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.     

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.     

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.     

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 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.     

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.     

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.     

Proliferative Activity and Glycogen Accumulation of Erythroblasts in β-Thalassaemia

S ummary . A combined microfluorometric, microspectrophotometric and autoradiographic method was used to determine the glycogen content, measured as the fluorescence intensity after a fluorescent periodic acid-Schiff reaction (F-PAS), of erythroblasts in homozygous β-thalassaemia. F-PAS-positive erythroblasts were found most frequently in the G₁-phase of early polychromatic erythroblasts and seem to account for the increased number of cells in this phase of DNA-synthesis in β-thalassaemia as compared to normal. The fraction of F-PAS-positive G₁-early polychromatic erythroblasts was higher and the glycogen content of F-PAS-positive cells was greater in severe as compared to less severe cases, which seems to be of prognostic importance. It is postulated that the accumulation of glycogen represents a storage of unutilized energy in erythroblasts that are blocked in the G₁-phase of the cell cycle.     

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.     

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.     

Globin Chain Synthesis in Haemoglobin New York (β 113 Valine→Glutamic acid

S ummary Five patients with Fanconi anaemia have been treated by bone marrow transplantation from HLA identical donors. Only one patient survived for more than 3 years. She is now perfectly healthy with complete haematological reconstitution with chimaerism and disparition of chromosomal abnormalities. In contrast, four patients died of acute severe GVHD soon after grafting. In addition, all had signs of severe cyclophosphamide toxicity. This evolution could be explained by a special sensitivity of FA cells to alkylating agents and may indicate the need to modify the conditioning regimen in FA patients.     

Haematological Data in 312 Cases of β-Thalassaemia Trait in Thailand

S ummary . The haematological data are presented in 312 individuals of Thai or Chinese extraction with the high A₂β-thalassaemia trait. The mean haemoglobin concentrations were 12.1 g% among males and 10.8 g% among eemalcs; 54% of the subjects were not anaemic and all were asymptomatic. Low MCV, MCH and MCHC values were found in 67%, 81% and 6% respectively; erythrocytosis was detected in 34%. Variable hypochromia and microcytosis were usually present, but leptocytosis and red cell basophilic stippling were often absent. Decreased red cell osmotic fragility was always detected. Fifteen cases were found to have normal red cell morphology and 17 normal red cell osmotic fragility. The reticulocyte counts, although ranged up to 12%, were normal in the majority. The serum iron levels and unsaturated iron binding capacity were normal except in 11% of the cases where the scrum iron levels were lower than 50 μg%. Haemoglobin A₂ levels varied from 3.4 to 7.8% with a normal distribution and a mean of 5.2%. Haemoglobin F levels varied from o to 7.8% with 58% elevated values. Analysis of variance revealed intrafamilial segregation of both haemoglobin A₂ and haemoglobin F levels. The literature is reviewed and findings are compared.     

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.     

β-Thalassaemia in Campania: DNA polymorphism analysis in βA and βthat chromosomes and its usefulness in prenatal diagnosis

In order to evaluate the feasibility of first trimester prenatal diagnosis of beta-thalassaemia by restriction fragment length polymorphism (RFLP) in Campania, one of the most affected regions in Southern Italy, DNA polymorphism analysis was performed on 40 unrelated patients, affected with homozygous beta-thalassaemia, and on their parents. Frequency of the presence of the Hinc II epsilon, Hind III G gamma and A gamma, Hinc II psi beta and 3' psi beta, Ava II psi beta, Ava II beta and Bam HI 3' beta sites have been determined in the beta A and beta thal chromosome samples. In 31 families (over 75%), RFLPs enabled tracing the beta-thalassaemia mutations in both father and mother (100% diagnosis). In the remaining nine families, RFLPs enabled tracing only one of the two mutations (50% diagnosis) because the other parent was found to be homozygous in all the analysed polymorphic sites. Restriction haplotypes, assembled on the basis of linkage analysis, were most heterogeneous, hence a wide heterogeneity of mutations is expected.     

Chelation Studies with 2,3-Dihydroxybenzoic Acid in Patients with β-Thalassaemia Major

S ummary . 2,3-Dihydroxybenzoic acid was evaluated as a potentially useful, orally effective iron-chelating drug by performing iron balance studies in patients with β-thalassaemia major. The administration of this substance at 25 mg/kg/d to five patients for 8 d caused an average increase in iron excretion of 4.5 mg/d. When the drug was administered at 25 mg/kg q.i.d. to eight patients for 21 d, iron excretion increased to 6.5 mg/d. Chelation was highly specific for iron with changes in magnesium and calcium excretion being insignificant. The drug was well tolerated with side effects limited to gastrointestinal complaints which ameliorated when the drug was taken with food. These studies provide a rationale for further evaluation of 2,3-dihydroxybenzoic acid in patients with iron overload.     

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.