Association of a novel high oxygen affinity haemoglobin variant with δβ thalassaemia

We report an uncommon association of δβ thalassaemia and a haemoglobin (Hb) variant with high oxygen affinity in an Asian Indian family. Minimal polycythaemia was seen in a heterozygote for this novel Hb variant, Hb Headington (β72 (E16) Ser→Arg), while compound heterozygosity for Hb Headington and the Indian ^Gγ (^Aγδβ) thalassaemia produces a marked increase in erythrocytosis with a concomitant increase in the level of the variant Hb. The HbF in such compound heterozygotes remains at a level consistent with that usually observed in individuals heterozygous for the ^Gγ (^Aγδβ)° thalassaemia alone. The purified Hb variant showed an increased oxygen affinity, moderately decreased co-operativity and a normal Bohr effect. Results of functional studies suggest that the high oxygen affinity of Hb Headington is due to the Ser→Arg substitution which disrupts the normal and tight interaction between A. B and E helices leading to a destabilization of the T deoxy-structure of the abnormal haemoglobin.     

Haemoglobin Manukau β67[E11] Val→Gly: transfusion-dependent haemolytic anaemia ameliorated by coexisting alpha thalassaemia

Summary Haemoglobin Manukau (β67 Val→Gly) is a novel haemoglobin variant presenting in two brothers as non-spherocytic haemolytic anaemia which became transfusion dependent by 6 months of age. The severity of clinical expression seems to be modulated by coexisting alpha thalassaemia: the severely affected children have a normal complement of alpha globin genes with an unusual genotype (-α^3,7/ααα^3,7), while their father, who carries the abnormal gene with minimal symptoms, has homozygous α⁺ thalassaemia (-α^3,7/ -α^3,7) Another unusual feature of this case is the association of the β67 Val→Gly mutation with modification of β141 Leu to a residue (believed to be hydroxyleucine) that is not detected by standard amino acid analysis. This finding offers an explanation for the previous report of an association of another mutation at this site (Hb Sydney β67 Val→Ala) with Hb Coventry (deletion of β141 Leu).     

Defective Synthesis of an Unstable Haemoglobin: Haemoglobin Koln (β98Val-Met)

S ummary . The in vitro synthesis of haemoglobin A and the unstable haemoglobin, haemoglobin Köln, has been studied using reticulocytes from a patient heterozygous for these two haemoglobins.
The proportions of the two haemoglobins synthesized were: 80% haemoglobin A and 20% haemoglobin Köln. The small amount of haemoglobin Köln synthesized has been found to be due to a deficiency in the synthesis of β^Koln-chains. The deficiency of β^Koln-chains results in an excess production of α-chains by the reticulocytes, which can be detected as a free α-chain pool. The excess α-chains are available to combine with β^A-chains to synthesize haemoglobin A.
The overall result is a preferential synthesis of haemoglobin A over haemoglobin Köln. The deficiency in the synthesis of the unstable haemoglobin probably explains the comparatively mild degree of haemolysis found in haemoglobin Köln disease.     

Haemoglobin Constant Spring has an unstable α chain messenger RNA

Haemoglobin Constant Spring (Hb CS) is a variant with an elongated α-chain associated with an α⁺ thalassaemia phenotype. The amount of α mRNA relative to β mRNA in reticulocytes was reduced in carriers of Hb CS by an amount equivalent to the reduction observed in carriers of α⁺ thalassaemia. In a patient with Hb CS-H disease there was greater α/β mRNA ratio in bone marrow nuclear RNA than in the peripheral blood. Furthermore, all the α mRNA in the patient's peripheral blood was derived from the α1 (α^A) gene. The data suggest that α^CS mRNA is unstable and degraded in the cytoplasm. This instability may be due to destabilization of a specific sequence in the 3'non-coding region during translation.     

Idiopathic Heinz Body Anaemia: Hb-Bristol (β67 (E11) Val→Asp)

S ummary . It is shown that the haemolytic anaemia in the original patient with 'idiopathic Heinz body anaemia' is due to the presence of 36% unstable haemoglobin: Hb-Bristol (β67 (E11) Val→Asp). This haemoglobin has a low oxygen affinity which accounts for the normal development of this patient in spite of the presence of permanent severe anaemia. The relative rates of synthesis of β^Bristol- to β^A-chains were identical, indicating that the reduced amount of Hb-Bristol found in the haemolysate is due to precipitation of the abnormal protein. The inability to separate this haemoglobin by electrophoresis is due to interaction of the abnormal aspartic acid with the adjacent histidine at β63 (E7).     

