Absence of beta mRNA in beta0-thalassemia in Kurdish Jews

We report the characterization of the amount of beta mRNA in a Kurdish Jewish population with beta0-thalassemia using the same methods employed for characterization of the Catania and Ferrara beta0 patients. We found very low amounts of beta mRNA sequences, consistent with the presence of beta0-thalassemia of the beta mRNA-negative population type. In addition, no globin gene deletion was detected that could account for the absence of beta mRNA.     

Nucleotide sequences of the 3'-terminal untranslated region of messenger RNA for human beta globin chain.

In normal messenger RNA for the human beta-globin chain, nucleotide sequences have been identified which can be matched to the amino-acid sequence of the abnormally long segment of the beta-chain of hemoglobin Cranston. The finding of these sequences strengthens the hypothesis that the betaCranston chain arose by a frameshift mutation allowing the "readthrough" of the normal termination codon and translation of usually untranslated portions of the messenger RNA for the beta-globin chain. The oligonucleotides which match the amino-acid sequence of hemoglobin Cranston provide a sequence of 36 nucleotides which follows the normal beta-chain termination codon UAA.     

Detection of sickle alpha- or beta0-thalassemia by studies of globin biosynthesis

Globin synthesis studies are useful in the analysis of thalassemia syndromes. We have applied globin synthesis and free alpha-chain pool studies of peripheral blood to characterize hematologic disorders where alpha- or beta-thalassemia was present in combination with HbS or HbC. In 60 non-thalassemic controls, the beta/alpha specific activity ratio was 1.01 +/- 0.06 (SD). In three patients with HbS-beta0-thalassemia, the (betas + gamma)/alpha ratios were 0.48-.067. In four patients with HbSS-alpha-thalassemia, the (BETAS/ALPHA RATIO was 1.26 +/- 0.18 (1.13- 1.53). The radioactive free alpha-chain pool in three patients with HbS- beta0-thalassemia was elevated (35.1%-53.0%), while three patients with HbSS-alpha-thalassemia had decreased free radioactive alpha-chain pools (3.2%-6.4%); both were significantly different from the mean (15.1% +/- 2.6%) of the 17 iron-sufficient controls. Simultaneous studies of the fraction of newly synthesized alpha chain contained in the free alpha- chain pool in peripheral blood and bone marrow demonstrated that this fraction was larger in peripheral blood than in marrow, and that the differences between thalassemia patients and controls previously found in bone marrow using these methods were also present in peripheral blood. The results indicate that even when family studies are not possible, patients with HbS in combination with alpha- or beta0- thalassemia can be differentiated from those with homozygous sickle cell disease by globin synthesis and free alpha-chain pool studies using peripheral blood.     

A 65 bp duplication/insertion in exon II of the beta globin gene causing beta0-thalassemia

We describe a patient originating from Ghana who had combined heterozygous alpha (4.2)thalassemia, alpha alpha alpha anti3.7 triplication, the common delta globin variant HbA2' and a new 65 bp duplication/insertion in exon II of the b globin gene causing beta (0)-thalassemia.     

Cloning and direct examination of a structurally abnormal human beta 0-thalassemia globin gene.

Restriction endonuclease mapping permitted identification of a form of beta 0-thalassemia in which a partial deletion of the beta-globin structural gene occurred [Orkin, S. H., Old, J. M., Weatherall, D. J. & Nathan, D. G. (1979) Proc. Natil. Acad. Sci. USA 76, 2400-2404]. To analyze its structure more directly, this abnormal human gene has now been cloned in bacteriophage lambda gtWES. Restriction mapping of the cloned gene and of a normal beta-globin gene contained in the phage H beta G1 confirmed previous findings regarding the presence of a deletion toward the 3' end of the gene but could not establish its position more accurately. Electron microscopy of the hybrid of the beta-thalassemia gene with globin RNA (R-loop analysis) provided unequivocal evidence for a deletion with the beta-globin structural gene. Hybridization of restriction fragments of the mutant gene with homologous fragments of H beta G1 (heteroduplex analysis) revealed a continuous, internal deletion of about 0.6 kilobase of DNA in the mutant gene and permitted its localization within the beta-globin gene region. This deletion removed the terminal third of the large intervening sequence within the beta-globin gene, the entire 3' coding block (extending from codon 105 to the end of the gene), and approximately 150 base pairs of DNA past the end of the normal globin gene.     

A mouse model for beta 0-thalassemia.

