allele (70% of A gamma I), but the percentage of A gamma-globin was the same whether or not A gamma T was present. The total percentage of Hb F, however, was significantly lower in the group with the A gamma T allele (77.1% vs. 87.4%, P < 0.01). These results indicate that the four base pair deletion is not only associated with reduced expression of the A gamma T allele, but also of the G gamma allele in cis, further suggesting a possible role of this region in the modulation of the expression of the linked gamma-globin genes.

Hereditary persistence of fetal hemoglobin (HPFH) can be generally classified into deletional and nondeletional forms. The family described in the present study has characteristics of both types of HPFH. The proband is a healthy 30-year-old black woman. Analysis of her hemoglobin revealed 40.4% HbS, 40.9% HbF (G gamma/A gamma ratio 0.53), 16.8% HbA, and 1.9% HbA2. All of her hematologic indices were normal, and the distribution of HbF in her red cells was pancellular. Family studies demonstrated that the proband has one chromosome 11 bearing the beta s-globin gene and the other bearing a G gamma A gamma (beta+) HPFH determinant in cis to the beta A-globin gene. Gene mapping studies of the region between the G gamma- and beta-globin genes were normal. However, when the A gamma and G gamma promoters were amplified by polymerase chain reaction (PCR) and sequenced, the A gamma promoter was found to have the T-->C mutation at -175, and the G gamma promoter region was found to have the C-->T mutation at -158. The -158 C-->T mutation has been associated with elevated G gamma levels and high HbF in hemolysis, although its role in causing these effects is unclear. The present study suggests that this mutation can also enhance G gamma-globin expression in cis to the -175 T-->C mutation in the absence of hemolysis. We suggest that the alteration of the A gamma gene octamer binding site by the -175 mutation, as well as the loss of a putative G gamma "silencer" caused by the -158 mutation may account for this phenotype. We propose calling these linked mutations the G gamma A gamma(beta+) HPFH.

We report a relatively mild phenotype associated with two siblings who are compound heterozygotes for Hb S and a beta zero-thalassemia mutation due to a approximately 1.4-kb deletion of the 5' region of the beta-globin gene. Each is found to have unusually high levels of Hb A2 and Hb F, accounting for more than 20% of the total hemoglobin. These may interfere with intracellular Hb S polymerization, thus leading to a mild clinical course.

We studied the molecular basis of transfusion-dependent hemolytic anemia in an infant who rapidly developed the phenotype of beta thalassemia major. DNA sequence of one beta-globin gene of the proband revealed two mutations, one for the moderately unstable hemoglobin (Hb) Koln and another for a novel codon 32 cytosine-thymidine-guanine-->cytosine-adenine-guanine transversion encoding a leucine-->glutamine mutation. A hydrophilic glutamine residue at beta 32 has an uncharged polar side chain that could potentially distort the B helix and provoke further molecular instability. This new hemoglobin was called Hb Medicine Lake. Biosynthesis studies showed a deficit of beta-globin synthesis with early loss of beta-globin chains. An abnormal unstable hemoglobin, globin chain, or tryptic globin peptide was not present, demonstrating the extreme lability of this novel globin. Hb Medicine Lake mRNA was present, but an aberrantly spliced message was not. Absence of an abnormal beta-globin gene in the mother makes it likely that a de novo mutation occurred in the proband. The molecular pathogenesis of Hb Medicine Lake illustrates a mechanism whereby the phenotype of a genetic disorder, like the mild hemolytic anemia associated with a hemoglobinopathy, can be modulated by a coincident mutation in the same gene.

