Globin Chain Synthesis Studies Research Articles

Why Are Hemoglobin F Levels Increased in HbE/β Thalassemia?

AbstractTo try to further define the mechanisms that increase the levels of hemoglobin F (HbF) in the blood of patients with severe forms of β thalassemia, we have studied two comparable populations of hemoglobin E (HbE)/β thalassemics, one regularly transfused and one receiving only occasional blood transfusions. Regular transfusion was associated with a significant decrease in soluble transferrin receptor and erythropoietin levels. Globin chain synthesis studies also show a highly significant decrease in HbF synthesis relative to HbE in the transfused patients. This effect was confirmed by sequential data on one patient, studied before and after the commencement of regular blood transfusion; blood transfusion was followed by a marked increase in the ∝/γ, βE/γ, and HbE/HbF ratios. These data suggest that the high HbF levels in HbE/β thalassemia, and other β thalassemia syndromes, result from increased erythropoietin levels leading to bone marrow expansion, and possibly increased F-cell production, combined with ineffective erythropoiesis giving a survival advantage to F cells. This study also suggests that alteration in blood transfusion regimes must be taken into account when interpreting changes in HbF levels seen in trials of HbF-promoting drugs.

Why Are Hemoglobin F Levels Increased in HbE/β Thalassemia?

To try to further define the mechanisms that increase the levels of hemoglobin F (HbF) in the blood of patients with severe forms of β thalassemia, we have studied two comparable populations of hemoglobin E (HbE)/β thalassemics, one regularly transfused and one receiving only occasional blood transfusions. Regular transfusion was associated with a significant decrease in soluble transferrin receptor and erythropoietin levels. Globin chain synthesis studies also show a highly significant decrease in HbF synthesis relative to HbE in the transfused patients. This effect was confirmed by sequential data on one patient, studied before and after the commencement of regular blood transfusion; blood transfusion was followed by a marked increase in the /γ, βE/γ, and HbE/HbF ratios. These data suggest that the high HbF levels in HbE/β thalassemia, and other β thalassemia syndromes, result from increased erythropoietin levels leading to bone marrow expansion, and possibly increased F-cell production, combined with ineffective erythropoiesis giving a survival advantage to F cells. This study also suggests that alteration in blood transfusion regimes must be taken into account when interpreting changes in HbF levels seen in trials of HbF-promoting drugs.

Is Hemoglobin Instability Important in the Interaction Between Hemoglobin E and β Thalassemia?

Hemoglobin E (HbE; α2β226glu-lys), globally the commonest hemoglobin variant, is synthesized at a slightly reduced rate and has a homozygous phenotype similar to heterozygous β thalassemia. Yet, when it is inherited together with a β thalassemia allele, the resulting condition, HbE/β thalassemia, is sometimes characterized by a severe, transfusion-dependent thalassemia major. The severity of this interaction has not been explained. We have explored the possibility that it may reflect the instability of HbE consequent upon globin chain imbalance imposed by the β thalassemia allele. Time-course and pulse-chase globin chain synthesis studies at 37°C on peripheral blood and bone marrow suggest that hemoglobin instability is not significant in steady-state HbE/β thalassemia; this is confirmed by density-gradient centrifugation studies that show no decrease in HbE levels relative to HbA as HbE/β+ thalassemia red blood cells age. Globin binding to membranes was assessed and only α globin chains were found, in contrast to other unstable hemoglobins in which both α and β chains were present. However, in experiments performed on blood from HbE/β thalassemics in the temperature range 39°C to 41°C, there was evidence of instability of HbE, a finding that was also observed in homozygous HbE. These findings suggest that the phenotype of HbE/β thalassemia is primarily the result of the interaction of two β thalassemia alleles; however, hemoglobin instability may be important during febrile episodes, contributing to worsening anemia.© 1998 by The American Society of Hematology.

Is Hemoglobin Instability Important in the Interaction Between Hemoglobin E and β Thalassemia?

