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.     

Stimulation of fetal hemoglobin synthesis in bone marrow cultures from adult individuals.

The regulation of fetal hemoglobin in adult erythroid cells was investigated with bone marrow cultures. Fetal hemoglobin (Hb F) was identified in individual erythroid colonies with fluorescent antibodies against Hb F and synthesis of gamma chains was determined with analyses of radioactive globins. The appearance of fetal hemoglobin in erythroid colonies was clonal. All the cells of the Hb F synthesizing colonies contained fetal hemoglobin. The frequency of erythroid colonies showing Hb F was higher than expected compared to the frequency of Hb F containing cells in the blood. Production of Hb F in culture, as shown by analysis of the radioactive globins, was 5 to 14 times higher than baseline Hb F synthesis. These results suggest that the ability for gamma chain synthesis in erythroid cells is determined at or above the level of the precursor cell from which the erythroid colonies, in vitro, derive (probably an erythropoietin responsive stem cell), and that stimulation of fetal hemoglobin synthesis in adult erythroid cells is possible.     

Synthesis of the minor fetal hemoglobin Fic in colonies of erythropoietic precursors isolated from human umbilical cord blood

The biosynthesis of the minor hemoglobin F_Ic, which contains acetylated γ chains, and the major hemoglobin F_o was studied during erythroid cell differentiation and maturation in cultures of erythroid precursors isolated from five human umbilical cord blood samples. A gradual decrease in the synthesis of Hb F_Ic was observed during the maturation of the erythroid cells when cultured from 7 to 15 days. The synthesis of Hb F_o did not show a consistent change; however, the relative synthesis of Hb F_Ic was the highest in the 7-day-old colonies and decreased at day 15 to levels observed in the reticulocytes. The addition of sodium butyrate, which is known to promote histone acetylation, significantly increased the synthesis of Hb F_Ic in 7-day-old colonies.     

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.     

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.     

The synthesis of the Gγ and Aγ chains of human fetal hemoglobin in erythroid colonies cultured from peripheral blood BFUe's of normal adults and newborn and of subjects with an Aγ or a Gγ chain abnormal fetal hemoglobin

Peripheral blood mononuclear cells from normal adults, normal newborn infants, and from newborn and adult subjects with one of three γ chain variants (^GγF-Malta I, ^GγF-Port Royal, ^AγF-Hull) were cultured in vitro with erythropoietin. The ³⁵S-methionine-labelled hemoglobin from 13- to 15-day-old BFUe-derived colonies was studied by chromatography on columns of DEAE-cellulose and the quantities of Hbs A₂, F_x, and F₀ determined. The percentages of ^Gγ and ^Aγ chains in isolated Hb F_x and Hb F₀ were determined using high-performance liquid chromatography (HPLC) of the tryptic peptides of these proteins. Calculation of these percentages was based on the total activities of the ^GγT-15 and ^AγT-15 peptides which contain one (³⁵S-labelled) methionyl residue each and can be separated by the HPLC procedure. The data show an increased synthesis of Hb F in the ?adult”? colonies and a decreased synthesis in the ?newborn”? colonies. The ^Gγ to ^Aγ ratio of the Hb F from adult colonies varied greatly. The percentages of ^Gγ and ^Aγ chains in the Hb F from adult colonies correlated with the percentages in the Hb F isolated from the Hb F of circulating red blood cells. The ^Gγ to ^Aγ ratio in the Hb F from newborn colonies was high as in the Hb F from cord blood samples. ^Gγ and ^Aγ chain abnormal Hb F variants were readily detectable in colonies from both adults and newborn. The ^Gγ to ^Aγ ratio in the Hb F of colonies of adult Hb F-Malta I and Hb F-Hull heterozygotes approached 1, but that of adult Hb F-Port Royal heterozygotes remained about as high as in colonies from newborn heterozygotes. The percent Hb F-Port Royal in the Hb F of adult colonies was twice that in the Hb F of newborn colonies. These results are discussed in the light of information from recent detailed studies of genomic DNA assuming controlling functions for the segments of DNA that are interspersed between the various structural genes.     

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.     

