Fetal hemoglobin synthesis in sickle cell anemia: Some molecular considerations

An ability to maintain high levels of fetal hemoglobin (Hb F) has been associated with the amelioration of the clinical severity of the sickle cell disease (SS). Clinical efforts to increase the Hb F level of the patients have, however, yielded variable therapeutic response. In an attempt to further elucidate the underlying molecular basis, in vitro Hb F synthesis was studied in erythroid progenitor (BFU-E) cells obtained from SS patients and their heterozygous (AS) relatives with varying genetic backgrounds. This allows us to study the Hb F biosynthesis pattern uncomplicated by the influence of the preferential survival of “F cells” In vivo. The Hb F levels and the relative concentrations of its constituent gamma globin chains, G_γ and A_γ, were assayed using the reversed phase HPLC method. A percentage increase in the fetal hemoglobin content was observed in the lysates of the erythroid progenitor cells relative to the circulating peripheral blood erythrocyte values in SS patients and their AS relatives with different β_s haplotypes reflecting the intrinsic capacity of fetal hemoglobin synthesis in these subjects. No such increase was observed in the patient with the Mor haplotype. Furthermore, the Hb F synthesized in the BFU-E colonies was more of the adult type, as evidenced by the decrease in the percent G_γlevel relative to the corresponding peripheral blood values of the subjects in all the haplotype groups studied. The Mor haplotype was again an exception, synthesizing fetal hemoglobin more of the fetal type. © 1994 Wiley-Liss, Inc.     

Studies of Hb F in adult nonanemic baboons: Hb F expression in erythroid colonies decreases as the level of maturation of erythroid progenitors advances

The expression of fetal hemoglobin was studied in erythroid cultures of bone marrow cells from normal baboons using globin biosynthesis and double immunofluorescent labeling with anti-gamma and anti-beta-chain monoclonal antibodies. Baboon erythroid cultures differed from human erythroid cultures in several respects. Erythroid clusters, CFUe, and to a lesser extent BFUe, could be grown without added erythropoietin (Epo), and in vitro maturation time of both CFUe and BFUe was shorter than of their human equivalents. In both CFUe and BFUe cultures Hb F synthesis increased following addition of Epo and reached a plateau at 0.5-1.0 IU. The biosynthetically determined levels of Hb F (mean, SD, and range of gamma/gamma + beta ratios) were: 0.84 +/- 0.09 (0.68-0.89) in BFUe colonies grown in 1.0-2.0 IU of Epo; 0.61 +/- 0.13 (0.45-0.85) in BFUe colonies grown in 0.2 IU of Epo; 0.28 +/- 0.12 (0.21-0.46) in erythroid clusters and CFUe-derived colonies grown at 0.2-0.5 IU of Epo; and 0.04 +/- 0.02 (0.02-0.06) in CFUe colonies and e-clusters grown in the absence of added Epo. Immunofluorescent labeling revealed a pancellular expression of Hb F in all BFUe-derived colonies and bimorphism of Hb F expression in CFUe colonies and in a few erythroid clusters. These results show that, in the baboon, the level of Hb F expression in culture decreases as the presumed differentiative state of cells that produce the erythroid colonies advances.     

The synthesis of fetal hemoglobin types in red blood cells and in BFU-E derived colonies from peripheral blood of patients with sickle cell anemia, beta+ - and delta beta-thalassemia, various forms of hereditary persistence of fetal hemoglobin, normal adults and newborn.

