Variability in the fetal hemoglobin level of the normal adult

We analyzed blood samples from more than 200 normal adults, and quantified their Hb F by cation-exchange high-performance liquid chromatography. In several subjects with slightly elevated Hb F (0.4–4.3%), we determined the ^Gγ levels in the Hb F and DNA sequence variations in the locus control region II and in the ^Gγ and ^Aγ promoters. About 25% of the ∼200 normal teenaged high school students had elevated Hb F; detailed analyses of some 20 students, selected at random, identified most as females with a homozygosity for the C→T variation at position -158 (^Gγ). One 11-year-old boy was heterozygous for the A→G change at position -161 (^Gγ); he and two of his relatives had ∼4% Hb F, high ^Gγ values, and a high level of (mainly) ^Gγ-mRNA. Nearly 40 normal adults from Macedonia and from Georgia (mostly Caucasians) were tentatively identified as Swiss HPFH heterozygotes because slightly elevated Hb F levels were observed at least once. Many of these persons were heterozygous or homozygous for the C→T mutation at -158 (^Gγ), and a few carried a γ-globin gene triplication. The C→T change appears to be an important factor predisposing the adult to increased Hb F production. Evidence suggests a gene dose effect in (mildly) anemic adults; however, other factors besides the C→T change at -158 (^Gγ), including factors not linked to the β-globin region, may cause an increase in γ-chain synthesis. © 1996 Wiley-Liss, Inc.     

The use of globin chain electrophoresis in polyacrylamide gels for the quantitation of the G gamma to A gamma ratio in fetal hemoglobin

Polyacrylamide gel electrophoresis (PAGE) in the presence of urea, acid, and Triton X-100 was used for determination of the G gamma to A gamma ratio in human Hb F. The data compared most favourable with results obtained by a HPLC procedure and by a chemical procedure. Moreover, its accuracy and reproducibility was determined. The presence of Hb A2 in a sample with Hb F level below 10% interferes with the determination because of the nearly identical electrophoretic mobilities of the delta and G gamma chains. Thus, the removal of Hb A2 is required. Samples free fo Hb A2 but with a Hb F level as low as 2% can be analyzed with an accuracy of about 5%. The PAGE method has been applied to the Hb F of 63 subjects with beta thalassemia and related conditions, and the results of these analyses are included in this communication.     

The G gamma:A gamma composition of fetal hemoglobin in fetuses and newborns.

To determine whether the G gamma:A gamma HbF switch is coordinated in development with the HbF:HbA switch, hemoglobin F Gly/Ala (G gamma:A gamma) heterogeneity was evaluated by electrophoresis on polyacrylamide gels. The mean G gamma was 70% in 19 newborns, similar to the published value of 73% obtained by amino acid analysis of the gamma CG-3 peptide. In 29 fetuses at 18-20-wk gestation, G gamma was 71%. The values were similar in normal fetuses and newborns and in those with hemoglobinopathies. The synthetic ratio of 3H-leucine-labeled G gamma:A gamma was identical to the ratio found in the globin protein accumulated by 20 wk. The proportion of G gamma was also identical in 6 paired samples, at 20-wk gestation and at birth. Thus, G gamma production does not decline with respect to A gamma in utero at the time of onset of the HbF:HbA switch.     

The occurrence of different levels of G gamma chain and of the A gamma T variant of fetal hemoglobin in newborn babies from several countries

