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.     

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.     

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.     

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.     

α and β thalassaemia among Chinese children in Guangxi Province, P.R. China: molecular and haematological characterization

Summary We have studied nearly 100 patients with β thalassaemia major and 60 patients with Hb H disease who were attending the Haematology Clinic of Guangxi Medical College. Treatment of the patients was limited and only a few patients with β-thalassaemia major received blood transfusion(s). As a result, the severe anaemia has led to early death at 3–4 years for β⁺-thalassaemia homozygotes, and 8–12 years for β⁺-thalassaemia homozygotes. Four β-thalassaemia alleles are responsible for nearly 90% of all β-thalassaemia chromosomes. This information has resulted in the initiation of a prenatal testing programme at the local level. The patients with Hb H disease maintained a haemoglobin level of 6–10 g/dl and early death was infrequently observed. The ^SEA deletion was the major type of α-thalassemia-1, while three smaller deletions (−2.7, −3.7 and −4.2 kb) and two nondeletional α-thalassaemia determinants (Hbs Constant Spring and Quong Sze) were the α-thalassaemia-2 types.     

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.     

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.     

Immunophenotypic and clinical features of T-cell receptor γδ+ T-lineage acute lymphoblastic leukaemia

The immunophenotypic and clinical features of TCR-γδ⁺ T-lineage acute lymphoblastic leukaemia (T-ALL) were prospectively analysed in 52 children with membrane CD3⁺ T-ALL. We observed a relatively high incidence of TCR-γδ⁺ T-ALL (26/52 patients). Leukaemic blasts from 22 children demonstrated TCR-αβ positivity, and simultaneous expression of the TCR-β and -δ chain was found in four children. Clinical and haematological features of TCR-αβ and γδ⁺ T-ALL did not differ significantly, except that haemoglobin levels were significantly lower in TCR-γδ⁺ cases. Event-free survival at 4 years was significantly better in TCR-γδ⁺ compared with TCR-αβ⁺ T-ALL, but expression of TCR molecules did not emerge as an independent prognostic factor in multivariate analysis.     

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.     

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.     

Atypical HbH disease in a Surinamese patient resulting from a combination of the −SEA and −α3.7 deletions with HbC heterozygosity

The first case of haemoglobin H (HbH) disease in combination with haemoglobin C (HbC) is reported in a man of Surinamese origin. Only haemoglobin A (HbA) and HbC were detected by electrophoresis. The amount of HbC was much less than expected in HbC heterozygotes. The synthesis ratio (β^A +β^C/α) indicated an α-thalassaemia defect with two non-functional α genes, which did not correlate with the degree of haemolysis and anaemia displayed by the patient. The DNA analysis of the α-genes clusters revealed a defect combination −SEA/−α^3.7. The haematological data and the physiopathology of this atypical case are compared with the typical HbH disease found in a first cousin of the propositus. Data on the globin chains expression and on the formation of β^A and β^C homotetramers in HbH/HbC disease are presented.     

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 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.     

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.     

Increase in Vδ1+γδ T cells in the peripheral blood and bone marrow as a selective feature of HIV-1 but not other virus infections

Dysregulation of T-cell receptor (TCR) αβ bearing lymphocytes and an increase in Vδ1⁺γδ T cells are typical features of HIV-1 infection. However, the role of γδ T cells remains unclear. Therefore, peripheral blood mononuclear cells (PBMC) of 103 HIV-1-infected patients were investigated with respect to expression of Vδ1. These results were compared to the Vδ1 expression of bone marrow mononuclear cells (BMMC). In contrast to healthy controls, Vδ1⁺ cells dominated among both PBMC and BMMC in HIV-1-infected patients. Analysis of the coexpression of CD25, CD8, HLA-DR and CD45RO revealed a high prevalence of Vδ1/CD45RO and Vδ1/HLA-DR double-positive PBMC only in HIV-1-infected patients but not in healthy donors. Furthermore, analysis of the γδ TCR repertoire in patients infected with hepatitis B virus, hepatitis C virus, herpes simplex virus (HSV)-1 and HSV-2 showed that the selective enhancement of Vδ1⁺ cells was restricted to HIV infection and not observed in other virus diseases. Our data provide further support for the involvement of γδ T cells in immunosuppression and progression of HIV infection.     

