Molecular heterogeneity of beta thalassaemia in the Italian population

S ummary Fifty-one subjects originating from Southern Italy and affected by Cooley's anaemia have been studied in order to define the degree of heterogeneity of β thalassaemia mutations in this high incidence area. Restriction endonuclease mapping has been carried out on genomic DNA by the Southern blot technique both to exclude the existence of gross deletions or rearrangements and to establish the relative frequency of four polymorphic restriction sites (i.e. ^Gγ and ^Aγ Hind III, β Ava II and β Bam HI) within the γδβ gene region. In 28 subjects unequivocal linkage of the four polymorphic sites has been determined leading to the identification of seven different chromosome haplotypes, six of which had previously been reported associated with specific β⁰ and β⁺ thalassaemia mutations. Globin chain synthesis studies on peripheral blood reticulocytes indicated that subjects carrying the same genotype may behave differently as far as the β chain production is concerned relative to both the a and the non-α chains. Thus, β thalassaemia turns out to be quite heterogeneous even in this limited geographical area. β⁺ mutations appear to be predominant, particularly those affecting nuclear precursor RNA splicing to mature β globin mRNA.     

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).     

Comparison of Haematological Features of the β0 and β+ Thalassaemia Traits in Jamaican Negroes

Haematological characteristics have been compared in 29 subjects with heterozygous β⁰ thalassaemia and in 33 subjects with heterozygous β⁺ thalassaemia, identified by the type of sickle cell-β thalassaemia among close relatives, in a Jamaican Negro population. Total haemoglobin, MCV and MCH were significantly lower in the β⁰ type but the level of Hb A₂ was not significantly different. Individual values for MCV, MCH and Hb A₂ in the β⁺ type occasionally overlapped those in the normal population casting doubt on the adequacy of these criteria in identifying all cases of heterozygous β⁺ thalassaemia. The haematological differences are those which would be expected on theoretical grounds. The inability to confidently differentiate the two types of heterozygous β thalassaemia has implications for genetic counselling. The inability to distinguish heterozygous β⁺ thalassaemia from normals on any single haematological index suggests that surveys depending on estimations of Hb A₂ or on MCV alone may have underestimated the prevalence of the β⁺ thalassaemia gene.     

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.     

Homozygous deletional α+ thalassaemia associated with unequal expression of the two remaining α1 genes (α1A and α1Q)

S ummary . A Cambodian family presenting several haemoglobinopathies, Hb E, Hb Q and α⁺ thalassaemia, has been investigated. DNA analysis showed that the thalassaemia syndrome corresponds to a leftward type (4.2 kb) deletional from of α⁺ thalassaemia. Genotypes found in the family are: propositus -α^A/-α^Q, β^A/β^E, mother and older sister α^Aα^A/ -α^Q, β^A/β^E; father α^Aα^A/-α^A, β^A/β^A. The propositus consistently presents an α^Q/α^A chain ratio of 60/40 although both chains are products of α₁ loci. The relatively higher expression of the α^Q chain is not observed in the mother and therefore makes it unlikely to reflect anything other than differential expression of the maternal -α^Q/ and paternal -α^A/ haplotypes. This observation raises the possibility that both haplotypes are not strictly identical and that the region of the cross-over event is important for α gene expression.     

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.     

Rapid analysis of -α3.7 thalassaemia and αααanti 3.7 triplication by enzymatic amplification analysis

Summary In this report we describe a PCR-based method for the diagnosis of the most common form of α thalassaemia, the –α^3.7 deletion which occurs throughout all tropical and subtropical regions of the world. The same procedure also identifies the reciprocal recombinant chromosome (ααα^{anti 3.7}). Restriction mapping of the PCR products has enabled us to distinguish between the type I (–α^3.7I), type II (–α^3.7II) and type III (–α^3.7III) deletions. This strategy will be very useful in screening programmes of α thalassaemia occurring on its own or in association with β thalassaemia and sickle cell disease.     