A four base pair deletion 5' to A gamma T-globin gene at positions -222 to -225 has been reported to reduce the expression of this gene. To evaluate the prevalence and effect of this deletion, PCR-based methods were employed. The deletion had a gene frequency of 0.06 in a sample of African-American individuals with sickle cell trait, 0.18 in adult African-Americans with normal Hb AA, and 0.36 in caucasians. Seventy cord blood samples from African-American newborns with Hb AA were evaluated by both HPLC and PCR. The frequency of the A gamma T allele was 0.13. The A gamma T-globin chain was always present in a lower proportion than the A gamma I allele (70% of A gamma I), but the percentage of A gamma-globin was the same whether or not A gamma T was present. The total percentage of Hb F, however, was significantly lower in the group with the A gamma T allele (77.1% vs. 87.4%, P0.01). These results indicate that the four base pair deletion is not only associated with reduced expression of the A gamma T allele, but also of the G gamma allele in cis, further suggesting a possible role of this region in the modulation of the expression of the linked gamma-globin genes.

We describe an African-American child with beta-thalassemia intermedia. Molecular studies revealed that the proband is a compound heterozygote for the -29 (A--G) beta (+)-thalassemia mutation and an extensive deletion involving the delta- and beta-globin genes. The proband's mother is a simple carrier of the deletion and exhibits the phenotype of delta beta-thalassemia rather than hereditary persistence of fetal hemoglobin. The deletion spans 11,767 bp, with the 5' deletion endpoint located 2,455 bp upstream of the delta-globin gene mRNA Cap site and the 3' endpoint located 441 bp downstream of the termination codon of the beta-globin gene. Based on this information, we have developed a polymerase chain reaction strategy for the rapid detection of this delta beta-thalassemia deletion.

Seventeen unrelated beta-thalassemia patients or carriers from Southwestern Iran were examined for the beta-globin gene mutations by polymerase chain reaction amplification of the beta-globin gene and direct genomic sequencing, or by the method of allele-specific oligonucleotide hybridization. Their clinical and hematological characteristics were also recorded. Of 26 potential thalassemia-causing chromosomes examined, 10 different mutations were found. The IVS-II-1 (G--A) mutation was the most frequent (31%) followed by the IVS-I-6 (T--C) mutation (15%). Eight mutations were initially described in Mediterranean populations and two were of Kurdish origin. Four of these mutations, both initially described in the Mediterranean region, are reported here for the first time in Iranians. The unexpectedly high number of different mutations that account for beta-thalassemia in this region of Iran suggest migration of chromosomes from distant places and genetic admixture.

Hereditary persistence of fetal hemoglobin (HPFH) can be generally classified into deletional and nondeletional forms. The family described in the present study has characteristics of both types of HPFH. The proband is a healthy 30-year-old black woman. Analysis of her hemoglobin revealed 40.4% HbS, 40.9% HbF (G gamma/A gamma ratio 0.53), 16.8% HbA, and 1.9% HbA2. All of her hematologic indices were normal, and the distribution of HbF in her red cells was pancellular. Family studies demonstrated that the proband has one chromosome 11 bearing the beta s-globin gene and the other bearing a G gamma A gamma (beta+) HPFH determinant in cis to the beta A-globin gene. Gene mapping studies of the region between the G gamma- and beta-globin genes were normal. However, when the A gamma and G gamma promoters were amplified by polymerase chain reaction (PCR) and sequenced, the A gamma promoter was found to have the T--C mutation at -175, and the G gamma promoter region was found to have the C--T mutation at -158. The -158 C--T mutation has been associated with elevated G gamma levels and high HbF in hemolysis, although its role in causing these effects is unclear. The present study suggests that this mutation can also enhance G gamma-globin expression in cis to the -175 T--C mutation in the absence of hemolysis. We suggest that the alteration of the A gamma gene octamer binding site by the -175 mutation, as well as the loss of a putative G gamma "silencer" caused by the -158 mutation may account for this phenotype. We propose calling these linked mutations the G gamma A gamma(beta+) HPFH.

