Five haplotypes in Black β-thalassaemia heterozygotes: three are associated with high and two with low Gγ values in fetal haemoglobin

Genotypes at seven different polymorphic restriction sites (5'to the ° gene, at the ^G γ, at the ^A γ, at the Ψβ , 3'to the Ψβ , at the β , and 3'to the β genes) were analysed by restriction endonuclease mapping of the DNA from 66 Black β -thalassaemia heterozygotes from Georgia and several of their normal relatives. Five different haplotypes were observed. Three of these were associated with high ^G γ values in the small amount of Hb F (0.8-8.3%) present in the blood of these patients and two with low ^G γ values. One haplotype [- + - ++++] that occurred on two of every three β thalassaemia chromosomes was associated with high ^G γ levels, and is the same as that found in some Black SS patients also having high ^G γ values (Gilman & Huisman, 1984). Two others [- ++ - + - +] and [−+−−+++] were also associated with high ^G γ, while two [−−−−+++] and [+−−−−++] were associated with low ^G γ. Variation in haematological data, mainly MCV and MCH values, was found to be caused in part by the type of β -thalassaemia (defined by its haplotype) and by the presence of an additional α-thalassaemia-2 heterozygosity or homozygosity.     

Haematological and clinical features of beta-thalassaemia associated with Hb Dhofar

Hb Dhofar is a variant haemoglobin (β^{29 (GGC–GGT) gly-gly}, β^{58 (CCT–CGT) pro-arg}) associated with a thalassaemic phenotype and unique to the Sultanate of Oman. We report clinical and haematological data on 54 subjects with Hb Dhofar (37 heterozygotes, 14 homozygotes and three compound heterozygotes with a different β-thalassaemia mutation). In heterozygotes, the level of Hb Dhofar ranged from 8.8% to 21.5%. All heterozygotes had Hb A₂ > 3.5%, consistent with β-thalassaemia trait. Hb Dhofar in homozygotes and compound heterozygotes ranged from 26% to 59.7%, with a peripheral film consistent with homozygous β-thalassaemia. Age at presentation in homozygotes ranged between 6 months and 8 yr, with a majority presenting before 5 yr of age. All had splenomegaly and six (43%) had undergone splenectomy. All had some degree of frontal bossing and in particular, two patients with infrequent transfusions had marked thalassaemic facies and stunting of growth. Hb Dhofar can be mistaken for Hb D as the electrophoretic mobility is similar, but differs from it by a variable and reduced quantity of variant Hb in both heterozygotes and homozygotes. Clinical and haematological data suggest that this mutation behaves like a moderately severe β⁺ thalassaemia allele resulting in a thalassaemia intermedia phenotype.     

The interaction of α° thalassaemia with Hb Icaria: three unusual cases of haemoglobinopathy H

The clinical, haematological, biosynthetic and molecular data of three Greek haemoglobin H (HbH) disease patients with a distinctive clinical phenotype are described. During infancy all three patients had unusually severe clinical manifestations for HbH disease, with anaemia necessitating blood transfusions, signs of bone changes, growth impairment, and splenomegaly. Molecular analysis identified a rare α-thalassaemia genotype (− −^Med/α ^Icα) . Splenectomy resulted in marked amelioration of the clinical signs; post splenectomy all three patients preserve adequate haemoglobin levels (9–10 g/dl) with growth restored to normal. Despite the initial severe clinical phenotype in these patients, our experience indicates that splenectomy modifies the clinical course to that of mild thalassaemia intermedia. This observation should be considered carefully when giving genetic counselling to families carrying the rare Hb Icaria mutation and an α° thalassaemia mutation.     

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.     

