The silent carrier allele: beta thalassemia without a mutation in the beta-globin gene or its immediate flanking regions

A molecular genetic analysis has been performed using as subjects an Albanian family in which the father is a silent carrier, the mother has high Hb A2-beta thalassemia trait, and both children have beta thalassemia. Nucleotide sequence analysis of the daughter's paternal beta-globin gene and its flanking regions failed to reveal any base changes of known functional significance. When introduced into HeLa cells the gene was expressed at normal levels with proper processing of RNA. Haplotype analysis revealed that the affected son and daughter inherited different epsilon gamma delta beta-globin gene clusters from the father. The silent carrier allele is not due to a mutation within the beta-globin structural gene or its flanking regions and as such represents a novel form of beta+ thalassemia.     

Variability in the amount of beta-globin mRNA in beta0 thalassemia

Globin mRNA isolated from a number of beta0 thalassemia patients of different ethnic origins was analyzed by RNA-cDNA hybridization and, in two cases, by fingerprint analysis of 125I-labeled mRNA. Quantitation of the relative amounts of alpha- and beta-mRNA by hybridization to purified alpha-and beta-cDNA revealed that in approximately half the cases, there was less than 1% as much beta-mRNA as alpha-mRNA. In the rest of the cases, low levels of beta-like mRNA were detected in amounts 4-12% as abundant as alpha-mRNA. There was variability in the yield of beta-like mRNA in patients of the same racial group, in the same patient at different times and in similarly affected siblings: beta-mRNA was virtually absent in some samples, whereas low but significant levels were found in other samples. In one patient, beta-like mRNA was not detected in peripheral blood RNA, but was present in the RNA of bone marrow cells. In one case, the thermal stability of the beta0 thalassemia mRNA-beta-cDNA hybrid was measured and found to be slightly lower than that of the authentic beta-mRNA-beta-cDNA hybrid. In none of the cases tested was there synthesis of beta-globin chains directed by beta0 thalassemia mRNA in a cell-free protein-synthesizing system, even when beta-like mRNA was detected in the sample by hybridization assays. mRNA from two patients was labeled in vitro with 125I, digested with T1 RNAase and fractionated in two dimensions. Analysis of the resulting fingerprints revealed the presence of prominent alpha chain-specific oligonucleotides without detectable beta chain-specific oligonucleotides, and thereby confirmed the results of hybridization assays showing absent or very low levels of beta-mRNA in the same RNA samples. Our results support the concept that beta0 thalassemia is heterogeneous in its molecular basis even within the same racial group: in some patients, it is associated with absent beta globin mRNA, whereas in other patients, it is associated with low but significant levels of nonfunctional beta or beta-like globin mRNA. The variable amounts of beta-like mRNA detected in different samples from the same patient, and in patients with the same genotype, indicate that as yet undefined factors can influence the yield of beta-like mRNA observed in beta0 thalassemia.     

Expression of a beta thalassemia gene with abnormal splicing.

Expression of a cloned human beta thalassemia gene with a single base change at position 5 of IVS 1 has been analyzed 48 hours after transfer of the gene into HeLa cells (transient expression). Little or no normal beta globin mRNA accumulates in the presence of the abnormal beta gene in contrast to significantly more normal beta mRNA produced with other mutations at this same position. By contrast, large amounts of an abnormal beta globin mRNA are present; this is due to the use of a cryptic 5' splice site in exon 1 rather than the normal 5' splice site of IVS 1. The results indicate the variability of the effect on RNA splicing of different single base defects within IVS.     

Molecular causes of thalassemia. IV. Cloning of the beta-globin gene in a patient with beta-thalassemia from Azerbaijan and determination of the point mutation in a minor intron

On the basis of DNA from a beta-thalassemic patient, human gene library has been obtained using bacteriophage lambda EMBL4 as a vector. The recombinants contain human DNA insertions of 15 to 20 kb. The lambda A1 beta 1 clone has been isolated by the method of hybridization of phage plaque replicas to the HhaI fragment of JW102 plasmid containing human beta-globin cDNA. Restriction mapping revealed the presence of a 22 kb human DNA fragment comprising a portion of the beta-globin gene system. Subcloned fragments of beta-globin gene (within the pUC19 plasmid or phage MI3mp10) were sequenced using the Maxam and Gilbert method as well as that of Sanger. 2150 nucleotides in total were analysed. We have detected the point substitution G----A in the 110 nucleotide of minor intron, leading to the formation of an additional splicing site.     