Small deletions of the 5' portion of the beta-globin gene that remove the promoters but stop 3' to the delta-globin gene are recognized as the sole cause of beta-thalassemia with exceptionally high hemoglobin A2 (HbA2) levels. Two patients with beta-thalassemia intermedia and exceptionally high levels of HbA2 (10.4 and 12.0%) were examined. One patient was a combined heterozygote for the -88 C----T and a novel -87 C----A mutation, while the other was homozygous for the -29 A----G beta(+)-thalassemia mutation. The remainder of the beta genes were normal. There was no evidence for deletions involving the 5' portion of the beta gene or the region between the beta and delta genes. Gene mapping studies excluded the possibility of a beta delta-anti-Lepore hemoglobin gene with beta promoters and delta coding sequences. There were no mutations in the promoters of the G gamma or A gamma-globin genes that have been associated with the hereditary persistence of HbF phenotype. The delta-globin gene promoters were normal from codon 17 to position -145 relative to the mRNA capping site. There appears to be considerable heterogeneity of HbA2 and HbF levels in patients who are homozygous or mixed heterozygotes for mutations in the TATA box and other promoter elements of the beta-globin gene. The capacity for proteolysis within the erythrocyte may vary among individuals. The authors hypothesize that in the exceptionally high HbA2 beta-thalassemia intermedia phenotype, proteolysis of superfluous alpha-globin chains is less efficient than in patients with customary levels of HbA2.(ABSTRACT TRUNCATED AT 250 WORDS)

We report a relatively mild phenotype associated with two siblings who are compound heterozygotes for Hb S and a beta zero-thalassemia mutation due to a approximately 1.4-kb deletion of the 5' region of the beta-globin gene. Each is found to have unusually high levels of Hb A2 and Hb F, accounting for more than 20% of the total hemoglobin. These may interfere with intracellular Hb S polymerization, thus leading to a mild clinical course.

The initial report of Hb Indianapolis described two affected individuals with the phenotype of severe beta-thalassemia that was dominantly inherited. The structure of this variant could not be deduced by standard techniques because of its extreme instability. Because of this limitation, the structure was ascertained by analysis of the abnormal globin chain, which had been radioactively labeled. These studies strongly suggested that the structure of this variant was cysteine beta 112 to arginine. Subsequent to this report, two additional families with Hb Indianapolis were found. The carriers were minimally affected and the abnormal hemoglobin was only mildly unstable. This major difference in phenotypic expression suggested that further investigation of the original family should be carried out. Unfortunately, both of the original carriers of the variant succumbed to their severe anemia prior to the subsequent reports. However, by the use of the polymerase chain reaction, enough DNA was obtained to sequence the third exon of the beta-globin gene in the original family from the DNA scraped off a 10-year-old bone marrow microscope slide. These studies revealed a substitution of leucine to arginine at position 106 of the beta-globin chain. The polymerase chain reaction results may be consistent with the original protein structural data, if incomplete tryptic cleavage of this arginine residue occurred in the original sample. We have renamed this variant Hb Terre Haute in an attempt to avoid confusion with the Cys beta 112----Arg substitution.

Hemoglobin (Hb) Suan-Dok (alpha 109Arg) is a rare alpha-globin structural mutation that is linked to an alpha-thalassemia (alpha-thal) determinant. When inherited in trans to an alpha-thal-1 mutation (-), it results in Hb H disease associated with low levels (9%) of the Suan-Dok Hb. The nature of the thalassemic defect associated with the alpha SD mutation has been investigated by structural and functional studies. Sequence analysis of the cloned Suan-Dok allele showed a missense mutation (T----G) at codon 109 in an otherwise normal alpha 2-globin gene. When the alpha 2SD-globin gene was introduced into mouse erythroleukemia cells, the steady state alpha-globin messenger RNA (mRNA) level was equivalent to the alpha A-globin gene control. Although in vitro translation of a synthetic alpha 2SD-globin mRNA generated levels of alpha globin equivalent to alpha 2A-globin mRNA at early time points, the ratio of alpha SD to alpha A globin decreased markedly at later time points. These data suggest that the thalassemic defect associated with the Suan-Dok mutation results from a significant instability of the alpha SD globin.