Haemoglobin Inkster (α285 aspartic acid → valineβ2) Coexisting with β-Thalassaemia in a Caucasian Family

S ummary . Haemoglobin Inkster, a new α-chain variant, was discovered in a family which also had the gene for β-thalassaemia. The amino acid abnormality was in αTp-9 which contains 29 amino-acid residues. Structural studies were facilitated by cleavage of the abnormal α-chains with cyanogen bromide followed by tryptic digestion. The substitution was shown to be valine for aspartic acid at position 85 in the α-chain. Affected individuals had no haematological abnormalities. Individuals with both β-thalassaemia and Hb Inkster had slightly lower percentages of Hb Inkster than those found in persons heterozygous for the Hb Inkster gene alone. 'Interaction' between thalassaemia and variant haemoglobin genes involving different haemoglobin loci has been reported in another family with β-thalassaemia and an α-chain haemoglobin mutant, as well as in the converse situation of coexisting β-thalassemia and a β-chain haemoglobin mutant. This decrease in the mutant haemoglobin percentage differs from the more common 'interaction' of thalassaemia and mutant haemoglobin genes involving the same haemoglobin locus, in which the mutant haemoglobin percentage is increased. The mechanism for the 'interaction' is unknown, but the presence of an unusually low percentage of a haemoglobin variant should warrant investigation for coexisting thalassaemia involving a different haemoglobin locus.     

Haemoglobin Tak: a β-Chain Elongation

In Haemoglobin Tak two normal α-chains are combined with two β-chains elongated by 11 residues beyond the C-terminus. Unlike in the α-chain abnormal Hb Constant Spring, the elongation cannot result from a point mutation of a stop codon. It is probably due to an unequal crossing-over near the 3' end of the β-chain structural gene. This could cause either a deletion of the normal stop codon or a shift in the reading frame. Oxygen dissociation of purified Hb Tak shows no cooperativity but in Hb A + Tak haemolysates there is no interaction between the two above 40% O₂ saturation. Heterozygotes for Hbs A and Tak show an imbalance of globin chain synthesis (α/non α= 1.5), the synthesis of β Tak resembles that of the β-chain in β⁺ thalassaemia.     

The dominant β-thalassaemia in a Spanish family is due to a frameshift that introduces an extra CGG codon (=arginine) at the 5' end of the second exon

We have discovered a Spanish family with a dominant type of β-thalassaemia. Carriers are characterized by mild anaemia, hypochromia, microcytosis, elevated Hb A₂ and Hb F levels, reticulocytosis, and splenomegaly. The molecular basis of this condition is the introduction of a CGG triplet between codons 30 and 31 of the β gene; this was determined by sequencing of amplified DNA and confirmed by dot-blot analysis. The abnormal mRNA (β^Th-mRNA) is stable and present in quantities similar to that of normal β^A-mRNA. cDNA fragments derived from β^Th- and β^A-mRNAs can be separated on a denaturing polyacrylamide gel electrophoresis because the β^Th fragment is three nucleotides (nts) longer than the β^A fragment. The β^Th-mRNA translates into a β chain that is 147 amino acid residues long and carries an extra arginine residue between residues 30 and 31. This β^X chain has not been detected. It may be unstable and does not bind to the α chain. It probably is continuously digested by proteolytic enzymes in red cell precursors in the bone marrow. The abnormal chain probably binds haem that is excreted after proteolysis causing a darkening of the urine.     

The Clinical and Haematological Findings in Children Inheriting Two Types of Thalassaemia: High-A2 Type β-Thalassaemia, and High-F Type or δβ-Thalassaemia

S ummary . Eleven children who are double heterozygotes for β- and δβ-thalassaemia are described. Of their parents one was always heterozygous for β-(A₂) thalassaemia (increased Hb A₂), and the other for the high F variant or δβ-thalassaemia (increased Hb F). The clinical syndrome resulting from the combination of β- and δβ-thalassaemia shows some heterogeneity, but in general is of intermediate severity. Red cell abnormalities were considerable, Hb F was very high (mean 70.3 ± 12.6%), Hb A₂ was low or normal (mean 2.36 ± 1.52%), and Hb A was absent in five patients. Hb F was nearly homogeneously distributed in the red cells of most patients. These findings are explained as the outcome of a mutation which suppresses δ- and β -chain synthesis which is associated with a genetically determined increased production of γ- chains.     