A short review of human gamma-globin gene anomalies

This paper summarizes various anomalies involving the duplicated gamma-globin genes which are normally arranged as -G gamma-A gamma-. Variations include G gamma----A gamma and A gamma----G gamma replacements; newborn heterozygotes for these anomalies have low G gamma (A gamma A gamma/G gamma A gamma) or high G gamma values (G gamma G gamma/G gamma A gamma). Additional abnormalities include the deletion of one gamma-globin gene (-A gamma-; gamma-thalassemia), gamma-globin gene triplications (G gamma-AG gamma-A gamma; G gamma-G gamma-A gamma) and gamma-globin gene quadruplications (G gamma-G gamma-G gamma-A gamma-); several of these conditions are found in different populations but at low frequencies. In adults, the G gamma to A gamma ratio in the Hb F and often also the Hb F level, are directly related to specific structural characteristics of the chromosome; specific mutations in the promoter sequences 5' to G gamma or to A gamma, for instance, may result in increased G gamma or A gamma chain production with an increase in Hb F levels (nondeletional G gamma- or A gamma-HPFH) or with only modest changes in the total Hb F (normal adults; Swiss-HPFH). Similar variations have been observed in SS patients.     

The percentages of Hb F and of G gamma and A gamma chains in the Hb F synthesized by reticulocytes and BFUe-derived colonies of patients with sickle cell anemia

The synthesis of Hb F was studied in the BFUe-derived colonies of 20 SS patients after incubation with 35S methionine for 24 hours, 13 days after the start of the experiments. Similar analyses were made for isolated reticulocytes from 28 SS patients; these cells were incubated for 2 hours and occasionally for 24 hours. The G gamma and A gamma percentages were determined in these two Hb F preparations and in the Hb F of circulating red cells using two different HPL chromatographic procedures. The data show an increased production of Hb F in the colonies mainly of patients with low blood Hb F values. A close correlation was present between the % G gamma chain in peripheral red cells and the G gamma percentages observed for the Hb F synthesized in the BFUe-derived colonies and that in reticulocytes. However, the values for the reticulocytes were significantly higher than those for the colonies. These data indicate a considerable variability among SS patients in their ability to produce Hb F in in vitro colonies which may in part be due to differences in the numbers of BFUe types in circulation. The variation in G gamma and A gamma levels in Hb F synthesized in reticulocytes and BFUe-derived colonies could be the result of slight differences in the stability of the corresponding mRNA's.     

DNA sequence variation associated with elevated fetal G gamma globin production

The percentage of G gamma chains in the Hb F of SS patients and beta-thalassemia heterozygotes is generally 40%, but some have 60% to 70% G gamma. To test the hypothesis that DNA sequence variation 158 base pairs 5' of the G gamma gene is associated with this variation in G gamma values, DNA was analyzed using the restriction endonuclease Xmn I (gamma IVS-II probe). Xmn I recognizes the sequence from -157 to -166 only if T is at position -158. Individuals from five families had T at -158 for G gamma genes in both chromosomes, and the mean G gamma value was 69.7% +/- 4.6% (SD). For 13 families, individuals with T at -158 for the G gamma gene of one chromosome had a G gamma value of 60.6% +/- 5.7%. With one exception, lack of T at -158 was associated with low G gamma values (39.6% +/- 4.0%). In low Hb F G gamma-beta+-HPFH, the Xmn I site was seen 5' to both G gamma and A gamma genes, which suggests that T at -158 is associated with elevated Hb F; Pst I digestion showed that the A gamma gene T producer G gamma globin, which accounts for high levels of G gamma (87-88%). Calculations show that T at -158 is associated with a three- to 11-fold increase in production per G gamma gene, which is an order of magnitude less than that associated with the previously identified -202 C----G substitution of high Hb F G gamma-beta+-HPFH.     

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.     

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.     

An A gamma type of nondeletional hereditary persistence of fetal hemoglobin with a T----C mutation at position -175 to the cap site of the A gamma globin gene

The nondeletional types of hereditary persistence of fetal hemoglobin (ndHPFH) concern the continued synthesis of hemoglobin (Hb) F with either G gamma or A gamma chains in amounts varying from 5% to 30%. Several mutations have been identified in either the A gamma or G gamma promoter which are considered causative to the continued production of one of the two gamma chains because the substitutions occur in sequence motifs essential for the expression characteristics of the gamma-globin gene in the 3' position. We report the discovery of a T----C mutation at position -175 in the A gamma promoter which was associated with a greatly increased level of Hb F (with mainly A gamma) and a decreased level of Hb A in the one (Black) heterozygote who had a beta c gene in trans. The same mutation has been observed in the G gamma promoter of a Black heterozygote who had high levels of Hb F with G gamma chains only. A detailed comparison between these two individuals indicated significant differences in the levels of Hb F and Hb A which may result from an additional mutation at position -158 in the G gamma promoter.     

