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.     

Some Features of Bone Marrow Macrophages in Patients with Homozygous β-Thalassaemia

The bone marrow macrophages of patients with homozygous beta-thalassaemia were frequently situated adjacent to collagen fibres and sometimes formed intrasinusoidal cytoplasmic protrusions. They also appeared to phagocytose processes of erythroblast cytoplasm (at times containing precipitated alpha-chains) which projected into them from neighbouring erythroblasts. The cytoplasm of the macrophages included large numbers of heavily-iron-loaded secondary lysosomes of various sizes and shapes in addition to phagocytosed erythroblasts, erythrocytes and extruded erythroblast nuclei. Numerous ferritin molecules were found in the cytoplasmic matrix but there were hardly any in the mitochondria, endoplasmic reticulum or golgi saccules. A small number of ferritin molecules were present within the nucleus. Electron microscope autoradiographs of marrow fragments which had been incubated with [3H]leucine for 1 h revealed the presence of newly-synthesized protein molecules in all types of secondary lysosomes. Light microscope autoradiographs showed the [3H]thymidine labelling index of the bone marrow macrophages was less than 1% and suggested that only a very small proportion of these cells were actively preparing for division.     

The Abnormal Haemoglobins in Homozygous α-Thalassaemia

S ummary The red cells from Chinese stillborn infants with erythroblastosis foetalis due to homozygous α-thalassaemia contain about 80% Hb-_γ4, the second haemoglobin always present does not contain α-chains and has the structure Hb_γ2X₂ identical to Hb-Portland 1. Hb-A, Hb-F, or Hb-A₂ were not detected.     

Observations on the Ultrastructure of Erythropoietic Cells and Reticulum Cells in the Bone Marrow of Patients with Homozygous β-Thalassaemia

An electron microscopic study of marrow fragments from patients with homozygous beta-thalassaemia has shown that 3% of early polychromatic erythroblast profiles and 20% of late polychromatic erythroblast profiles contain intracytoplasmic alpha-chain precipitates. Various nuclear abnormalities were found including the loss of parts of the nuclear membrane and the presence of intranuclear alpha-chain precipitates, and these abnormalities were virtually confined to the non-dividing, late polychromatic erythroblasts. As most profiles of the proliferating early polychromatic erythroblasts did not contain intracytoplasmic or intranuclear alpha-chain precipitates, it is suggested that the arrest of many of these cells in the G1 phase of the cell cycle may be related to the presence of an excess of free alpha-chains rather than to the presence of alpha-chain precipitates within them. The cytoplasm of the bone marrow reticulum cells contained early and late polychromatic erythroblasts at various stages of degradation, providing direct evidence of ineffective erythropoiesis.     

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.     

Epstein-Barr Virus Infection in Polytransfused Patients with Homozygous β-Thalassaemia

The frequency of Epstein-Barr virus (EBV) and hepatitis B virus (HBV) infection has been studied in 149 polytransfused thalassaemic patients and in healthy controls. Evidence for EBV infection was based on the detection of antibodies to viral capsid antigen (anti-VCA) and for HBV infection on the detection of either hepatitis B surface antigen (HBsAg) or hepatitis B surface antibody (anti-HBs). The frequency of anti-VCA was not significantly higher in the patients (16.4%) compared to the controls (69.8%) whereas HBV infection was more frequently observed in the patients (91.3%) than in the controls (17.3%). There was also no evidence of repeated infection or recent infection with EBV in the polytransfused patients. These data suggest that transfusion of stored blood does not represent a significant factor of spread for EBV.     

The Correlation between Red-Cell Survival and Excess of α-Globin Synthesis in β-Thalassaemia

In 13 subjects affected by β-thalassaemia major, in three subjects affected by β-thalassaemia minor and in five normal healthy persons haemoglobin synthesis and the survival of red cells transfused into normal, group compatible, healthy recipients has been studied. The existence of an excess of newly synthesized α-chains and of a negative correlation between the excess α-chain and the red-cell survival has been demonstrated. The harmful role of the α-chain excess on the erythrocyte and the implications of this finding are discussed.     

Haemoglobin Synthesis in β-Thalassaemia

S ummary . The incorporation of radioactive amino acids into the polypeptide chains of haemoglobin has been studied in reticulocyte preparations from five patients with β-thalassaemia and four control patients. In both groups of patients an intracellular pool of free α-chains has been found, but the pool is much larger in patients with β-thalassaeniia major. The α-chains exist in two forms: α-dimers and α-monomers, but the results suggest that they are released as dimers from the ribosomes and that the α-chains for Hb-A and Hb-F synthesis are derived from the α-dimer pool. The α-dimers are also gradually converted to the monomers, and this may be the first stage in the denaturation of this protein. The results also show that the α-dimer can exchange with the α-chains of Hb-A and that this exchange is more rapid in the presence of in vivo aged Hb-A. In one patient with thalassaemia it was shown that there was no pool of unreleased β-chains on the ribosomes. There is also no evidence for the release of incompleted β-chains in our patients.     

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.     