The β+ IVS, I-NT no. 6 (T→C) thalassaemia in heterozygotes with an associated Hb Valletta or Hb S heterozygosity in homozygotes from Malta

Summary. In vitro DNA amplification and dot blot analysis with synthetic allele specific oligonucleotides (ASO) identified the β IVS, I-6 (T→C) thalassaemia in 78% of 32 chromosomes from 16 β-thalassaemia homozygotes in Malta. The preponderance of a single thalassaemia mutation in one population is unusual. The β⁺ IVS, I-6C thalassaemia mutation was also found in three carriers who had an associated β globin heterozygosity, i.e. Hb Valletta (or α₂β₂^87PRO) or Hb S (or α₂β₂^6VAL). The proportion of Hb A in these cases (av. = 29.7%) provided objective documentation of the relatively mild effect of this mutation on in vivo globin gene expression. However, the expression of homozygous disease was more severe in developing children compared to adults. The β⁺ IVS, I-6C mutation complicates population testing because heterozygotes can have Hb A₂ levels below those classically associated with β thalassaemia.     

Congenital Methaemoglobinaemia due to NADH Methaemoglobin Reductase Deficiency: Successful Treatment with Oral Riboflavin

S ummary . A Japanese family with congenital methaemoglobinaemia is described. The family pedigree was compatible with autosomal recessive type of inheritance. The increased methaemoglobin concentration was ascribed to the red cell NADH diaphorase deficiency associated with the almost complete lack of one of the two peaks of the diaphorase activity as separated by DEAE Sephadex column chromato-graphy. The NADH diaphorase and NADH methaemoglobin reductase deficiency was limited to the red cells. The methaemoglobin content in the blood of the propositus was 17.8% and isoelectric focusing analysis on a polyacrylamide gel plate showed that the haemoglobin consisted of 65.2% oxyhaemoglobin (α²⁺β²⁺)₂, 29.6% half-oxidized forms, 20.9% (α+β²+)₂ and 8.7% (α²⁺β⁺)₂, and 3% full-oxidized methaemoglobin (α⁺β⁺)₂. Oral administration of riboflavin 120 mg/d resulted in a gradual but significant decrease in the level of the met-form haemoglobins in parallel with a gradual increase in the red cell flavin content. Riboflavin is considered to be effective by activating the NADPH diaphorase (NADPH flavin reductase) system and appears to be useful for the treatment of congenital methaemoglobinaemia.     

α-Thalassaemia in the population of Cyprus

We have determined the α-thalassaemia (α-thal) determinants in 78 patients with Hb H disease from Cyprus; 25 were Turkish Cypriots and 53 were Greek Cypriots. Four deletional and three non-deletional α-thal alleles were present; the -α(3.7 kb) α-thal-2 and the —^MED-1α-thal-1 were most frequently seen; —^MED-II and -(α)^20.5 deletions occurred at considerably lower frequencies. About 15% of all chromosomes carried a non-deletional α-thal-2 allele; of these the 5 nucleotide (nt) deletion at the first intervening sequence (IVS-I) donor splice site was present in ˜ 8% of all chromosomes. Two types of polyadenylation signal (poly A) mutations were observed. No striking frequency differences were seen between Greek and Turkish Cypriot patients. Combinations of the various types of α-thal resulted in eight different forms of Hb H disease. The phenotypes were comparable except for great variations in the level of Hb H which was highest (average ˜ 22%) in the 12 patients with the α^5ntα/—^MED-I combination. One patient with the same form of Hb H disease but with an additional β-thal (IVS-I-110, G → A) heterozygosity had a most severe microcytosis and hypochromia with < 1% Hb H. Variations in the level of Hb H might correlate with the severity of the disease, although this was not evident from the haematological data.     

The Development of Haemoglobin A2 in Normal Negro Infants and in Sickle Cell Disease

The development of haemoglobin A₂ levels from birth to 3 years has been compared in normal, β-thalassaemia trait, sickle cell (SS) disease, and S-β-thalassaemia genotypes. Hb A₂ levels were almost identical in normals and in children with SS disease at 1, 2 and 3 years. The most rapid increases in Hb A₂ levels occurred before 6 months but levels were still rising at the end of the third year. Sickle cell-β⁺ thalassaemia could be differentiated from SS disease by the higher Hb A₂ levels between 6 months and 1 year. Insufficient data were available on S-β° thalassaemia but since Hb A₂ levels in this condition are generally higher than those in S-β⁺ thalassaemia, differentiation from SS disease may also be possible from the age of 6 months.