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.     

Haplotype analysis and Aγ gene polymorphism associated with the Brazilian type of hereditary persistence of fetal hemoglobin

We have identified three unrelated individuals and three members of a family with the non-deletion form of ^Aγ–hereditary persistence of fetal hemoglobin (HPFH). Molecular analysis showed that each individual is a heterozygote for a previously described −195 ^Aγ (C→G) mutation. The β-globin gene cluster was studied using the polymerase chain reaction and related techniques. Haplotyping using nine restriction sites identified two closely related chromosomes with the −195 ^Aγ mutation, differing only in a single site 3′ to the β-globin gene. Further analysis of β-globin framework indicated that the HPFH allele segregates with haplotype V, according to Orkin's classification. The second haplotype probably originated by a point mutation or DNA rearrangement of a pre-existing −195^Aγ chromosome. We also determined the sequences from −622 to +55 bp upstream to the ^Aγ gene and part of the ^Aγ IVS-2. We found four polymorphisms associated to the −195^Aγ promoter region. All −195^A γ chromosomes had a G at positions −588 and +25 relative to the ^Aγ gene. One individual was also homozygous for polymorphisms at −398 (G→A), and another at −369 (C→G). Cloning and sequencing of the polymorphic patterns of the 3′ region of ^Aγ IVS-2 showed that the mutated allele is linked to β-globin chromosome B. Some correlations between chromosome characteristics and ^Aγ point mutations were also observed. Am. J. Hematol. 58:49–54, 1998. © 1998 Wiley-Liss, Inc.     

Two Novel Mutations (HBG1: c.-250C>T and HBG2: c.-250C>T) Associated With Hereditary Persistence of Fetal Hemoglobin

Mutations within the promoters of either of the γ-globin genes [^Gγ (HBG1) and ^Aγ (HBG2)] lead to variably increased levels of fetal hemoglobin (Hb) (Hb F, α2γ2) in the syndrome of hereditary persistence of fetal Hb (HPFH). Carriers of such mutations are clinically asymptomatic and the mutations are usually detected as part of routine screening or family studies. We describe two new nondeletional HPFH mutations, both C>T substitutions at position c.-250, one in the HBG1 and the other in the HBG2 globin gene promoters.     

Association of Hb S/Hb lepore and δβ-thalassemia/Hb lepore in Sicilian patients: Review of the presence of Hb lepore in Sicily

Abstract: The hemoglobin (Hb) lepore-Boston is a β-globin structural variant, produced in a reduced amount and formed from the fusion of N-terminus δ-(residues 1–87) and C-terminus β-chains (residues 116–146). This type of fusion protein is quite common in Southern Italy (Campania, Calabria, and Sicily). We report here the hematological and hemoglobin data on 96 unrelated Sicilians with Hb lepore trait. Particularly interesting are the subjects where Hb lepore occurs with Hb S or Sicilian type δβ-thalassemia. In these individuals, striking features are clinical variability and different hematological pictures. These observations underscore the importance of thalassemia screening in these geographic areas, such as Southern Italy, principally Sicily, where the mutations in globin gene clusters are especially prevalent. Moreover, as from the second half of the last century, owing to high migratory flux from Sicily to Northern Europe, North and South America, and Australia, the Hb lepore, as well as other hemoglobin variants, have become prevalent, making the identification of the heterozygotes a problem of general interest.     

β-thalassemia and hemoglobin types in Argentina: Determination of most frequent mutations

In order to know the spectrum of β-thalassemia alleles and other mutations affecting the β-globin gene, we analyzed the hemoglobin abnormalities in 24 patients from the Province of Córdoba in Argentina. Molecular screening of samples was performed by the polymerase chain reaction (PCR), using six sets of oligonucleotides to amplify fragments encompassing the whole β-globin coding region and splice junctions, as well as the promoter and 3′ untranslated regions. The altered fragments were determined by denaturing gradient gel electrophoresis (DGGE), and the corresponding mutations were identified by restriction enzyme analysis or by direct sequencing of PCR products. Using this approach, three different β-thalassemia mutations were detected, codon 39 (C→T), IVS-1-110 (G→A), and IVS-1-1 (G→A), and also the hemoglobin S trait. This is the first report of β-thalassemia mutations described in Argentina. Our results show that these mutations are similar to those found in Spain and Italy, possibly due to the important Mediterranean migratory stream received in our country, and could be important for prenatal diagnosis of these diseases in Córdoba, Argentina. Am. J. Hematol. 54:160–163, 1997 © 1997 Wiley-Liss, Inc.     

