The interaction of α thalassaemia with heterozygous β thalassaemia

S ummary . The α globin genotypes of 55 β thalassaemia heterozygotes have been determined by restriction endonuclease analysis to identify those with interacting α thalassaemia genes. A comparison of the haematological and haemoglobin synthesis findings of individuals with normal α genotypes (αα/αα) with those with one (- α/αα) or two (-α/-α) α genes deleted shows that the latter two groups have more balanced globin chain synthesis ratios, higher haemoglobin levels, and larger, better haemoglobinized red cells. This suggests that the degree of globin chain imbalance is a significant factor in determining the red cell characteristics in heterozygous β thalassaemia. Screening programmes for thalassaemia, based on the detection of low MCVs, could miss cases of the interaction of α and β thalassaemia.     

Improvement of mouse β thalassaemia by hydroxyurea

Summary. The present report provides evidence that hydroxyurea (HU) improves the β 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 β minor/α 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 α chains decreased, the proportion of spectrin and ankyrin increased and red cell deformability also increased.     

Characterization of an Indian (δβ)° thalassaemia

The molecular basis of delta beta thalassaemia in an Indian family is shown here to be due to a previously undescribed deletion within the beta globin gene complex. Starting 3 kilobases from the 3' end of the A gamma gene, the deletion removes the delta and beta globin genes and continues to an unknown extent in the 3' direction. Heterozygotes for this deletion have about 25% Hb F with a G gamma:A gamma ratio of 70:30 while interaction with beta+ thalassaemia results in the clinical picture of thalassaemia intermedia.     

The Corfu δβ thalassaemia mutation in Greece: haematological phenotype and prevalence

The Corfu delta beta thalassaemia mutation, a 7.2 kb deletion partially removing the delta-globin gene and a single nucleotide mutation (G----A) at intervening sequence I (IVSI-n5) in the beta-globin gene in cis, was first described in a family from Corfu; the carriers for this mutation had the unusual haematological phenotype of heterozygous beta-thalassaemia with normal levels of HbA2. To investigate the frequency and haematological characteristics of Corfu delta beta thalassaemia in Greece we analysed 25 unrelated normal HbA2 type 2 beta-thalassaemia heterozygotes and their 23 clinically affected offspring. Gene mapping demonstrated that nine (36%) of the 25 normal HbA2 beta-thalassaemia heterozygotes were in fact Corfu delta beta thalassaemia heterozygotes and of the 23 patients, two were Corfu delta beta thalassaemia homozygotes and five compound heterozygotes for Corfu delta beta thalassaemia and another beta-thalassaemia defect. Detailed haematological analysis demonstrated that: the Corfu delta beta thalassaemia mutation does not completely abolish the expression of the beta-globin gene; the HbA2 levels are slightly lower (P less than 0.01) and the HbF levels slightly higher (P less than 0.01) in Corfu delta beta thalassaemia heterozygotes compared to beta-thalassaemia heterozygotes with the normal HbA2-type 2 phenotype who do not have the Corfu delta beta chromosome.     

The spectrum of β-thalassaemia mutations in Sicily

To characterize beta-thalassaemia genes among the Sicilian population we have previously determined the DNA haplotypes in the beta-globin gene cluster of 99 beta-thal chromosomes. We found seven haplotypes, although 95 of 99 beta-thal chromosomes contained framework 1 and framework 3 beta genes. We have now determined the mutation in all 99 of these beta-thal genes by the use of oligonucleotide hybridization. PCR-amplification and direct genomic sequencing, and direct restriction analysis. Our results indicate that (1) the beta (0)-39 mutation is most frequent (35%); (2) beta(0)-39, IVS-1 nt 110 and IVS-1 nt 6 mutations account for 90% of beta-thal genes: (3) the IVS-1 nt 6 mutation is more frequent in thalassaemia intermedia (77%) than in Cooley's disease (34%): (4) the association between haplotypes and specific mutations is imperfect, but mutation spread has occurred within haplotypes containing the same beta-gene framework: (5) the beta(0)-39 and the IVS-1 nt 6 mutations, with a mutation spread to two major haplotypes, may be older than the IVS-1 nt 110 mutation: (6) these data make possible first-trimester prenatal diagnosis in many families (85%) in Sicily using only three pairs of oligonucleotides. In addition, a new mutation, a frameshift at codon 76 due to loss of a C residue, was found in a single beta-thal chromosome.     

