We report a Swiss-Spanish family three members of which have the clinical picture of thalassemia intermedia. Restriction endonuclease mapping of the alpha-globin cluster and digestion with Mae I of the in vitro amplified 5' segment of the beta-globin gene shows a combination of triplicated alpha globin locus, anti-3.7 kb type, with heterozygous codon 39 C----T beta (0) thalassemic mutation. These, as well as 16 similar cases reported in the literature, permit the following conclusion: a single extra alpha-globin gene gives rise to a clinically significant degree of dyserythropoietic anemia only when it interacts with a severe beta(+) or beta(0) thalassemic mutation.

Summary.We describe a new case of an association of aglobin gene quadruplication of the anti-4.2 type with b Њ-thalassaemia. The patient, a young woman of mixed Brazilian-Portuguese origin, suffered from chronic haemolytic anaemia with splenomegaly. Bone marrow supravital staining with brilliant cresyl blue and electron microscopy studies showed large inclusion bodies in about 3% of erythroblasts. Upon immunofluorescent staining these inclusions reacted with a monoclonal antibody to a-but not to b-globin. Analysis of a-globin cluster by Southern blotting showed the presence of pathologic fragments specific for the anti-4.2 a-globin gene quadruplication. a/b mRNA ratio was higher than in cases combining a-globin triplication and bЊ-thalassaemia or in cases of bЊ-thalassaemia heterozygous state alone (18, 14·7 and 10·1 respectively). Our data confirmed the hypothesis that the clinically detectable haemolysis in this bЊ-thalassaemic patient was due to an unusually high amount of precipitated a-globin in erythroid precursors. This considerable excess of a-globin chains was due partly to the b-globin deficit caused by the presence of the bЊ-thalassaemic gene, but also to the presence of 6 active a-globin genes resulting from a-globin gene quadruplication in one chromosome.Keywords: a-globin gene quadruplication, inclusion body bthalassaemia, a/b mRNA ratio, globin mRNA quantification, thalassaemia intermedia.Rearrangements in the human a-globin cluster are amongst the most common genetic abnormalities observed in human populations. Deletions are the most common type of modification and involve the a-genes or larger areas of the cluster. However a-globin triplications are also encountered, and are a result of non-reciprocal crossing-over events (Weatherall, 1994).Association of such a-globin haplotypes (aaa/) with bЊ-thalassaemias strongly suggests that all genes are functioning, since these patients present a thalassaemia intermedia phenotype (Beris et al, 1991b;Ho et al, 1998). a-globin gene quadruplication is exceptional. To date, clinical and haematological data have been reported for six cases belonging to two families presenting this haplotype, but the information given was scanty and incomplete (Gu et al, 1987).We report a new case of a-globin gene quadruplication in association with a bЊ-thalassaemia, in a patient presenting large erythroblastic inclusions, severe ineffective erythropoiesis and haemolysis.The inclusion bodies were investigated by electron microscopy and immunofluorescence staining, using monoclonal antibodies against the a-and b-globin chains. Only the first antibody was found to react with the inclusions. Globin mRNA ratio quantification was performed, revealing, as expected, a great excess of a-globin mRNA. CASE REPORT, MATERIALS AND METHODSThe patient, a young woman of mixed Brazilian-Portuguese origin, was initially referred to our service because of chronic fatigue. Upon examination chronic haemolytic anaemia and splenomegaly were revealed with: Hb 8·4 g/dl, RBC 4·09 ×

We have sequenced the 5' hypersensitive-2 (5'HS-2) site of the locus control region (LCR) and the promoters of the two gamma-globin genes located on chromosome 11 of a black patient with mild beta-thalassemia (beta-thal) major due to a homozygosity for the C----T mutation at position -88 of the beta promoter and with a high Hb F level. Sequence variations in the 5'HS-2 were the same as observed for the beta s chromosome with haplotype number 3, while most of the G gamma promoter and the A gamma promoter had sequences similar to that of the beta S chromosome with haplotype number 19. This atypical haplotype (number 19A) is apparently associated with an increased gamma chain production which is particularly evident during periods of severe hematopoietic stress. Additional studies on relatives of the proband and on 10 unrelated black beta-thal homozygotes with either the C----T mutation at -88 or the A----G mutation at -29, confirm the possible importance of the sequence differences in the 5'HS-2, and also suggest that at least two additional factors, namely a C----T mutation at position -158 of the G gamma promoter and a relative deficiency in alpha chain synthesis play a (perhaps less important) role in the increased Hb F synthesis in these patients.

A method of identifying delta chain variants using relatively small volumes of blood is described. The procedure consists of the amplification of two segments of genomic DNA with two sets of delta chain specific primers and sequencing of the three exons (exons 1, 2, and 3) which are part of the amplified DNA segments. Data for three delta chain variants present in seven adult heterozygotes are presented.