Influence of hydroxyurea on fetal hemoglobin production in vitro

Cytotoxic drugs increase circulating fetal hemoglobin levels. We examined the mechanism by measuring the fetal hemoglobin produced per BFU-E-derived erythroblast following hydroxyurea treatment in vivo and in vitro. Treatment of four sickle cell patients increased the percentage of circulating F reticulocytes. The frequencies of bone marrow or peripheral blood BFU-E or CFU-E-derived colonies and their fetal hemoglobin content were unaffected. In all cases, the number of erythroid cells/progenitor-derived colony increased. To explore further the effect of hydroxyurea on fetal hemoglobin production, we added 50 mumol/L hydroxyurea to cultures of peripheral blood BFU-E-derived erythroblasts on 1 of 9 days (day 5 through 13) to nine samples. These BFU-E were derived from the peripheral blood of normal donors, sickle trait donors, and sickle cell anemia patients and from the bone marrows of monkeys. This concentration of hydroxyurea was selected so that the frequency of BFU-E and their size was moderately decreased. Addition of hydroxyurea to these progenitor-derived erythroid cells had no effect on fetal hemoglobin content per cell. Neither did transient exposure of progenitors to hydroxyurea prior to culture in nontoxic concentrations (0 to 500 mumol/L) result in a significant increase in fetal hemoglobin content in progenitor-derived erythroblasts. These data suggest that hydroxyurea does not directly alter the HbF program expressed by progenitor-derived erythroid cells. Instead, it enhances hemoglobin F content secondarily, possibly by inducing alterations in erythropoiesis.     

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.     

The gamma chain heterogeneity of fetal hemoglobin in black beta- thalassemia and HPFH heterozygotes

High pressure liquid chromatography (HPLC) was applied to the HbF isolated from blood of numerous black patients with beta-thalassemia trait or homozygosity, G gamma-delta beta-thalassemia trait, G gamma A- gamma HPFH heterozygosity, or the G gamma-[delta+ beta+]-HPFH condition. The method allowed an accurate evaluation of the relative quantities of three types of gamma-chain (G gamma, A gamma I, A gamma T) in the fetal hemoglobins. The results have shown the following. (A) The incidence of the A gamma T-chain in beta-thal heterozygotes and G gamma A gamma-HPFH heterozygotes is about the same as has been observed in black newborn; about one of five blacks are heterozygous for this A gamma-chain variant. The A gamma T-chain was not detected in the nine G gamma-delta beta-thal heterozygotes nor in the eight G gamma-[delta+ beta+]-HPFH heterozygotes. (B) In most cases, the A gamma T-chain was produced by the A gamma gene in trans to the beta-thal or HPFH determinant. The contribution by the gamma-chain genes in trans to the beta-thal or HPFH determinant is about 15% of the total gamma-chain production in both conditions. (C) Three black beta-thal heterozygotes (and five additional relatives) had the A gamma T gene in cis to the beta-thal determinant. Four of these patients had a low levels of G gamma-chain (the "adult" level), and the contribution by the A gamma gene in cis to the beta-thal determinant was about three times that of the A gamma gene in trans. The four additional patients, all members of one family, had a high level of G gamma-chain (the "newborn" level), and the contribution of the A gamma gene in cis was half of that seen in the previously mentioned four patients while that of the A gamma gene in trans was essentially the same. These limited data suggest that the genetic anomaly causing high high G gamma levels in adult beta-thal heterozygotes is linked to the beta-thal determinant and that one of its primary effects is a decreased synthetic expression of the A gamma gene in cis to the beta-thal determinant.     

Interaction of two different disorders in the beta-globin gene cluster associated with an increased hemoglobin F production: a novel deletion type of (G) gamma + ((A) gamma delta beta)(0)-thalassemia and a delta(0)-hereditary persistence of fetal hemoglobin determinant.