Erythrokinetics and iron status in heterozygous β thalassaemia, and the effect of interaction with α thalassaemia

Ferrokinetic studies, alpha globin gene mapping, and assessment of iron status have been carried out in 16 healthy subjects with heterozygous beta thalassaemia. Six subjects had coinherited alpha thalassaemia and had more balanced alpha/beta globin chain synthesis ratios than the remaining 10 subjects with uncomplicated heterozygous beta thalassaemia. The overall efficiency of erythropoiesis was significantly reduced in the latter group (mean 76 +/- 17 (SD)% of normal), but was indistinguishable from normal in subjects with coexistent alpha thalassaemia. Red cell survival was unimpaired in both groups, indicating that the defect was one of mild ineffective erythropoiesis rather than peripheral haemolysis. Values for total plasma iron turnover were normal or only slightly increased. This suggests a lack of any additional stimulus to erythropoiesis, which might normally be expected to compensate easily for the mild degree of anaemia. Uncomplicated heterozygous beta thalassaemia produces an extremely mild disorder of erythropoiesis, which is dependent on the imbalance between alpha and beta globin chain synthesis, and is not associated with a risk of serious iron overload.     

Thalassaemia intermedia in Cyprus: the interaction of α and β thalassaemia

Restriction endonuclease analysis has been performed on the alpha and beta globin gene clusters of 57 Cypriots homozygous for beta thalassaemia, 30 with the transfusion dependent form of the condition (thalassaemia major) and 27 who are less severely affected (thalassaemia intermedia). There was a significant difference in the incidence of alpha thalassaemia between the two groups: 14/27 of the patients with thalassaemia intermedia also had deletion forms of alpha thalassaemia, while only 4/30 of the patients with thalassaemia major were similarly affected. Thus in Cypriot patients who are homozygous for beta thalassaemia the co-inheritance of alpha thalassaemia is an important factor in determining the clinical course.     

Severe thalassaemia intermedia caused by interaction of homozygosity for α-globin gene triplication with heterozygosity for β thalassaemia

Summary A 3-year-old child was evaluated for β-thalassaemia intermedia. Molecular characterization including β-globin gene sequence analysis revealed heterozygosity for a single β-thalassaemia mutation, IVSI nt1 (GA). In addition the patient was found to be homozygous for α-globin gene triplication (ααα^anti3,7/ααα^anti3,7). The propositus has a significantly more severe phenotype than has been previously reported with this combination of genetic defects. In contrast, four individuals heterozygous for both triplicated α and for β-thalassaemia had a phenotype of thalassaemia minor, and a fifth had very mild thalassaemia intermedia.     

Association of a novel high oxygen affinity haemoglobin variant with δβ thalassaemia

We report an uncommon association of δβ thalassaemia and a haemoglobin (Hb) variant with high oxygen affinity in an Asian Indian family. Minimal polycythaemia was seen in a heterozygote for this novel Hb variant, Hb Headington (β72 (E16) Ser→Arg), while compound heterozygosity for Hb Headington and the Indian ^Gγ (^Aγδβ) thalassaemia produces a marked increase in erythrocytosis with a concomitant increase in the level of the variant Hb. The HbF in such compound heterozygotes remains at a level consistent with that usually observed in individuals heterozygous for the ^Gγ (^Aγδβ)° thalassaemia alone. The purified Hb variant showed an increased oxygen affinity, moderately decreased co-operativity and a normal Bohr effect. Results of functional studies suggest that the high oxygen affinity of Hb Headington is due to the Ser→Arg substitution which disrupts the normal and tight interaction between A. B and E helices leading to a destabilization of the T deoxy-structure of the abnormal haemoglobin.     