Predominant gain of promoter TATA box after gene duplication associated with stress responses

TATA box, the core promoter element, exists in a broad range of eukaryotes, and the expression of TATA-containing genes usually responds to various environmental stresses. Hence, the evolution of TATA-box in duplicate genes may provide some clues for the interrelationship among environmental stress, expression differentiation, and duplicate gene preservation. In the present study, we observed that the TATA box is significantly overrepresented in duplicate genes compared with singletons in human, worm, Arabidopsis, and yeast genomes. We then conducted an extensive functional genomic analysis to investigate the evolution of TATA box along over 700 yeast gene family phylogenies. After reconstructing the ancestral TATA-box states (presence or absence), we found that significantly higher numbers of TATA box gain events than loss events had occurred after yeast gene duplications-the overall gain-loss ratio is about 3-4 to 1. Interestingly, these TATA-gain duplicate genes on average have experienced greater expression divergence from the ancestral expression states than their most closely related TATA-less duplicate partners, but only under environmental stress conditions (asymmetric evolution); indeed, under normal physiological conditions, they have similar expression divergence (symmetric evolution). Moreover, we showed that TATA-gain duplicates are enriched in stress-associated functional categories but that is not the case for TATA-ancestral duplicates (those inherited from their ancestors prior to duplication). Together, we conclude that after the gene duplication, gain of the TATA box in duplicate promoters may have played an important role in yeast duplicate preservation by accelerating expression divergence that may facilitate the adaptive evolution of the organism in response to environmental changes.     

The beta-globin gene in Sardinian delta beta 0-thalassemia carries a C----T nonsense mutation at codon 39.

Sardinian delta beta 0-thalassemia is an inherited syndrome characterized by the inactivity of the beta-globin gene and the persistent activity of the fetal gamma-globin genes, particularly the A gamma-globin gene. Previous mapping studies with restriction enzymes failed to show any abnormality in the non-alpha globin gene cluster. We have now examined the possibility that this syndrome might result from a single rather than two different defects. Restriction enzyme polymorphisms linked to the delta beta 0-thalassemic non-alpha globin fragments were defined providing the basis for cloning the delta beta 0-thalassemic beta-globin gene from the DNA of a heterozygous patient. This gene appears to carry a C----T single mutation causing the appearance of a stop codon at amino acid position 39 of the beta-globin gene. This mutation was previously reported in beta 0-thalassemic patients, in linkage with different haplotypes. We conclude that Sardinian delta beta 0-thalassemia is the result of two separate mutations, the former one (unknown) responsible for persistent expression of gamma-globin genes, the latter for beta 0-thalassemia.     

Testing of G----A mutation in position 110 of a minor intron of beta-globin genes in patients with thalassemia in Azerbaijan

A point mutation G-A in the 110 position of the beta-globin gene small intron has been revealed by cloning and sequencing from the material of a homozygote beta-thalassemia patient in Azerbaijan. In the present study two allele-specific oligonucleotide probes for testing the mutation have been synthesized. Assessment frequency of the mutation among the beta-thalassemia patients in Azerbaijan has been performed with the use of the amplified beta-globin gene fragments obtained by using the thermostable DNA-polymerase from T. thermophilus with the subsequent dot-hybridization in gel of the amplified material with the oligonucleotide probes. The possibility to test the mutation by hybridization of the oligonucleotide probes with the donors and beta-thalassemia patients restricted genomic DNA has been analyzed. Only one of 50 thalassemia alleles of beta-globin genes under study has been shown to possess the mutation mentioned.     

Alpha and beta thyroid hormone receptors bind immediately adjacent to the rat growth hormone gene TATA box in a negatively hormone-responsive promoter region

We report that alpha and beta type rat thyroid hormone receptors bind specifically and with high affinity to the 10-base pair sequence immediately 3' of the rat growth hormone TATA box (positions -25 to -16) in a region of the rat growth hormone promoter which can be negatively hormone responsive (nTRE). The receptors have approximately 7-fold lower affinity in vitro for the nTRE than for the thyroid hormone-responsive enhancer of the rat growth hormone gene (TRE). Proteins extracted with high salt concentration from rat pituitary cell nuclei enhance binding of the receptors to both the TRE and nTRE. A modification of the avidin-biotin complex DNA binding assay which enhances the sensitivity of the assay approximately 100-fold was used in these studies. The immediate proximity of a receptor binding site to the rat growth hormone TATA box suggests that direct interaction between receptor and TFIID (the TATA binding protein) mediates nTRE activity.     

A novel beta thalassemia gene with a single base mutation in the conserved polypyrimidine sequence at the 3' end of IVS 2

An adult Algerian patient with homozygous beta thalassemia was found to have a unique beta thalassemia gene. Cloning and sequencing revealed that the only abnormality present in this beta gene is a transversion in the polypyrimidine stretch at the 3' end of the large intervening sequence (IVS 2) six bases 5' to the consensus AG dinucleotide sequence (CCGCCCACAG instead of CCTCCCACAG). In addition, digestion of the cloned fragment by the enzyme Mnl I demonstrates the disappearance of a restriction site as expected. This is the first example of a defect in the consensus sequence at the 3' end of an IVS leading to a thalassemia phenotype presumably due to decreased splicing.     

