The level of Hb F-Sardinia (alpha 2A gamma 2(75)Ile----Thr) in the fetal hemoglobin of Sardinian beta-thalassemic homozygotes determined by isoelectric focusing

A simple thin-layer isoelectric focusing technique was used to separate Hb F-Sardinia, containing the A gamma T-globin chain, from the Hb F containing the G gamma- and the A gamma I-globin chains. The identity of the slow-moving Hb F fraction as Hb F-Sardinia was verified by PAGE. A negative correlation (R2 = 0.747, p less than 0.001) was found between the percent Hb F-Sardinia and percent G gamma-chain in homozygotes for beta-thalassemia. Of 31 Sardinian beta-thalassemic patients studied, 21 were homozygous and eight heterozygous for the A gamma T polymorphism with a gene frequency of 0.823. The mean values of Hb F-Sardinia were 39.1 +/- 5.9% for the homozygotes and 17.1 +/- 3.6% for the heterozygotes. The percentage of Hb F-Sardinia found in beta o-thalassemic newborns was similar to that of corresponding normal newborns who also had the A gamma T polymorphism. No measurable differences in the percent Hb F-Sardinia level were observed among beta o-thal patients who were polytransfused, beta o-thal patients studied before transfusion, and beta o-thal patients exhibiting the intermediate form of the disease who had never been transfused.     

Sequence variations in the 5' hypersensitive site-2 of the locus control region of beta S chromosomes are associated with different levels of fetal globin in hemoglobin S homozygotes.

We have compared the sequence of the 5' hypersensitive site-2 (5'-HS-2) of the locus control region (LCR) from a sickle cell anemia (SS) patient homozygous for haplotype 19 and with low levels of fetal hemoglobin (HbF), with the same sequence from an SS patient homozygous for haplotype 3 and with high levels of HbF. Several nucleotide variations were present in the 5'HS-2 of the haplotype 19 individual. One is the A----G at position -10905 that creates an Sp1 binding site GCCCC (A----G)CCCC. A second is the T----G at position -10924 in a sequence that binds both erythroid and ubiquitous factors and exhibits high homology to the long terminal repeat of the Moloney leukemia viruses and Friend murine leukemia virus. Other differences were in the two AT-rich stretches of DNA, and an A----T substitution at position -10390. Dot-blot analyses of amplified DNA from several SS patients showed that these variations are specific for beta S chromosomes with haplotype 19. We also examined the 5'HS-2 sequence from an SS patient who is homozygous for haplotype 19, but has abnormally high levels of HbF (greater than 20%). We observed a cross-over that has placed sequences similar to the 5'HS-2 of haplotype 3 in juxtaposition to the 5' flanking regions of haplotype 19. Thus, a beta S chromosome with haplotype 19 but having a 5'HS-2 (LCR) characteristic for haplotype 3 is associated with high gamma-chain expression. We postulate that factors produced under conditions of hematopoietic stress, together with genetic determinants on the haplotype 3-like LCR sequences, allow for high level expression of gamma-globin genes.     

Study of the single nucleotide polymorphism (SNP) at the palindromic sequence of hypersensitive site (HS)4 of the human beta-globin locus control region (LCR) in Indian population

LCR, a genetic regulatory element, was examined in beta-thalassemia patients who do not show any mutation in the beta-globin genes. We sequenced LCR-HS2, HS3, and HS4 in samples from 16 such patients from the Indian population and found only one SNP A-G in the inverted repeat in HS4. A significant association was observed between the G allele and occurrence of beta-thalassemia by Fisher's exact test. The AG and GG genotypes showed higher relative risk as compared to the AA genotype. We also observed linkage disequilibrium between the A/G polymorphism and the AT-rich motif of the LCR HS2 region, suggesting that the G allele could be an evolutionarily new mutation in the study population.     

Variation in the level of fetal hemoglobin in (delta beta) (0)-thalassemia heterozygotes with different numbers of alpha-globin genes.