We have defined the clinical and laboratory characteristics of a group of patients with HbS-β°-thalassemia plus α-thalassemia, by analysis of erythrocyte indices, hemoglobin A 2 and F levels, globin biosynthesis studies and α-globin gene mapping. Patients with HbS-β° + α-thalassemia closely resembled individuals with HbS-β°-thalassemia except for balanced globin synthesis ratios and a lower HbF level. The frequency of painful crises, leg ulceration, aseptic necrosis of bone and acute chest syndrome was similar in HbS-β° + α-thalassemia patients and controls with sickle cell anemia (HbSS), HbSS-α-thalassemiaand HbS-β°-thalassemia. These findings are consistent with previous work which failed to show a reduction in the vaso-occlusive severity of sickle cell disease by the coexistence of α-thalassemia.

We studied erythrocyte calcium levels and uptake in a group of patients with sickle haemoglobinopathies of different clinical severity in an attempt to relate these measurements to the production of irreversibly sickled cells and disease severity. Erythrocyte calcium levels were measured by atomic absorption spectroscopy and calcium uptake by isotopic means. In sickle cell anaemia, erythrocyte calcium content was elevated and the uptake of isotopic calcium increased under both oxygenated and deoxygenated conditions. There was a direct correlation between the numbers of irreversibly sickled cells and calcium uptake and an inverse relationship between calcium uptake and red cell potassium level. The clinical course of disease was milder in patients with high fetal haemoglobin levels, but there was no relationship between clinical course and calcium levels, calcium flux or irreversibly sickled cells. Our observations suggest that calcium accumulation and irreversibly sickled cell formation are related processes. The absence of good correlation between various biochemical and clinical parameters emphasizes the complexity of factors which modify the clinical course of this disorder.

In HbSC disease, as in sickle cell anaemia, there is a spectrum of clinical severity. A reduced mean corpuscular volume and haemoglobin concentration, traits typical of thalassaemia, might retard sickling. We therefore ascertained the prevalence of alpha-thalassaemia in 53 adults with HbSC disease and related alpha-globin gene deletion to the haematologic and clinical findings. Alpha-globin genotype was identified by restriction endonuclease gene mapping. Indirect ophthalmoscopy and fluorescein angiography were used to document the presence of proliferative retinopathy. Bone necrosis and infarction were determined roentgenographically or by radionuclide scanning. Either heterozygous or homozygous alpha-thalassaemia-2 was present in 26% of patients. There was no relationship between alpha-globin genotype and haematocrit, pain crises, bone lesions, proliferation retinopathy or clinical severity score.

Some patients with Diamond-Blackfan syndrome may have suppressor T-cells responsible for the failure of normal erythropoiesis. The capacity of Diamond-Blackfan syndrome mononuclear cells to react in mixed leukocyte culture to the stimulus of nucleated red cells was tested using bone marrow as the cell source. In all but one instance, Diamond-Blackfan mononuclear cells and cells from normal or a multiply-transfused control showed similar degrees of stimulation. Patient mononuclear cells reacted normally to phytohemagglutinin (PHA). The presence of a cytotoxic effect of Diamond-Blackfan mononuclear cells on normal nucleated red cells was examined using a 59Fe-release assay. There was no evidence for lymphocyte-mediated erythroid cytotoxicity. Immunoglobulins (Ig) were removed from Diamond-Blackfan serum by affinity chromatography. Erythroid burst-forming units (BFU-E) failed to grow normally in Ig-depleted patient autologous serum, but growth failure was reversed when Ig eluted from the affinity column was added back to the culture. Diamond-Blackfan Ig added to autologous mononuclear cells in the absence of autologous serum normalized colony growth. T-cells of some Diamond-Blackfan syndrome patients may suppress BFU-E growth, but they do not react abnormally in mixed leukocyte culture to nucleated erythroid cells and are not cytotoxic for erythroblasts under our conditions of study. The serum-blocking factor in our patients appears to be an Ig that promotes BFU-E generation despite the presence of suppressor T-cells. Although it cannot be proved, this may be related to the remission of disease in our patients.

