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.     

Disparate lympho-erythroid donor to recipient chimaerism in a β°-thalassaemia bone marrow transplant recipient with red cell indices indicative of apparent full engraftment

A 4-year-old girl with transfusion-dependent β°-thalassaemia received an HLA-identical bone marrow transplant (BMT) from her β°-thalassaemia trait sister. Prior to BMT, chromosomal analysis revealed the recipient to have 46,XX,9qh+, a polymorphic variant of the heterochromatin region of chromosome 9, which her donor did not have. Within 1 month post-BMT, 89% of nucleated bone marrow cells were of donor origin. One year later, donor engraftment had decreased to 44% and 34% in nucleated bone marrow cells and blood lymphocytes, respectively. By 2 years, donor lymphocyte engraftment fell to 5%, raising concern of possible graft rejection. To examine erythroid chimaerism, globin synthesis by individual erythroid progenitor cell derived colonies (BFU-E) was analysed. On days 1000 and 1130 post-BMT, 79% and 77% of colonies, respectively, synthesized β-globin and therefore were of donor origin.     

Homozygous β-thalassaemia resulting in the β-thalassaemia carrier state phenotype

Summary. This paper describes the phenotypic manifestations of a very mild β-thalassaemia mutation detected in several members of two families of Italian descent. The molecular defect, defined by denaturing gradient gel electrophoresis analysis and direct sequencing. consists of a C G substitution at position 844 of IVSII of the β-globin gene within the consensus sequence of IVSII acceptor splice site. Heterozygotes for this mutation show a haematological phenotype ranging in severity from silent β-thalassaemia to that of a mild β-thalassaemia carrier silent β-thalassaemia to that of a mild β-thalassaemia carrier state, whereas homozygotes have the typical manifestations commonly resulting from heterozygosity for a β-thalassaemia mutation. Compound heterozygotes for the IVSII nt844 (C G) mutation and a severe β-thalassaemia mutation have the phenotype of thalassaemia intermedia.
This paper indicates that the presence of borderline red blood cell indices or HbA₂ values should make one suspect the presence of a very mild or silent β-thalassaemia.     

A novel deletion causing (epsilon gamma delta beta) degrees thalassaemia in a Chilean family

We describe a novel deletion causing (epsilongammadeltabeta) degrees thalassaemia segregating in three generations of a Chilean family of Spanish descent. Heterozygotes for the deletion were all affected by neonatal haemolytic anaemia. The deletion of 152,569 bp extends from 77 kb upstream of the epsilon gene to 31 kb downstream of the beta gene, and includes the entire beta-globin gene cluster and two upstream olfactory receptor genes. Comparison of the sequences of the deletion junction with those of the flanking normal DNA suggests that the deletion results from a non-homologous recombination event. The insertion of 16 'orphan' nucleotides in the deletion junction creates a perfect inverted repeat of 12 nucleotides, forming a 12-bp stem with a four-nucleotide loop that could have contributed to the illegitimate recombination. The 3' breakpoint is located within an L1 family repeat that contains a perfect 160-bp palindrome, and is in close proximity to the 3' breakpoints of five other deletions in the beta cluster - Indian (HPFH-3), Italian (HPFH-4) and Vietnamese GgammaAgamma (deltabeta) degrees HPFH, German and Belgian Ggamma (Alphagammadeltabeta) degrees thalassaemia.     

A novel amber mutation in a β°-thalassaemia gene (β37TGG→TAG), with direct detection by mapping the restriction fragments in amplified genomic DNA

Summary. A novel amber mutation, a G to A substitution at the second position of codon 3 7 in the β-globin gene that changes the tryptophan coding triplet (TGG) to a termination codon (TAG), was found in a Chinese β-thalassaemia carrier. The mutant gene creates an additional Dde I recognition site and eliminates the Ava II site, so this point mutation can be directly identified by restriction enzyme analysis.     