Atypical HbH disease in a Surinamese patient resulting from a combination of the −SEA and −α3.7 deletions with HbC heterozygosity

The first case of haemoglobin H (HbH) disease in combination with haemoglobin C (HbC) is reported in a man of Surinamese origin. Only haemoglobin A (HbA) and HbC were detected by electrophoresis. The amount of HbC was much less than expected in HbC heterozygotes. The synthesis ratio (β^A +β^C/α) indicated an α-thalassaemia defect with two non-functional α genes, which did not correlate with the degree of haemolysis and anaemia displayed by the patient. The DNA analysis of the α-genes clusters revealed a defect combination −SEA/−α^3.7. The haematological data and the physiopathology of this atypical case are compared with the typical HbH disease found in a first cousin of the propositus. Data on the globin chains expression and on the formation of β^A and β^C homotetramers in HbH/HbC disease are presented.     

The β+ IVS, I-NT no. 6 (T→C) thalassaemia in heterozygotes with an associated Hb Valletta or Hb S heterozygosity in homozygotes from Malta

Summary. In vitro DNA amplification and dot blot analysis with synthetic allele specific oligonucleotides (ASO) identified the β IVS, I-6 (T→C) thalassaemia in 78% of 32 chromosomes from 16 β-thalassaemia homozygotes in Malta. The preponderance of a single thalassaemia mutation in one population is unusual. The β⁺ IVS, I-6C thalassaemia mutation was also found in three carriers who had an associated β globin heterozygosity, i.e. Hb Valletta (or α₂β₂^87PRO) or Hb S (or α₂β₂^6VAL). The proportion of Hb A in these cases (av. = 29.7%) provided objective documentation of the relatively mild effect of this mutation on in vivo globin gene expression. However, the expression of homozygous disease was more severe in developing children compared to adults. The β⁺ IVS, I-6C mutation complicates population testing because heterozygotes can have Hb A₂ levels below those classically associated with β thalassaemia.     

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.     

The triplicated α-globin gene locus in β-thalassaemia heterozygotes: clinical, haematological, biosynthetic and molecular studies

Excess α-globin chains play a major role in the pathophysiology of homozygous β-thalassaemia. In β-thalassaemia carriers a minor effect of α-globin chain excess is reflected in a minimal or mild anaemia without clinical symptoms. Factors that increase α-chain excess in heterozygotes are expected to accentuate the severity of the clinical and haematological phenotype.
We report the clinical, haematological, biosynthetic and molecular data in three β-thalassaemia heterozygotes with the rare interaction of homozygosity for α-globin gene triplication, and in 17 heterozygotes with a single additional α-globin gene. The three patients homozygous for the α-globin gene locus (anti 3.7 kb arrangement) had β °-thalassaemia mutations and a diagnosis of thalassaemia intermedia, preserving haemoglobin levels around 7–8 g/dl. Of the 17 β-thalassaemia heterozygotes (six children and 11 adults), 16 had severe β-thalassaemia mutations interacting with an additional α-globin gene (13 with ααα^anti-37 and four with ααα^anti-42). Compared to simple β-thalassaemia heterozygotes, they had lower haemoglobin levels and red cell indices, but higher α/β biosynthesis, HbF levels and reticulocytes.
Our results suggest that homozygous α-gene triplication interacts with a severe β-thalassaemia mutation to cause an α-chain excess equivalent to that observed in homozygous β-thalassaemia intermedia. In heterozygotes for severe β-thalassaemia mutations with one additional α-globin gene, the α-chain excess causes a more pronounced degree of anaemia than is usually seen in simple β-thalassaemia heterozygotes.     

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.     

Expression of CD8β and alteration of cell surface phenotype in adult T-cell leukaemia cells

Typical adult T-cell leukaemia (ATL) cells have a CD4⁺CD8⁻ cell surface phenotype, but atypical phenotypes such as CD4⁺CD8⁺ and CD4⁻CD8⁺ have also been reported. The CD8 molecule is composed of α and β chains and commonly used monoclonal antibodies against CD8 molecule detect only CD8α. Since it has been reported that CD8α can be induced in mature CD4⁺ T cells by cell activation, but not CD8β, we studied whether ATL cells which express CD8α may also express CD8β. We found some cases of CD8α⁺ ATL were also positive for CD8β. Furthermore, we experienced a case whose ATL cell surface phenotype changed from CD4⁺CD8α⁺CD8β⁺ to CD4⁻CD8α⁺CD8β⁺ and finally to CD4⁺CD8α⁻CD8β⁻. Southern blot analysis revealed that the monoclonal integration of human T lymphotropic virus type I (HTLV-I) was identical throughout the course of the study, indicating that a single clone had demonstrated the alterations. These data suggest that peripheral CD4⁺CD8⁺ ATL cells can express not only CD8α, but also CD8β and that a single ATL cell clone has the potential to change its surface phenotype in vivo as well as in vitro .     