The characteristic clinical heterogeneity of sickle cell anemia (HbSS) may be, in part, a result of its interactions with alpha-thalassemia. Although alpha-thalassemia clearly affects some hematologic features of HbSS, its role in modulating the vasoocclusive severity of disease is not clear. To further explore this relationship, we examined the incidence of painful episodes, acute chest syndrome, aseptic bone necrosis, and leg ulcers in 3 patient groups with sickle cell disease: (1) 2,147 patients over age 2 yr, stratified according to mean corpuscular volume (MCV); (2) 183 patients selected on the basis of microcytosis and elevated HbA2, on whom globin biosynthesis studies were done; and (3) 125 patients who had alpha-globin genotype assigned by restriction endonuclease gene mapping. When patients were stratified by MCV, there was a reciprocal relationship between HbA2 levels and MCV, reflecting the presence of patients with beta o and alpha- thalassemia in the low MCV groups. The erythrocyte indices and HbA2 levels in patients classified as HbSS-alpha-thalassemia, by either globin synthesis studies or gene mapping, were very similar to those previously reported by others. Neither microcytosis, beta o, or alpha- thalassemia appeared to provide any clear protection from the vasoocclusive complication evaluated, and the prevalence of aseptic necrosis was increased in patients with microcytosis over age 20 yr and in groups with alpha-thalassemia. The effects of a reduced MCV and mean corpuscular hemoglobin concentration (MCHC), of possible benefit by themselves, when accompanied by a reduction in hemolysis and rise in hemoglobin concentration, as in HbSS-alpha-thalassemia, may cause sufficient rise in blood viscosity in critical vascular beds to impair blood flow and negate any amelioration of vasoocclusive complications in HbSS.

The characteristic clinical heterogeneity of sickle cell anemia (HbSS) may be, in part, a result of its interactions with alpha-thalassemia. Although alpha-thalassemia clearly affects some hematologic features of HbSS, its role in modulating the vasoocclusive severity of disease is not clear. To further explore this relationship, we examined the incidence of painful episodes, acute chest syndrome, aseptic bone necrosis, and leg ulcers in 3 patient groups with sickle cell disease: (1) 2,147 patients over age 2 yr, stratified according to mean corpuscular volume (MCV); (2) 183 patients selected on the basis of microcytosis and elevated HbA2, on whom globin biosynthesis studies were done; and (3) 125 patients who had alpha-globin genotype assigned by restriction endonuclease gene mapping. When patients were stratified by MCV, there was a reciprocal relationship between HbA2 levels and MCV, reflecting the presence of patients with beta o and alpha-thalassemia in the low MCV groups. The erythrocyte indices and HbA2 levels in patients classified as HbSS-alpha-thalassemia, by either globin synthesis studies or gene mapping, were very similar to those previously reported by others. Neither microcytosis, beta o, or alpha-thalassemia appeared to provide any clear protection from the vasoocclusive complication evaluated, and the prevalence of aseptic necrosis was increased in patients with microcytosis over age 20 yr and in groups with alpha-thalassemia. The effects of a reduced MCV and mean corpuscular hemoglobin concentration (MCHC), of possible benefit by themselves, when accompanied by a reduction in hemolysis and rise in hemoglobin concentration, as in HbSS-alpha-thalassemia, may cause sufficient rise in blood viscosity in critical vascular beds to impair blood flow and negate any amelioration of vasoocclusive complications in HbSS.