Clinical and laboratory effects of long-term administration of hydroxyurea to patients with sickle-cell/β-thalassaemia

Hydroxyurea (HU), a widely used cytostatic, has been given over a long period of time to 14 adult Caucasian compound heterozygotes for β^s and various β-thalassaemia genes. All patients had severe pain crises and other complications prior to receiving the drug. After 4-8 weeks on high 'sub-toxic'doses of HU all patients responded with a multifold increase of fetal haemoglobin (HbF) and a marked increase of MCV and MCH; they also felt significantly better and ceased having pains or other complaints. Haematological toxicity was minimal and rapidly reversible. Follow-up of the patients has now exceeded 100 weeks and goes up to 180 weeks in two of them. Pain crises have never recurred. Maintenance of high levels of HBF requires continuous administration of high doses of HU; whenever the latter were decrease in various attempts to avoid potential long-term toxicity, the observed changes gradually faded. The effect of HU in HbS/β-thalassaemia may be better than that reported for homozygous HbS disease because the synthesized λ-chains not only inhibit the sickling process but they also neutralize the noxious effects of the excess α-chains and cut down the ineffective erythropoiesis of the patients.     

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.     

A novel β-thalassaemia mutation in the 5'untranslated region of the β-globin gene

Summary. A thymidine deletion at position +10 of the 5'untranslated region of the β-globin gene was detected in a β-thalassaemia intermedia patient carrying a β; 39 stop codon mutation on the other chromosome; this new mutation, +10(-T), was detected by automated fluorescent DNA sequencing and verified by dot-blot allele-specific hybridizations.
The +10(-T) mutation is a 'silent carrier', is associated with a reduced amount of steady-state β-globin mRNA, and establishes a connection between the 5'untranslated region of the β-globin gene and the regulation of its expression.     

Severe inclusion body β-thalassaemia with haemolysis in a patient double heterozygous for β°-thalassaemia and quadruplicated α-globin gene arrangement of the anti-4.2 type

We describe a new case of an association of alpha-globin gene quadruplication of the anti-4.2 type with beta(0)-thalassaemia. The patient, a young woman of mixed Brazilian-Portuguese origin, suffered from chronic haemolytic anaemia with splenomegaly. Bone marrow supravital staining with brilliant cresyl blue and electron microscopy studies showed large inclusion bodies in about 3% of erythroblasts. Upon immunofluorescent staining these inclusions reacted with a monoclonal antibody to alpha- but not to beta-globin. Analysis of alpha-globin cluster by Southern blotting showed the presence of pathologic fragments specific for the anti-4.2 alpha-globin gene quadruplication. Alpha/beta mRNA ratio was higher than in cases combining alpha-globin triplication and beta(0)-thalassaemia or in cases of beta(0)-thalassaemia heterozygous state alone (18, 14.7 and 10.1 respectively). Our data confirmed the hypothesis that the clinically detectable haemolysis in this beta(0)-thalassaemic patient was due to an unusually high amount of precipitated alpha-globin in erythroid precursors. This considerable excess of alpha-globin chains was due partly to the beta-globin deficit caused by the presence of the beta(0)-thalassaemic gene, but also to the presence of 6 active alpha-globin genes resulting from alpha-globin gene quadruplication in one chromosome.     

Possible factors influencing the haemoglobin and fetal haemoglobin levels in patients with β-thalassaemia due to a homozygosity for the IVS-I-6 (T→C) mutation