Homozygous deletional α+ thalassaemia associated with unequal expression of the two remaining α1 genes (α1A and α1Q)

S ummary . A Cambodian family presenting several haemoglobinopathies, Hb E, Hb Q and α⁺ thalassaemia, has been investigated. DNA analysis showed that the thalassaemia syndrome corresponds to a leftward type (4.2 kb) deletional from of α⁺ thalassaemia. Genotypes found in the family are: propositus -α^A/-α^Q, β^A/β^E, mother and older sister α^Aα^A/ -α^Q, β^A/β^E; father α^Aα^A/-α^A, β^A/β^A. The propositus consistently presents an α^Q/α^A chain ratio of 60/40 although both chains are products of α₁ loci. The relatively higher expression of the α^Q chain is not observed in the mother and therefore makes it unlikely to reflect anything other than differential expression of the maternal -α^Q/ and paternal -α^A/ haplotypes. This observation raises the possibility that both haplotypes are not strictly identical and that the region of the cross-over event is important for α gene expression.     

Absence of Haemoglobin A in an Individual Simultaneously Heterozygous in the Genes for Hereditary Persistence of Foetal Haemoglobin and β-Thalassemia°

S ummary . Haemoglobins F and A₂ are the only haemoglobins present in an American Negro adult who is simultaneously heterozygous in the genes for hereditary persistence of foetal haemoglobin (HPFH) and β-thalassaemia°. About 15% of the haemoglobin F in descendants who are heterozygous only for HPFH is of the ^Gγ-chain type. About 73% of the haemoglobin F in the HPFH-β-thalassaemia° heterozygote is of the ^Gγ-chain type, and the fraction attributable to control cis to the β-thalassaemia° gene in this individual is calculated to be entirely of the ^Gγ-chain type. Thus synthesis of ^Aγ-chain as well as of β chain is absent under control cis to this β-thalassaemia° gene.     

δβ-Thalassaemia in a Chinese Family

S ummary . δβ-Thalassaemia has been observed for the first time in individuals of Chinese origin. The clinical and haematological features have been characterized in the heterozygous state and in the double heterozygous state withβ-thalassaemia. Studies of haemoglobin synthesis indicate that the degree of globin chain imbalance in β-thalassaemia is less than that found in β-thalassaemia. Analysis of the foetal haemoglobin has shown that all individuals carrying the δβ-thalassaemia gene in this family synthesize only the α₂γ₂^{136 glycine} variety. This type of foetal haemoglobin has been found previously only in two Negro individuals heterozygous for hereditary persistence of foetal haemoglobin (HPFH). The genetic relationships between δβ-thalassaemia and HPFH are discussed in the light of these new findings.     

Molecular analysis of the Turkish form of deletion-inversion (δβ)° thalassaemia

Two unrelated (δβ)°-thalassaemia patients from Southern Turkey are presented. DNA studies indicated that both of them are homozygous for the Turkish type of (δβ)°-thalassaemia characterized by one large deletion of 11.5 kb including the δ and β globin genes at the 5' end and one small deletion of 1.6 kb at the 3' end, which are separated by an inverted 7.6 kb long DNA segment that includes L1 repetitive sequence. In the present study a PCR-based method was performed to produce a unique deletion-specific product and subjected to sequence analysis for the determination of the breakpoint. DNA polymorphisms in the β-globin gene cluster of deletion-inversion type of (δβ)°-thalassaemia, IVS-I-6 and β-39 globin genes were examined. Analysis of sequence variations in regulatory regions including the 5' hypersensitive site-2 of the locus control region (LCR), the δ, ^Gγ and ^Aγ 5' flanking regions and the second intervening sequence (IVS-II) of ^Aγ and ^Gγ genes indicated the presence of close similarities between the chromosome carrying the Turkish form of deletion-inversion δβ)°-thalassaemia and the chromosome associated with β-39 nonsense mutation in haplotype II. These two chromosomes are characterized by the presence of a 4 base pair deletion in the ^Aγ^T globin gene promoter. A C → T alteration at position −199 5' to the δ gene was also found to be associated with the Turkish type of (δβ)°-thalassaemia and β-39 chromosome.     

