A. J. Dimovski

βs haplotypes in various world populations

SummaryWe have determined the βs haplotypes in 709 patients with sickle cell anemia, 30 with SC disease, 91 with S-β-thalassemia, and in 322 Hb S heterozygotes from different countries. The methodology concerned the detection of mutations in the promoter sequences of the Gγ- and Aγ-globin genes through dot blot analysis of amplified DNA with 32P-labeled probes, and an analysis of isolated Hb F by reversed phase high performance liquid chromatography to detect the presence of the AγT chain [Aγ75 (E19) Ile→Thr] that is characteristic for haplotype 17 (Cameroon). The results support previously published data obtained with conventional methodology that indicates that the βs gene arose separately in different locations. The present methodology has the advantage of being relatively inexpensive and fast, allowing the collection of a vast body of data in a short period of time. It also offers the opportunity of identifying unusual βs haplotypes that may be associated with a milder expression of the disease. The numerous blood samples obtained from many SS patients living in different countries made it possible to compare their hematological data. Such information is included (as average values) for 395 SS patients with haplotype 19/19, for 2 with haplotype 17/17, for 50 with haplotype 20/20, for 2 with haplotype 3/3, and for 37 with haplotype 31/31. Some information on haplotype characteristics of normal βA chromosomes is also presented.

The G→A Mutation at Position +22 31 to the Cap Site of the β-Globin Gene as a Possible Cause for a β-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 β-thalassemia. All patients had a common type of β-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 β 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 β-thalassemia chromosomes and 180 normal chromosomes. Hetero-zygotes have the features of a high Hb A2-β-thalassemia hetero-zygosity, although the hematological parameters might be less abnormal than observed in heterozygotes for the more common β-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 m...

Possible factors influencing the haemoglobin and fetal haemoglobin levels in patients with β‐thalassaemia due to a homozygosity for the IVS‐I‐6 (T→C) mutation

Summary. We have collected haematological, haemoglobin (Hb) and DNA sequence data for 29 patients with a homozygosity for the IVS‐I‐6 (TC) mutation with the intention of identifying factors contributing to the observed variability in the severity of the disease. None of the patients had received blood transfusion therapy for at least 6 months prior to the study. Hb levels varied from 5·0 to 9·9 g/dl. Patients with high Hb F (more than 1·5 g/dl or <20%) had high total Hb levels (7·5–9·7 g/dl) but some with low Hb F also had high total Hb levels; two had a concomitant α‐thalassaemia‐2 (α‐thal‐2) heterozygosity. An inverse correlation between the Hb F and Hb A2 levels was observed. The majority of the patients were homozygous for haplotype VI (49/58 chromosomes) but haplotypes IV (2/58) and VII (7/58) were also present. The only haplotype IV homozygote had high Hb F levels with high Gγ values and the CT mutation at position – 158 in the Gγ promoter, while both high and low Hb F levels were observed among patients with haplotypes VI and VII. Analysis of sequence variations in regulatory regions included the 5 hypersensitive sites (HS) 4, 3 and 2 of the locus control region (LCR), the Gγ and Aγ 5 flanking regions, the second intervening sequence (IVS‐II), and the 5 β‐globin gene region in two patients with high Hb F (one homozygote each for haplotypes VI and IV), and in two patients with low Hb F levels (one homozygote each for haplotypes VI and VII). Haplotype specific differences were observed in the LCR 5 HS‐2 and in the Gγ and Aγ flanking and IVS‐II regions; however, no differences were present between the low and high Hb F‐producing haplotype VI chromosomes, suggesting a major role for factors which are not linked to the β‐globin gene cluster in mediating γ‐globin gene expression in patients with this type of β‐thal.

