Hb Jambol: a new hyperunstable hemoglobin causing severe hemolytic anemia.

We describe a new hyperunstable beta-chain variant due to a complex genomic rearrangement. The abnormal hemoglobin (Hb) was found as a de novo mutation in a 2-year-old Bulgarian girl with severe hemolytic anemia. The mutation was detected through RNA/DNA analysis. It represents a complex genomic rearrangement involving an insertion of 23 nts after IVS-II-535 (derived by triplication of the 12-nts adjacent sequence and subsequent deletion of 1 nt), a deletion of 310 nts extending from IVS-II-550 to the first nt of Cd 108 and an insertion of 28 nts at the deletion junctions (derived from the inverted sequence between nts +3,707 and +3,734 3' to the beta-globin gene termination codon). At the protein level this mutation leads to a deletion of 4 amino acid residues (Leu-Leu-Glu-Asn) at positions 105-108 and an insertion of 9 residues (Val-Pro-Ser-Val-Thr-Leu-Phe-Phe-Asp) at the same location, creating an abnormal elongated beta-chain of 151 amino acid residues. This highly unstable variant was named 'Hb Jambol' after the geographic location in which the patient resides.

An Alu insert as the cause of a severe form of hemophilia A.

Alu sequences represent a specific human family of interspersed repetitive DNA, with a copy number in excess of 500,000 within the human genome. Alu repeats are rarely present in protein-coding regions of mature RNA, and only a few Alu insert mutations have been described so far. In this paper we present an Alu retroposition event in a family with a severe form of hemophilia A. The inserted Alu element belonging to the youngest Yb8 subfamily disrupts the reading frame at methionine 1224, exon 14 of the factor VIII gene, leading to a stop codon within the inserted sequence. This observation indicates that the retroposition of Alu elements is a continuing process possibly generating various human genetic defects.

Alpha-, beta-, and gamma-mRNA levels in beta-thalassemia; transcriptional and translational differences in heterozygotes, homozygotes, and compound heterozygotes.

We have determined the relative levels of alpha-, beta-, and gamma- (G gamma- and A gamma-) mRNAs in the reticulocytes of patients with mild beta-thalassemia intermedia due to combinations of promoter mutations and a classical type of beta-thalassemia, as well as in their relatives. The expected differences in the alpha/beta-mRNA ratio confirmed the mild suppression of beta-mRNA synthesis, particularly in heterozygotes for the -101 (C-->T) promoter mutation and the large increase in the relative gamma-mRNA level in compound heterozygotes. A significant discrepancy between Hb F and gamma-mRNA levels, observed in previously published studies, was confirmed indicating a less efficient gamma-mRNA translation. When the two different gamma-mRNA (G gamma- and A gamma-) levels were determined it was observed that in beta-thalassemia heterozygotes the extra gamma-mRNA was primarily of the G gamma type suggesting a more efficient translation of the A gamma-mRNA. This difference disappeared in homozygotes and compound heterozygotes: both mRNAs (G gamma- and A gamma-) translate with an equal efficiency.

The G----A mutation at position +22 3' to the Cap site of the beta-globin gene as a possible cause for a beta-thalassemia.

We describe the occurrence of a chromosome with a G----A mutation at position +22 relative to the Cap site that was found in five patients with beta-thalassemia. All patients had a common type of beta-thalassemia mutation on the second chromosome, namely the frameshift at codon 8 (-AA), the IVS-I-110 (G----A) and the IVS-II-1 (G----A) mutations. The beta genes of two patients, including the 5' and 3' untranslated regions, were completely sequenced and no other mutations, except a few polymorphic sites, were observed. Dot-blot analyses failed to demonstrate this G----A mutation at +22 in nearly 400 beta-thalassemia chromosomes and 180 normal chromosomes. Heterozygotes have the features of a high Hb A2-beta-thalassemia heterozygosity, although the hematological parameters might be less abnormal than observed in heterozygotes for the more common beta-thalassemia mutations. The possibility has been presented suggesting that this mutation might impair the binding of mRNA to ribosomes. Another mutation in this segment of DNA, i.e. a C----G mutation at position +20, is observed exclusively on a chromosome which also carries the C----G mutation at IVS-II-745. It is postulated that the +20 C----G mutation accentuates the beta-thalassemia condition caused by the IVS-II-745 mutation; the mechanism might be similar to that suggested for the G----A at +22 mutation.

Beta-thalassemia in Bulgaria.

Analyses of DNA from 64 patients with thalassemia major using the hybridization technique of amplified DNA with radiolabeled synthetic oligonucleotide probes identified 13 different beta-thalassemia mutations. The codon 39 (C----T) and IVS-I-110 (G----A) mutations occurred most frequently but seven additional mutations were observed which were present at frequencies of 3.9 to 10.2%. This broad spectrum of beta-thalassemia alleles complicates the analyses for institutions involved in prenatal diagnosis. Promoter mutations were rare and the frequencies of two other mild mutations [IVS-I-6 (T----C) and the poly A mutation] were relatively low indicating that beta-thalassemia is a severe disease among Bulgarians. The high frequencies of 4.7-5.5% for the four frameshifts at codons 5, 6, 8, and 8/9 may be specific for this population.