Interaction between the Glucose-6-Phosphate Dehydrogenase Deficiency and Thalassaemia Genes at Phenotype Level

Summary. No significant differences were observed in the mean values of Hb A₂ levels and red cell indices between G6PD^- and G6PD+β thalassaemia carriers apart from the MCV, which was significantly higher in β thalassaemia G6PD^- subjects, but still in the thalassaemia carrier range. No difference was seen between G6PD+ and G6PD^-α thalassaemia carriers. G6PD+β thalassaemia carriers show a significant increase in G6PD levels expressed as activity per g of Hb and to lesser extent as activity per number of red cells x 10⁹; in G6PD+α thalassaemia carriers this increase is statistically significant only when the enzyme levels are expressed as activity per g of Hb. G6PD^-β thalassaemia carriers had enzyme levels higher than non-thalassaemic G6PD^- subjects only when the activity is expressed per g of Hb. G6PD activity was found to be increased in G6PD+ and G6PD^- Hb H disease patients.     

The Development of Haemoglobin A2 in Normal Negro Infants and in Sickle Cell Disease

The development of haemoglobin A₂ levels from birth to 3 years has been compared in normal, β-thalassaemia trait, sickle cell (SS) disease, and S-β-thalassaemia genotypes. Hb A₂ levels were almost identical in normals and in children with SS disease at 1, 2 and 3 years. The most rapid increases in Hb A₂ levels occurred before 6 months but levels were still rising at the end of the third year. Sickle cell-β⁺ thalassaemia could be differentiated from SS disease by the higher Hb A₂ levels between 6 months and 1 year. Insufficient data were available on S-β° thalassaemia but since Hb A₂ levels in this condition are generally higher than those in S-β⁺ thalassaemia, differentiation from SS disease may also be possible from the age of 6 months.     

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 .     

Red blood cell phenotypes in the α+ thalassaemias from early childhood to maturity

The α⁺ thalassaemias are the most common single gene disorders of humans, yet little is known about their haematological characteristics in childhood. Blood samples have been collected randomly from more than 2000 individuals in village communities in Vanuatu in the South West Pacific and analysed for α thalassaemia and associated haematological changes. Here we describe the haematological effects of the α⁺ thalassaemias from early childhood through to maturity in this population. Mean cell volume (MCV) and mean cell haemoglobin (MCH) levels in individuals of normal, heterozygous and homozygous genotype differed significantly from one another throughout the entire age range (2 P  < 0.05). In contrast, haemoglobin levels in heterozygous and homozygous individuals were well maintained throughout development. Adults of normal genotype attain Hb levels which are indistinguishable from Caucasian reference values, a finding made all the more remarkable given the high frequency of clinical malaria in this population. It is clear from these findings that haematological data are valuable in screening for carriers of α⁺ thalassaemia in this population. MCH is clearly the most sensitive discriminator. None of the homozygous adults tested had an MCH of >27 pg, whereas <10% of normals had a value of <27 pg. These data provide reference values for areas in which the α⁺ thalassaemias are common and often confused with iron-deficiency anaemia.     

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.     

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.     

Congenital Methaemoglobinaemia due to NADH Methaemoglobin Reductase Deficiency: Successful Treatment with Oral Riboflavin

S ummary . A Japanese family with congenital methaemoglobinaemia is described. The family pedigree was compatible with autosomal recessive type of inheritance. The increased methaemoglobin concentration was ascribed to the red cell NADH diaphorase deficiency associated with the almost complete lack of one of the two peaks of the diaphorase activity as separated by DEAE Sephadex column chromato-graphy. The NADH diaphorase and NADH methaemoglobin reductase deficiency was limited to the red cells. The methaemoglobin content in the blood of the propositus was 17.8% and isoelectric focusing analysis on a polyacrylamide gel plate showed that the haemoglobin consisted of 65.2% oxyhaemoglobin (α²⁺β²⁺)₂, 29.6% half-oxidized forms, 20.9% (α+β²+)₂ and 8.7% (α²⁺β⁺)₂, and 3% full-oxidized methaemoglobin (α⁺β⁺)₂. Oral administration of riboflavin 120 mg/d resulted in a gradual but significant decrease in the level of the met-form haemoglobins in parallel with a gradual increase in the red cell flavin content. Riboflavin is considered to be effective by activating the NADPH diaphorase (NADPH flavin reductase) system and appears to be useful for the treatment of congenital methaemoglobinaemia.     

