Homozygous β-thalassaemia resulting in the β-thalassaemia carrier state phenotype

Summary. This paper describes the phenotypic manifestations of a very mild β-thalassaemia mutation detected in several members of two families of Italian descent. The molecular defect, defined by denaturing gradient gel electrophoresis analysis and direct sequencing. consists of a C G substitution at position 844 of IVSII of the β-globin gene within the consensus sequence of IVSII acceptor splice site. Heterozygotes for this mutation show a haematological phenotype ranging in severity from silent β-thalassaemia to that of a mild β-thalassaemia carrier silent β-thalassaemia to that of a mild β-thalassaemia carrier state, whereas homozygotes have the typical manifestations commonly resulting from heterozygosity for a β-thalassaemia mutation. Compound heterozygotes for the IVSII nt844 (C G) mutation and a severe β-thalassaemia mutation have the phenotype of thalassaemia intermedia.
This paper indicates that the presence of borderline red blood cell indices or HbA₂ values should make one suspect the presence of a very mild or silent β-thalassaemia.     

Molecular, haematological and clinical studies of the -101 C --> T substitution of the beta-globin gene promoter in 25 beta-thalassaemia intermedia patients and 45 heterozygotes

We report the clinical, haematological, biosynthetic and molecular data of 25 double heterozygote beta-thalassaemia intermedia patients and 45 beta-thalassaemia heterozygotes with the C --> T substitution at nucleotide position -101 from the Cap site, in the distal CACCC box of the beta-globin gene promoter. This mutation is considered the most common amongst the silent beta-thalassaemia mutations in Mediterranean populations. Of the 25 compound heterozygotes for the beta -101 C --> T and common severe beta-thalassaemia mutations, all but one had mild thalassaemia intermedia preserving haemoglobin levels around 9.5 g/dl and haemoglobin F levels < 25%. The only transfused patient was characterized to have an additional alpha-globin gene. Strict assessment of haematological and biosynthetic findings in the heterozygotes for the beta -101 C --> T mutation (excluding six cases with an alpha-globin gene defect) demonstrated that less than half of them had completely normal (silent) haematology; the remainder had either high haemoglobin A2 values (in the range of 3.7-5.1%) and/or low red cells indices and/or raised haemoglobin F values. The alpha/non-alpha-globin chain synthesis ratios were generally raised, with mean 1.44 (1.07-2.10). Amongst the parents of the compound heterozygotes, who were not selected for molecular analysis following haematological screening, half of the cases were completely silent. Interaction with severe beta-thalassaemia mutations always resulted in the clinical phenotype of mild non-transfusion-dependent thalassaemia intermedia.     

A novel β-thalassaemia mutation in the 5'untranslated region of the β-globin gene

Summary. A thymidine deletion at position +10 of the 5'untranslated region of the β-globin gene was detected in a β-thalassaemia intermedia patient carrying a β; 39 stop codon mutation on the other chromosome; this new mutation, +10(-T), was detected by automated fluorescent DNA sequencing and verified by dot-blot allele-specific hybridizations.
The +10(-T) mutation is a 'silent carrier', is associated with a reduced amount of steady-state β-globin mRNA, and establishes a connection between the 5'untranslated region of the β-globin gene and the regulation of its expression.     

A novel amber mutation in a β°-thalassaemia gene (β37TGG→TAG), with direct detection by mapping the restriction fragments in amplified genomic DNA

Summary. A novel amber mutation, a G to A substitution at the second position of codon 3 7 in the β-globin gene that changes the tryptophan coding triplet (TGG) to a termination codon (TAG), was found in a Chinese β-thalassaemia carrier. The mutant gene creates an additional Dde I recognition site and eliminates the Ava II site, so this point mutation can be directly identified by restriction enzyme analysis.     

Severe inclusion body β-thalassaemia with haemolysis in a patient double heterozygous for β°-thalassaemia and quadruplicated α-globin gene arrangement of the anti-4.2 type

We describe a new case of an association of alpha-globin gene quadruplication of the anti-4.2 type with beta(0)-thalassaemia. The patient, a young woman of mixed Brazilian-Portuguese origin, suffered from chronic haemolytic anaemia with splenomegaly. Bone marrow supravital staining with brilliant cresyl blue and electron microscopy studies showed large inclusion bodies in about 3% of erythroblasts. Upon immunofluorescent staining these inclusions reacted with a monoclonal antibody to alpha- but not to beta-globin. Analysis of alpha-globin cluster by Southern blotting showed the presence of pathologic fragments specific for the anti-4.2 alpha-globin gene quadruplication. Alpha/beta mRNA ratio was higher than in cases combining alpha-globin triplication and beta(0)-thalassaemia or in cases of beta(0)-thalassaemia heterozygous state alone (18, 14.7 and 10.1 respectively). Our data confirmed the hypothesis that the clinically detectable haemolysis in this beta(0)-thalassaemic patient was due to an unusually high amount of precipitated alpha-globin in erythroid precursors. This considerable excess of alpha-globin chains was due partly to the beta-globin deficit caused by the presence of the beta(0)-thalassaemic gene, but also to the presence of 6 active alpha-globin genes resulting from alpha-globin gene quadruplication in one chromosome.     

