Beta-thalassemia in Yugoslavia.

This study concerned the evaluation of beta-thalassemia alleles in nearly 50 patients with beta-thalassemia major and in 130 -thalassemia heterozygotes using gene amplification and dot-blot hybridization with synthetic probes. Fourteen different mutations were observed; of these, three (IVS-I-110; IVS-I-6; IVS-I-1) account for some 75% of all beta-thalassemia alleles. Newly discovered variants, i.e. T----C in the initiation codon and AATAAA----AATGAA in the poly A site were observed in a few patients. The poly A mutation with classical beta-thalassemia alleles result in thalassemia intermedia. Hb Lepore is a rather common abnormality and combinations of this variant with beta-thalassemia often result in severe disease; a search for beta-thalassemia mutations among patients affected with this disease should include an analysis to detect this hemoglobin abnormality.

Variation in clinical severity among patients with Hb Lepore-Boston-beta-thalassaemia is related to the type of beta-thalassaemia.

Clinical and haematological observations, made for 10 Yugoslavian patients with the Hb Lepore-beta-thalassaemia condition, suggested a considerable variation from severe disease and complete blood transfusion dependency to a moderate, compensated, anaemia without major complications and without a need for regular blood transfusions. As the type of Hb Lepore was the same in all patients (Lepore-Boston-Washington) and an alpha-globin gene deficiency was absent, it was concluded that the type of beta-thalassaemia determined the severity of the disease. Six patients with severe disease had one of the following three beta-thalassaemia determinants: IVS-1 position 110 G----A, exon 2 codon 39 C----T, and IVS-1 position 1 G----A, while the three patients with mild disease had the Portuguese type of thalassaemia which is caused by the T----C substitution at position 6 of the IVS-1. In one patient with severe disease the beta-thalassaemia determinant remained unknown. Our observations are consistent with those made for thalassaemia patients with a homozygosity for these determinants.

One haplotype is associated with the Swiss type of hereditary persistence of fetal hemoglobin in the Yugoslavian population.

Blood samples from normal adults and from members of seven families with the Swiss type of hereditary persistence of fetal hemoglobin (HPFH) from Yugoslavia were analyzed for their fetal hemoglobin (Hb F) and G gamma levels, while haplotyping defined the chromosomes at eight or nine polymorphic restriction sites. The data indicate that Swiss-HPFH, characterized by slightly elevated Hb F and G gamma levels and no recognizable hematological abnormality, is associated with a chromosome whose restriction enzyme haplotype is identical to the no. 3 (Senegal) haplotype found in black sickle cell (SS) patients. Many adults with this chromosome have high G gamma but normal Hb F levels. It is suggested that the Swiss-HPFH phenotype results from the action of more than one factor; one is linked to the beta-globin gene cluster and causes high G gamma values, while others result in an increased Hb F production and are perhaps of different origins.

G gamma A gamma(delta beta)zero-thalassaemia and a new form of gamma globin gene triplication identified in the Yugoslavian population.

Among several hundred apparently healthy Yugoslavian adults with slightly elevated levels of fetal haemoglobin, we have identified two distinct abnormalities. (a) A G gamma A gamma(delta beta)0-thalassaemia heterozygosity with an approximately 15 kb deletion which involves part of the delta globin gene and the beta globin gene. This deletion is probably the same as that seen among Italians (Ottolenghi et al, 1982; Carè et al, 1984). (b) A nondeletion form of hereditary persistence of Hb F which is caused by a gamma globin gene triplication of the (+)G gamma.(+)G gamma.A gamma type. It is characterized by the presence of some 5% Hb F in the heterozygote containing nearly 100% G gamma chains. The C----T mutation at position--158 5' to the G gamma chain [(+)G gamma], identified through analyses of Xmn I digests, was present at both G gamma globin genes. This mutation is known to be associated with increased G gamma chain production (Gilman & Huisman, 1985), and thus is responsible for the increased G gamma chain production in these heterozygotes. The condition is different from the (+)G gamma.(+)G gamma nondeletion type of HPFH which has been observed in heterozygotes of two Black families, and is associated with the presence of 3-4% Hb F (with mainly G gamma chains) in heterozygotes.

[Alpha thalassemia in Macedonia]. / Alfa talasemija u SR Makedoniji.

The frequency of alpha thalassemia in SR Macedonia was determined with studies of Hb Bart's in 1.140 newborn babies. Hb Bart's was found in 83 infants. Distribution of the levels of Hb Bart's in these neonates, as determined by column chromatography on CM Sephadex, was trimodal. The mean values for Hb Bart's in the three groups were 0.5% (SD = 0.22), 1.61% (SD = 0.48), and 4.88% (SD = 0.81). The first group is believed to result from asynchronism of the neonatal "switch off" of gamma chains and activation of beta chain production. The second and the third group represent alpha thal2 and alpha thal1, respectively. Thus, the incidence of beta thal2 in SR Macedonia is 2.4%, and that of alpha thal1 0.8%. Hb H disease was found in three out of 16.000 school children. This form of alpha thalassemia was also found in five out of 2.800 patients examined for the course of anemia. Biosynthetic studies of family members of the eight individuals with Hb H disease showed that one parent is heterozygous alpha thal1 while the other is heterozygous alpha thal2. All individuals with alpha thalassemia had a reduced rate of synthesis of the alpha chains. The mean alpha/beta total activity ratio in individuals with Hb H disease was 0.51 (+/- 0.08), in heterozygous alpha thal1 0.74 (+/- 0.06), and in heterozygous alpha thal2 0.86 (+/- 0.06). Experiments with in vitro translation of globin mRNK isolated from patients with Hb H disease showed lower alpha/beta ratios (0.06) than the intact cell ratios. These results support previously published data that in Hb H disease and alpha thalassemia trait, there is quantitative deficit in alpha globin mRNK, which is a consequence of deleted alpha globin genes.

The use of globin chain electrophoresis in polyacrylamide gels for the quantitation of the G gamma to A gamma ratio in fetal hemoglobin.

Polyacrylamide gel electrophoresis (PAGE) in the presence of urea, acid, and Triton X-100 was used for determination of the G gamma to A gamma ratio in human Hb F. The data compared most favourable with results obtained by a HPLC procedure and by a chemical procedure. Moreover, its accuracy and reproducibility was determined. The presence of Hb A2 in a sample with Hb F level below 10% interferes with the determination because of the nearly identical electrophoretic mobilities of the delta and G gamma chains. Thus, the removal of Hb A2 is required. Samples free fo Hb A2 but with a Hb F level as low as 2% can be analyzed with an accuracy of about 5%. The PAGE method has been applied to the Hb F of 63 subjects with beta thalassemia and related conditions, and the results of these analyses are included in this communication.