Molecular characterization of β-thalassaemia in 174 Greek patients with thalassaemia major

The mutations producing beta-thalassaemia in 174 Greek patients with thalassaemia major were investigated by dot-blot hybridization of oligonucleotide probes to genomic DNA amplified by the polymerase chain reaction procedure, by direct sequencing of amplified DNA, and by gene mapping. beta-thalassaemia in Greeks was found to be very heterogeneous at the molecular level as 17 different mutations were observed: 86.6% of the beta-thalassaemic genes, however, could be identified with five probes: IVS-I-110 (G----A) (42.5%), codon 39 (C----T) (17%), IVS-I-1 (G----A) (13.2%), IVS-I-6 (T----C) (7.2%) and IVS-II-745 (C----G) (6.9%). Several mutations which had not previously been reported in the Greek population and which occurred at an incidence of 2% or lower were observed in this study. The information obtained will facilitate the prenatal diagnosis of beta-thalassaemia in Greece.     

Diagnosis of thalassaemia by non-isotope detection of α/β and ζ/α mRNA ratios

Summary. The α/β and ζ/α messenger RNA (mRNA) ratios in the thalassaemia syndromes were investigated by polymerase chain reaction (PCR) with silver staining of the PCR products. In this study we used the PCR to amplify cDNA copies of circulating erythroid cell mRNA in order to measure the relative amounts of α-, β- and ζ-globin contained within. Quantitation was performed by scanning the silver stain of specific globin cDNA bands. We found that there were significant differences of α/β-mRNA and ζ/α-mRNA in patients with Hb H disease and α-thalassaemia-1 compared to normal subjects. There was a marked increase in the α/β-mRNA ratio but not in the ζ/α-mRNA ratio in patients with β-thalassaemia. In two β-thalassaemia cases abnormal increases of ζ-globin bands were noted and they were confirmed through DNA analysis to be combined with α-thalassaemia-1. This method provides a simple, rapid and non-radioactive approach to detect thalassaemia syndromes, and can help to screen cases of β-thalassaemia with α-thalassaemia-1.     

α- and β-haemoglobin chain induced changes in normal erythrocyte deformability: comparison to β thalassaemia intermedia and Hb H disease

Summary The α and β-thalassaemias are characterized by decreased erythrocyte deformability. To determine what effects excess α and β-haemoglobin (globin) chains have on cellular and membrane deformability, purified haem-con-taining α and β-chains were entrapped within normal erythrocytes. Entrapment of purified α-chains in normal erythrocytes resulted in a significant decrease in cellular and membrane deformability similar to that observed in β-thalassaemia intermedia. The decreased deformability was correlated with α-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 α-chain deposition. The membrane changes and the loss of membrane deformability appeared to account for the loss of cellular deformability in the α-chain loaded cells. While both β-chain loaded and Hb H erythrocytes demonstrated a significant loss of cellular deformability, this loss was less pronounced than in the α-chain loaded and β-thalassaemic cells and may arise from either the increased intracellular viscosity of the β-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.     

Molecular analysis of the Turkish form of deletion-inversion (δβ)° thalassaemia

Two unrelated (δβ)°-thalassaemia patients from Southern Turkey are presented. DNA studies indicated that both of them are homozygous for the Turkish type of (δβ)°-thalassaemia characterized by one large deletion of 11.5 kb including the δ and β globin genes at the 5' end and one small deletion of 1.6 kb at the 3' end, which are separated by an inverted 7.6 kb long DNA segment that includes L1 repetitive sequence. In the present study a PCR-based method was performed to produce a unique deletion-specific product and subjected to sequence analysis for the determination of the breakpoint. DNA polymorphisms in the β-globin gene cluster of deletion-inversion type of (δβ)°-thalassaemia, IVS-I-6 and β-39 globin genes were examined. Analysis of sequence variations in regulatory regions including the 5' hypersensitive site-2 of the locus control region (LCR), the δ, ^Gγ and ^Aγ 5' flanking regions and the second intervening sequence (IVS-II) of ^Aγ and ^Gγ genes indicated the presence of close similarities between the chromosome carrying the Turkish form of deletion-inversion δβ)°-thalassaemia and the chromosome associated with β-39 nonsense mutation in haplotype II. These two chromosomes are characterized by the presence of a 4 base pair deletion in the ^Aγ^T globin gene promoter. A C → T alteration at position −199 5' to the δ gene was also found to be associated with the Turkish type of (δβ)°-thalassaemia and β-39 chromosome.     