Hemoglobin E (HbE; 2β226glu-lys), globally the commonest hemoglobin variant, is synthesized at a slightly reduced rate and has a homozygous phenotype similar to heterozygous β thalassemia. Yet, when it is inherited together with a β thalassemia allele, the resulting condition, HbE/β thalassemia, is sometimes characterized by a severe, transfusion-dependent thalassemia major. The severity of this interaction has not been explained. We have explored the possibility that it may reflect the instability of HbE consequent upon globin chain imbalance imposed by the β thalassemia allele. Time-course and pulse-chase globin chain synthesis studies at 37°C on peripheral blood and bone marrow suggest that hemoglobin instability is not significant in steady-state HbE/β thalassemia; this is confirmed by density-gradient centrifugation studies that show no decrease in HbE levels relative to HbA as HbE/β+ thalassemia red blood cells age. Globin binding to membranes was assessed and only  globin chains were found, in contrast to other unstable hemoglobins in which both  and β chains were present. However, in experiments performed on blood from HbE/β thalassemics in the temperature range 39°C to 41°C, there was evidence of instability of HbE, a finding that was also observed in homozygous HbE. These findings suggest that the phenotype of HbE/β thalassemia is primarily the result of the interaction of two β thalassemia alleles; however, hemoglobin instability may be important during febrile episodes, contributing to worsening anemia. © 1998 by The American Society of Hematology.

Clonal Hematopoiesis and Acquired Thalassemia in Common Variable Immunodeficiency

Common variable immunodeficiency (CVID) is defined by hypogammaglobulinemia and increased susceptibility to infections. The gene defect responsible for CVID remains unknown. During the course of their CVID disease, a female and three male patients developed microcytic anemia. The investigation of this anemia forms the basis for this report. Reticulocyte globin chain synthesis studies revealed the abnormal alpha/beta ratios that are pathognomonic of thalassemia. Through transcriptional analysis of the glucose-6-phosphate-dehydrogenase (G6PD) locus of the active X-chromosome in blood cells, we determined that the female patient has clonal reticulocytes, platelets, granulocytes, and B and T lymphocytes. The simultaneous presence of globin synthesis abnormalities and panhypogammaglobulinemia suggests that a common insult at the stem cell level could contribute to the development of CVID and acquired thalassemia.

Rapid HPLC techniques for globin chain synthesis studies.

Globin chain synthesis and alpha/beta ratios were determined in a number of normal subjects, alpha-thalassemia-2 homozygotes, and beta-thalassemia trait using three different techniques. Cation exchange high performance liquid chromatography on a Pharmacia Mono-S, HR 5/5 and reversed phase high performance liquid chromatography on a semi-preparative Vydac C4 column were compared with the traditional carboxymethylcellulose chromatography. Both high performance liquid chromatography columns give excellent results when 2 mg of hemoglobin was chromatographed in each analysis. By modifying the protocols for column equilibration and gradient shape for preparative Vydac C4 columns, conditions were found yielding excellent resolutions of the labeled globin chains in less than an hour without the need for substantial increase of the flowrate. This method was found to be superior to other methods and may be a suitable alternative for the classical carboxymethylcellulose chromatography. Up to five specimens could easily be analyzed in a single day with this system.

Occurrence of hereditary spherocytosis and beta thalassaemia in the same family: globin chain synthesis and visco diffractometric studies.

Hereditary spherocytosis and beta thalassaemia are rarely inherited together. We have studied a large family of Caucasian extraction in whom these two diseases segregate independently over four generations. The diagnosis rested on specialized laboratory findings and in a number of subjects on the measurement of alpha and beta globin chain synthesis. In addition, a viscometric method (osmotic gradient ektacytometry) was used to evaluate the rheological function of the erythrocytes. In patients inheriting both diseases, the results indicate that the clinical and biological expression of hereditary spherocytosis is modulated by the degree of imbalanced globin chain synthesis. The opposite properties of spherocytes (decreased surface/volume ratio, increased haemoglobin concentration) and thalassaemic red cells (increased surface/volume ratio, decreased haemoglobin concentration) may explain the antagonistic influence of each genotype.