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.     

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.     

Interaction between Homozygous β0 Thalassaemia and the Swiss Type of Hereditary Persistence of Fetal Haemoglobin

The interaction between β⁰ thalassaemia and an heterocellular form of hereditary persistence of fetal haemoglobin (HPFH), presumably of the Swiss type, has been studied in three generations of a family in which both traits occur. The haematological parameters and the segregation of the two characters in the family suggest that the propositus, a 52-year-old male from southern Sardinia, is homozygous for β⁰ thalassaemia and carrier of the HPFH.
In spite of the complete suppression of adult haemoglobin synthesis, the patient is not anaemic and shows only morphological abnormalities of the red cells associated with a moderate decrease of the erythrocyte life span. Studies of the synthesis of haemoglobin chains in vitro have revealed only a mild degree of unbalance in the propositus, with a γ/α ratio of 0–67, and a very slight unbalance in a 3-year-old child heterozygous for β thalassaemia and HPFH.
Preliminary analysis of the linkage between this kind of heterocellular HPFH and the β Hb locus has been performed, utilizing all the suitable families reported in the literature. Although positive lod scores (1.535) have been obtained at a recombination fraction of 0.20, the data available are not sufficient to conclude in favour or against the linkage between the β Hb locus and the heterocellular type of HPFH.     

Alpha thalassaemia is associated with increased soluble transferrin receptor levels

Although α⁺ thalassaemia is the commonest haemoglobinopathy in the world, it is not known if it is associated with significant ineffective erythropoiesis, a fact of importance in interpreting its complex interaction with malaria. To study this problem, we have measured the concentrations of soluble transferrin receptor (sTfR) and ferritin in 181 children from Vanuatu with heterozygous (68) and homozygous (46) α⁺ thalassaemia, and normal controls (67). sTfR concentrations were significantly higher in both homozygotes (mean 3.1 mg/l, range 2.8–3.4) and heterozygotes (2.86 mg/l, 2.6–3.2) compared to the normal controls (2.48 mg/l, 2.3–2.7), suggesting that although globin chain imbalance is minimal, there is ineffective erythropoiesis in both these conditions. Age was also shown to significantly affect sTfR, with peak levels occurring in the 5–9 years age group. Ferritin concentrations showed a similar trend, being higher in the thalassaemic groups, although this did not reach statistical significance. No individuals had low ferritin concentrations, although two had significantly elevated sTfR levels. These observations suggest that the α⁺ thalassaemia phenotype includes an expansion of the erythron, and may suggest possible mechanisms for the increased susceptibility in babies with α thalassaemia to both P. falciparum and P. vivax malaria.     

The Genetics and Molecular Basis of Alpha Thalassaemia in Association with Hb S in Jamaican Negroes

S ummary . We have studied seven Jamaican Negro families in whom the genes for α thalassaemia and the sickle cell mutation (β^s) were independently segregated. Using a combination of techniques we identified two α thalassaemia phenotypes which resemble the severe (α thalassaemia 1) and mild (α thalassaemia 2) determinants previously described in Orientals. This study has enabled us to clearly correlate the phenotype of α thalassaemia with the genotype in this population. Furthermore, since in each family α thalassaemia was present in association with the gene for the sickle cell mutation we have determined the proportion of Hb S in the peripheral blood of individuals with the αα/αα, -α/αα and -α/-α genotype who are also heterozygous for the β^s mutation. Genetic analysis in these families shows that in each case subjects with the α thalassaemia 1 phenotype are homozygous for the α thalassaemia 2 defect (-α/ -α). We have found no instances of the genotype -/αα in this population which may explain the rarity of the severe α thalassaemia syndromes in Jamaica. Restriction mapping data in the α thalassaemia 2 homozygotes from this population shows that the (-α/) haplotype results from a deletion of one of the linked pair of α globin genes and that this has probably arisen by an unequal crossover between non-homologous α genes.