The Sicilian type of (delta beta) (0)-thalassemia characterized by a approximately 13 kb deletion, was present in a Turkish boy who is a homozygote and in his heterozygous parents who are first cousins. The father with approximately 21% Hb F had five alpha-globin genes (alpha alpha/alpha alpha alpha) and the mother with approximately 10% Hb F had an alpha-thal-2 heterozygosity (alpha alpha/-alpha). The difference in Hb F level is explained by a decreased formation of alpha 2 gamma 2 tetramers in the mother with an alpha-chain deficiency while the extra alpha-globin gene in the father will promote Hb F production.     

Treatment of severe beta-thalassemia (patients) with myleran.

We previously reported that myleran, a cell cycle nonspecific drug, can stimulate gamma-globin gene expression in anemic adult rhesus monkeys. This finding prompted us to treat two patients with severe beta-thalassemia with myleran. Both patients received an initial course of therapy, constantly of myleran at a dosage of 0.2 mg/kg/d for 9 days followed by 0.15 mg/kg/d for the next 11 days. One patient received an additional 20-day course of myleran at a dosage of 0.2 mg/kg/d beginning 44 days after completion of the first course. No severe ill effects related to the drug were observed during or after drug administration. After 20 days of myleran treatment, levels of HbF and reticulocytes increased in both patients and level of F cells increased in patient 1. In patient 1, Hb concentration rose from 42g/L (9 days after transfusion) before treatment to a maximum of 65g/L afterward; in patient 2, it rose from 74g/L to a maximum of 106g/L. A value of 15g/L above baseline lasted for about 5 months in both patients. Hypomethylation of bone marrow DNA near the gamma-globin gene was demonstrated in patient 1.     

Molecular characterization of hemoglobin C in Sicily.

Analysis of polymorphisms of the beta-globin gene cluster was performed on 12 families and on one unrelated individual of Sicilian origin who carried hemoglobin C (Hb C). Two different haplotypes were found in association with beta c Sicilian alleles, corresponding to haplotypes I and II previously described in American blacks. In our population, the more frequent one (haplotype I) was linked to the lack of a polymorphic HpaI site 3' to the beta gene (13.0-kb fragment), similarly to haplotype I in blacks, while the less frequent one was linked to a 7.0-kb HpaI fragment attributable to a site that had never been previously described in linkage with beta c alleles. In Italy, these two haplotypes have been found in rare cases in association with beta A alleles. These findings provide new insights into the origin of Hb C present in Sicily, suggesting that (1) the beta c mutation detected in Sicily derived from African black chromosomes and does not represent a new mutation; and (2) Hb C may have originated either by multiple mutational events on separate chromosomes or by mutation in the HpaI site 3' to the beta gene in a pre-existing beta c chromosome.     

Spectrum of beta-thalassemia mutations and their association with allelic sequence polymorphisms at the beta-globin gene cluster in an Eastern Indian population

In this report, the spectrum of beta-thalassemia mutations and genotype-to-phenotype correlations were defined in large number of patients (beta-thalassemia carriers and major) with varying disease severity in an Eastern Indian population mainly from the state of West Bengal. The five most common beta-thalassemia mutations were detected, which included IVS1-5 (G-->C), codon 15 (G-->A), codon 26 (G-->A), codon 30 (G-->C), and codon 41/42 (-TCTT). These accounted for 85% in 80 beta-thalassemic alleles deciphered from 56 patients, including beta-thalassemia major and carriers, and 15% of alleles remained uncharacterized in these patients. Expression of the human beta-globin gene is regulated by an array of cis-acting DNA elements, including five DNase I hypersensitive sites (HSs) in the locus control region (LCR), promoters that incorporate certain silencer elements, and enhancers at 3' of the beta-globin gene. For detailed studies and to understand the molecular basis of beta-thalassemia, we studied two groups of subjects: a group of 12 patients from four families having beta-thalassemia major and carrier phenotype and a control group of 26 healthy individuals. In these two groups, we examined portions of the beta-globin gene locus control region HSs 1, 2, 3, and 4, which included the (CA)(x)(TA)(y) repeat motif, the (AT)(x)N(y)(AT)(z) repeat motif, the inverted repeat sequence TGGGGACCCCA, the promoter region of the (G)gamma-globin gene, an (AT)(x)(T)(y) repeat 5' of the silencer region, and the beta-globin gene and its 3' flanking region. We investigated the allelic sequence polymorphisms in these regions and their association with the beta-thalassemia mutations to know the possible genotype-phenotype relationship in beta-thalassemia patients. An analysis of cis-acting regulatory regions showed varied sequence haplotypes associated with some frequent beta-thalassemia mutations in this Eastern Indian population.     