The biosynthesis of two types of human fetal hemoglobin (Hb F), namely Hb F with G gamma chains having glycine in position 136 and Hb F with A gamma chains having alanine in position 136, was studied in blood samples and in cultures of erythroid precursors from blood of patients with different hemoglobinopathies. High pressure liquid chromatography (HPLC) was adapted to allow the separation of the methionyl-containing tryptic peptides G gamma T-15 and A gamma T-15 (which include the Gly leads to Ala polymorphism at position 136) from a digest of microquantitites of 35S-methionyl labelled Hb F. This method was sensitive enough to quantitate the relative production of the G ygamma and A gamma chains by erythroid colonies derived from cloned Burst Forming Units (bfu-e) which were cultured for 16 days on methylcellulose. The production of Hb F in these colonies was generally higher than the level of Hb F in blood except for subjects with the G gamma A gamma-HPFH heterozygosity. The G gamma to A gamma ratio in the Nb F produced in cultures of cells from G gamma delta beta-thalassemia or G gamma-HPFH heterozygotes was lower and that from A gamma-HPFH heterosygotes was higher than the ratios in the Hb F of the corresponding peripheral blood cells. Mixtures of G gamma and A gamma chains were present in cell cultures of SS patients, beta+-thalassemia homozygotes and G gamma A gamma-HPFH heterozygotes in a ratio similar to that in the Hb F of mature red cells. These data suggest that erythroblasts in BFU-E derived colonies reactivate all available gamma chain structural genes, both in cis and in trans to the abnormal determinant. Hb F biosynthesis by adult blood samples concerns primarily the G gamma chains. This was particularly striking for blood samples in which erythroblasts were absent and the biosynthesis took place in fetal reticulocytes. Thus, the F-retuculocytes in blood of A gamma-HPFH heterozygotes with about 5% Hb F of the A gamma type produced primarily Hb F with G gamma chains. Similar differences were observed for G gamma A gamma-HPFH heterozygotes and, less strinkingly, for SS patients. A satisfactory explanation for this observation has not yet been obtained.     

Direct evidence for interaction between human erythroid progenitor cells and a hemoglobin switching activity present in fetal sheep serum.

An activity that induces Hb F to Hb A switching in human cells is present in fetal sheep serum. To test directly the role of cell-to-environment interactions in hemoglobin switching and to define the level of erythroid cell differentiation at which this activity operates, colony transfer experiments were done. Clones grown in the presence of switching activity-containing medium (fetal sheep serum) or control medium (fetal calf serum) were transferred, at the 16- to 30-cell stage, to either fetal sheep serum or fetal calf serum plates and Hb F synthesis was determined in the fully mature erythroid bursts. Fetal calf serum-to-fetal calf serum transfers produced colonies with the high Hb F levels characteristic of undisturbed fetal calf serum-grown clones. Fetal sheep serum-to-fetal calf serum transfers resulted in significant decrease in Hb F synthesis, revealing an interaction between hemoglobin switching activity and cells at an early stage of progenitor cell development. The reduction of Hb F synthesis in fetal calf serum-to-fetal sheep serum transfers indicated that hemoglobin switching activity interacts with cells at later stages of progenitor cell development. Maximal decrease in Hb F synthesis was observed in fetal sheep serum-to-fetal sheep serum transfers, indicating that optimal effects on Hb switching are obtained when the environment that induces Hb switching is present throughout the development of progenitor cells. By splitting single early clones into two parts and transferring them to either a fetal sheep serum or a fetal calf serum environment, these interactions were further demonstrated in the progeny of a single erythroid burst-forming unit. Since all clone transfers were done on cell-free plates, the results of fetal calf serum-to-fetal sheep serum and of fetal sheep serum-to-fetal sheep serum transfers indicated that the switching activity does not require helper cells for its action. These studies show directly that (i) Hb F synthesis is controlled at the level of progenitors and (ii) it involves interactions between progenitor cells and their environment.     

Hemoglobin F synthesis in vitro: evidence for control at the level of primitive erythroid stem cells.

The in vitro regulation of fetal hemoglobin (HbF was investigated in clones of cultured adult human erythroid cells by in situ immunofluorescent identification of the hemoglobins synthesized. Formation of Hb F-containing clones was enhanced by erythropoietin and by culture conditions favoring the proliferation of less-differentiated stem cells of the burst-forming-unit type. Burst-forming units differed in their capacity to direct Hb F synthesis in their terminally differentiated progeny. A class of early precursors that can produce descendent stem cells with or without commitment to Hb F production was identified. The findings suggest that the capability for expression of Hb F in terminally differentiated cells of the adult is determined at the level of less-differentiated erythroid stem cells with characteristics of burst-forming units. It is proposed that the regulation of Hb F synthesis in vivo is also linked to the process of differentiation of the erythroid stem cells and that the patterns of Hb F synthesis during ontogeny reflect the attainment of progressively higher levels of differentiation of erythroid stem cells as development proceeds.     