Hemoglobin A2 levels in normal adults are rarely greater than 3.5%. In patients heterozygous for beta-thalassemia, they average about 5% but do not usually exceed 7%. We studied a family in which four patients with heterozygous beta-thalassemia had HbA2 levels of 8.4% to 11.2%. Globin biosynthesis studies and restriction endonuclease mapping of the alpha-globin loci showed homozygous or heterozygous alpha-thalassemia-2 as well as beta-thalassemia in some family members. The delta- and beta-globin genes were examined by using the restriction enzymes Eco RI, Pvu II, and Xba I, which cut both within and outside the coding portions of the delta- and beta-loci. Only the expected delta- and beta-globin gene containing fragments were present, excluding a crossover event producing a fusion gene that would code for delta-globin but possibly be under the regulatory influence of nucleotide sequences that control the expression of the beta-gene. This kindred provides evidence that in the presence of beta-thalassemia, expression of the delta-gene, beyond that commonly seen, is possible. This could be a direct result of the gene defect producing beta-thalassemia or be due to differences in the delta-globin gene linked to this beta-thalassemia gene. The interactions of alpha- and beta-thalassemia may alter tetramer assembly and increase HbA2 levels; however, this possibility seems less likely.

An American black woman was found to have the phenotype of moderately severe alpha-thalassemia normally associated with the loss of two to three alpha-globin genes despite an alpha-globin gene map that demonstrated the loss of only a single alpha-globin gene (-alpha/alpha alpha). Several individuals in her kindred with normal alpha-globin gene mapping studies (alpha alpha/alpha alpha) had mild alpha-thalassemia hematologic values consistent with the loss of one to two alpha-globin genes. These data suggested the presence of a nondeletion alpha-thalassemia defect in this family which segregates with the intact alpha alpha gene cluster. An abnormally migrating and highly unstable alpha-globin gene product was demonstrated by in vitro translation of the reticulocyte mRNA from the proposita and this mutant alpha-globin protein was mapped to the alpha 2-globin gene by hybrid-selected translation. The abnormal alpha 2-globin gene was cloned and sequenced. A single base mutation that results in a premature termination codon was identified at codon 116 (GAG----UAG). The defined alpha-globin genotype of the proposita (-alpha/alpha 116UAG alpha) and the positioning of this nonsense mutation at the alpha 2-globin gene locus are fully consistent with the observed alpha-thalassemia phenotype.

A rapid and simple procedure has been developed for the isolation of Plasmodium berghei parasites from infected-mouse erythrocytes employing the heat stable hemolysin produced by Pseudomonas aeruginosa. Using parasites isolated by this method, the presence of a parasite specific hexokinase has been demonstrated, providing an explanation for the increased glucose consumption observed with infected cells. Enzyme assays and serology were employed in determining the purity and yield of purified parasites. The enzyme assays showed that about 25% of the parasites in infected RBCs were recovered in the purified state. The purified parasites were not agglutinated by rabbit-anti-mouse RBC serum which indicated the purified parasites were not contaminated by RBC components.

A novel deletion of at least 26 kilobase of DNA, including both alpha-globin genes, the psi alpha- and psi zeta-globin genes, but sparing the functional zeta-gene was found in a 10-year-old black boy with HbH disease and sickle cell trait. This particular deletion has not previously been described in blacks. Its existence makes it likely that the absence of Hb Barts hydrops fetalis in blacks is due to the rarity of the chromosome lacking two alpha-globin genes rather than a result of early embryonic death due to the failure to synthesize embryonic hemoglobins because of deletion of functional zeta-globin genes.

We have defined the clinical and laboratory characteristics of a group of patients with HbS-beta o-thalassemia plus alpha-thalassemia, by analysis of erythrocyte indices, hemoglobin A2 and F levels, globin biosynthesis studies and alpha-globin gene mapping. Patients with HbS-beta o + alpha-thalassemia closely resembled individuals with HbS-beta o-thalassemia except for balanced globin synthesis ratios and a lower HbF level. The frequency of painful crises, leg ulceration, aseptic necrosis of bone and acute chest syndrome was similar in HbS-beta o + alpha-thalassemia patients and controls with sickle cell anemia (HbSS), HbSS-alpha-thalassemia and HbS-beta o-thalassemia. These findings are consistent with previous work which failed to show a reduction in the vaso-occlusive severity of sickle cell disease by the coexistence of alpha-thalassemia.