In vitro synthesis of hemoglobin and hemoglobin chains in the BFUe-derived colonies form person with alpha- or beta-thalassemia

The synthesis of alpha and non-alpha chains of human hemoglobin (Hb) was studied in reticulocytes and in BFUe-derived cell colonies from patients with alpha chain or beta chain deficiencies. The subjects included normal adults (alpha alpha/alpha alpha) with or without a beta chain variant (Hb S, Hb Leslie) or an alpha chain variant (Hb G-Georgia); alpha-thalassemia-2 heterozygotes (alpha 0 alpha/alpha alpha) with an alpha chain variant (G-Georgia or G-Philadelphia); an alpha-thalassemia-1 heterozygote (alpha 0 alpha 0/alpha alpha); alpha-thalassemia-2 homozygotes (alpha 0 alpha 0/alpha 0 alpha) with a beta chain variant (Hb S), an alpha chain variant (G-Philadelphia), a Hb S homozygosity with Hb G-Philadelphia, or a Hb G-Philadelphia homozygosity; and three black beta +-thalassemia homozygotes. Data from reticulocyte in vitro synthesis analysis showed the expected deficiencies. However, similar analyses of the Hb synthesized in cell colonies (even from the black beta-thalassemia homozygotes) gave (nearly) balanced sigma alpha/sigma non-alpha ratios. It is speculated that this balanced synthesis is due to a most effective proteolysis in the immature erythroblast which rapidly removes free alpha or beta chains. The levels of Hb F and Hb A2 were considerably increased in these proerythroblasts; a two- to threefold increase in the synthesis of Hb A2 was observed over that seen in the reticulocytes.     

Concordant fetal hemoglobin response to hydroxyurea in siblings with sickle cell disease

Fetal hemoglobin (HbF) level and the HbF responses to hydroxyurea (HU) vary among patients with sickle cell disease and are, at least in part, genetically regulated. We hypothesized that siblings with sickle cell disease are likely to share the same parental beta-like globin gene clusters with their cis-acting regulatory sequences and therefore, if regulation of this response is linked to the beta-globin gene cluster, might have concordant HbF responses to HU. Accordingly, we studied 26 families (30 sib pairings), 20 with sickle cell anemia (three families had three siblings) and 6 families with HbS-beta-thalassemia (one family had three siblings, and one family consisted of monozygotic twins), to see if siblings with sickle cell disease had discordant or concordant changes in HbF during HU treatment. Intraclass correlation coefficients (r) showed a high, positive correlation between sibs for HbF levels before and during HU treatment and a concordant change in HbF response from baseline to treatment-associated levels. Changes in mean corpuscular volume (MCV) paralleled HbF levels, while the expected correlations between treatment-associated fall in leukocyte count and increase in MCV were also present. Our results provide additional evidence that some elements that regulate HbF expression are linked to the beta-globin gene cluster.     

Two mutations in the locus control region hypersensitivity site-2 (5′HS-2) of haplotype 19 βs chromosomes alter binding of trans-acting factors

There are five major haplotypes associated with sickle cell anemia (SS). Individuals homozygous for haplotypes 3 (Senegal) and 31 (Saudi Arabian) have high fetal hemoglobin (HbF) levels (15 to 30% of total hemoglobin) whereas individuals homozygous for haplotypes 17 (Cameroon), 19 (Benin), and 20 (Bantu) have low HbF levels (1 to 10%). We previously identified several point mutations in the LCR 5′HS-2 that were specific for haplotype 19 β^s chromosomes (compared to the GenBank HUMHBB reference sequence, T→G at position 8580, A→G at position 8598, and A→T at position 9114). We postulated that one or more of these mutations may alter the binding of specific trans-acting factors and ultimately affect the expression of HbF in these sickle cell patients. We performed gel mobility shift assays using ³²P-end-labeled double-stranded 19mers corresponding to each of the LCR 5′HS-2 normal (GenBank) and mutant sequences. Nuclear extracts prepared from HeLa and HEL cells were used in our experiments and neither the normal nor mutant sequence at position 8580 bound trans-acting factors in either nuclear extract. The 8598 mutant increased binding of Sp1; using purified protein and both nuclear extracts. HEL extracts were used to quantify the increase in Sp1 binding to the 8598 mutation and we found an increase in binding of 66 and 47%, respectively, in two shifted bands. The 9114 mutation sharply decreased binding of an unknown trans-acting factor by 74%. This factor was present in both HeLa and HEL nuclear extracts. © 1996 Wiley-Liss, Inc.     