Cellular regulation of hemoglobin switching: evidence for inverse relationship between fetal hemoglobin synthesis and degree of maturity of human erythroid cells.

To investigate whether the level of maturity of human erythroid cells influences the expression of the fetal hemoglobin program, we studied the relative production of fetal (Hb F) and adult (Hb A) hemoglobins during the maturation of erythroid clones produced in vitro by adult or neonatal erythroid stem cells. In both the adult and the neonatal cell cultures, clones composed of immature erythroblasts showed a significantly higher Hb F/Hb A ratio compared to the mature clones. Culture conditions enhancing erythroid cell maturity (such as an increase in the level of erythropoietin or culture time) decreased the relative synthesis of Hb F in the maturing erythroid cells. Direct immunofluorescence studies demonstrated earlier production of Hb F compared with Hb A during maturation of adult-origin HbF-synthesizing clones. The findings show that the final expression of Hb F is influenced by the degree of maturity of the terminally differentiated cells and suggest that, in addition to regulation at the level of erythroid stem cells, there is control of Hb F expression during erythroblast maturation. The inverse relationship between Hb F expression and level of cell maturity suggests that a regulatory mechanism operating throughout the process of erythroid stem cell differentiation/erythroblast maturation decreases the potential of Hb F expression as the development of the erythroid cell advances.     

Adult''fetal-like''erythropoiesis characterizes recovery from bone marrow transplantation

The transient fetal-like erythropoiesis which appears during recovery from bone marrow transplantation has now been examined at the level of erythroid progenitor cells. A 7-year-old boy with beta +-thalassaemia major was studied during engraftment from his beta-thalassaemia trait sister. Hb F and i antigen rose as expected. Macrocytosis never developed, but red cell size distribution became very heterogeneous. Bone marrow CFU-E and BFU-E were detected by 30 d, prior to the appearance of reticulocytes. Marrow erythroid progenitor cell numbers were normal by 146 d, while those in the blood became normal by 360 d. After transplantation globin synthesis ratios in erythroid colonies were diagnostic of thalassaemia trait, indicating engraftment. Individual erythroid colonies derived from both blood and marrow at all times during reconstitution showed no correlation of G gamma and gamma. Thus the fetal-like stress erythropoiesis of marrow expansion following transplantation was derived from adult and not fetal progenitor cells.     

Fetal calf serum contains activities that induce fetal hemoglobin in adult erythroid cell cultures.

Cultures of peripheral blood or bone marrow erythroid progenitors display stimulated production of fetal hemoglobin. We investigated whether this stimulation is due to factors contained in the sera of the culture medium. Comparisons of gamma/gamma + beta biosynthetic ratios in erythroid colonies grown in fetal calf serum (FCS) or in charcoal treated FCS (C-FCS) showed that FCS-grown cells had significantly higher gamma/gamma + beta ratios. This increase in globin chain biosynthesis was reflected by an increase in relative amounts of steady-state gamma-globin mRNA. In contrast to its effect on adult cells, FCS failed to influence gamma-chain synthesis in fetal burst forming units-erythroid (BFU-E) colonies. There was a high correlation of gamma-globin expression in paired cultures done with C-FCS or fetal sheep serum. Dose-response experiments showed that the induction of gamma-globin expression is dependent on the concentration of FCS. These results indicate that FCS contains an activity that induces gamma-globin expression in adult erythroid progenitor cell cultures.     

Fetal hemoglobin synthesis in erythroid cultures in hereditary persistence of fetal hemoglobin and beta o-thalassemia

To determine whether hemoglobin regulation is normal in diseases affecting beta-globin gene expression, globin synthesis was examined in members of a family of a patient with hereditary persistence of fetal hemoglobin/beta o-thalassemia (HPFH/beta o-thal). The HPFH defect is the Ghanian type II, with a deletion from psi beta 1 to at least 20 kb 3' to beta. The beta o-thal gene has the haplotype II restriction enzyme pattern and has the beta 39 nonsense mutation. Erythroid colonies from blood BFU-E were radiolabeled, and globin chains were separated by gel electrophoresis. Colonies from the beta o-thal heterozygote had non-alpha/alpha ratios more balanced than in the reticulocytes. Gamma synthesis was 11% of non-alpha, which is higher than in reticulocytes, but within the range seen in normal adult colonies. Both HPFH heterozygotes produced 20%-30% gamma in erythroid colonies as well as reticulocytes, although non-alpha/alpha was more balanced in the colonies. The HPFH/beta o-thal patient produced 100% gamma in reticulocytes and in colonies. G gamma and gamma-synthetic proportions were not correlated at the individual colony level in the heterozygotes, suggesting that they had "adult" and not "fetal" progenitor cells. The Hb expression of these adult progenitors is presumably modulated normally in vivo in beta o-thal, but the normal decrease in HbF production does not occur in gene deletion HPFH.     