Genetic and Biochemical Heterogeneity of β-Thalassaemia in Naples

S ummary We have investigated 32 children with Cooley's anaemia from Naples, Italy. Criteria for inclusion in the study were: (a) typical clinical and haematological findings; (b) absolute transfusion requirement; and (c) elevated Hb A₂ in both parents. From biosynthetic studies we have established that five children (including two sets of sibs) had β° thalassaemia, while the rest had β⁺ thalassaemia. Thus, the frequency of β⁺ thalassaemia among unrelated patients was about 90%. The distribution of β/α ratios among β⁺ patients ranged from 0·01 to 0·16 and it was bimodal, consistent with some of them having a β°/β⁺ genotype and others a β⁺/β⁺ genotype. The distribution of β/α ratios of the patients' parents (obligate heterozygotes) ranged from 0·24 to 0·73, and it was plurimodal, consistent with the coexistence in this population of multiple β thalassaemia alleles, of which one must be β° and at least one is β⁺. A systematic analysis of 20 families indicates that the β/α ratio is to some extent quantitatively inherited, and its suggests non-randomness in the assortment of β thalassaemia alleles that can give rise to a Cooley's phenotype.     

The Clinical and Haematological Findings in Children Inheriting Two Types of Thalassaemia: High-A2 Type β-Thalassaemia, and High-F Type or δβ-Thalassaemia

S ummary . Eleven children who are double heterozygotes for β- and δβ-thalassaemia are described. Of their parents one was always heterozygous for β-(A₂) thalassaemia (increased Hb A₂), and the other for the high F variant or δβ-thalassaemia (increased Hb F). The clinical syndrome resulting from the combination of β- and δβ-thalassaemia shows some heterogeneity, but in general is of intermediate severity. Red cell abnormalities were considerable, Hb F was very high (mean 70.3 ± 12.6%), Hb A₂ was low or normal (mean 2.36 ± 1.52%), and Hb A was absent in five patients. Hb F was nearly homogeneously distributed in the red cells of most patients. These findings are explained as the outcome of a mutation which suppresses δ- and β -chain synthesis which is associated with a genetically determined increased production of γ- chains.     

Effect of α-Chain Precipitates on Bone Marrow Function in Homozygous β-Thalassaemia

S ummary . An autoradiographic study of protein synthesis by erythropoietic cells in liomozygous β-thalassaemia has shown that a high proportion of non-dividing, late polychromatic erythroblasts fail to become labelled when incubated with radioactive amino acids. It is possible that this abnormality and the previously described disturbance of cell proliferation in the early polychromatic erythroblasts result from damage by intracellular α-chain precipitates. This possibility has been investigated by correlating the extent of α-chain precipitation with protein or DNA synthetic activity in individual cells. Over 80% of late polychromatic cells with the largest α-chain inclusions failed to label with ³H-leucine or ³H-phenylalanine but 10–23% of cells with no inclusions were also unlabelled. None of the dividing, early polychromatic cells with moderate or large quantities of insoluble α-chains incorporated ³H-thymidine. These results support the hypothesis that a-chain precipitates are associated with and possibly responsible for the ineffectiveness of erythropoiesis in thalassaemia, provided it is assumed that such precipitates are continuously degraded or extruded. The latter assumption is necessary to account for the presence of several metabolically abnormal cells with no α-chain precipitates.     