Summary. We have collected haematological, haemoglobin (Hb) and DNA sequence data for 29 patients with a homozygosity for the IVS-I-6 (TC) mutation with the intention of identifying factors contributing to the observed variability in the severity of the disease. None of the patients had received blood transfusion therapy for at least 6 months prior to the study. Hb levels varied from 5·0 to 9·9 g/dl. Patients with high Hb F (more than 1·5 g/dl or <20%) had high total Hb levels (7·5–9·7 g/dl) but some with low Hb F also had high total Hb levels; two had a concomitant α-thalassaemia-2 (α-thal-2) heterozygosity. An inverse correlation between the Hb F and Hb A₂ levels was observed. The majority of the patients were homozygous for haplotype VI (49/58 chromosomes) but haplotypes IV (2/58) and VII (7/58) were also present. The only haplotype IV homozygote had high Hb F levels with high ^Gγ values and the CT mutation at position – 158 in the ^Gγ promoter, while both high and low Hb F levels were observed among patients with haplotypes VI and VII. Analysis of sequence variations in regulatory regions included the 5 hypersensitive sites (HS) 4, 3 and 2 of the locus control region (LCR), the ^Gγ and ^Aγ 5 flanking regions, the second intervening sequence (IVS-II), and the 5 β-globin gene region in two patients with high Hb F (one homozygote each for haplotypes VI and IV), and in two patients with low Hb F levels (one homozygote each for haplotypes VI and VII). Haplotype specific differences were observed in the LCR 5 HS-2 and in the ^Gγ and ^Aγ flanking and IVS-II regions; however, no differences were present between the low and high Hb F-producing haplotype VI chromosomes, suggesting a major role for factors which are not linked to the β-globin gene cluster in mediating γ-globin gene expression in patients with this type of β-thal.     

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.     

Enhanced adherence of β-thalassaemic erythrocytes to endothelial cells

The adherence of red blood cells (RBC) to endothelial cells (EC), shown to correlate with microvascular occlusion in sickle cell disease and malaria, is considered a major contributor to microcirculatory disorders. In the present study the adherence to EC was markedly enhanced with RBC from beta-thalassaemia major (TM) patients, and even more so with RBC from beta-thalassaemia intermedia (TI) patients (10-fold and 25-fold higher than normal, respectively). It is proposed that enhanced RBC/EC adherence may contribute to the microcirculatory disorders observed in thalassaemia, especially in TI patients who are particularly known to suffer from leg ulcers.     

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.     

Rapid diagnosis of β-thalassaemia by mutagenically separated polymerase chain reaction (MS-PCR) and its application to prenatal diagnosis

Summary. We have developed a rapid and simple PCR-based method which is modified from the mutagenically separated polymerase chain reaction (MS-PCR) to detect the molecular defects of β-thalassaemia. We can use this technique to amplify normal and mutant alleles of the β-globin gene in the same reaction tube, using different-sized allele-specific primers. This mutagenesis separates the amplification reactions of alleles performed in the same tube, Subsequent gel electrophoresis shows at least one of the two allelic products at the same locus or at least two of the several allelic products at different loci. Therefore, in addition to simple handling, MS-PCR provides a within-assay quality control for the exclusion of false negative results. The five most common mutations of β-thalassaemia and haemoglobin E which occur in the Taiwanese population were tested, and 14 prenatal samples were checked with accurate results. This method is simple, rapid and accurate, and can be used routinely in prenatal diagnosis. The principle used here can also be applied to other genetic diseases.     

α-Thalassaemia in the population of Cyprus

We have determined the α-thalassaemia (α-thal) determinants in 78 patients with Hb H disease from Cyprus; 25 were Turkish Cypriots and 53 were Greek Cypriots. Four deletional and three non-deletional α-thal alleles were present; the -α(3.7 kb) α-thal-2 and the —^MED-1α-thal-1 were most frequently seen; —^MED-II and -(α)^20.5 deletions occurred at considerably lower frequencies. About 15% of all chromosomes carried a non-deletional α-thal-2 allele; of these the 5 nucleotide (nt) deletion at the first intervening sequence (IVS-I) donor splice site was present in ˜ 8% of all chromosomes. Two types of polyadenylation signal (poly A) mutations were observed. No striking frequency differences were seen between Greek and Turkish Cypriot patients. Combinations of the various types of α-thal resulted in eight different forms of Hb H disease. The phenotypes were comparable except for great variations in the level of Hb H which was highest (average ˜ 22%) in the 12 patients with the α^5ntα/—^MED-I combination. One patient with the same form of Hb H disease but with an additional β-thal (IVS-I-110, G → A) heterozygosity had a most severe microcytosis and hypochromia with < 1% Hb H. Variations in the level of Hb H might correlate with the severity of the disease, although this was not evident from the haematological data.     

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.     

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.