Definitive Estimate of Rate of Hemoglobin Switching: Measurement of Percent Hemoglobin F in Neonatal Reticulocytes

The transition from fetal to adult erythropoiesis starts at 32-36 wk postconception. The rate of this switchover has been controversial. Studies of globin chain synthesis by reticulocytes in vitro have indicated gradual switching with a time for 50% reduction in fetal Hb synthesis ("half-time") of more than 6 wk. This may not accurately reflect fetal/adult Hb synthetic balance in vivo. By contrast, histochemical studies and also indirect mathematical analysis of postnatal changes in circulating fetal Hb and adult Hb may imply abrupt patterns of switching with half-times of less than 1 wk. We have resolved this discrepancy by direct measurement of the changing proportions of fetal and adult Hb in reticulocytes prepared by flourescence activated cell sorting from 10 full-term cord and 45 preterm postnatal blood samples. This method overcomes problems both of extrapolation from in vitro measurements and of mathematical analysis. We find a gradual transition from fetal Hb to adult Hb synthesis. The half-time is approximately 16-18 wk. Values of fetal Hb in reticulocytes were on average higher than predicted from in vitro synthesis studies. We find considerable individual variation. Infants differ in their switching behaviour, many showing prolonged dependence on fetal Hb.

FEASIBILITY OF PRENATAL DIAGNOSIS OF β-THALASSAEMIA WITH SYNTHETIC DNA PROBES IN TWO MEDITERRANEAN POPULATIONS

A feasibility study in two Mediterranean populations showed that prenatal diagnosis of β-thalassaemia with a limited number of synthetic oligonucleotide probes would have been possible in about 70% of cases. To provide a comprehensive programme of prenatal diagnosis for the thalassaemias it would be necessary, in most populations, to combine fetal DNA analysis with fetal blood sampling and globin-chain synthesis studies.

Molecular heterogeneity of beta thalassaemia in the Italian population.

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 beta 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. G gamma and A gamma Hind III, beta Ava II and beta Bam HI) within the gamma delta beta 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 beta(0) and beta(+) thalassaemia mutations. Globin chain synthesis studies on peripheral blood reticulocytes indicated that subjects carrying the same genotype may behave differently as far as the beta chain production is concerned relative to both the alpha and the non-alpha chains. Thus, beta thalassaemia turns out to be quite heterogeneous even in this limited geographical area. Beta(+) mutations appear to be predominant, particularly those affecting nuclear precursor RNA splicing to mature beta globin mRNA.

β + Thalassaemia—Portuguese type: clinical, haematological and molecular studies of a newly defined form of β thalassaemia

Summary. We have characterized 14 patients in 10 families with a mild form of homozygous β thalassaemia which has not been previously well defined. As these patients originate from a small area of northern Portugal we propose to call this β+ thalassaemia—Portuguese type. Clinically, the homozygotes range from asymptomatic to thalassaemia intermedia and they are characterized by low levels of HbF, <20%, indicating only a mild deficit in β globin production. Heterozygotes are indistinguishable from those with the more common types of β thalassaemia as regards red cell morphology, haemoglobin analysis and globin chain synthesis studies. Globin gene mapping excluded the presence of α thalassaemia in these patients and demonstrated no abnormalities in the βM‐like globin gene cluster. Restriction enzyme site polymorphisms around the β gene cluster are identical on both chromosomes in all of the homozygotes, confirming their homogeneity.

Beta + thalassemia--Portuguese type: clinical, haematological and molecular studies of a newly defined form of beta thalassaemia.

We have characterized 14 patients in 10 families with a mild form of homozygous beta thalassemia which has not been previously well defined. As these patients originate from a small area of northern Portugal we propose to call this beta + thalassaemia--Portuguese type. Clinically, the homozygotes range from asymptomatic to thalassaemia intermedia and they are characterized by low levels of HbF, less than 20%, indicating only a mild deficit in beta globin production. Heterozygotes are indistinguishable from those with the more common types of beta thalassaemia as regards red cell morphology, haemoglobin analysis and globin chain synthesis studies. Globin gene mapping excluded the presence of alpha thalassaemia in these patients and demonstrated no abnormalities in the beta-like globin gene cluster. Restriction enzyme site polymorphisms around the beta gene cluster are identical on both chromosomes in all of the homozygotes, confirming their homogeneity.

Hemoglobin E diseases: hematological, analytical, and biosynthetic studies in homozygotes and double heterozygotes for alpha-thalassemia.