β-Thalassaemia in south-western Spain: high frequency of G → A (IVS I-1) mutation

Huelva province is situated in southwestern Spain; its historic and geographic characteristics contribute to the existence of erythrocyte genetic anomalies, such as β thalassaemia. We have carried out a prenatal study of microcytosis, with a preliminary β-gene analysis. Our findings show a β-thalassaemia trait prevalence of 0.81%. In the molecular research there was a high frequency of G → A (IVS I-1) injury: 55%. By comparing the rates of this mutation in neighbouring Cáceres province in western Spain (47%) and Algarve in south Portugal (32%), it demonstrates that this β-gene defect has a high frequency in the southwest of the Iberian Peninsula.     

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.     

Clinical and laboratory effects of long-term administration of hydroxyurea to patients with sickle-cell/β-thalassaemia

Hydroxyurea (HU), a widely used cytostatic, has been given over a long period of time to 14 adult Caucasian compound heterozygotes for β^s and various β-thalassaemia genes. All patients had severe pain crises and other complications prior to receiving the drug. After 4-8 weeks on high 'sub-toxic'doses of HU all patients responded with a multifold increase of fetal haemoglobin (HbF) and a marked increase of MCV and MCH; they also felt significantly better and ceased having pains or other complaints. Haematological toxicity was minimal and rapidly reversible. Follow-up of the patients has now exceeded 100 weeks and goes up to 180 weeks in two of them. Pain crises have never recurred. Maintenance of high levels of HBF requires continuous administration of high doses of HU; whenever the latter were decrease in various attempts to avoid potential long-term toxicity, the observed changes gradually faded. The effect of HU in HbS/β-thalassaemia may be better than that reported for homozygous HbS disease because the synthesized λ-chains not only inhibit the sickling process but they also neutralize the noxious effects of the excess α-chains and cut down the ineffective erythropoiesis of the patients.     

Haemoglobin Dhofar is linked to the codon 29 C –→ T (IVS-1 nt – 3) splice mutation which causes /3+ thalassaemia

Investigations of a young man with apparent thalassaemia minor showed that he was a heterozygote for a rare abnormal haemoglobin variant, Hb Dhofar. The amino acid replacement is in the /3-globin chain (j358 Pro – Arg) and is therefore not consistent with the observed proportion of Hb Dhofar, as in both this and the original case, it constituted only 15% of the total haemoglobin. We have determined the basis of the low expression of this mutant, which is due to its linkage to a thalassaemic splicing mutation on the same /3-globin gene at codon 29 (C –> T). The finding of this thalassaemia mutation linked to Hb Dhofar not only explains the low level of Hb Dhofar, but also provides evidence that the codon 29 C –> T, IVS-1 splice junction mutation causes a (3 ⁺ form of thalassaemia.     

A 5' splice region G → C mutation in exon 3 of the human β-spectrin gene leads to decreased levels of β-spectrin mRNA and is responsible for dominant hereditary spherocytosis (spectrin Guemene-Penfao)

We studied a family with autosomal dominant hereditary spherocytosis (HS) associated with a mild spectrin deficiency. Linkage analysis using two microsatellite markers (D14S63 and D14S271) very close to the β-spectrin gene (SPTB) showed that HS co-segregated with alleles of these microsatellite markers and the linkage between the marker and HS was statistically significant. The presence of a β-spectrin protein polymorphism (β-spectrin Vay; A1880V) in trans of the HS allele was not itself deleterious, but allowed the detection of decreased membrane expression of the spherocytic β-spectrin allele in two HS-affected subjects. Direct sequencing of the coding exons of the β-spectrin gene in one affected subject showed the presence of a G → C transversion at the terminal nucleotide of exon 3, which did not change the leucine codon 100 (CTG → CTC). The presence of the mutation was confirmed by restriction enzyme digestion at the DNA level in all affected SH members of the family. The G → C mutation severely reduced the utilization of the 5' splice site and resulted in aberrant mRNA splicing with intron 3 retention.     

GγAγ(β+) hereditary persistence of fetal hemoglobin: The Gγ – 158 C → T mutation in cis to the − 175 T → C mutation of the Aγ-globin gene results in increased Gγ-globin synthesis