The gamma chain compositions of the fetal hemoglobins of 2453 newborn babies from East Asian countries (1350 babies), from Italy, Yugoslavia, Bulgaria, and Georgia (417 Caucasian babies), and 686 black babies from Georgia were determined by high pressure liquid chromatography. Unusual results for a limited number of babies were confirmed by chemical analyses, and were evaluated further by family studies. Statistical analyses indicated high gene frequencies for the A gamma T chain in Italian (f = 0.237), Yugoslavian and Bulgarian (f = 0.238), and white Georgia babies (f = 0.224), a lower frequency in Japan (f = 0.178), and India (f = 0.173), and particularly in mainland China (f = 0.079). The A gamma T gene frequency in normal (AA) Black babies was 0.102. When a beta S or beta C mutation was also present this frequency was greatly decreased, particularly in babies with the AC condition (f = 0.036). These results suggest the near absence of the A gamma T mutation on the chromosome also carrying the beta C determinant. Most babies had the expected G gamma values which vary between 60 and 80%, but several (mainly black) babies had higher values (between 80 and 90%), while one normal black baby had a G gamma value of (nearly) 100%. This condition may be a form of A gamma +1-thalassemia and has been discussed in detail elsewhere (Blood 58:491-500, 1981). Thirty-five clinically normal (mainly Chinese, Indian, and Japanese) babies had G gamma values of about 40%. Twenty-six babies had A gamma I values of about 60%, while the remaining nine babies had A gamma T and A gamma I chains in a ratio of either 1 to 2 or 1 to 1. Two additional newborns did not produce any G gamma chains, but had only A gamma I chains or A gamma T chains. Family studies failed to indicate a specific hematological abnormality. These unusual ratios between the G gamma and A gamma (either A gamma I or A gamma T) chains have led to speculations regarding possible genetic abnormalities present in these infants.     

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.     

Complete sequence of the gamma chain from the fetal hemoglobin of the baboon, Papio cynocephalus

The amino acid sequence of the hemoglobin gamma chain from a baboon, Papio cynocephalus, was determined by automated sequencing of the intact chain and six fragments generated by specific clevage reactions. The existence of structural heterogeneity at position 75, where both valyl and isoleucyl residues were found, is suggestive of the presence of nonallelic V gamma- and I gamma-chain genes in this species, and further emphasizes the extent to which the genetic basis of hemoglobin production among many higher primates is similar. Comparison of the sequences of those gamma chains from Homo sapiens, Pan troglodytes, Macaca nemestrina and P. cynocephalus that have been well characterized attests to the conservative nature of gamma-chain evolution among the Anthropoidea, the differences in sequence between any two of these chains ranging from none (between the A gamma and G gamma chains of P. troglodytes and H. sapiens) to no more than five (between the V gamma chains of P. cynocephalus and the A gamma chains of H. sapiens).     

Concordance of a point mutation 5' to the G gamma globin gene with G gamma beta +. Hereditary persistence of fetal hemoglobin in the black population

Hereditary persistence of fetal hemoglobin (HPFH) is a genetically heterogeneous and clinically benign condition characterized by persistent expression of fetal hemoglobin (Hb F) into adulthood. In the G gamma beta + type, no major deletions in the globin gene cluster occur; adult heterozygotes produce approximately 20% Hb F, which results from overproduction of G gamma chains, with no apparent increase in production from the adjacent A gamma gene. We have recently described a point mutation 202 base pairs 5' to the cap site of the G gamma gene in an individual with G gamma beta + HPFH. This mutation abolishes a normal ApaI restriction endonuclease site, and thus can be detected by blotting of genomic DNA. We present here further data on the ApaI mutation: (1) It occurs in six of seven families with G gamma beta + HPFH. (2) In three families, detailed haplotype analysis using 11 polymorphic restriction sites in the beta globin cluster has been done. The two that carry the missing ApaI site are identical but the third, which has a normal ApaI pattern, differs from the other two in at least two sites, one of which is a new polymorphic Nco I site between the delta and beta globin genes. This suggests the possibility of a different HPFH mutation in the third family. (3) The haplotype of the G gamma beta + HPFH chromosome carrying the ApaI mutation is different from that of 108 beta A chromosomes of black individuals that have been tested. (4) The G gamma ApaI site is normal in 61 beta A and 109 beta S alleles from non-HPFH black individuals, including 22 who share the same haplotype for the intragenic G gamma, A gamma HindIII polymorphisms. These data add support to the possibility that the -202 mutation is actually causative of the G gamma beta + HPFH phenotype.     

Haplotypes and levels of fetal hemoglobin and G gamma to A gamma ratios in Mediterranean patients with thalassemia minor and major