We examined globin chain synthesis in erythroid bursts (BFU-E) of patients with heterozygous alpha or beta thalassaemia. BFU-E were cloned from circulating mononuclear cells, labelled with [3H]leucine and globin chains purified by gel filtration and column chromatography. In six patients heterozygous for beta thalassaemia, globin synthesis in BFU-E was nearly balanced, with an alpha/non alpha ratio of 1.05 +/- 0.12. These BFU-E produced 33.8 +/- 12.7% gamma globin chain, an amount similar (P > 0.05) to that found in 10 controls with sickle cell anaemia (25.6 +/- 6.7) but greater (P > 0.05) than that of five normal controls (17.2 +/- 2.2). The balanced globin synthesis appeared due to the large amounts of gamma chain made by BFU-E. In two alpha thalassaemia carriers, who also had sickle cell trait, the BFU-E alpha/non-alpha ratio was 0.67 and 0.79. These BFU-E produced 15% and 20% gamma chain and 39% and 45% betaS globin. The synthesis of betaS globin in BFU-E exceeded the erythrocyte levels of 20% and 29% HbS and indicated nearly equal expression of betaA and betaB globin genes in these proliferating erythroid precursors. This provides further evidence that the low levels of HbS in sickle cell carriers with alpha thalassaemia are due to post-translational events resulting from the differing affinity of betaS and betaA globin for alpha chain and the destruction of excessive betaS chain.

We studied the growth of erythroid burst-forming units (BFU-E) and erythroid colony forming units (CFU-E) from bone marrow and blood in six patients with erythroleukemia. Five patients grew CFU-E, while BFU-E were found in the marrow of two and in the peripheral blood of only one patient. In all cases with colony growth, the numbers of colonies were markedly decreased with respect to normal controls. Patient BFU-E were composed of fewer clusters than those of controls. BFU-E and CFU-E growth was dependent on the addition of erythropoietin to the medium, and no growth was observed in absence of erythropoietin. At present it is not known if the growth obtained is derived from residual normal erythropoietic stem cells or from abnormal erythroid precursors of the leukemic cells.

Colonies derived from erythroid burst-forming units (BFU-E) synthesize fetal hemoglobin (HbF) in amounts that far exceed in vivo levels. There is some evidence that HbF synthesis is controlled at the level of a primitive erythroid precursor cell. Dexamethasone may potentiate the development of BFU-E. Since a means of augmenting HbF production in sickle cell anemia or severe beta-thalassemia would be of great therapeutic value, we studied the effects of dexamethasone on HbF and gamma-globin chain synthesis in BFU-E from patients with sickle cell anemia and controls. HbF was measured by radioimmunoassay of BFU-E lysate and gamma-chain synthesis by the incorporation of 3H-leucine into globin, which was then purified by gel filtration and column chromatography. Dexamethasone (10(-9) M) produced an increase in the number of BFU-E in 16 of 19 subjects when compared with numbers of BFU-E cultured with only erythropoietin. The individual BFU-E were larger and contained more subcolonies. Dexamethasone did not increase HbF or gamma-chain synthesis, and there was no relationship between proliferation of BFU-E and augmented HbF production. Thus, although dexamethasone augmented the development of erythroid bursts, there was no increment in HbF.

A rapid and simple procedure has been developed for the isolation of Plasmodium berghei parasites from infected-mouse erythrocytes employing the heat stable hemolysin produced by Pseudomonas aeruginosa. Using parasites isolated by this method, the presence of a parasite specific hexokinase has been demonstrated, providing an explanation for the increased glucose consumption observed with infected cells. Enzyme assays and serology were employed in determining the purity and yield of purified parasites. The enzyme assays showed that about 25% of the parasites in infected RBCs were recovered in the purified state. The purified parasites were not agglutinated by rabbit-anti- mouse RBC serum which indicated the purified parasites were not contaminated by RBC components.