Fetal hemoglobin expression in the compound heterozygous state for -117 (G-->A) Agamma HPFH and IVS-1 nt 110 (G-->A) beta+ thalassemia: a case study

The splicing defect at IVS-I-110 is by far (43.15%) the most common beta-thalassaemia mutation in Greece. The - 117 (G-->A) Agamma hereditary persistence of fetal hemoglobin (Greek HPFH) is also the most frequent nondeletional HPFH in Greece. We report a case in which these two defects co-segregates. She is a healthy female where the total Hb is 12.3 g/dl with 51% HbF and normal HbA2. Her Ggamma/Agamma ratio is 35:65 differing from that of 10 simple heterozygotes for the Greek HPFH who have ratio of 8:92. Molecular analysis of the beta-globin genotype revealed the presence of the IVS-I-110 beta+ mutation in trans to the -117 G-->A Greek HPFH. Both mutations are linked to Ia. Her father has Greek HPFH in trans to the -158 C-->T on the Ggamma promoter, which is linked with haplotype IIIalpha. He has 13% HbF with a Ggamma/Agamma ratio 32:68. Her sister is a compound heterozygote for the IVS-I-110 mutation in trans to the - 158 C-->T, with HbF levels of 3% and a Ggamma/Agamma ratio 72:28.     

Genetic interactions in thalassemia intermedia: Analysis of β-Mutations, α-Genotype, γ-Promoters,and β-LCR hypersensitive sites 2 and 4 in Italian patients

In order to verity the genetic factors influencing the clinical expression of β-thalassemla we have studied 292 Kalian patients, 165 with thalassemia intermedia and 127 with thalassemia major. The β-globin gene mutations were defined in all cases. The number of α-globin genes and the integrity of specific control regions of the β-globin cluster—γ promoters and β-Locus Control Region (β-LCR)—were studied in selected cases. Homozygosity for mild mutations (group I) accounts for 24% of the intermedia patients and it is not represented among major patients. Forty-four percent of intermedia patients had combinations of mild/severe (group II) mutations and 32% had homozygosity or double heterozygosity for severe mutations (group III). Seventy-six percent of patients with thalassemia major were classified in group III end 24% in group II. Deletion type —α^3.7 thalassemia, assessed in a part of the cases, was found in 5% of thalassemia major and 19.5% of Intermedia patients in groups II and III. Structural analysis of γ promoters and β-LCR HS2 and HS4 regions, carried out in order to look for alterations associated with Hb F increase, did not reveal new mutations. Only rare polymorphic changes were observed at the HS2 and HS4 level. The ? 158 γ C T change was found with an increased incidence in intermedia patients in groups II and III. A subset of 10 β-thalassemia heterozygotes with mild intermedia phenotype resulted from coinheritance of a triplicated α-locus. We have been unable to find a molecular basis for the benign clinical course in approximately 20% of patients with thalassemia intermedia. Other genetic or acquired factors must be hypothesized which ameliorate the clinical condition.     

De novo initiation codon mutation (ATG→ACG) of the β-globin gene causing β-thalassemia in a swiss family

Investigation of microcytic anemia with normal ferrous status in two members (father and daughter) of a Swiss family originating from Bern revealed high levels of HbA₂ (4%, 7.3%) and HbF (3.2%, 3.1%). Direct sequence analysis of asymmetrically amplified DNA showed the ATGǎG mutation in the initiation codon of the β-globin gene. Heterozygous β-thalassemia was not found in either of the propositus's parents or in any of his brothers and sisters. Extended restriction fragment length polymorphism haplotyping of the β chromosomes led us to the conclusion of a recent spontaneous mutation in the paternal germ cell. The results of routine HLA and blood group testing supported the stated paternity. We also found that the intragenic sequence polymorphisms (frameworks) are not always in linkage disequilibrium with the Bam HI polymorphism downstream from the β-globin gene as previously observed. This is the second family found to carry this initiation codon mutation in the β-globin gene. Unlike the first reported family, of Yugoslavian origin, our patients have high HbF levels and this in the absence of a C→T substitution at-158 site 5′to Gγ. © 1993 Wiley-Liss, Inc.     

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.     

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.     

