GΓ and AΓ globin chain synthesis in bone marrow and peripheral blood of β-thalassaemia homozygotes

S ummary . ^Gγ, ^Aγ and β globin chain synthesis has been investigated in the peripheral blood and bone marrow from eight β-thalassaemia homozygotes. In five out of eight cases total γ chain synthesis was higher in the peripheral blood than in the bone marrow; in seven out of eight cases ^Aγ chain synthesis was markedly higher in the marrow than in the peripheral blood. These data suggest that ineffective erythropoiesis selects F-cells synthesizing the largest amounts of ^Gγ chains, while ^Aγ producing cells are preferentially destroyed in the marrow.     

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.     

Severity differences in β-thalassaemia/haemoglobin E syndromes: implication of genetic factors

Summary. Genetic factors determining the difference in severity of anaemia in β-thalassaemia/HbE disease were studied in 90 patients who had haemoglobin levels, at steady state, ranging from 4.2 to 12.6 g/dl. Co-inheritance of α-thalassaemia 2 and haemoglobin Constant Spring could significantly decrease the severity of the disease. Inheritance of a β-thalassaemia chromosome with Xmn I cleavage site at position — 158 of the ^Gγ-globin gene which was linked to the haplotype - + - ++ or ++ - ++, was associated with a milder anaemia. Two copies of these alleles were necessary to produce a significant clinical effect. Increased expression of the ^Gγ-globin gene and higher production of haemoglobin F. which could reduce the overall globin chain imbalance, were also associated with homozygosity for the Xmn I cleavage site and thus with less severe anaemia. However, this effect was not seen in Xmn I site heterozygotes. Whether the effects of the Xmn I polymorphism, HbF concentration and ^Gγ/^Aγ ratio act separately or through common mechanisms in reducing anaemia remains to be ascertained.     

Multiplex ligation-dependent probe amplification screening of isolated increased HbF levels revealed three cases of novel rearrangements/deletions in the β-globin gene cluster

Investigations of naturally occurring mutations, such as the deletional thalassaemias and hereditary persistence of fetal haemoglobins (HPFHs), have brought many insights into human globin switching, but limited data have been reported so far. We selected 15 individuals with elevated fetal haemoglobin (HbF) levels (>5%) from a previous screening of 27 006 Korean individuals and analysed dosage changes of the globin gene cluster using multiplex ligation-dependent probe amplification (MLPA). Dosage changes detected by the MLPA probes were followed up with gap-polymerase chain reaction and sequence analysis. Three subjects were found to have deletions in the globin gene cluster, including a β-thalassaemia due to deletion of HBB (β-globin gene), an HPFH due to deletions of HBD (δ-globin gene) and HBB , and an HPFH due to a novel HBG2–HBG1 fusion gene consisting of exons 1 and 2 of HBG2 (^Gγ-globin gene) and exon 3 of HBG1 (^Aγ-globin gene). The case with the HBG2–HBG1 fusion suggested the existence of another mechanism for the reactivation of HBG2 and HBG1 . The IVS2 of HBG2 and HBG1 might play a role in HbF regulation, and combinations of specific polymorphisms could influence the reactivation of these genes in adults.     

A NEW DNA POLYMORPHISM IN THE β-GLOBIN GENE CLUSTER CAN BE USED FOR ANTENATAL DIAGNOSIS OF β-THALASSAEMIA

S ummary . Restriction endonuclease analysis of the human β-globin gene cluster has revealed a new DNA polymorphism at a Pvu II recognition site approximately 3.5 kilobases from the 3' end of the ^Aγglobin gene. In patients from the Mediterranean area, the Pvu II polymorphism was associated equally with both normal and β-thalassaemia chromosomes. In patients of Indian and Pakistani origin the polymorphism was almost exclusively associated with only the normal chromosome. Therefore this site may prove very useful for the antenatal diagnosis of β-thalassaemia by acting as a genetic marker for the normal chromosome in linkage analysis of family members.     