γ‐mRNA and Hb F levels in β‐thalassaemia

Summary. The Hb F levles in β‐thalassaemia can be affected by factors both linked and unlinked to the β‐globin gene cluster. We have recently analysed a group of patients with a homozygosity for the IVS‐1–6 (T → C) mutation, showing a wide variation in Hb F levels (2–47%) which could not be accounted for by any sequence variation within regulatory elements of the β‐globin gene cluster. In order to further investigate factors underlying this phenotypic difference we have developed a competitive reverse trascription/polymerase chain reaction procedure and used this method to determine the relative amounts of γ‐and β‐mRNAs in 10 patients with the IVS‐1–6 homozygosity and 15 heterozygous parents, two IVS‐I‐6δβ‐thalassaemia compound heterozygotes, five homozygotes for the βd́; IVS‐I‐110 (G → A) mutation, and in two with a homozygosity for the β† codon 39 (C → T) mutation. Three heterozygotes were also included. The percentages of γ/(γ+β) mRNA were 10–73% in the IVS‐I‐6 homozygotes and <2% to 10% in their heterozygous parents. A direct relationship existed between the level of mRNA and the % Hb F. However, the relative γ‐mRNA levels in the IVS‐I‐6 homozygotes were higher than their Hb F levels, indicating a possible competition between the γ and β transcripts for translational factors with a less effcient initiation of protein synthesis on the γ‐mRNA or a preferential survival of cells with mainly β‐globi gene expression at the post‐reticulocye stage. The γ‐mRNA levels in the two IVS‐I‐6/δβ‐thalassaemia compound heterozygotes were 71% and 62%, similar to their Hb F levels (63% and 59%), and averaged 82% (range 65–91%) in the five IVS‐I‐110 homozygotes and 97.5% in the two codon 39 homozygotes. The correlation between these values and the % Hb F could not be evaluated because of the transfusion regimens; however, the levels, of γ‐mRNA were as expected for patients with these β‐thalassaemia alleles.

The relative levels of βA and βS mRNAs in Hb S heterozygotes and in patients with Hb S-β+ -thalassaemia or Hb S-β+ -HPFH combinations

SUMMARY. We have used a quantitative reverse transcription/ polymerase chain reaction (RT/PCR) procedure to evaluate the relative amounts of βA and βS mRNA transcripts in eight subjects with a simple Hb S heterozygosity, in six with Hb S‐β+ ‐thalassaemia (thal), and in three individuals with Hb S‐β+ ‐HPFH [hereditary persistence of fetal haemoglobin (Hb)] [two with the Atlanta type and one with the Gγ‐202 (C ± G) substitution]. A balanced synthesis of βA and βS mRNAs was observed in all Hb S heterozygotes, whereas the βA mRNA levels were reduced to ˜ 16% of that of the βS mRNA in the six Hb S‐β+ ‐thal compound heterozygotes, to ˜43% in the two subjects with Hb S‐β+ ‐HPFH (Atlanta type), and to 23.8% in the one individual with Hb S‐β+ ‐HPFH [Gγ‐202 (C ± G) substitution]. The higher Hb A versus Hb S levels observed in all groups of the patients studied, further confirm a post‐translational control mechanism in determining the levels of Hb A and Hb S in the peripheral blood of these individuals. The procedure described here provides an accurate and easy method for studying the relative expression of particular globin genes at the transcriptional level in patients with various haemoglobinopathies.

Sickle cell anemia, sickle cell β-thalassemia, and thalassemia major in Albania: characterization of mutations

We have analyzed the hemoglobin abnormalities in nearly 50 Albanian patients with a significant hemoglobinopathy and included 37 relatives in this study. Sickle cell anemia (SS) is a common disorder; all 15 sickle cell anemia patients had the complications expected for this disease. The βs haplotype was type 19 (Benin); α-thalassemia-2 was rare. Three β-thalassemia alleles (IVS-I-110, G→A; codon 39, C→T; IVS-I-6, T→C) were present in nearly 85% of the β-thalassemia alleles; their frequencies were intermediate between those observed in the populations of neighboring countries. A few rare mutations were also found, which might have originated in India, Turkey, Macedonia, and Greece. Nearly all patients with Hb S-β-thalassemia had the IVS-I-110 (G→A) mutation. The frequencies of 11 β-thalassemia mutations in 17 mostly Mediterranean countries have been reviewed.

The in vivo expression of the globin genes of theβ cistron in γ-,δ-, andδβ-thalassemia heterozygotes

There is considerable evidence suggesting that the switch from γ to δ and β chain production after birth is due, in part, to silencing of the γ genes by stage-specific factors which bind to their promoters and to the competition from the adult (δ and β) genes for a common enhancer element located in the locus control region. As a consequence one can expect that the increased Hb F production in adults with hereditary persistence of fetal hemoglobin or δβ-thalassemia is directed mainly by γ-globin genes in cis to the deletion(s) responsible for these conditions. Here we review data on heterozygotes with γ-, δ-, or δβ-thalassemia, who also had anAγT mutation, in cis or in trans, which was used as a marker of γ gene expression. The results show that a deletion affecting adult β genes favors the expression of γ genes in cis, while the deletion of a single γ gene does not affect the expression of the β gene in cis but leads to a faster γ→β switch postnatally.