Physical mapping of yeast artificial chromosomes containing sequences from the human beta-globin gene region

The recently developed technique for cloning genomic DNA fragments of several hundred kilobases or more into yeast artificial chromosomes (YACs) makes it possible to isolate gene families while preserving their structural integrity. We have analyzed five independent yeast clones identified by PCR screening using oligonucleotides derived from the adult human beta-globin gene. Analysis of the five clones containing YACs by conventional and pulsed-field gel electrophoresis revealed that all of the clones include a YAC with sequences from the adult beta-globin gene as expected. One of the clones contains multiple, unstable YACs. Two other clones carry single YACs in which there are at least two unrelated human genomic inserts. The remaining two clones contain single YACs, 150 and 220 kb in size, that contain the entire beta-globin gene family and flanking regions in a single, structurally intact genomic fragment. These should prove useful in future studies of the regulation of expression of genes in the beta-globin gene cluster.     

Rabbit globin pseudogene psi beta 2 is a hybrid of delta- and beta-globin gene sequences

The evolutionary history of the rabbit globin pseudogene psi beta 2 was studied by completing its nucleotide sequence and aligning the sequence with that of the rabbit adult globin gene beta 1 and the human minor adult globin gene delta. The 5' flanking region and exon 1 of psi beta 2 were most similar to rabbit beta 1, but the large intervening sequence and the 3' untranslated region were most similar to human delta. Intron 1 and exon 2 were equally similar to both delta and beta 1. This pattern indicates that psi beta 2 was originally a delta-like gene that acquired the 5' portion of gene beta 1 by intrachromosomal gene conversion. The presence of a delta-globin gene sequence in both rabbits and humans shows that it is an ancient gene, predating the mammalian radiation that occurred over 85 Myr ago. Delta has shown a pronounced tendency to be altered in its 5' end during the course of mammalian evolution. Quantitative divergence analysis shows that the ancestor to rabbit psi beta 2 was active until 20-30 Myr ago, during which time the lagomorph beta-globin gene family apparently functioned without a pseudogene.      

Scanning of beta-globin gene for identification of beta-thalassemia mutation in Romanian population

Beta-thalassemia is uncommon (0.5%) in the Romanian population, but it must be considered in the differential diagnosis of hypochromic anemia. The molecular characterization of beta-thalassemia is absolutely necessary for molecular diagnosis, as well as any genetic epidemiological study in this region. Molecular analyses consist of mutation detection by molecular scanning of beta-globin gene. This gene has 3 exons and 2 introns, involved in beta-thalassemic pathogenesis. Clinical application of DNA analysis on beta-thalassemic chromosomes allowed characterization of 29 persons with different beta-thalassemia mutations among 58 patients with anemia. The experimental strategy was based on sequential PCR amplification of most of the beta-globin gene and running on denaturing gradient gel electrophoresis of amplification products. Definitive characterization of mutations in samples identified with shifted DGGE patterns was performed ARMS-PCR and/or PCR-restriction enzyme analysis methods. Eight different beta-thalassemia alleles were identified, the most common being IVS I-110 (G-A) and cd 39 (C-T). Comparison of overall frequency of mutations in the neighboring countries, shows that these results are in the frame of overall distribution of these mutations in Mediterranean area, especially in Greece and in Bulgaria. Molecular diagnosis is useful for differentiating mild from severe alleles, for genetic counseling, as well as for mutation definition in carriers, identified by hematological analysis necessary for prenatal testing and genetic counseling.     

Five nucleotide changes in the large intervening sequence of a beta globin gene in a beta+ thalassemia patient.

A beta globin gene from a patient with homozygous beta+ thalassemia has been cloned and completely sequenced. No changes from normal are found in the 200 nucleotides 5' to the cap site, in the 3' untranslated region up to the poly A addition site, in the small intervening sequence (IVS 1), or in the coding sequence except for a third base change in codon 2. The only other differences are in the large intervening sequence (IVS 2). One of these, at a position 16 nucleotides from the 5' end of IVS 2, has been reported previously in normal individuals, and is probably a polymorphism. Four other changes, at positions 74, 81, 666, and 705 are also seen in IVS 2. Abnormal beta globin mRNA precursors detected in the bone marrow cells of this patient, and abnormal beta globin RNA splicing observed when this gene is transcribed in a tissue culture system taken together with these IVS 2 changes, suggest that the beta+ thalassemia phenotype is produced by a decrease in normal beta globin mRNA processing.