Level and composition of fetal hemoglobin expression in normal newborn babies are not dependent on beta cluster DNA haplotype.

Interindividual variations in the level and composition of fetal hemoglobin observed in 604 cord blood samples from normal white and nonwhite newborns, unlike those in adults, are not dependent on beta gene cluster DNA haplotype. The data suggest that the mechanism(s) involved in the neonatal expression of Hb F is distinct from that at the adult stage.     

Hb S/beta zero-thalassemia due to the approximately 1.4-kb deletion is associated with a relatively mild phenotype.

We report a relatively mild phenotype associated with two siblings who are compound heterozygotes for Hb S and a beta zero-thalassemia mutation due to a approximately 1.4-kb deletion of the 5' region of the beta-globin gene. Each is found to have unusually high levels of Hb A2 and Hb F, accounting for more than 20% of the total hemoglobin. These may interfere with intracellular Hb S polymerization, thus leading to a mild clinical course.     

G gamma-196 C-->T, A gamma-201 C-->T: two novel mutations in the promoter region of the gamma-globin genes associated with nondeletional hereditary persistence of fetal hemoglobin in Greece

The increased level of fetal hemoglobin in nondeletional hereditary persistence of fetal hemoglobin (nd-HPFH) is associated with several single base substitutions in the promoter region of either the ^Gγ- or the ^Aγ-globin genes. In this study, we report two new forms of nd-HPFH found in two unrelated Greek adults with high HbF production (8.6% and 10.2% respectively) and positive for the ^Gγ-158 C→T substitution. Scanning by DGGE analysis and direct sequencing of the γ-globin gene 5′ promoter region revealed the presence of a ^Gγ-196 C→T in the first case and an ^Aγ-201 C→T in the second. These mutations seem to reactivate γ-genes and cause their high expression in the adult period.     

Linkage analysis of the hemoglobin F determinant(s) in an australian hemoglobin lepore (Boston) kindred

Genetic determinants that influence the levels of fetal hemoglobin (Hb F) in a single Australian kindred with heterozygous Hb Lepore (Boston) were sought. There were 22 affected individuals, some of whom had high Hb F and others with Hb F levels within the normal range. Family members were typed for restriction fragment length polymorphisms (RFLPs) associated with the β-globin gene complex and the nearby genetic markers D11S12, INS, HRAS, and PTH. Prior to linkage analysis, a cohort of 54 unrelated Hb Lepore heterozygotes was analyzed to establish the distribution of Hb F levels measured by alkaline denaturation (Hb F_AD). An Hb F level of >2.0% was used as the cutoff point for linkage analysis of the putative hereditary persistence of fetal hemoglobin (HPFH) determinant(s) in this kindred. Positive peak lod scores were obtained for the entire pedigree between the HPFH determinant and Hb Lepore (Z_m+1 = 2.35 at θ = 0.15) and the β-globin cluster (HBBC) (Z_m+1 = 2.38 at θ = 0.20) marker loci, indicating the possibility of an independent HPFH gene at some distance from the β-globin gene cluster. However, most of these lod scores result from the non-Hb Lepore members of the family who, with one exception, do not have high Hb F, and when only those affected with Hb Lepore were analyzed the lod score values at these loci fell to small positive values (<1.0). These data do not support an independent cosegregating HPFH determinant separate from the Hb Lepore locus in this pedigree. The results favor a pleiotropic effect of the Hb Lepore lesion itself influencing Hb F levels by genetic or environmental factors not yet elucidated.     