Hereditary persistence of fetal hemoglobin (HPFH) can be generally classified into deletional and nondeletional forms. The family described in the present study has characteristics of both types of HPFH. The proband is a healthy 30-year-old black woman. Analysis of her hemoglobin revealed 40.4% HbS, 40.9% HbF (^Gγ/^Aγ ratio 0.53), 16.8% HbA, and 1.9% HbA₂. All of her hematologic indices were normal, and the distribution of HbF in her red cells was pancellular. Family studies demonstrated that the proband has one chromosome 11 bearing the β^s-globin gene and the other bearing a ^Gγ^Aγ(β⁺) HPFH determinant in cis to the β^A-globin gene. Gene mapping studies of the region between the ^Gγ- and β-globin genes were normal. However, when the ^Aγ and ^Gγ promoters were amplified by polymerase chain reaction (PCR) and sequenced, the ^Aγ promoter was found to have the T→C mutation at ?175, and the ^Gγ promoter region was found to have the C→T mutation at ?158. The ?158 C→T mutation has been associated with elevated ^Gγ levels and high HbF in hemolysis, although its role in causing these effects is unclear. The present study suggests that this mutation can also enhance ^Gγ-globin expression in cis to the ?175 T→C mutation in the absence of hemolysis. We suggest that the alteration of the ^Aγ gene octamer binding site by the ?175 mutation, as well as the loss of a putative ^Gγ “silencer” caused by the ?158 mutation may account for this phenotype. We propose calling these linked mutations the ^Gγ^Aγ(β⁺) HPFH. © 1993 Wiley-Liss, Inc.     

Novel point mutations in the αIIb subunit (Phe289 → Ser, Glu324 → Lys and Gln747 → Pro) causing thrombasthenic phenotypes in four Japanese patients

We analysed the molecular basis of Glanzmann thrombasthenia (GT) in four Japanese patients with type I or type II disease. Polymerase chain reaction (PCR) and subsequent direct sequencing of platelet RNA and genomic DNA revealed three single nucleotide substitutions of the αIIb gene, which were confirmed by allele-specific PCR or restriction analysis. One patient with type I GT had a T to C base substitution in exon 11 resulting in a Phe (TTT)-289 to Ser (TCT) mutation (F289S) of the subunit. Another type I patient had a G to A base substitution in exon 12 resulting in a Glu (GAA)-324 to Lys (AAA) mutation (E324K). Interestingly, two unrelated patients with type II GT shared an A to C base substitution in exon 23, a region previously not associated with GT, resulting in a Gln (CAA)-747 to Pro (CCA) mutation (Q747P). To analyse the effects of these mutations on αIIbβ3 surface expression, the wild-type αIIb cDNA or mutant αIIb cDNAs were transfected into Chinese hamster ovary (CHO) cells together with a wild-type β3 cDNA. Flow cytometric analysis using an anti-αIIbβ3 complex antibody revealed that 50.6% of CHO cells with wild-type αIIbβ3 expressed complexes, whereas only 1.6%, 7.7% and 31.3% of cells, with αIIb(F289S)β3, αIIb(E324K)β3 and αIIb(Q747P)β3 expressed complexes, respectively. Our data indicate that these three novel point mutations in the αIIb subunit may hamper surface expression of the αIIbβ3 complex, thus resulting in the quantitative GT phenotypes of platelets from these patients.     

Two new unstable haemoglobins leading to chronic haemolytic anaemia: Hb Caruaru [β122 (GH5) Phe→Ser], a probable case of germ line mutation,and Hb Olinda [β22 (B4) ‐ 25 (B7)], a deletion of a 12 base‐pair sequence

We describe here two new unstable β‐globin variants, Hb Caruaru and Hb Olinda, found in northeastern Brazil, both associated with chronic haemolytic anaemia. Haemoglobin Caruaru is caused by a single base substitution at codon 122 (TT C→TC C), possibly originating from the germ line cells of the patient’s grandmother. Haemoglobin Olinda is also a de novo mutation, caused by a 12 bp deletion leading to the removal of the 22nd to the 25th residues of the normal β‐globin chain.     

A randomized phase I/II trial of HQK‐1001, an oral fetal globin gene inducer,in β‐thalassaemia intermedia and HbE/β‐thalassaemia

β‐thalassaemia intermedia (BTI) syndromes cause haemolytic anaemia, ineffective erythropoiesis, and widespread complications. Higher fetal globin expression within genotypes reduces globin imbalance and ameliorates anaemia. Sodium 2,2 dimethylbutyrate (HQK‐1001), an orally bioavailable short‐chain fatty acid derivative, induces γ‐globin expression experimentally and is well‐tolerated in normal subjects. Accordingly, a randomized, blinded, placebo‐controlled, Phase I/II trial was performed in 21 adult BTI patients (14 with HbE/β⁰ thalassaemia and seven with β⁺/β⁰ thalassaemia intermedia, to determine effective doses for fetal globin induction, safety, and tolerability. HQK‐1001 or placebo were administered once daily for 8 weeks at four dose levels (10, 20, 30, or 40 mg/kg per day), and subjects were monitored for laboratory and clinical events. Pharmacokinetic profiles demonstrated a t_1/2 of 10–12 h. Adverse events with HQK‐1001 treatment were not significantly different from placebo treatment. The 20 mg/kg treatment doses increased median HbF above baseline levels by 6·6% and 4·4 g/l (P < 0·01) in 8/9 subjects; total haemoglobin (Hb) increased by a mean of 11 g/l in 4/9 subjects. These findings identified a safe oral therapeutic which induces fetal globin in BTI. Further investigation of HQK‐1001 with longer dosing to definitively evaluate its haematological potential appears warranted.