Characterization of the erythropoietic precursors (BFU-E) in a patient with juvenile chronic myelogenous leukaemia by the analysis of G gamma and A gamma globin chains

S ummary . In order to investigate the pathogenesis of juvenile chronic myelogenous leukaemia (J-CML) we examined the biosynthetic rates of ^Gγ and ^Aγ globin chains in the erythropoietic bursts from the bone marrow of a patient with J-CML. Globin chains were labelled with ¹⁴C-labelled amino acids, separated by isoelectric focusing and quantitated by fluorography. The synthesis of γ-chains in the erythropoietic bursts comprised 89·0% of the total non-α-chains. The ^Gγ: ^Aγ ratio was 0·67, which is within the ratios obtained in newborns. Furthermore, individual erythropoietic bursts contained varying ratios of both γ and β chains and all revealed more ^Gγ than ^Aγ chain synthesis. The relative proportions of ^Gγ and total γ chain biosynthesis in 62 separate erythropoietic bursts were 0·69·0·06 and 0·86·0·06, respectively. Cumulative frequency distributions of individual bursts differing in the ratios of γ/(γ+β) and ^Gγ/(^Gγ+^Aγ) approached normal frequency distributions. These results suggest that the levels of Hb F in J-CML are controlled by qualitative changes in a single population of erythropoietic precursors, in which normal switching of the ^Gγ:^Aγ ratio has not occurred, rather than by the abnormal proliferation of an F-cell clone.     

A Second Type of Hereditary Persistence of Foetal Haemoglobin in India

S ummary . Six individuals in four Indian families have 25–30% foetal haemoglobin in which the two types of γ chains (the ^Gγ chain with glycine in position 136 and the ^Aγ chain with alanine in that position) are present in a ratio of 70:30. It is suggested that these heterozygotes form a distinct subgroup of the Hb_Gγ Hb_Aγ class of the hereditary persistence of foetal haemoglobin. In three relatives this HPFH condition occurs together with β-thalassaemia. It is concluded that the β-thalassaemia is of the type in which the ratio of ^Gγ:^Aγ chains is 3:1 as in the newborn.     

Nature of Foetal Haemoglobin in F-Thalassaemia

The nature of Hb-F was studied in 32 heterozygotes for F-thalassaemia, in four homozygotes, and in four persons who have F-thalassaemia in combination with β-thalassaemia or Hb-S. Analysis of the cyanogen bromide fragment γCB-3 indicated that, in all heterozygotes, both ^Gγ and ^Aγ chains were present in Hb-F in an average ratio of about 2:3. In the homozygotes and the double heterozygotes, both ^Gγ and ^Aγ chains were observed in an approximate ratio somewhat higher than 1:1. This pattern of γ chain synthesis is nonspecific for F-thalassaemia but similar to that observed in the traces of Hb-F of normal adults. In conjunctionwith existing information from other genetic studies, it may be concluded that the mutation in F-thalassaemia is associated with a complete deficiency of β and σ chains from cis position together with an increased synthesis of γ chains that is directed by both ^Aγ and ^Gγ loci.     

Nondeletional type of hereditary persistence of fetal haemoglobin: molecular characterization of three unrelated Thai HPFH

The β-globin gene clusters of three unrelated Thai families with a nondeletional type of hereditary persistence of fetal haemoglobin (HPFH) were studied using polymerase chain reaction-related techniques. All appeared to have normal nucleotide sequences from the Cap site to position -400 of both the ^Gγ- and ^Aγ-globin genes. Two individuals suspected of having a β-thalassaemia gene linked to the high HbF condition also had a normal β-globin gene sequence, spanning from position -108 from the Cap site to the polyadenylation site. Deletion of four nucleotides, AGCA, at positions -225 to -222 of one ^Aγ-globin allele was detected in one subject and was confirmed by dot-blot hybridization. Restriction fragment length polymorphisms in the β-globin gene cluster showed that the 5' haplotype (- + - ++) and the presence (+) of an Xmn 1 polymorphic site at -158 of the ^Gγ-globin gene are associated with the high F phenotype in these families. Direct sequencing of the 5' hypersensitive-2 (5' HS-2) site of the locus control region (LCR) showed that this Xmn , 1 (+) site is also linked to a specific rearrangement of TA repeats (TA)₉CACATATACG(TA)₁₀, in HS-2 segment.     