Two families with Hb E diseases are described. In the Laotian family S, three homozygous Hb E were found. In the Vietnamese family H, double heterozygous Hb E-alpha-thalassemia-2 and Hb E-Hb H diseases were found. Anemia or hemolysis was absent in Hb E carriers, unless complicated by iron deficiency, the presence of severe alpha-thalassemia gene (Hb H disease), or oxidative drug (paraaminosalicylic acid). Moreover, iron deficiency or concurrent alpha-thalassemia genes resulted in a decreased amount of Hb E in its heterozygous carriers. Mild microcytosis and hypochromia were observed in Hb E heterozygotes, whereas the microcytosis and hypochromia were more pronounced in Hb E homozygotes. Globin chain synthesis studies yielded unbalanced alpha/non-alpha ratios in both heterozygotes and homozygotes (average ratios were 1.13 and 1.56, respectively). The unbalanced biosynthetic ratios with microcytosis and hypochromia in Hb E carriers represented a beta-thalassemia phenotype, which could be a result of reduced synthesis of beta E-globin mRNA, as suggested by recent hybridization studies.

Haemoglobin D Los Angeles, D-β<sup>+</sup>-Thalassaemia, and D-β°- Thalassaemia

The first 2 Canadian cases of combinations of Hb D Los Angeles and beta-thalassaemia were found in an Italian family and an East Indian family in Hamilton, Ont. The structure of the Hb D variant was identified by radioimmunoassay. The beta +-thalassaemia trait in the Italian family and the beta o-thalassaemia trait in the East Indian family were confirmed by studies of globin chain synthesis. Previous cases of Hb D-beta-thalassaemia were summarized and family studies of the 2 new cases were presented.

Unstable triosephosphate isomerase (TPI)

This study was undertaken in the investigation of a child with chronic non-spherocytic haemolytic anaemia. B.C. was born in 1977 of unrelated parents. His father has β-thalassaemia minor. He required an exchange transfusion for neonatal jaundice and subsequently has had persistent anaemia, splenomegaly and intermittent jaundice. Delayed gross motor development was noted at 6 mth of age, and increasing spasticity from 14 mth. Haemoglobin electrophoresis and globin chain synthesis studies established that B.C. also had thalassaemia minor. Microassays of 23 enzymes of the glycolytic and pentose phosphate pathways, and some enzymes and metabolites involved in glutathione metabolism were undertaken. Since his red cell population was invariably young, it was anticipated that increased activity of many enzymes known to be increased in reticulocyte-rich blood would be detected. This was found to be so, with the exception of TPI which was present at normal to low levels. This enzyme was shown to be unstable when haemolysate was stored on ice for 1 h (activity fell from 1973 I.U./g Hb to zero, control 3695 to 3096 I.U./g Hb) and when whole red cells were stored at –20°C (B.C. 1937 to 64 I.U/g Hb, control 3695 to 3068 I.U./g Hb). However, these phenomena did not clearly distinguish between the homozygous individual and presumed heterozygotes in a family study. Heat inactivation studies showed a clearer distinction, allowing for the possibility of antenatal diagnosis of this defect. Methods have been established for measurement of TPI in cultured foetal fibroblasts, and it is now planned to examine cultured fibroblasts from B.C. and his parents. The problems of identification of a TPI deficiency variant and the association with thalassaemia minor are discussed.

Interaction of Alpha and Beta Thalassaemia Genes in Two Sardinian Families

Our paper describes two Sardinian families with alpha-beta thalassaemia interaction. In the first (family S), the propositus, whose haemoglobin pattern at birth consisted of about 25% Hb Bart's and 75% Hb F, successively developed a clinical and haematological picture typical of Cooleys anaemia. Haematological and globin chain synthesis studies together with these findings suggest that he is homozygous for beta 0 thalassaemia and heterozygous for alpha thalassaemia-1 and alpha thalassaemia-2. This conclusion is further substantiated by the finding of various combination of alpha and beta thalassaemia among his family members. In the P family two twins whose haemoglobin pattern and synthesis data at birth were similar to those of the proband of family S died in the neonatal period. The mother was assumed to be a compound heterozygte for alpha thalassaemia-2 and beta 0 thalassaemia and the father for alpha thalassaemia-1 and beta 0 thalassaemia. The homozygous state for beta 0 thalassaemia in association with the alpha thalassaemia 1 and alpha thalassaemia 2 genes results in a severe clinical picture similar to that of a homozygous beta 0 thalassaemia. The interaction between the heterozygous state for beta 0 thalassaemia and the alpha thalassaemia 1 or alpha thalassaemia 2 genes, or the combination of both, results in a haematological picture similar to that of a beta thalassaemia heterozygote.