Sodium butyrate enhances fetal globin gene expression in erythroid progenitors of patients with Hb SS and beta thalassemia

Increasing the expression of the gamma globin genes is considered a useful therapeutic approach to the beta globin diseases. Because butyrate and alpha-amino-n-butyric acid (ABA) augment gamma globin expression in normal neonatal and adult erythroid progenitors, we investigated the effects of sodium butyrate and ABA on erythroid progenitors of patients with beta thalassemia and sickle cell anemia who might benefit from such an effect. Both substances increased fetal hemoglobin (Hb F) expression in Bfu-e from 7% to 30% above levels found in control cultures from the same subjects with sickle cell anemia. The fraction of cultured erythroblasts producing Hb F increased more than 20% with sodium butyrate treatment in 70% of cultures. In most cultures, this produced greater than 20% total Hb F and greater than 70% F cells, levels which have been considered beneficial in ameliorating clinical symptoms. Alpha: non-alpha (alpha-non-alpha) imbalance was decreased by 36% in erythroid progenitors of patients with beta thalassemia cultured in the presence of butyrate compared with control cultures from the same subjects. These data suggest that sodium butyrate may have therapeutic potential for increasing gamma globin expression in the beta globin diseases.     

Stochastic expression of fetal hemoglobin in adult erythroid cells.

The expression of fetal hemoglobin, Hb F, in the adult cells is cellularly restricted both in vivo and in culture. Because, in cultures of erythroid progenitors, subclones that express or fail to express Hb F are derived from the same erythroid stem cell, a mechanism must exist whereby Hb F expression segregates in the progeny of erythroid progenitors during their differentiation. We present mathematical analyses of experimental data which suggest that expression of Hb F in human adult erythroid cells occurs on a stochastic basis. We quantified Hb F expression among the subclones of erythroid bursts (clones) in vitro by labeling subclones with fluorescent anti-Hb F antibodies. The observed data were compared with predictions from a stochastic model with the assumption that the expression of Hb F in a subclone occurs with a probability P equal to the frequency of Hb F-expressing subclones in an experiment. There was good fit between the observed and predicted data with respect to: (i) the relative frequencies of monomorphic F+, monomorphic F-, and bimorphic F+/F- bursts, respectively; (ii) the size distributions among F+, F- and F+/F- bursts; and (iii) the proportions of subclones expressing Hb F within bimorphic F+/F- bursts. Given the hypothesis that erythroid progenitors which have an active Hb F program are less differentiated than cells which do not proceed to express Hb F, the stochastic event indicated by our analyses may be the probability that adult erythroid progenitor cells undergo terminal differentiation at an earlier stage than usual.     

Haemoglobin Synthesis in Human Erythroid Bursts during Ontogeny: Reproducibility and Sensitivity to Culture Conditions

Patterns of haemoglobin (Hb) synthesis were analysed in erythroid bursts grown in vitro from human fetal, umbilical cord and adult blood to determine the reproducibility of the system and its sensitivity to various alterations in culture conditions. The proportion of Hb F synthesis in duplicate plates from the same sample showed little variation but when an individual was studied under the same conditions but at different times, far more variability was observed. We have confirmed that the synthesis of Hb F and Hb A in the cultures is asynchronous, relatively more Hb F being synthesized earlier in the maturation of the colony. Hence factors affecting colony maturation are likely to change the apparent pattern of Hb synthesis. Removal of adherent cells prior to plating resulted in the presence of more mature colonies and lower levels of Hb F synthesis at any fixed time in culture. Furthermore, the overall ratio of gamma:beta chain synthesis measured in the whole plate was dependent on the proportion of large, immature multicluster bursts present. The significance of measurements of Hb synthesis in erythroid bursts can be assessed only if all of these factors are recognized and taken into account.     