This study concerned the gamma chain composition of Hb F and the haplotypes of 44 patients with beta-thalassemia major or intermedia and many of their relatives. Seventeen patients came from Northern (Turkish) Cyprus, 12 from the Istanbul area, and 15 from Macedonia and Bulgaria. Analysis of the A gamma T-G gamma-A gamma I ratio was made by HPLC, while haplotyping involved seven restriction sites. Specific haplotypes were present in certain populations; haplotype I [1] is the dominant type among North Cypriot thalassemia patients. Numerous types were seen in the patients from the Balkan countries. A direct relationship between the A gamma to G gamma ratios and the haplotypes, which exists among black beta-thalassemia heterozygotes [3], was also observed among these Mediterranean patients, although such analyses were considerably complicated by extensive blood transfusion therapy. Haplotypes without the Hinc II restriction site within the psi beta gene were associated with lower G gamma values than those that had this polymorphic site. The A gamma T chain was observed in a small number of beta-thalassemia homozygotes and heterozygotes. Three thalassemia chromosomes with slightly different haplotypes and one normal chromosome with a related haplotype were associated with the gamma 75 Ile----Thr substitution. A few patients with a thalassemia intermedia were heterozygotes for beta-thalassemia with either haplotypes V or VII [1] while the "nonthalassemic" chromosome had a haplotype I, which is the most common "beta-thalassemic" haplotype among the Mediterranean population(s). Detailed analyses of this chromosome have not been completed.     

Granulocyte-macrophage colony-stimulating factor reactivates fetal hemoglobin synthesis in erythroblast clones from normal adults

Reactivation of fetal hemoglobin (HbF, alpha 2 gamma 2) synthesis was previously reported in normal human adult erythroblast colonies ("bursts") generated by erythroid progenitors (BFU-E) in fetal calf serum-supplemented (FCS+) semisolid cultures stimulated with erythropoietin (Ep). Our studies focused on the reactivation of HbF synthesis in normal adult erythroid bursts generated by peripheral blood mononuclear cells (PBMCs) seeded in FCS+ methylcellulose culture. Reactivation is almost totally suppressed when (a) PBMCs are grown in optimized FCS- culture, or (b) PBMCs are first stringently depleted of monocytes and then plated in FCS+ medium (ie, BFU-E growth in FCS+ Mo- culture). In both experimental conditions, the proliferation of lymphocytes and macrophages interspersed among colonies is drastically reduced, and the cloning efficiency of granulocyte-macrophage (GM) progenitors is sharply diminished. In either case, addition of biosynthetic GM colony-stimulating factor (GM-CSF) induces a dose-related increase of HbF synthesis up to the level in FCS+ culture, with even more elevated values on delayed addition of Ep. A dose-related increase was also observed in erythroblast clones generated by highly purified BFU-E. These results suggest that reactivation of HbF synthesis in normal adults is at least in part mediated by GM-CSF. Furthermore, they imply intriguing hypotheses on the mechanism(s) of perinatal Hb switching. Finally, they raise the possibility of reactivation of HbF synthesis in beta-thalassemia and sickle cell anemia by GM-CSF therapy.     

The homozygous state of G to A--117A gamma hereditary persistence of fetal hemoglobin

During a study of Sardinian families with hereditary persistence of fetal hemoglobin (HPFH), two unrelated subjects with unusually elevated Hb F levels were identified. By selective amplification of the A gamma gene promoter and hybridization to synthetic oligonucleotides, we demonstrate that these subjects are homozygous for the -117A gamma G---- A substitution that is responsible for a form of nondeletional HPFH. The hemoglobin synthetic pattern of these patients is discussed.     

Characterization of two types of fetal hemoglobin: alpha 2G gamma 2 and alpha 2A gamma 2

The effect of differences in G gamma and A gamma fractions of fetal hemoglobin (HbF) on the kinetics of polymerization of HbS-HbF mixtures was studied. We also examined their effect on oxygen affinity, surface hydrophobicity, mechanical stability, and solubility of HbF. Differences in G gamma:A gamma ratio did not affect the polymerization of mixtures of HbF and HbS, suggesting that the inhibitory effect of HbF on the polymerization of HbS is independent of the G gamma:A gamma ratio of HbF and is totally dependent on the fraction of HbF in the mixture. The oxygen equilibrium curve of HbF was not affected by differences in the ratios of G gamma and A gamma in HbF. In contrast, surface hydrophobicity, mechanical stability, and solubility of HbF were affected by differences in the G gamma:A gamma ratio. The higher the G gamma:A gamma ratio, the smaller the elution volume on a TSK Gel SW hydrophobic column in high phosphate buffer. The mechanical stability of HbF was also dependent on the ratio of G gamma:A gamma; stability was greater at higher fractions of A gamma. Differences in the G gamma:A gamma ratio also affected solubility of HbF: HbF containing the higher fraction of G gamma was the more soluble. These data indicate that although alanine at the 136th position of the gamma chains has a stronger surface hydrophobicity than does glycine, this difference does not affect either the polymerization of HbS or the oxygen affinity of HbF.     