Globin Biosynthesis in Erythroid Bursts of Heterozygous α or β Thalassaemia

S ummary . We examined globin chain synthesis in erythroid bursts (BFU-E) of patients with heterozygous α or β thalassaemia. BFU-E were cloned from circulating mononuclear cells, labelled with [H]leucine and globin chains purified by gel filtration and column chromatography. In six patients heterozygous for β thalassaemia, globin synthesis in BFU-E was nearly balanced, with an α/non α ratio of 1.05 0α12. These BFU-E produced 33.8 12.7%γ globin chain, an amount similar ( P >1 0.05) to that found in 10 controls with sickle cell anaemia (25.6 6.7) but greater ( P <0.05) than that of five normal controls (17.2 2.2). The balanced globin synthesis appeared due to the large amounts of γ chain made by BFU-E. In two α thalassaemia carriers, who also had sickle cell trait, the BFU-E α/non-α ratio was 0.67 and 0.79. These BFU-E produced 15% and 20%γ chain and 39% and 45%β^S globin. The synthesis of β^S globin in BFU-E exceeded the erythrocyte levels of 20% and 29% HbS and indicated nearly equal expression of β^A and β^B globin genes in these proliferating erythroid precursors. This provides further evidence that the low levels of HbS in sickle cell carriers with α thalassaemia are due to post-translational events resulting from the differing affinity of β^S and β^A globin for α chain and the destruction of excessive β^S chain.     

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.     

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.     

α- and β-haemoglobin chain induced changes in normal erythrocyte deformability: comparison to β thalassaemia intermedia and Hb H disease

Summary The α and β-thalassaemias are characterized by decreased erythrocyte deformability. To determine what effects excess α and β-haemoglobin (globin) chains have on cellular and membrane deformability, purified haem-con-taining α and β-chains were entrapped within normal erythrocytes. Entrapment of purified α-chains in normal erythrocytes resulted in a significant decrease in cellular and membrane deformability similar to that observed in β-thalassaemia intermedia. The decreased deformability was correlated with α-chain membrane deposition, an alteration in membrane proteins and a decrease in membrane reactive thiol groups. These changes in membrane and cellular deformability were time dependent and closely correlated with membrane α-chain deposition. The membrane changes and the loss of membrane deformability appeared to account for the loss of cellular deformability in the α-chain loaded cells. While both β-chain loaded and Hb H erythrocytes demonstrated a significant loss of cellular deformability, this loss was less pronounced than in the α-chain loaded and β-thalassaemic cells and may arise from either the increased intracellular viscosity of the β-chain loaded cells or to the smaller amount of membrane bound globin. In summary, these studies demonstrate that alteration of cellular and membrane deformability occurs very rapidly and as a direct consequence of the autoxidation and membrane binding of the unpaired globin chains.     

Hb Arta [β45 (CD4) Phe→Cys]: a new unstable haemoglobin with reduced oxygen affinity in trans with β-thalassaemia

Summary. The interaction of rare Hb variants with β°-thalassaemia results in a quasihomozygous state where the erythrocytes contain the variant as the only major adult Hb component. Such a situation is a unique model that enables functional studies even in the case of a neutral variant that could not be isolated from Hb A. We report here an unusual patient carrying Hb Arta, a novel Hb variant [β45 (CD4) Phe→Cys], in trans with β°-thalassaemia gene (β° 39). The aminoacid substitution at the critical CD corner of this Hb molecule renders the molecule unstable. In addition, haem is displaced in a position that favours the deoxy (T) conformation of the variant, but less than in Hb Cheverly [β45 (CD4) Phe-Ser], and results in a p50 of 43 mmHg (pH 7-4, 37°C) in the red cells with preservation of cooperativity. Solution studies of the almost pure Hb Arta show a 50% decrease in oxygen affinity and normal cooperativity; the Bohr effect and the interaction with organic phosphates are similar to those of Hb A. Hb Arta retains both normal homo- and hetero-tropic effects allowing a well-preserved oxygen transport in vivo despite a mild anaemia.