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.     

Polymorphic pattern of the (AT)x(T)y motif at −530 5′ to the β-globin gene in over 40 patients homozygous for various β-thalassemia mutations

Nucieotlde sequence analysis of the 5′ β-globin gene flanking region has been carried out for numerous homozygous β-thalassemla patients with difterent mutations and of various ethnic backgrounds. Four different rearrangements were found associated with numerous β-thalassemia mutations. The (AT)_x(T)_y repeat motif at -530 showed polymorphic patterns among these patients as follows: All ten IVS-II-1 (G→A) chromosomes and the two with the -87 (C→G) mutation are associated with the (AT)₉(T)₅ rearrangement, while the 30 IVS-I-6 (T→C), the 16 codon 39 (C→T), the slx codon 8 (-AA) chromosomes, and 12 chromosomes with different promoter mutations had the (AT)₇(T)₇ motif. Six chromosomes with the promoter mutation at position ? 29 (A→ G) had the (AT)₆(T)₆ motif, while an (AT)₅(T)₄ motif appears characteristic for two IVS-I-5 (G→A and G→T). No direct association between any of the (AT)_x(T)_y arrangements and an increased γ gene expression [^gγ and fetal hemoglobin (Hb F)] levels could be demonstrated, suggesting that variations In the (AT)_x(T)_y motif are common polymorphisms. © 1994 Wiley-Liss, Inc.     

Dissection of the association status of two polymorphisms in the β-globin gene cluster with variations in F-cell number in non-anemic individuals

Expression of fetal hemoglobin (Hb F) is under polygenic control involving determinants both linked and unlinked to the β-globin gene cluster on chromosome 11. Variations in the DNase I-hypersensitive site 2 of the locus control region (LCR-HS2) and a C → T change at position −158 from the Gγ-gene (detected as an XmnI polymorphism) correlate with the high level of Hb F expression in patients with sickle-cell anemia and β-thalassemia. Interpretation of data under these conditions of anemic stress is difficult because the preferential survival of Hb F-containing erythrocytes (F-cells) may not reflect the true status of Hb F expression. We investigated the relationship between these markers and Hb F expression in terms of F-cell levels in 48 unrelated non-anemic AS heterozygotes from Sicily. The β^S-chromosome of all these individuals was of the Benin haplotype and they differed only by their β^A chromosomes. We demonstrate that F-cell expression is more strongly associated with LCR-HS2 polymorphism than with XmnI polymorphism. The observed association between XmnI polymorphism and Hb F expression is very likely to be due to linkage disequilibrium with LCR-HS2 sequences. Am. J. Hematol. 56:239–243, 1997. © 1997 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.     

Effects of human locus control region elements HS2 and HS3 on human beta-globin gene expression in transgenic mouse

The locus control region (LCR) is the most important cis-element in the regulation of beta-globin gene expression. DNaseI-hypersensitive site (HS) 2 and HS3 are two significant components of beta-LCR. To examine the effect of HS2, HS3, and HS2-HS3 (combination of HS2 and HS3) on the spatial and temporal expression of the human beta-globin gene, we have produced transgenic mice with constructs, in which the gene encoding enhanced green fluorescent protein (EGFP) is driven by beta-globin promoter and under the control of HS2, HS3, and HS2-HS3, respectively. The results showed that HS2 and HS3 each had the same enhancement activity in regulation of beta-globin gene expression in transgenic mice. When HS2 and HS3 were in combination (HS2-HS3), the two cis-elements showed a marked synergy in regulating beta-globin gene spatial and temporal expression as well as its expression level in transgenic mice although the EGFP expression varied largely among different transgenic mouse litters. The results also showed that HS2 was able to confer beta-globin gene expression in embryonic yolk sac, fetal liver, and adult bone marrow, which was not developmentally stage-specific, while HS3 could confer the same beta-globin gene expression in the adult. Thus, HS3 was different from HS2, the former being more important for specific expression of beta-globin gene in the developmental stages and the switch of gamma-->beta-globin genes. Our results indicate that the mechanism of gamma-->beta switch could be best explained by the "divided model."