Molecular characterization of β-globin gene mutations in Malay patients with Hb E-β-thalassaemia and thalassaemia major

This study concerned the identification of the beta-thalassaemia mutations that were present in 27 Malay patients with Hb E-beta-thalassaemia and seven Malay patients with thalassaemia major who were from West Malaysia. Nearly 50% of all beta-thalassaemia chromosomes carried the G----C substitution at nucleotide 5 of IVS-I; the commonly occurring Chinese anomalies such as the frameshift at codons 41 and 42, the nonsense mutation A----T at codon 17, the A----G substitution at position -28 of the promoter region, and the C----T substitution at position 654 of the second intron, were rare or absent. Two new thalassaemia mutations were discovered. The first involves a frameshift at codon 35 (-C) that was found in two patients with Hb E-beta zero-thalassaemia and causes a beta zero-thalassaemia because a stop codon is present at codon 60. The second is an AAC----AGC mutation in codon 19 that was present on six chromosomes. This substitution results in the production of an abnormal beta chain (beta-Malay) that has an Asn----Ser substitution at position beta 19. Hb Malay is a 'Hb Knossos-like' beta +-thalassaemia abnormality; the A----G mutation at codon 19 likely creates an alternate splicing site between codons 17 and 18, reducing the efficiency of the normal donor splice site at IVS-I to about 60%.     

Arterial elastorrhexis in β-thalassaemia intermedia, sickle cell thalassaemia and hereditary spherocytosis

Arterial and stromal elastorrhexis, an elastic tissue disorder, was recently described in beta-thalassaemia major. Histopathological material from 10 patients with thalassaemia intermedia, 14 with sickle cell thalassaemia and 18 with hereditary spherocytosis was examined in order to investigate the specificity of the arteriopathy. Histological re-examination was made in a total of 42 spleens with parasplenic lymph nodes in 14 cases, 26 surgical liver biopsies and 16 gallbladders with associated regional lymph nodes. Arteriopathy, qualitatively similar to that seen in beta-thalassaemia major, was found in up to 90% of extrasplenic muscular arteries. Elastorrhexis lesions were also found in intrasplenic arteries and in stromal elastic tissue of spleens and parasplenic lymph nodes, in the absence of tissue iron overload. The arteriopathy appears in the first decade of life even in spleens of normal weight, and seems unrelated to the severity of permanent anaemia. It is suggested that patients suffering from hereditary chronic haemolytic diseases are subject to an elastic tissue disorder which is similar to hereditary pseudoxanthoma elasticum, the earliest and most frequent manifestation of which is arterial elastorrhexis of muscular extrasplenic arteries.     

The C–T substitution in the distal CACCC box of the β-globin gene promoter is a common cause of silent β thalassaemia in the Italian population

This paper describes four families of Italian descent in each of which the propositus had the clinical phenotype of thalassaemia intermedia, resulting from the compound heterozygous state for high HbA2 beta thalassaemia and type I silent beta thalassaemia. Direct sequencing on amplified DNA and/or oligonucleotide analysis detected, in all families but one, the compound heterozygous state for codon 39 nonsense mutation and the C-T substitution at position -101 in the distal CACCC box of the beta-globin gene promoter (beta th-101). Members of these families who are heterozygous for high HbA2 beta thalassaemia showed the codon 39 nonsense mutation, while those with the clinical phenotype of silent beta thalassaemia had the beta th-101 mutation. In the remaining family, the propositus and one of his siblings had the compound heterozygous state for a molecularly undefined high HbA2 beta thalassaemia and the beta th-101 mutation in combination with the triple alpha globin gene arrangement. These patients showed a more severe thalassaemia intermedia like clinical phenotype as compared to those with the same beta-globin genotype and a normal alpha-globin gene arrangement. In the families investigated the beta th-101 was always associated with haplotype I. A group of patients with thalassaemia intermedia from Southern Italy, either homozygous or heterozygous for haplotype I and in whom previous studies had failed to define the mutation in one of the beta thalassaemia globin genes, were screened by oligonucleotide analysis for the presence of the beta th-101. Three out of nine were positive. These results indicate that the beta th-101 mutation is a common cause of the type I silent beta thalassaemia phenotype in the Southern Italian population.     

