Haemoglobin Lleida: a new α2-globin variant (12 bp deletion) with mild thalassaemic phenotype

Molecular studies of α-thalassaemias have revealed defects at different steps in the process of α-gene expression. It is not surprising, therefore, that in some cases a single mutation or small deletion can result in a structurally abnormal haemoglobin that produces the α-thalassaemia phenotype. In this report we describe a new unstable α-globin variant, Hb Lleida, in a Spanish patient with α-thalassaemia trait. The mutation was detected by single-strand conformation polymorphism in the third exon of the α₂-globin gene. Direct sequence analysis of the α-globin gene showed a 12 bp deletion as the only defect of the α₂- and α₁-globin genes. The propositus was revealed to be a heterozygous carrier, and two alleles were separated by electrophoresis. This deletion causes the loss of four aminoacid residues (from codon 113 to 116) and would be expected to produce an unstable haemoglobin, as a shorter α-globin chain variant is created with 137 amino acids instead of 141 amino acids present in a normal α-globin chain. However, no abnormal haemoglobin was found by either isoelectric focusing or haemoglobin electrophoresis. Since the deletion affects an aminoacid residue (114 Pro) involved in α₁-β₁-globin chain contacts, the interaction required for efficient Hb assembly is also compromised. The resulting unstable α-globin chain is rapidly catabolized and unsuitable for haemoglobin tetramer formation, causing an α-thalassaemia trait phenotype in the heterozygous patient.     

FIRST DESCRIPTION OF A FRAMESHIFT MUTATION IN THE α1-GLOBIN GENE ASSOCIATED WITH α-THALASSAEMIA

A frameshift mutation in the α₁-globin gene, responsible for a clinically mild α-thalassaemia phenotype, has been characterized in a Spanish woman. After excluding the most common forms of α-thalassaemia found in the Mediterranean area, both α-globin genes (α₁ and α₂) were amplified and analysed selectively by non-radioactive single-strand conformation polymorphism (SSCP). An abnormal SSCP mobility was present in the second exon of the α₁-globin gene and direct sequence analysis revealed a 13 bp deletion (between codons 51 and 55) affecting a single allele. The consequence of this mutation is a reading frameshift leading to a novel amino acid coding sequence from codons 51–61 and a premature stop signal at new position 62, which results in a net reduction of the affected α-globin chain output. The presence of this new mutation was confirmed by restriction enzyme analysis of the specific PCR product.     

A new α chain variant Hb Sallanches [α2 104(G11) Cys→Tyr] associated with HbH disease in one homozygous patient

Summary. We identified a new α-chain variant (α^Sal) associated with haemolytic anaemia and low level of HbH in one homozygous patient. This new mutation is located in codon 104 (TGC → TAC) of the a2 globin gene and results in a Cys→Tyr replacement. In vitro and in vivo biosynthetic studies suggest that the mechanism leading to HbH disease in this homozygous patient is mostly related to a significant instability of α^Sal:β dimers rather than to the hyper-instability of the α^Sal chain itself only.     

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.     

The C-->G transition in the alpha 2-globin gene of a normal alpha alpha-chromosome is responsible for the Hb G-Philadelphia variant in Sardinians

Sequencing of alpha-globin genes of 18 Sardinian heterozygotes for the Hb G-Philadelphia [alpha 68(E17)Asn-->Lys] variant, with four active alpha genes and circulating level of the variant of about 27%, showed the AAC-->AAG change at codon 68 of the alpha 2-globin gene (alpha(G)alpha/alpha alpha). Two heterozygotes with level of about 37% were the carriers of the same mutation on the same alpha 2 gene, and of the alpha 2 alpha 1 hybrid gene, because of the 3.7-kb deletion, in trans (alpha(G)alpha/-alpha(3.7)). In Black people, the same C-->G mutation occurs on the hybrid gene (-alpha(G)3.7), whereas in Caucasians the Lys for Asn change is because of the C-->A transversion occurring on the alpha 2 gene of a normal alpha alpha arrangement. The identification of the C-->G mutation on the normal alpha alpha chromosome points to an undescribed genotype for this rather common variant, which is probably because of the high rate of recombination between the duplicated alpha-globin genes.     

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.     

Spectrin Cosenza: a novel β chain variant associated with Sp αI/74 hereditary elliptocytosis

A Calabrian family (Southern Italy) with Sp α^I/74 hereditary elliptocytosis (HE) in the heterozygous state was studied. Sp α^I/74 HE is associated with asymptomatic elliptocytosis, a defect in spectrin dimer self association and an increase of the α^I/74 kD fragment from the α chain after partial tryptic digestion of spectrin. To identify the underlying molecular defect, we analysed exons V, W, X, Y, Z of the β gene and exon 2 of the α gene by single-strand conformational polymorphism (SSCP) of the amplification products. Direct DNA sequencing of the mutant exon showed a C →G substitution at position 6284 of the β gene. The corresponding substitution at the protein level was Arg → Pro in the 2064 position of the β-spectrin chain.     