Partial repression of human γ-globin genes by LCR element HS3 when linked to β-globin genes and LCR element HS2 in MEL cells

Clues for overcoming fetal (γ-) globin gene repression in adult human erythroid cells may come from understanding why repression of isolated γ-globin genes has not previously been achieved in the adult erythroid environment of mouse erythroleukemia cells (MEL). Repression of human γ-globin genes has been demonstrated in MEL cells when transferred as part of the entire β-globin gene cluster packaged in chromatin. Major differences in these approaches are prior packaging into chromatin and the presence of additional sequences, notably from the locus control region (LCR). In this report we focus on the contribution to γ-globin gene repression that multiple elements of the LCR may have. We first show preferential activation of β-globin genes over γ-globin genes in MEL cells when linked to each other and to LCR sequences containing the core elements of DNase I hypersensitive sites 4, 3, and 2. Removal of the HS4 element had no effect, however, removal of the 225 bp HS3 core element resulted in a five-fold increase in γ-globin gene expression. The enhancer 3′ to the Aγ-globin gene also had no apparent effect on γ-globin gene expression. These results provide first evidence of γ-globin gene repression involving the core region of HS3 in the presence of the core region of HS2 and a β-globin gene. A mechanism for repression involving sequestration of the γ-promoter away from the strong enhancer activity of HS2 is proposed. © 1996 Wiley-Liss, Inc.     

The molecular heterogeneity of beta-thalassemia in Greece

β-thalassemia is the most predominant genetic defect in Greece. In this study, we investigated the heterogeneity and the frequency of β-thalassemia mutations among 3796 heterozygotes detected in the course of DNA based diagnoses. The diagnostic strategy included Denaturing Gradient Gel Electrophoresis (DGGE), Allele Specific Oligonucleotide Hybridization (ASO), GAP PCR, Restriction Enzyme (RE) analysis and direct sequencing and led to 100% identification of the underlying molecular lesion. Six out of 33 different β-globin defects identified accounted for more than 91.4% of the total β-thalassemia chromosomes in Greece. The β-globin gene mutations cd29 C→T, IVS-I-2 T→C, IVS-I-5 G→T, cd37 G→A and poly A Kurdish AATAAA→AATAAG are for the first time reported in Greece, whereas cd7 GAG→TAG is a new β⁰-thalassemia mutation detected in an adult man from Albania residing in Greece. Three DNA single nucleotide polymorphisms (IVS-I-85 T→C, IVS-I-91 C→T and IVS-I-108 T→C) were also revealed; among these, IVS-I-85 T→C and IVS-I-91 C→T are new and described for the first time worldwide.     

A new unstable variant of the fetal hemoglobin HBG2 gene: Hb F‐Turritana [Gγ64(E8)Gly→Asp,HBG2:c.194G>A] found in cis to the Hb F‐Sardinia gene [Aγ(E19)Ile→Thr,HBG1:c.227T>C]

A new variant of the fetal hemoglobin (Hb) was observed in a newborn baby subjected to phototherapy due to jaundice, by means of electrophoretic and chromatographic techniques. The variant Hb resulted unstable by the isopropanol stability test. After HBG2 gene sequencing, the G to A transversion at codon 64, position eight of the E helix, was found, which corresponds to the Asp for Gly amino acid substitution. The new variant was called Hb F‐Turritana [^Gγ64(E8)Gly→Asp, HBG2:c.194G>A]. Incoming aspartic acid residue, bulky and negatively charged, may be responsible for alteration of the heme pocket steric configuration and for instability. The new abnormal HBG2 gene was found to be associated in cis with the mutated HBG1 gene, which characterizes the Hb F‐Sardinia [^Aγ (E19)Ile→Thr, HBG1:c.227T>C] variant.     

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.     

Maximal γ-globin expression in the compound heterozygous state for –175 Gγ HPFH and β°39 nonsense thalassaemia: a case study

The –175 (T→C) ^Gγ hereditary persistence of fetal haemoglobin is a very rare promoter mutation occurring in Caucasians as well as in African-Americans. Heterozygotes for this non-deletional HPFH show 20% HbF, mostly of ^Gγ type. We describe here a healthy Sardinian man who coinherited –175 (T→C) ^Gγ HPFH with the β-thalassaemia codon 39 nonsense mutation in trans; he showed 64% HbF, 100% of ^Gγ type. Although the β-globin haplotype pattern (II/II) was indicative of the presence of the ^Aγ^T allele on both chromosomes, the ^Aγ^T expression was undetectable by HPLC even in red cell populations separated by age. The proband was, moreover, homozygous for the –4 bp deletion at position -225 to -222 of ^Aγ promoter which has recently been associated with decreased ^Aγ^T globin expression. These findings suggest that this maximal overexpression of ^Gγ-globin probably reflects intensified stimulation of the mutated ^Gγ promoter in this hitherto undescribed genetic condition.