We have used a "plug and socket" targeting technique to generate a mouse model of beta 0-thalassemia in which both the b1 and b2 adult globin genes have been deleted. Mice homozygous for this deletion (Hbbth-3/Hbbth-3) die perinatally, similar to the most severe form of Cooley anemia in humans. Mice heterozygous for the deletion appear normal, but their hematologic indices show characteristics typical of severe thalassemia, including dramatically decreased hematocrit, hemoglobin, red blood cell counts, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration, as well as dramatically increased reticulocyte counts, serum bilirubin concentrations, and red cell distribution widths. Tissue and organ damage typical of beta-thalassemia, such as bone deformities and splenic enlargement due to increased hematopoiesis, are also seen in the heterozygous animals, as is spontaneous iron overload in the spleen, liver, and kidneys. The mice homozygous for the b1 and b2 deletions should be of great value in developing therapies for the treatment of thalassemias in utero. The heterozygous animals will be useful for studying the pathophysiology of thalassemias and have the potential of generating a model of sickle cell anemia when mated with appropriate transgenic animals.     

Stability of alpha and beta globin messenger RNA during induced differentiation of mouse erythroleukemia cells.

Murine erythroleukemia cells (MELC) are induced to express erythroid differentiation when cultured with hexamethylene bisacetamide (HMBA). Newly synthesized alpha and beta globin mRNA are both relatively stable, half-life (t1/2) greater than 50 hr, early in the course of induced differentiation. In fully induced cells there is a decrease in stability of both newly synthesized alpha and beta globin mRNA. The decay of alpha mRNA is faster, (t 1/2, 10--12 hr) than beta globin mRNA (t1/2, 20--22 hr). Thus, differences in stability of alpha and beta globin mRNA plays a role in determining the ratio of alpha to beta mRNA content in differentiated erythroid cells.     

Decreased globin messenger RNA in thalassemia detected by molecular hybridization

In previous studies of patients with β thalassemia, mRNA extracted from reticulocytes in peripheral blood when added to cell-free systems reproduces the deficient β-chain synthesis characteristic of intact cells. The present studies with specific probes for α and β mRNA were designed to decide whether the decreased β mRNA activity is due to the presence of abnormal or reduced β globin mRNA in these cells. Purified α and β complementary DNAs (cDNAs) have been synthesized with RNA-instructed DNA polymerase; α and β mRNAs isolated from heavy (β-producing) and light (α-producing) polyribosomes of rabbit reticulocytes were used as templates. Each of the cDNAs is more than 80% pure by the criterion of biological activity. The α cDNA labeled with [³²P]dCTP and the β cDNA labeled with [³H]dCTP have been added simultaneously to reaction mixtures containing various concentrations of mRNA from thalassemic and nonthalassemic subjects. The extent and rate of hybridization were determined, permitting a comparison of relative α and β mRNA content in the same annealing mixture. In six nonthalassemic patients, relatively equal amounts of hybridizable α and β mRNA appear to be present. In five of seven patients with β-thalassemia, significantly decreased amounts of β mRNA compared to α mRNA can be demonstrated. In two patients with Hemoglobin H disease, there is a decreased amount of α mRNA compared to β mRNA.     

"Silent" nucleotide substitution in a beta+-thalassemia globin gene activates splice site in coding sequence RNA.

A beta+-thalassemia globin gene was isolated from the genome of a Black individual by molecular cloning. DNA sequence analysis revealed only a single difference between this gene and the normal human beta-globin gene--adenine is substituted for thymine in the third position of codon 24. Codon 24 in both the normal gene (GGT) and the beta+-thalassemia gene (GGA) encodes glycine. The function of this beta+-thalassemia gene was compared to the function of the normal human beta-globin gene in monkey kidney cells by using plasmid expression vectors. The codon 24 substitution activates a 5' splice site that involves the guanine-thymine dinucleotide present in codon 25, 16 nucleotides upstream from the normal exon 1-intron I boundary. The splice, involving the abnormal 5' site in codon 25, is completed with the normal 3' splice site at the end of intron I. This splicing abnormality leads to a 75% decrease in the accumulation of normally processed beta-globin mRNA, thereby causing the beta+-thalassemia phenotype.     

Rapid HPLC techniques for globin chain synthesis studies

Globin chain synthesis and alpha/beta ratios were determined in a number of normal subjects, alpha-thalassemia-2 homozygotes, and beta-thalassemia trait using three different techniques. Cation exchange high performance liquid chromatography on a Pharmacia Mono-S, HR 5/5 and reversed phase high performance liquid chromatography on a semi-preparative Vydac C4 column were compared with the traditional carboxymethylcellulose chromatography. Both high performance liquid chromatography columns give excellent results when 2 mg of hemoglobin was chromatographed in each analysis. By modifying the protocols for column equilibration and gradient shape for preparative Vydac C4 columns, conditions were found yielding excellent resolutions of the labeled globin chains in less than an hour without the need for substantial increase of the flowrate. This method was found to be superior to other methods and may be a suitable alternative for the classical carboxymethylcellulose chromatography. Up to five specimens could easily be analyzed in a single day with this system.     