Globin chain synthesis and clinical and hematologic findings in double heterozygotes for the silent and classical ß-thalassemia genes, and their parents

Detailed hematologic and globin chain synthesis studies were performed in a special group of six patients with thalassemia syndrome who were offsprings of segregation of classical β-thalassemia trait (increased HbA2 and normal or slightly elevated HbF), to "silent" β-thalassemia trait (normal HbA2 and HbF, but impaired β-chain synthesis). Clinically these patients varied widely, from those severely affected which necessitated regular transfusions since the first year of life, to others with an intermediate clinical course without transfusion requirements. The levels of both HbA (28-80%), and HbF (18-70%), varied widely. Of interest were the results of globin chain synthesis in the patients with the "silent" β-thalassemia gene and their siblings; the ratio of a to non-a chains ranged from 1.43 to 3.02 indicating a considerable variation in the degree of impairement of β-chain synthesis. This favors the assumption that a heterogeneity in "silent" β-thalassemia gene may exists, and that the extreme variation in the clinical severity observed in double heterozygotes patients, could be associated with heterogeneity of the "silent" thalassemia gene.

Hemoglobin Suan-Dok (α2109(G16)LEU-ARGβ2). an Unstable Variant Associated with α-Thalassemia

An abnormal hemoglobin variant was detected in a seven-year old Thai girl. The clinical and hematological pictures were indistinguishable from hemoglobin H disease. Upon cellulose acetate electrophoresis at pH 8.5 an abnormal hemoglobin variant was found together with hemoglobins A and H. The variant moved slightly behind HbF and amounted to approximately 9%. Globin chain synthesis studies of peripheral blood reticulocytes incubated with tritiated leucine revealed that the total radioactivity ratio alpha-chain + variant alpha-chain/beta-chain was 0.59, while the ratio variant alpha-chain/beta-chain was 0.12. The variant hemoglobin was somewhat unstable under heat denaturation and in the isopropanol test. Structural studies by component isolation, globin chain separation, peptide mapping and aminoacid analysis of abnormal peptides showed that the leucine residue 109 of the alpha-chain was replaced by arginine. This hemoglobin mutant has not been described before and is named Hemoglobin Suan-Dok.

615 HIGH HOMOGLOBIN A2 LEVELS IN HOMOZYGOUS SICKLE CELL ANEMIA

Elevated hemoglobin (Hb) A2 levels are seen in heterozygous β thalassemia and rarely in pernicious anemia, Hb Zurich, and Hb Tacoma but have not been reported in homozygous sickle cell anemia (HSCA) in the English literature. We measured Hb A2 by cellulose acetate Hb electrophoresis and densitometry in 34 children with HSCA. Nine of them (26%) displayed Hb A2 levels of 3.2 to 4.7% (normal values for our laboratory: 0.7 - 3.0%). Their Hb values (7.4 to 9.2 g/dl) and reticulocyte counts (468 to 700 x 109/1) did not differ from those of the other 25 patients. Eight of these nine children showed normal to high MCVs ranging from 77 to 100 fl; one had an MCV of 63 fl. Globin chain separation by carboxymethylcellulose chromatography following the method of Cleggs and Weatherall was performed in five of the nine high HB A2 children including the one with the low MCV, and all showed a normal α/β chain ratio ranging from 0.91 to 1.05. The fetal Hb in these five subjects ranged from 1.2 to 6.6% and was not different from the values of the other 29 HSCA patients. An elevated Hb A2 level may be found in a significant proportion of HSCA patients. In addition to careful analysis of the MCV, globin chain synthesis studies may be necessary to differentiate these patients from subjects with sickle-β thalassemia.

Fetal haemoglobin in early malignant osteopetrosis.

The characteristics of the fetal haemoglobin (HbF) in two children with osteopetrosis and high levels of HbF have been studied. The structural analysis of the gamma chains demonstrated a fetal Ggamma/Agamma ratio. HbF was distributed inside only 30% of the peripheral red blood cells. In vitro globin chain synthesis studies showed that there was balanced globin chain production, despite the increased level of HbF.