The molecular basis of HPFH in a British family identified by heteroduplex formation

Summary. Single-base substitutions in the immediate 5'-flanking region of the fetal ^Gγ and ^Aγ globin genes have been associated with non-deletional forms of hereditary persistence of fetal haemoglobin (HPFH). Previously, the sole promoter mutation associated with HPFH in British individuals has been the T to C substitution at position – 198 relative to the ^Aγ globin gene CAP site.
We have investigated a British family with ^Gγ HPFH and found a T to C substitution at position – 175 of the ^Gγ globin gene. The mutation was first detected by examining the amplified 5'regions of both the ^Gγ and ^Aγ globin genes for heteroduplex formation after electrophoresis in a Hydrolink gel. The potential of such a system for the study of sequence variations in the γ gene promoter regions associated with elevated HbF expression has been evaluated.
Previously reported cases of an identical mutation in an American-Black and a Sardinian family display haematological phenotypes remarkably similar to that of the British family described here.     

The Interaction of α-Thalassaemia and Haemoglobin G Philadelphia

An American Negro woman was found to have HbH disease in association with HbG Philadelphia (α68-asnlys). Starch gel electrophoresis failed to reveal the presence of any HbA or HbA₂ and studies of globin chain synthesis indicated absence of α^A production. The α^G/β synthesis ratio was 0.63. The woman's son and her two half-sibs had α-thalassaemia trait with no HbH and α/β synthesis ratios of 0.84, 0.84 and 0.76. The data indicate that there is no functioning α^A gene linked to the α^G gene. The absence of α^A synthesis by the propositus also indicates that the α-thalassaemia gene trans to the α^G gene completely suppresses α chain production, the first evidence for such a gene in Negroes.     

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.     

The Australian type of nondeletional Gγ-HPFH has a C→G substitution at nucleotide — 114 of the Gγ gene

Nondeletional hereditary persistence of fetal haemoglobin (HPFH) results in the continued production of 2–25% haemoglobin F (Hb F) in the adult who is heterozygous for this mutation. This increase is associated with single-base mutations in the promoter region of either the ^Gγ- or ^Aγ- globin genes. Affected positions include – 202, – 175, – 161, – 158 and – 114 of the ^Gγ gene, and – 202, – 198, – 196, – 195, – 175, and – 117 of the ^Aγ gene. There is now evidence that these mutations produce their effect by changing the binding of certain regulatory proteins. We describe a novel C→G transversion at position – 114 of the ^Gγ gene which is associated with the phenotype of ^Gγ- HPFH.     

The British type of non-deletion HPFH: characterization of developmental changes in vivo and erythroid growth in vitro

Further studies of a unique British family with a rare type of hereditary persistence of fetal haemoglobin (HPFH) have confirmed that this condition appears to result from a regulatory defect affecting globin gene expression. Adult heterozygotes for this disorder have 3.5–10.0% Hb F while homozygotes have 18–21% Hb F and in both cases the Hb F contained predominantly ^Aγ chains (89–94%). In all other respects these individuals were haematologically normal. Two obligate heterozy-gotes were studied from birth, at which time both the proportion of Hb F and the ^Gγ/^Aγ ratio were completely normal. There was a delayed decline in Hb F which continued well into childhood and a parallel decline in the proportion of ^Gγ chains. The growth of erythroid bursts showed no difference in number, size or erythropoietin responsiveness between homozygous HPFH, heterozygous HPFH or normal family members. Haemoglobin synthesis in these bursts showed the normal pattern of asynchrony between γ and β chain synthesis but there was an overall increase in γ chain production in the HPFH cases, particularly the homozygotes, compared to normal. In addition, the abnormal ^Gγ/^Aγ ratio was faithfully reproduced in vitro . This condition appears to result, therefore, from a regulatory abnormality affecting the postnatal repression of γ chain synthesis.     