Ultrastructural Studies in β-Thalassaemia Major

S ummary . The peripheral red blood cells of 11 patients with β-thalassaemia major were studied by electronmicroscopy, and a wide spectrum of intracellular changes was observed. Findings were striking in the six splenectomized individuals and most prominent in reticulocytes and late normoblasts. There was marked accumulation of iron in different forms, either as free particles or as aggregates of ferritin and haemosiderin within membrane-bound particles or mitochondria. Glycogen accumulation was often found in normoblasts. Cells were grossly distorted and deformed, showing indentations and infolding of the plasma membrane with marked vacuole formation.
The most striking finding was the presence of Heinz bodies in various stages of development. These were unlike those encountered in phenylhydrazine induced haemolytic anaemia; no large marginated Heinz bodies were found attached to the cell membrane. The implications of this finding in relation to the present concept of sequestration of Heinz bodies in the spleen are discussed. Another prominent feature was the presence of many bizarre membrane forms and myclin figures which may represent attempts at autodigestion of excess intracellular inclusions, particularly in the absence of the spleen.
The wide spectrum of ultrastructural changes found may be an expression of multiple intracellular defects involving the biosynthesis of globin, haem, glycoprotein, and membranes in β-thalassaemia.     

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.     

Five Families with Homozygous δ-Thalassaemia in Japan

Five families with delta-thalassaemia discovered in Ehime, Japan, are presented. The delta-thalassaemia was associated with a slight elevation of the level of Hb F in two families and with normal Hb F levels in three. Complete absence of HbA2 was found in the homozygous probands. No abnormal clinical or haematological findings were noted in the individuals with delta-thalassaemia.     

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.     

Haemoglobin Tak: a β-Chain Elongation

In Haemoglobin Tak two normal α-chains are combined with two β-chains elongated by 11 residues beyond the C-terminus. Unlike in the α-chain abnormal Hb Constant Spring, the elongation cannot result from a point mutation of a stop codon. It is probably due to an unequal crossing-over near the 3' end of the β-chain structural gene. This could cause either a deletion of the normal stop codon or a shift in the reading frame. Oxygen dissociation of purified Hb Tak shows no cooperativity but in Hb A + Tak haemolysates there is no interaction between the two above 40% O₂ saturation. Heterozygotes for Hbs A and Tak show an imbalance of globin chain synthesis (α/non α= 1.5), the synthesis of β Tak resembles that of the β-chain in β⁺ thalassaemia.     

Abnormal Red-Cell Metabolism of Pyridoxine Associated with β-Thalassaemia

Red-cell conversion of pyridoxine to pyridoxal phosphate was studied in control subjects, and patients with heterozygous and homozygous beta-thalassaemia. In 7% of control subjects the rate of pyridoxine conversion was well below the range found in the other control subjects (5.0-8.6%, mean 6.5%/g Hb x 10(-2)) but in heterozygous beta-thalassaemia was below that range in 63% of the patients. The conversion rate was also slow or borderline in the majority of patients with severe transfusion-dependent homozygous beta-thalassaemia, in spite of the presence of some donor cells; but was normal, or fast as in other anaemias, in all but one patient with mild homozygous thalassaemia. There was a much higher incidence of a slow conversion rate in the parents of the severe homozygotes than in parents of the mild homozygotes, illustrating the familial pattern. This supports our view that the red-cell conversion rate of pyridoxine is an inherited characteristic, independent of thalassaemia. The cause of a reduced rate of pyridoxine conversion was investigated. The increase to a normal rate following riboflavin ingestion suggests a defect in the activity of the flavin mononucleotide (FMN)-dependent pyridoxine phosphate oxidase.     

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.     

The Erythropoietic Response to Pregnancy in β-Thalassaemia Minor

S ummary . In 15 women with β-thalassaemia minor, the haemoglobin concentration fell to a greater extent during pregnancy than in normal women. The lower haemoglobin level in the thalassaemics was caused by limited expansion of their red-cell mass.
Although the increase in red-cell mass in iron-sufficient thalassaemics was not influenced by iron supplementation, two of the unsupplemented women were in negative iron balance. Ideally, each thalassaemic patient should receive individual assessment prior to iron therapy. Iron supplementation is recommended, when there is evidence of a negative iron balance.
There was great variability in the rise of red-cell mass observed in the thalassaemic women. The rise correlated inversely with the morphological abnormality of the red cells, suggesting that the underlying disorder in haemoglobin synthesis was the main limiting factor.
In the thalassaemic women foetal haemoglobin was significantly greater in the first half of pregnancy than in the puerperium. This raises the possibility that the increased production of γ-chains could favourably influence haemoglobin synthesis in pregnant women with thalassaemia minor.     

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 Interaction of α-Thalassaemia and Haemoglobin G Philadelphia

An American Negro woman was found to have HbH disease in association with HbG Philadelphia (α68-asnlys). Starch gel electrophoresis failed to reveal the presence of any HbA or HbA₂ and studies of globin chain synthesis indicated absence of α^A production. The α^G/β synthesis ratio was 0.63. The woman's son and her two half-sibs had α-thalassaemia trait with no HbH and α/β synthesis ratios of 0.84, 0.84 and 0.76. The data indicate that there is no functioning α^A gene linked to the α^G gene. The absence of α^A synthesis by the propositus also indicates that the α-thalassaemia gene trans to the α^G gene completely suppresses α chain production, the first evidence for such a gene in Negroes.