Haemoglobin Constant Spring has an unstable α chain messenger RNA

Haemoglobin Constant Spring (Hb CS) is a variant with an elongated α-chain associated with an α⁺ thalassaemia phenotype. The amount of α mRNA relative to β mRNA in reticulocytes was reduced in carriers of Hb CS by an amount equivalent to the reduction observed in carriers of α⁺ thalassaemia. In a patient with Hb CS-H disease there was greater α/β mRNA ratio in bone marrow nuclear RNA than in the peripheral blood. Furthermore, all the α mRNA in the patient's peripheral blood was derived from the α1 (α^A) gene. The data suggest that α^CS mRNA is unstable and degraded in the cytoplasm. This instability may be due to destabilization of a specific sequence in the 3'non-coding region during translation.     

Haemoglobin Manukau β67[E11] Val→Gly: transfusion-dependent haemolytic anaemia ameliorated by coexisting alpha thalassaemia

Summary Haemoglobin Manukau (β67 Val→Gly) is a novel haemoglobin variant presenting in two brothers as non-spherocytic haemolytic anaemia which became transfusion dependent by 6 months of age. The severity of clinical expression seems to be modulated by coexisting alpha thalassaemia: the severely affected children have a normal complement of alpha globin genes with an unusual genotype (-α^3,7/ααα^3,7), while their father, who carries the abnormal gene with minimal symptoms, has homozygous α⁺ thalassaemia (-α^3,7/ -α^3,7) Another unusual feature of this case is the association of the β67 Val→Gly mutation with modification of β141 Leu to a residue (believed to be hydroxyleucine) that is not detected by standard amino acid analysis. This finding offers an explanation for the previous report of an association of another mutation at this site (Hb Sydney β67 Val→Ala) with Hb Coventry (deletion of β141 Leu).     

Immunophenotypic and clinical features of T-cell receptor γδ+ T-lineage acute lymphoblastic leukaemia

The immunophenotypic and clinical features of TCR-γδ⁺ T-lineage acute lymphoblastic leukaemia (T-ALL) were prospectively analysed in 52 children with membrane CD3⁺ T-ALL. We observed a relatively high incidence of TCR-γδ⁺ T-ALL (26/52 patients). Leukaemic blasts from 22 children demonstrated TCR-αβ positivity, and simultaneous expression of the TCR-β and -δ chain was found in four children. Clinical and haematological features of TCR-αβ and γδ⁺ T-ALL did not differ significantly, except that haemoglobin levels were significantly lower in TCR-γδ⁺ cases. Event-free survival at 4 years was significantly better in TCR-γδ⁺ compared with TCR-αβ⁺ T-ALL, but expression of TCR molecules did not emerge as an independent prognostic factor in multivariate analysis.     

Increased erythropoiesis of β-thalassaemia/Hb E proerythroblasts is mediated by high basal levels of ERK1/2 activation

β - thalassaemia is one of the most common inherited anaemias, arising from a partial or complete loss of β-globin chain synthesis. In severe cases, marked bone marrow erythroid hyperplasia, believed to result from erythropoietin (EPO)-mediated feedback from the anaemic condition is common, however, as yet, no study has investigated EPO-mediated signal transduction in thalassaemic erythroid cells. Using proerythroblasts generated from peripheral blood circulating CD34⁺ haematopoietic progenitor cells, the activation of the mitogen-activated protein kinase/extracellular signal-regulated kinases (MAPK/ERKs) pathway was examined under conditions of steady state growth, cytokine deprivation and post-EPO stimulation. Levels of cellular cyclic adenosine monophosphate (cAMP) and Ca²⁺ were determined as was the degree of erythroid expansion. A significantly higher basal level of phosphorylation of ERK1/2 was observed in β-thalassaemia/Hb E proerythroblasts as compared to normal controls, which was coupled with significantly higher levels of both cAMP and Ca²⁺. Modulation of either cAMP or Ca²⁺ or direct inhibition of MAPK/ERK kinase (MEK) reduced basal levels of ERK1/2 phosphorylation, as well as significantly reducing the level of erythroid expansion. These results suggest that, in contrast to current models, hyper proliferation of β-thalassaemia/Hb E proerythroblasts is an intrinsic process driven by higher basal levels of ERK1/2 phosphorylation resulting from deregulation of levels of cAMP and Ca²⁺.