Complete amino acid sequence of gamma chain of fetal hemoglobin of Japanese macaque (Macaca fuscata)

The complete amino acid sequence of the gamma chain of Japanese macaque (Macaca fuscata) was deduced from sequence analysis of its tryptic peptides and a few other peptides. Three amino acid residues are substituted compared to the human gamma chain, 77His (human) to 77Asn (Japanese macaque), 104Lys to 104Arg and 135Thr to 135 Ala. The primary structure of the gamma chain of Japanese macaque is the same as that of rhesus macaque (Macaca mulatta) (2) and yellow baboon (Papio cynocephalus) (3). Difference in the structure of the gamma chain among primate species is smaller than that of beta chain.     

Molecular diagnosis of A gamma hereditary persistence of fetal hemoglobin using polymerase chain reaction and oligonucleotide analysis.

By combining the polymerase chain reaction (PCR) of the gamma globin gene promoters with synthetic oligonucleotide analysis we have diagnosed the -196 C----T and the -117 G----A substitutions in heterozygous carriers of non deletional A gamma HPFH from two unrelated Italian families. The identification of the beta-thalassemic defect in a compound heterozygote for -196 A gamma HPFH/beta thalassemia allows us to discuss the effect of this gamma promoter mutation on the globin chain synthetic pattern, and to make a comparison with the mutation at the -117 position.     

Frequency and quantitative levels of T gamma chain (Hb F Sardinia) in the fetal hemoglobin of newborn and fetuses

The fetal hemoglobin of 137 Dutch newborn has been examined by peptide mapping after digestion with trypsin, in order to detect the T gamma chain (gamma chain bearing a replacement Ile replaced by Thr at position 75) and to assess its quantity relative to the total gamma chains. Forty-two (31%) individuals were carriers of this variant, with an average amount of T gamma chain equal to 18.89% +/- 4.12 (1 SD). Three babies had T gamma levels higher than 30%, outside, therefore, the upper limit of the distribution. If we consider these three children homozygous carriers and assume that the remaining thirty-nine are heterozygotes, the gene frequency of T gamma in this population is 0.16 and the observed frequencies of the three phenotypes (T gamma-/-, T gamma+/- and T gamma +/+) agree closely with the expected ones. The occurrence and levels of T gamma chain are apparently not subject to variations during the last 25 weeks of gestation. In a group of twenty-nine fetuses investigated between the 12th and 20th week gestation, seven were, in fact, positive and showed T gamma percentages (12-26%) within the limits found in newborn. Also, the relative amounts of G gamma and A gamma chains were identical to those calculated in babies born at term. The composition of fetal hemoglobin remains, thus, uniform from the 12th week of gestation to birth. A comparison of the distribution of T gamma and A gamma percentages in T gamma positive newborn supports indirectly the identification of T gamma chain as a polymorphic variant of A gamma chain.     

Expression of G gamma and A gamma globin genes in human adults

Expression of the human fetal G gamma and A gamma globin genes declines shortly after birth, and adults generally have less than 1% fetal hemoglobin or Hb F (alpha 2 gamma 2). However, some adults with hereditary persistence of fetal hemoglobin (HPFH) have elevated expression of either the G gamma or A gamma gene due to a mutation in its upstream promoter. Mutations with strong effects on expression have been found at -175 and -202 of the G gamma gene and at -117, -196, -198 and -202 of the A gamma gene. Mutations at -158 and -161 of G gamma have weaker effects, which are observable primarily as increases in the G gamma:A gamma ratio. Published data are reviewed which suggest that the -158 mutation may lead to observable elevations of Hb F in SS and beta(0)-thal patients and occasionally in normal non-anemic individuals. These data also suggest that additional high Hb F determinants are linked to Benin, Bantu and Asian beta S haplotypes in some instances. A model based on data from SV40 is presented which suggests that specific DNA sequence motifs of the gamma globin gene may bind regulatory proteins. It is proposed that the -158 and -161 mutations have weak effects because they are located on the fringe of regulatory sequence motifs.