Proportion of fetal hemoglobin synthesis decreases during erythroid cell maturation.

Peripheral blood mononuclear cells from pregnant and postpartum women were cultured in vitro with erythropoietin. Burst-forming unit (BFU-E)-derived erythroid colonies composed of immature erythroblasts with low hemoglobin contents were observed by day 8 of culture. By day 12 of culture, numerous BFU-E-derived erythroid colonies with high hemoglobin contents were present. The gamma/(gamma + beta) globin synthetic ratio was approximately 12% in the early cultures and 6% in the late cultures, indicating that the proportion of fetal hemoglobin synthesis decreases during erythroid cell maturation. These studies also reveal that the capacity fof fetal hemoglobin production by peripheral blood BFU-E in vitro is not altered during pregnancy.     

Patterns of Globin Chain Synthesis in Erythroid Colonies Grown from Sheep Marrow of Different Developmental Stages

Erythroid colonies were grown from fetal sheep bone marrow at different stages of development and from adult marrow. Colony numbers increased with erythropoietin concentration, but fetal erythroid cells were more sensitive to the hormone than adult cells. Haemoglobin synthesis was characterized in the colonies and compared to that in control marrow incubations. No beta chain synthesis was detectable in marrow incubations or erythroid colonies from fetal marrow at 87-96 d gestation. Incubations of 115-120 d marrow shoed 5% beta chain synthesis while erythroid colonies synthesised up to 20% beta chains. Incubations and erythroid colonies from 125 d marrows showed about 27% beta chain synthesis, despite a similar increase in colony numbers with erythropoietin as the other marrows. Later in gestation, beta chain synthesis was lower in erythroid colonies than in marrow incubations and declined further in erythroid colonies as erythropoietin concentration increased. Adult marrow showed no detectable gamma chain synthesis but erythroid colonies produced up to 12% gamma chains, the identity of which was confirmed by peptide mapping. All changes in globin synthesis in vitro were correlated with colony numbers. In adult marrow, the maximal level of gamma chain production was inversely related to the cloning efficiency of the marrow samples studied. Potential uses of this model for studying globin gene expression are discussed.     

Elevated Hb F associated with beta-thalassaemia trait: haemoglobin synthesis in reticulocytes and in blood BFU-E

The red cells of a patient heterozygous for beta-thalassaemia contained 19% fetal Hb. Study of his family suggested that the proband had inherited the Swiss type of hereditary persistence of fetal Hb (HPFH) from his mother who is not thalassaemic and possessed 1.37% of Hb and 11% F-cells. Studies of globin synthesis showed a similar imbalance in the heterocellular HPFH-beta-thalassaemia compound heterozygotes and in the heterozygous beta-thalassaemic members of the family. Age stratification of the red cells showed a slight enrichment in Hb F and a decreased Hb A2 level in the older cell populations. Hb F production in the BFU-E colonies of the proband was higher than that found in vivo and in other beta-thalassaemic heterozygotes in culture. Study of single erythroid burst colonies showed a marked heterogeneity in Hb F synthesis from one colony to another, while the pool of free alpha-chains remained of similar magnitude. It is suggested that in the proband, the HPFH gene, which is in trans with respect to the beta-thal-gene, increases the size of the F-cell population and its activity is carried on at the expense of the normal beta A gene.     

Hemoglobin switching in sheep: a comparison of the erythropoietin- induced switch to HbC and the fetal to adult hemoglobin switch