A patient of German descent with (delta beta)0-thalassemia carrying the Sicilian type deletion of the delta and beta globin genes

A deletion-type (delta beta)0-thalassemia with elevated production of fetal hemoglobin (Hb F) is described. The patient, homozygous for the disease, presented a clinical picture of beta-thalassemia intermedia. DNA analysis demonstrated that the deletion removed about 13 kb from the beta-globin cluster, including part of delta and the complete beta gene. The deletion appears to be identical to the previously described Sicilian deletion. Its presence in the homozygous state in a patient from Central Europe suggests that the deleted chromosome may be rather prevalent in that area.     

Phenotypic effect of heterozygous alpha and beta 0-thalassemia interaction

In this study, we carried out restriction endonuclease mapping in order to characterize the alpha-globin genotype of 10 Sardinian beta 0- thalassemia heterozygotes, all of whom presented with normal red blood cell indices and increased HbA2 levels. In 8 of these subjects, we found the deletion of two alpha-globin genes (-alpha/-alpha), and in the remaining two the deletion of a single alpha-globin gene (- alpha/alpha alpha). In three of these carriers with the (-alpha/-alpha) alpha-globin genotype and in one with the (-alpha/alpha alpha) genotype, we also found the glucose-6-phosphate dehydrogenase (G6PD) defect of the Mediterranean type. On the basis of these findings, we may conclude that the interaction of heterozygous beta 0-thalassemia with alpha-thalassemia, due to the deletion of either one or two alpha- globin genes, may lead to the production of red blood cells with normal indices. The association of the G6PD defect with this thalassemia gene complex may eventually contribute to this effect. We suggest, therefore, that screening programs for heterozygous beta-thalassemia in populations where alpha-thalassemia is also prevalent, should incorporate the determination of HbA2 in the first set of tests.     

Studies of globin chain synthesis and globin mRNA content in a patient homozygous for hemoglobin Lepore

Globin chain synthesis and globin mRNA content were studied in blood cells of a patient homozygous for Hb Lepore. Peripheral blood cells incubated with tritiated leucine synthesized approximately 1.5 to 3% as many Lepore globin chains as alpha chains. Globin mRNA in peripheral blood cell RNA was assayed by molecular hybridization assays using human alpha and beta cDNA, and the results indicated the presence of approximately 1% to 2% as much beta-like mRNA (presumably deltabeta Lepore mRNA) as alpha mRNA. The amount of Lepore deltabeta chain mRNA in peripheral blood cells is therefore proportional to the amount of Lepore globin chain synthesis in the same cells. An incidental observation was the finding that peripheral blood cell RNA of this patient, at a time when she was being heavily transfused, contained substantially higher levels of beta-like mRNA (relative to alpha mRNA) than in subsequent studies. Cell-free translation of this mRNA however revealed that it contained authentic beta chain mRNA which must have been derived in some way from the transfused blood cells.     

Molecular characterization of (deltabeta)(0)/beta(0)-thalassemia and (deltabeta)(0)-thalassemia/hemoglobin E in Thai patients

Two cases of the Thai thalassemia patients with compound heterozygosities for (deltabeta)(0)/beta(0)-thalassemia and (deltabeta)(0)-thalassemia/hemoglobin E have been reported. The first case was a 8-yr-old boy who had the following hematologic data: Hb 6.5 g/dL, Hct 20.5%, MCV 70.4 fL, MCH 22.3 pg and MCHC 31.7 g/dL. Hemoglobin analysis revealed 1.9% hemoglobin A2 and 91.7% hemoglobin F. The second case, with Hb 13.9 g/dL, Hct 41.5%, MCV 69.5 fL, MCH 22.5 pg and MCHC 32.2 g/dL, was a 16-yr-old male who had 46.1% hemoglobin E and 49.8% hemoglobin F. Globin gene analyses showed that both probands carried the same deletional type (deltabeta)(0)-thalassemia trans to the 4 bp deletions in codons 41/42 beta(0)-thalassemia and to the betaE-globin gene, respectively. Polymerase chain reaction and DNA sequence analyses demonstrated that the 5' breakpoint of the (deltabeta)(0)-thalassemia deletion was located in the second intron of the delta-globin gene and that the 3' breakpoint lay within a cluster of LI repetitive sequences at 4.7 kb 3' to the beta-globin gene.