Molecular heterogeneity of beta thalassaemia in the Italian population

S ummary Fifty-one subjects originating from Southern Italy and affected by Cooley's anaemia have been studied in order to define the degree of heterogeneity of β thalassaemia mutations in this high incidence area. Restriction endonuclease mapping has been carried out on genomic DNA by the Southern blot technique both to exclude the existence of gross deletions or rearrangements and to establish the relative frequency of four polymorphic restriction sites (i.e. ^Gγ and ^Aγ Hind III, β Ava II and β Bam HI) within the γδβ gene region. In 28 subjects unequivocal linkage of the four polymorphic sites has been determined leading to the identification of seven different chromosome haplotypes, six of which had previously been reported associated with specific β⁰ and β⁺ thalassaemia mutations. Globin chain synthesis studies on peripheral blood reticulocytes indicated that subjects carrying the same genotype may behave differently as far as the β chain production is concerned relative to both the a and the non-α chains. Thus, β thalassaemia turns out to be quite heterogeneous even in this limited geographical area. β⁺ mutations appear to be predominant, particularly those affecting nuclear precursor RNA splicing to mature β globin mRNA.     

Rapid identification by denaturing gradient gel electrophoresis of mutations in the γ-globin gene promoters in non-deletion type HPFH

Summary We have outlined a fast, non-radioactive strategy to identify point mutations in the 5'flanking region of the γ-globin genes using denaturing gradient gel electrophoresis (DGGE) of amplified DMA. In a sample of previously characterized carriers of non deletion-type hereditary persistence of fetal haemoglobin (HPFH) the different point mutations in both γ gene promoters could be easily identified by DGGE of a 327 bp fragment. A 4 bp deletion at position —225 to —222 of the ^Aγ globin gene was unexpectedly found in several samples and shown to represent a frequent polymorphism.
Analysis of a 681 bp fragment specific for the 5'region of the ^Aγ gene, showed that this can be used to determine the haplotype of the chromosome under study.
This technique may be useful in the study of sequence variations associated with high Hb F expression in physiological and pathological conditions.     

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.     

Restriction endonuclease maps of the β-like globin gene cluster in the British and Greek forms of HPFH, and for one example of Gγβ+ HPFH

We report here restriction endonuclease maps of the β-like globin gene cluster for the British form of HPFH and for a case of ^Gγβ⁺ HPFH, and also confirm and extend previous reports of the map for the Greek form of HPFH. These results show that all these conditions belong to a group lacking any substantial deletion or rearrangement of DNA sequence in this gene cluster. The absence of any gross disruption of the structure of this region of the genome, together with evidence that the HPFH genotype is either allelic with, or closely linked to, the β-like globin gene cluster, suggests that the responsible lesions are nearer to being purely regulatory in nature than in forms of HPFH due to substantial deletions. Thus these conditions promise to provide less equivocal evidence about the regulation of β-like globin gene expression than has so far been available.     

γ Chain Composition in Five Italian Newborns Heterozygous for Hb F Malta Gγ117 His → Arg

The percentage of Hb F Malta ^Gγ^{117His Arg} and the γ chain composition have been evaluated at birth and at different times after birth in five Italian newborns heterozygous for this variant. The percentage of Hb F Malta ranged at birth from 24% to 31% of the total Hb F, while the average ^Gγ/^Aγ chain ratio was about 7/3, overlapping the values observed in normal newborns. ^Tγ chains were detected in three out of five newborns, with a percentage of about 10% of the total Hb F. After birth the Hb F Malta declined faster than the total Hb F; thus, the Hb F decrease during the first 45 d of life is mainly due to the switch-off of the ^Gγ locus containing the Hb F Malta gene.