We report two different disorders of the beta-globin gene cluster segregating in a Belgian family: a novel deletion that results in (G) gamma + ((A) gamma delta beta)(0)-thalassemia (thal) and a heterocellular hereditary persistence of foetal hemoglobin of the Swiss type linked to a delta(0)-thal gene (delta (0)-HPFH). Heterozygosity for the heterocellular HPFH brings about a moderate (3.4% to 8.24%) increase of hemoglobin (Hb) F having a G gamma/A gamma ratio of 4:1, whereas carriers of the G gamma + ((A) gamma delta beta)(0)-thal deletion show in their peripheral blood a considerably higher (15%) percentage of Hb F. Both defects interact in the compound heterozygotes for G gamma + ((A) gamma delta beta)(0)-thal and delta(0)-HPFH producing a further increase (up to 24%) of fetal Hb consisting entirely of G gamma chains. Molecular characterization of the (G) gamma + ((A) gamma delta beta)(0)-thal by means of Southern analysis showed that the deletion spans about 50 kb, removing the 3' end of the A gamma-gene, the psi beta-, delta-, and beta-genes. A number of possible mechanisms leading to the overproduction of Hb F in HPFH and (G) gamma + ((A) gamma delta beta)(0)-thal will be discussed.     

Bromodeoxyuridine treatment of normal adult erythroid colonies: an in vitro model for reactivation of human fetal globin genes

We report that bromodeoxyuridine (BrdU) addition in semi-solid cultures of normal adult erythroid progenitors causes a sharp rise of gamma-globin gene expression in erythroid colonies. Control studies were carefully carried out to exclude the possibility of toxic effects exerted by the drug in these experimental conditions. In particular, BrdU addition induces a sharp increase in the level of relative gamma-globin synthesis and content in pooled BFU-E-derived colonies: this rise is clearly observed in single bursts of the mature type (largely composed of late erythroblasts) but not in immature ones (essentially comprising early erythroblasts). Furthermore, it is associated with an increase of the G gamma/G gamma + A gamma synthetic ratio from adult up to fetal like values. Reactivation of gamma-synthesis was observed even if BrdU was added to colonies composed essentially of early erythroblasts, ie, when BrdU was added to either bursts at day 10 of culture or late CFU-E-derived clones at day 1. These in vitro observations indicate modulation of gamma-synthesis at the stage of erythroblasts from normal adults. At the molecular level we suggest that BrdU, by replacing thymidine in DNA, may inhibit the switch from a fetal-like biosynthetic program expressed in early erythroblastic differentiation to the adult program expressed in later stages of maturation.     

Quantitation of three types of gamma chain of HbF by high pressure liquid chromatography; application of this method to the HbF of patients with sickle cell anemia or the S-HPFH condition

A modification of a high pressure liquid chromatographic (HPLC) procedure is described that enables the complete separation and quantitation of the A gamma T, A gamma I, and G gamma chains in human fetal hemoglobin. The method, which is fast and accurate, requires 5 to 2000 micrograms Hb F. The purity of the Hb F is not essential and admixture of up to 70% adult Hb does not interfere with the determination. The method has been applied to the Hb F of 64 Black SS patients and 7 persons with the Hb S-HPFH (G gamma A gamma type) conditions. (A) Both "adult" G gamma to A gamma (2:3) and "newborn" G gamma to A gamma (3:2) ratios were observed in adult SS patients, 8 yr and older. Only 12% of the SS patients had the "newborn" ratio. This high G gamma to A gamma ratio may be due to a modification of the genetic switch mechanism that regulates the change of this ratio after birth. (B) Intermediate G gamma to A gamma ratios were only found in young SS patients, 5 yr of age or less. The results suggest a delayed switch of the newborn leads to adult ratio in sickle cell anemia. (C) The A gamma T chain was present in only 6% of all SS patients. One patient is homozygous for this variant chain. (D) Three of the 7 subjects with Hb S-HPFH were positive for the A gamma T chain. Its percentage was low, which suggests that the A gamma T chain gene is in trans of the HPFH determinant. (E) Quantitation of the three gamma chain types is also possible in the Hb F from Hb S heterozygotes with (nearly) normal Hb F levels. Such an analysis is useful for an evaluation of genetic conditions involving variations in the production of (different types of) Hb F.