The mutations producing beta-thalassaemia in 174 Greek patients with thalassaemia major were investigated by dot-blot hybridization of oligonucleotide probes to genomic DNA amplified by the polymerase chain reaction procedure, by direct sequencing of amplified DNA, and by gene mapping. beta-thalassaemia in Greeks was found to be very heterogeneous at the molecular level as 17 different mutations were observed: 86.6% of the beta-thalassaemic genes, however, could be identified with five probes: IVS-I-110 (G----A) (42.5%), codon 39 (C----T) (17%), IVS-I-1 (G----A) (13.2%), IVS-I-6 (T----C) (7.2%) and IVS-II-745 (C----G) (6.9%). Several mutations which had not previously been reported in the Greek population and which occurred at an incidence of 2% or lower were observed in this study. The information obtained will facilitate the prenatal diagnosis of beta-thalassaemia in Greece.

The presence of various substitutions and deletions resulting in beta- thalassemia was studied in 19 black patients with homozygous beta- thalassemia and in numerous relatives; all patients were from Georgia, South Carolina, and Alabama. Methodology included gene mapping, amplification of genomic DNA with Taq polymerase, identification of known nucleotide substitutions or a single nucleotide deletion through hybridization with synthetic oligonucleotides, cloning and sequencing of a beta-globin gene, and sequencing of amplified genomic DNA. Of the 38 chromosomes tested, 21 (55%) had the A----G substitution at nt -29, eight (21%) had the C----T substitution at nt -88, three (8%) had the substitution at codon 24, while one each of the following abnormalities were also detected: frameshift at codon 6, a C----A mutation at nt 848 of the beta IVS-II (new), an A----T mutation at codon 61 (new), a deletion of 1.35 kilobases including the 5′ end of beta, a Ggamma(Agammadelta beta) degree-thalassemia, and one thalassemia determinant that remained unidentified. The C----A mutation at nt 848 of IVS-II occurred at a position 3 nucleotides 5′ to the third exon, adjacent to the invariant AG dinucleotide of the acceptor sequence. The A----T mutation in codon 61 (AAG----TAG) resulted in the creation of a stop codon and thus in beta degree-thalassemia. The various mutations occurred on chromosomes with different haplotypes; however, chromosomes with a specific mutation but with different haplotypes belonged to one specific framework, which suggested that crossovers were responsible for these different types. Hemoglobin (Hb) F levels were generally high (55% to 75% with 98.5% in one patient with beta degree/beta degree); a few patients with specific haplotypes and an alpha-thalassemia-2 heterozygosity had a lower Hb F level. The Ggamma in the Hb F was consistently high when the C----T mutation occurred at nt -158 to the Cap site of the Ggamma-globin gene; seven patients with +/+ at this site had an average Ggamma of 73.8%, eight patients with +/- had 64.8%, and one patient with -/- had 34.2%. Variations in hematologic values and in Hb F, Ggamma, and Hb A2 levels of relatives with a beta- thalassemia heterozygosity depended to some extent on the types of mutations or deletions and on the haplotypes of the chromosomes with the beta-thalassemia determinant.

Sequence analyses and dot-blot analyses with synthetic oligonucleotide probes have identified eight individuals in three Turkish families and one Bulgarian family with one chromosome having a C----T mutation at nucleotide position--101 relative to the Cap site of the beta-globin gene. This nucleotide is part of one of the conserved blocks of nucleotides within the promoter region; in vitro expression analyses with the chloramphenicol acetyltransferase system showed that this substitution will decrease the effectiveness of transcription. Five subjects had a thalassemia intermedia due to the additional presence of a known classical high hemoglobin (Hb) A2 beta-thalassemia mutation on the second chromosome; their hematologic condition was relatively mild. The three persons with a heterozygosity for the--101 C----T mutation had normal hematologic data without microcytosis but with high-normal levels of Hb A2 and a mild imbalance in chain synthesis. The newly discovered mutation is considered one of the silent types of beta- thalassemia. It is relatively rare because it was absent among several hundred normal and beta-thalassemia chromosomes.

Details are given of a study of blood samples from 24 patients with Hb H disease from different Mediterranean countries and from the Far East. Four different types of alpha-thal-1 (--) were observed, namely -(alpha) (approximately 20.5-kb deletion); --MED-I (approximately 17.5-kb deletion); --MED-II (greater than 26.5-kb deletion); and --SEA (approximately 18-kb deletion, in Orientals only). The alpha-thal-2 was mainly of the deletion type (16 with the 3.7-kb deletion; 1 with the 4.2-kb deletion), while 4 of the 7 patients with a nondeletional type had the five-nucleotide deletion at the donor splice site of the first intron of the alpha 2 gene. All patients had a mild-to-moderate hemolytic anemia; no significant differences in hematology were observed between the groups. Hb A2 was decreased to about one-third of the normal level. The Hb H formation varied considerably and its quantitation was not always satisfactory. Patients with Hb H disease due to any alpha-thal-1 combined with a nondeletional alpha-thal-2 had the highest Hb H levels and a more marked anemia. The zeta chain production was small and absent in patients with the MED-II type of alpha-thal-1 because this deletion included the zeta and psi zeta genes. The highest zeta chain levels were present in the four patients with the SEA type of alpha-thal-1. The gamma chain production was increased, particularly in patients with a mutation of C----T at position -158 to the G gamma globin gene. This gamma chain was primarily present as Hb Bart's (or gamma 4) and only about 15% was recovered as Hb F or alpha 2 gamma 2. The evaluation of the rate of gamma chains produced in these patients was greatly facilitated by data from one patient who had Hb H disease and a heterozygosity for the A gamma-beta+-HPFH. The low levels of Hb A2 and of Hb F (relative to Hb Bart's) can be explained by a decreased affinity of alpha chains for delta and gamma chains as compared with beta chains in conditions of severe alpha chain deficiency.

An extension of previous reports describing the molecular defects and hematological abnormalities in black patients with Hb S(C) beta-thalassemia living in the Southeastern United States is presented. As many as 58 patients with Hb S-beta(+)-thalassemia, 16 with Hb C-beta(+)-thalassemia and 12 with Hb S-beta(0) -thalassemia have been studied. Patients with Hb S(C) beta(+)-thalassemia type 2 (high Hb A values) were most common; the thalassemia was due to mutations in the promoter of the beta-globin gene [-88 (C----T) and -29 (A----G)] or at the polyadenylation signal (T----C). Two patients with lower Hb A values (type 1) carried a mutation in the first intron of the beta-globin gene (IVS-1-5: G----T). The simultaneous presence of an alpha-thalassemia -2(-alpha/) resulted in some modifications of the hematological parameters, but had a minimal effect on the clinical condition. Patients with Hb S-beta (0) thalassemia had lower hemoglobin values, lower number of red blood cells, and lower MCHC values and suffered more frequently from complications than the patients with Hb S-beta(+)-thalassemia. A total of 17 different beta-thalassemia mutations were observed in 128 chromosomes; two mild beta(+)-thalassemia mutations [-88(C----T) and -29(A----G)] account for more than 80% of the thalassemic chromosomes.