Stimulation of sheep erythropoietic progenitor cells by erythropoietin (epo) has been studied with regard to its effect on the pattern of hemoglobin production. An analysis of hemoglobin (Hb) synthesis in BFU- E- and CFU-E-derived colonies from fetuses either homozygous for HbA (AA) (homozygous also for the beta c gene responsible for HbC production) or HbB (BB) (lacking the beta c gene) indicated the following. Colonies derived from precursor cells from 51- and 89-day fetuses exhibited small but detectable increments of HbB synthesis with prolonged incubation in vitro. This response was not dependent on the epo concentration. Erythropoietic precursor cells from a 124-day BB fetus were already committed to HbB synthesis, since HbF production was replaced by HbB on successive days in vitro as erythroid colonies matured; this switch was not affected by varying the epo concentration. In contrast, progenitor cells from a 124-day AA fetus responded to higher doses of epo by forming colonies in which more HbC was made at the expense of both HbF and HbA. Erythropoietic stress did not result in induction of HbF in vivo or in erythroid colonies derived from CFU-E in young adult BB sheep, whereas our prior studies had shown induction of HbC synthesis under analogous conditions in colonies derived from young adult AA sheep. We conclude that the epo-induced HbF (or HbA) to HbC switch and the fetal to adult hemoglobin switch are regulated by different mechanisms.     

The Synthesis of Fetal Hemoglobin Types in red Blood Cells and in BFU-E Derived Colonies from Peripheral Blood of Patients with Sickle Cell Anemia, β+- and δβ-Thalassemia,Various forms of Hereditary Persistence of Fetal Hemoglobin,Normal Adults and Newborn

The biosynthesis of two types of human fetal hemoglobin (Hb F), namely Hb F with ^Gγ chains having glycine in position 136 and Hb F with Ay chains having alanine in position 136, was studied in blood samples and in cultures of erythroid precursors from blood of patients with different hemoglobinopathies. High pressure liquid chromatography (HPLC) was adapted to allow the separation of the methionyl-containing tryptic peptides ^GγT-15 and ^AγT-15 (which include the Gly → Ala polymorphism at position 136) from a digest of microquantities of ³⁵S-methionyl labelled Hb F. This method was sensitive enough to quantitate the relative production of the ^Gγ and ^Aγ chains by erythroid colonies derived from cloned Burst Forming Units (BFU-E) which were cultured for 16 days on methylcellulose. The production of Hb F in these colonies was generally higher than the level of Hb F in blood except for subjects with the GγAγ-HPFH heterozygosity. The Gγ to Aγ ratio in the Hb F produced in cultures of cells from Gγδβ-thalassemia or Gγ-HPFH heterozygotes was lower and that from Aγ-HPFH heterozygotes was higher than the ratios in the Hb F of the corresponding peripheral blood cells. Mixtures of Gγ and Aγ chains were present in cell cultures of SS patients, β-thalassemia homozygotes and GγAγ-HPFH heterozygotes in a ratio similar to that in the Hb F of mature red cells. These data suggest that erythroblasts in BFU-E derived colonies reactivate all available γ chain structural genes, both in cis and in trans to the abnormal determinant.

Hb F biosynthesis by adult blood samples concerns primarily the Gγ chains. This was particularly striking for blood samples in which erythroblasts were absent and the biosynthesis took place in fetal reticulocytes. Thus, the F-reticuloytes in blood of Aγ-HPFH heterozygotes with about 5% Hb F of the Aγ type produced primarily Hb F with Gγ chains. Similar differences were observed for GγAγ-HPFH heterozygotes and, less strikingly, for SS patients. A satisfactory explanation for this observation has not yet been obtained.     

High Prevalence of the −α3.7 Deletion Among Thalassemia Patients in Iran

Bone marrow cells from hematologically normal subjects were obtained by aspiration in 19 cases and by surgery in two cases; erythroid colonies were subsequently cultured from these cells in plasma clots. The hemoglobins synthesized in culture were studied either by incubation of the cells at the 6th-7th day of culture, or at the 13th-l4th day of culture with ³H-leucine, in order to determine whether the reactivation of fetal hemoglobin (Hb F) in vitro was related to the stage of differentiation of the erythroid precursors. Hemoglobins synthesized in culture were specifically purified from other proteins by affinity chromatography on Sepharose-haptoglobin and were analyzed by separation of globin chain on carboxy-methyl-cellulose in urea. In 14-day-old cultures, reactivation of Hb F synthesis was observed in all cases but one (mean value 8.2%, range 2.3% to 17.7%). In the 7-day-old cultures, a lower proportion of Hb F synthesis was observed (mean value 4.2%, range 1.6% to 7.6%).

These results indicate that the reactivation of Hb F synthesis is related to the stage of differentiation of the erythroid precursors.