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.     

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.     

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.     

Severity differences in β-thalassaemia/haemoglobin E syndromes: implication of genetic factors

Summary. Genetic factors determining the difference in severity of anaemia in β-thalassaemia/HbE disease were studied in 90 patients who had haemoglobin levels, at steady state, ranging from 4.2 to 12.6 g/dl. Co-inheritance of α-thalassaemia 2 and haemoglobin Constant Spring could significantly decrease the severity of the disease. Inheritance of a β-thalassaemia chromosome with Xmn I cleavage site at position — 158 of the ^Gγ-globin gene which was linked to the haplotype - + - ++ or ++ - ++, was associated with a milder anaemia. Two copies of these alleles were necessary to produce a significant clinical effect. Increased expression of the ^Gγ-globin gene and higher production of haemoglobin F. which could reduce the overall globin chain imbalance, were also associated with homozygosity for the Xmn I cleavage site and thus with less severe anaemia. However, this effect was not seen in Xmn I site heterozygotes. Whether the effects of the Xmn I polymorphism, HbF concentration and ^Gγ/^Aγ ratio act separately or through common mechanisms in reducing anaemia remains to be ascertained.     

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 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.     

Composition of the intra-erythroblastic precipitates in thalassaemia and congenital dyserythropoietic anaemia (CDA): identification of a new type of CDA with intra-erythroblastic precipitates not reacting with monoclonal antibodies to α- and β-globin chains

Ultrathin sections of bone marrow cells from two patients with homozygous β-thalassaemia, two patients with haemoglobin H (HbH) disease, a patient with congenital dyserythropoietic anaemia (CDA) type III and two patients with severe congenital dyserythropoietic anaemia of an unusual type were reacted with mouse monoclonal antibodies against various globin chains and the reaction visualized using a gold-labelled goat antibody against mouse IgG. The multiple rounded intra-erythroblastic inclusions found in homozygous β-thalassaemia reacted with the monoclonal antibody against α-globin chains but not β-globin chains, thus confirming that they consisted of precipitated α-globin chains. The branching intra-erythroblastic inclusions found in HbH disease and CDA type III reacted with the monoclonal antibody against β-globin chains but not α-globin chains, indicating that they consisted of precipitated β-globin chains. The two patients with severe CDA had been transfusion-dependent since infancy, had a normal α:β globin chain synthesis ratio or parents with normal red cell indices, displayed prominent dysplastic changes in their erythroblasts, and had intra-erythroblastic inclusions resembling those seen in homozygous β-thalassaemia. However, unlike those in β-thalassaemia, the inclusions in these two patients did not react with the monoclonal antibody against either α- or β-globin chains. The inclusions reacted with antibody against ζ-globin chains, but detailed studies in one of the patients indicated that the antigen involved was not ζ-globin. These patients have features not reported in the condition known as dominantly inherited inclusion body β-thalassaemia and appear to suffer from a novel type of CDA in which the intra-erythroblastic inclusions may consist of some non-globin protein or structurally-abnormal α-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.     

Relationship between transfusion regimen and suppression of erythropoiesis in β-thalassaemia major

In the management of β-thalassaemia major, different transfusion schemes are employed with baseline haemoglobin levels ranging from 8 to over 12 g/dl. We studied the relationship between transfusion regimen and suppression of erythropoiesis in 52 patients with β-thalassaemia major whose mean pretransfusion haemoglobin levels ranged from 8.6 to 10.9 g/dl. Multiple, regression analysis showed that serum transferrin receptor was the parameter more closely related to mean pretransfusion haemoglobin (r = -0.77, P < 0.001). As measured through serum transferrin receptor, erythroid activity was 1-2 times normal for pretransfusion haemoglobin levels between 10 and 11 g/dl, 1-4 times normal for levels from 9 to 10 g/dl, and 2-6 times normal for levels from 8.6 to 9 g/dl. Mean pretransfusion haemoglobin was also inversely related to serum erythropoietin (r = -0.72, P < 0.001), whereas it showed no or a weak relationship with Hb F, reticulocyte count, or circulating nucleated red cell count. This study suggests that serum transferrin receptor is a reliable indicator of suppression of erythropoiesis in β-thalassaemia major. On the basis of our findings, pretransfusion haemoglobin values of ≦ 9 g/dl should be adopted with caution, because these levels can be associated with an insufficient inhibition of erythroid marrow expansion. However, a transfusion programme, with a baseline haemoglobin of 9-10 g/dl, may provide enough suppression of erythropoiesis and allow a reduction in blood consumption as compared with the classic hyper- or supertransfusion schemes. Since fixed haemoglobin levels may not be the best target for transfusion treatment in all thalassaemic patients, assay of serum transferrin receptor may be helpful for individualizing the transfusion regimens.     

δβ-Thalassaemia in a Chinese Family

S ummary . δβ-Thalassaemia has been observed for the first time in individuals of Chinese origin. The clinical and haematological features have been characterized in the heterozygous state and in the double heterozygous state withβ-thalassaemia. Studies of haemoglobin synthesis indicate that the degree of globin chain imbalance in β-thalassaemia is less than that found in β-thalassaemia. Analysis of the foetal haemoglobin has shown that all individuals carrying the δβ-thalassaemia gene in this family synthesize only the α₂γ₂^{136 glycine} variety. This type of foetal haemoglobin has been found previously only in two Negro individuals heterozygous for hereditary persistence of foetal haemoglobin (HPFH). The genetic relationships between δβ-thalassaemia and HPFH are discussed in the light of these new findings.     