The molecular basis of β thalassaemia in Punjabi and Maharashtran Indians includes a multilocus aetiology involving triplicated α-globin loci

Summary We have analysed 201 β-thalassaemia (β-thal) genes from natives of the Punjab (156) and Maharashtra states of India and found the causative mutation in 200 of them. The most common β-globin gene mutations differed significantly between these two groups and between these groups and Indian immigrants in the U.S.A. and the U.K. In the Punjabi Indians the IVS-1, nt 1 (G–T) mutation accounted for nearly one-quarter of β-thal genes, whereas it was 5% or less in the other groups. Likewise, the cap+ 1 mutation was much more prevalent in the Punjabis, whereas the nonsense codon 15 allele had a higher frequency in the Maharashtrans of the Bombay region. The common IVS-1, nt5 allele had a frequency of 60% of β-thal genes in the Maharastrans, 35% in North American immigrants, and only 23% in the Punjabis. Two-thirds of all β-thal genes in Punjab were found in the merchant caste (Khatri-Arora), whereas the menial caste (Shudra) was highly represented among those with β-thal genes in Maharashtra. Two novel β-globin alleles were each found once; a frameshift codon 55 (+ A) in Maharashtrans and a frameshift codons 47–48 (+ ATCT) in Punjabis.
Of three Punjabi patients with β-thal intermedia in whom only a single severe β-globin gene mutation was found, two had six α-globin genes (homozygosity for a triplicated α-globin locus) instead of the normal α-globin gene number of four. Thus, these two individuals had a multilocus aetiology of β-thal and their parents have the unusual recurrence risk of 1 in 8 for conceiving a third with β-thal intermedia. Since 15% of 126 α-globin clusters studied in Punjabis contained either single (10%) or triplicated (5%) α-globin genes, the α-globin gene number is a frequent modifier of the phenotype of β-thal in this ethnic group.     

Feasibility of prenatal diagnosis of β thalassaemia by DNA polymorphisms in an Italian population

A feasibility study of prenatal diagnosis of beta thalassaemia in a northern Italian population has been carried out. Twenty-five families have been studied, each consisting of two parents and a homozygous beta thalassaemia child, thus enabling linkage analysis of restriction fragment length polymorphisms (RFLPs) to the normal and the thalassaemic chromosomes. Using seven standard RFLPs, 19/25 families could be offered prenatal diagnosis; inclusion of the recently described Ava II psi beta polymorphism increased this figure to 23/25 (92%) of the families.     

Immune function in patients with β thalassaemia receiving the orally active iron-chelating agent deferiprone

Short-term deferiprone may reduce body iron in some patients with thalassaemia major. Concerns regarding potential immunosuppressive effects of deferiprone have been raised from results of animal studies and case reports in humans. We studied immune function in 57 thalassaemia patients: 36 treated with deferiprone (L1; CP020) and 21 treated with desferrioxamine (DFO). Circulating B lymphocytes were increased in all patient groups. No differences were detected between treatment groups in percentages of circulating lymphocytes, concentrations of IgG, IgM or IgA, specific antibody titres, complement levels, or in vitro lymphocyte proliferation. No clinically important infections were observed in any patient. These data suggest that no clinical or laboratory changes consistent with immunosuppression or immunodeficiency are observed during deferiprone therapy.