Spectrin Anastasia (αI/78): a new spectrin variant (α45 Arg Thr) with moderate elliptocytogenic potential

We describe a white Italian kindred in which hereditary elliptocytosis (HE) is associated with abnormal level of α^1/78 peptide in spectrin digest. Clinical phenotype varied among the family members ranging from asymptomatic to mild haemolytic HE. The original mutation responsible is a G-C substitution of the spectrin α-gene: α45 Arg Thr (AGG ACG). The corresponding spectrin is designated spectrin Anastasia. Utilizing a secondary structure predictive method we suggest that this mutation has a poor capability to induce conformational changes of the tetramerization site and thus shows a moderate elliptocytogenic potential.     

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.     

Haemoglobin α2β223Val → Ile produced in Escherichia coli facilitates Hb S polymerization

The doubly substituted variant Hb S-Antilles (beta 6 Glu----Val, beta 23 Val----Ile) produces sickling in heterozygous carriers. The Csat value for pure deoxyHb S-Antilles is nearly half that of deoxyHb S. Dilute solutions of pure Hb S-Antilles have a lower oxygen affinity than those of Hb A or Hb S. The mutant Hb alpha 2 beta 2 23 Val----Ile was synthesized in E. coli. It exhibits a decreased oxygen affinity compared to Hb A and does not polymerize in 1.8 M phosphate buffer. Mixtures of equal amounts of Hb S + Hb beta 23 Val----Ile have a decreased Csat value compared to mixtures of Hb S + Hb A. The beta 23 Val in Hb S contributes to the axial contact joining molecules in each single filament. Substituting Ile for Val at this site increases the strength of this contact through hydrophobic interactions, allowing increased stability of the lateral contact between filaments in pair, which is the specific unit structure of polymers in deoxyHb S.     

Combination low-dose lymphoblastoid interferon and thymosin α1 therapy in the treatment of chronic hepatitis B

SUMMARY. This open label study was initiated to assess the safety and efficacy of lymphoblastoid interferon-α (IFN-α) and thymosin α₁ (Tα₁) in the treatment of 11 patients with chronic hepatitis B, who had failed to respond to standard IFN-α2b therapy, and in four interferon naive patients. These fifteen hepatitis B surface antigen (HBsAg) positive and serum hepatitis B virus (HBV) DNA positive patients were given Tα₁ (1 mg) subcutaneously (sc) on 4 consecutive days. Low-dose lymphoblastoid IFN-α (3 MU) was administered intramuscularly (IM) on the fourth day. Beginning with the second and for the subsequent 25 weeks, patients self-administered Tα₁ twice weekly in the morning followed, 12 h later, by 3 million units (MU) lymphoblastoid IFN-α. Patients were followed-up for 12 months. Nine (60%) of the 15 patients, including six (55%) of the 11 patients previously treated with IFN-α2b, responded by losing serum HBV DNA and normalizing alanine aminotransferase (ALT) values. Six of the nine responders seroconverted to HBsAg negativity. Significant improvements in the Knodell histological activity index were observed in the responders and no significant adverse effects were observed. Combination low-dose lymphoblastoid IFN-α and Tα₁ treatment may provide a safe and potentially effective therapeutic approach in chronic hepatitis B. These results require confirmation in future randomized controlled studies.     

α4β1 integrin-mediated adhesion of CD34+ cells from patients with chronic myeloid leukaemia: influence of IL-3

Interactions between integrins on haemopoietic progenitor cells and their stromal ligands have an important role in the control of haemopoiesis. Growth factors can modulate these interactions (so-called 'inside-out' signalling) resulting in changes in ligand binding activity. We have studied alpha4beta1 integrin-mediated adhesion to the H120 fragment of fibronectin (which contains the strongest alpha4beta1 binding site) in CD34+ cells from patients with chronic myeloid leukaemia (CML) and have determined the effect of IL-3 on the level of adhesion. Compared to normal CD34+ cells isolated from cord blood and peripheral blood progenitor harvests (mean of 61.4 +/- 14.9% of cells attached) the CML CD34+ cells showed reduced levels of adhesion (mean of 41.9 +/- 14.7%, P < 0.05). The effect of 10 ng/ml of IL-3 resulting in reduced adhesion of normal CD34+ cells at 30 min was absent in 6/7 patients with CML. Abnormalities of adhesion to fibronectin may thus be related to IL-3 pathways affected by BCR-ABL. These findings will have implications for understanding the dysregulation of growth and adhesion in CML.     

A variant of spectrin low-expression allele αLELY carrying a hereditary elliptocytosis mutation in codon 28

Summary. Allele α^LELY is a low-expression allele of the erythroid spectrin α-gene. It carries mutations in exon 40 (α^V/41 polymorphism) and intron 45, respectively, and is associated with partial skipping of exon 46. The latter phenomenon is thought to impair the recruitment of α-chains by β-chains, and would eventually account for the low-expression character. When it occurs in trans to an α-allele responsible for hereditary elliptocytosis (α^HE allele; α^HE/α^LELY diplotype), allele α^LELY enhances the severity of elliptocytosis. Because allele α^LELY is widespread, we anticipated that it would occasionally carry HE determinants. These variants of allele α^LELY will be designated α^HE-LELY alleles. We report two families with the same α^HE-LELY allele. The HE component was the known α28 Arg → His mutation. This α^HE-LELY allele was investigated within the α^HE-LELY/α^LELY diplotype, a diplotye not described before. Except for the neonatal period, the presentation was mild. In a consistent manner, the α^LELY component in cis of the α^HE mutation counteracted the like component in trans .