The Clinical and Biosynthetic Characterization of αβ-Thalassaemia

A Cypriot family is described in which three thalassaemia genes, α-thalassaemia 1, α-thalassaemia 2, and β–thalassaemia, are segregating. Two siblings are heterozygous for both α-thalassaemia 1 and β-thalassaemia while a third child has typical haemoglobin H disease. The α-thalassaemia β-thalassaemia combination, which is associated with an α/β chain production ratio of unity, produces a moderate degree of anaemia with marked hypochromia and morphological changes of the red cells together with increased rates of flux of potassium across the membranes, but the red cell survival is normal. These changes m red cell morphology and metabolism are very similar to those found in the sibling with haemoglobin H disease in whom there is gross imbalance of globin chain synthesis and shortened red cell survival. These results suggest that imbalanced globin chain production is the primary cause of shortened red cell survival in thalassaemia and that changes in membrane permeability are probably of secondary importance and may, at least in part, result from factors other than globin chain imbalance.     

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.     

α and β thalassaemia among Chinese children in Guangxi Province, P.R. China: molecular and haematological characterization

Summary We have studied nearly 100 patients with β thalassaemia major and 60 patients with Hb H disease who were attending the Haematology Clinic of Guangxi Medical College. Treatment of the patients was limited and only a few patients with β-thalassaemia major received blood transfusion(s). As a result, the severe anaemia has led to early death at 3–4 years for β⁺-thalassaemia homozygotes, and 8–12 years for β⁺-thalassaemia homozygotes. Four β-thalassaemia alleles are responsible for nearly 90% of all β-thalassaemia chromosomes. This information has resulted in the initiation of a prenatal testing programme at the local level. The patients with Hb H disease maintained a haemoglobin level of 6–10 g/dl and early death was infrequently observed. The ^SEA deletion was the major type of α-thalassemia-1, while three smaller deletions (−2.7, −3.7 and −4.2 kb) and two nondeletional α-thalassaemia determinants (Hbs Constant Spring and Quong Sze) were the α-thalassaemia-2 types.     

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.     

Multiplex ligation-dependent probe amplification screening of isolated increased HbF levels revealed three cases of novel rearrangements/deletions in the β-globin gene cluster

Investigations of naturally occurring mutations, such as the deletional thalassaemias and hereditary persistence of fetal haemoglobins (HPFHs), have brought many insights into human globin switching, but limited data have been reported so far. We selected 15 individuals with elevated fetal haemoglobin (HbF) levels (>5%) from a previous screening of 27 006 Korean individuals and analysed dosage changes of the globin gene cluster using multiplex ligation-dependent probe amplification (MLPA). Dosage changes detected by the MLPA probes were followed up with gap-polymerase chain reaction and sequence analysis. Three subjects were found to have deletions in the globin gene cluster, including a β-thalassaemia due to deletion of HBB (β-globin gene), an HPFH due to deletions of HBD (δ-globin gene) and HBB , and an HPFH due to a novel HBG2–HBG1 fusion gene consisting of exons 1 and 2 of HBG2 (^Gγ-globin gene) and exon 3 of HBG1 (^Aγ-globin gene). The case with the HBG2–HBG1 fusion suggested the existence of another mechanism for the reactivation of HBG2 and HBG1 . The IVS2 of HBG2 and HBG1 might play a role in HbF regulation, and combinations of specific polymorphisms could influence the reactivation of these genes in adults.     

A Unique Thalassaemic Syndrome: Homozygous α-Thalassaemia + Homozygous β-Thalassaemia

The disturbed balance of globin chain synthesis is a major factor in the pathophysiology of the thalassaemic disorders; this concept is strongly supported by the study of a patient displaying an extreme but symmetrical deficit of both major types of chains α and β. The patient had a mild clinical picture but presented a striking hypochromia (MCH 10 pg) with compensatory erythrocytosis (RBC 10¹²l.). Study of the propositus and his family by haematological, biochemical and biosynthetic techniques indicates that the patient carries two α- and two β-thalassaemia genes resulting in balanced globin chain synthesis; in addition, several members of the family carry two or three abnormal genes. During observation a change in the haematological pattern occurred with a shift towards more intensive β-chain and away from γ-chain synthesis; this appeared with be associated with improvement of his anaemia through more effective erythropoiesis.     

Ultrastructural Studies of Erythropoiesis in β-Thalassaemia Trait

The erythropoietic cells of six cases of β-thalassaemia trait were studied by electron microscopy and electron microscope autoradiography. Intracytoplas-mic and intranuclear α-chain precipitates were found in some late polycliromatic erythroblasts and intracytoplasmic precipitates were found in several marrow reti-culocytes. This provides direct morphological evidence of unbalanced globin chain synthesis in the marrow. Several of the polychromatic erythroblasts and marrow reticulocytes contained autophagic vacuoles and showed a variety of other dysery thropoietic changes. Erythroblast profiles containing moderate quantities of precipitated α-chains usually suffered from a marked depression of protein synthesis. The proportions of marrow cells containing α-chain precipitates and displaying dyserythropoietic changes varied considerably from patient to patient. It is proposed that this variation largely reflects variations in the proteolytic capacity of the erythropoietic cells in different individuals and leads to different degrees of ineffective erythropoiesis in β-thalassaemia trait.