Type and position of promoter elements in retroviral vectors have substantial effects on the expression level of an enhanced green fluorescent protein reporter gene

Purpose: Although gene transfer with retroviral vectors has already been applied to patients as part of clinical protocols, low expression of transgenes in target cells still remains a problem. Therefore, we compared various retroviral vectors using different promoters and backbones for expression of the enhanced green fluorescent protein (EGFP) reporter gene in fibroblasts and CD34⁺ cells. Methods: The N2A retroviral vector was used to test expression from the herpes simplex virus thymidine kinase promoter (vector N2A-TK-EGFP), a human phosphoglycerate kinase promoter (vector N2A-PGK-EGFP), and the SV40 promoter (vector N2A-SV-EGFP). Additional constructs used the spleen focus-forming virus (SFFV) long terminal repeat (LTR) as promoter and expressed EGFP alone (vector SFβ1-EGFP) or EGFP and a downstream (vector SFβ1-EGFP-IRES) or upstream (vector SFβ1-IRES-EGFP) internal ribosomal entry site. Results: For NIH 3T3 cells the fluorescence-activated cell sorting analysis revealed that the most active internal promoter was the SV40 promoter in the vector N2A-SV-EGFP (mean fluorescence intensity, MFI, 66.7 ± 0.4), followed by N2A-PGK-EGFP (26.3 ± 1.8 MFI), and N2A-TK-EGFP (4.8 ± 0.1 MFI). Expression from the SFβ1-EGFP vector (82.6 ± 6.7 MFI) and the SFβ1-EGFP-IRES vector (102.8 ± 6.2 MFI) was higher than from SFβ1-IRES-EGFP vector (15.5 ± 1.8 MFI). In human CD34-positive cells, the EGFP expression from all vectors was considerably lower than in fibroblasts with the SFβ1-EGFP vector still being four- to fivefold more active than the internal promoters tested. Conclusion: The SFFV LTR seems to allow a high expression of transgenes, as long as the transgene is not expressed downstream of an internal ribosomal entry site. Internal promoters may be useful for targeted gene expression in specific cell types, but the reduced level of expression from some internal promoters has to be taken into consideration. Received: 7 January 2000 / Accepted: 1 February 2000     

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.     

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.     

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.     

Efficient and persistent expression of β-glucuronidase gene in CD34+ cells from human umbilical cord blood by retroviral vector

Abstract: We succeeded in efficiently transferring the β-glucuronidase gene in a retroviral vector to human hematopoietic progenitor cells using a centrifugation enhancement protocol. The transduction efficiency in CFU–GM was highly variable (23–100%) with an average of 66.8%. In the case of BFU–E, efficiency was 83% and 76% in 2 separate experiments. In LTCIC (long-term culture-initiating cell), transduction efficiency were 20% and 50% in 2 experiments. The enzymatic activity of β-glucuronidase in transduced cells were increased above the control level up to 5 wk. Considering that correction of the enzyme deficiency in a small number of hematopoietic cells can be therapeutic for the Sly disease mouse, our data provide encouragement that human trials of gene therapy based on transferring β-glucuronidase gene to hematopoietic cells may be efficacious.     

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."     

Sickle cell disorder, β-globin gene cluster haplotypes and α-thalassemia in neonates and adults from Guadeloupe

We have studied haplotype of β^s chromosome and α-globin gene status in 534 patients (255 adults and 279 children of whom 159 neonates) from Guadeloupe with various sickle cell-related conditions, namely SS (n = 298), SC (n = 170), S-β -thal (n = 56), and other rare forms (n = 10). Haplotype data on β^s chromosomes confirm our previous observation that Benin type is the most prevalent (75%) β^s chromosome in Guadeloupe, in disagreement with the historical records. Comparison of the frequency of distribution of various β^s haplotypes between neonates and adults on the one hand and between SS and SC cases on the other shows that the current β^s haplotype distribution in this island is not distorted by haplotype-related differential survival. We also show that the frequency of α-thalassemia (-3.7 kb) in Guadeloupe is one of the highest recorded in this region involved in Atlantic slave trade and also failed to reveal any age-dependent increase in frequency. We conclude that the African component of Guadeloupe is distinct from that of Brazil and Cuba but is close to that of Jamaica. Am. J. Hematol. 55:24-27, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Haematological phenotypes in a family with triplicated α-globin gene, β∘39 and δ+27 thalassaemia mutations

Summary In this paper we report an unusual Sardinian family, in which the heterozygosity for β°₃₉-thalassaemia and for triple α-globin gene complex have been found in two members: the former showing a high HbA₂ mild thalassaemia intermedia syndrome, the latter, her daughter, showing a normal HbA₂ thalassaemia trait. Molecular analysis revealed the daughter to also be a carrier of a δ⁺_27-thalassaemia point mutation, which in trans to the β°₃₉ defect invariably normalizes the HbA₂ levels.     

Molecular characterization of β-thalassemia genes in an Argentine population

This study was designed to identity the β-thalassemia mutations in an Argentine population. Seventy-one pediatric patients and 101 available relatives were studied (85 chromosomes). Diagnosis of β-thalassemia was made by conventional hematological procedures. Molecular studies were carried out by dot-blot and restriction endonuclease analysis on amplified DNA to detect the eight most frequent mutations in the Mediterranean area. We were able to identify 95.3% of the β-thalassemia mutations in the subjects under study. The four common defects (C-39, 47%; IVS-I nt 110, 22.4%; IVS-I nt 1, 9.4%; and IVS-I nt 6, 5.9%) account for 84.7% of the β-thalassemia alleles. The alleles and their distributions showed a close similarity to the spectrum of alleles in Italy. The differences might represent the influence of other immigrations, especially from Spain. We conclude that β-thalassemia in Argentina originated mainly from Italian immigrants. This study will enable us to design an adequate approach to genetic counseling and/or prenatal diagnosis for couples at risk. Am. J. Hematol. 54:179–182, 1997 © 1997 Wiley-Liss, Inc.     

Beta-globin haplotypes from blood spots for follow-up of newborn hemoglobinopathy screening

The inheritance of sickle-cell anemia upon the background of the major β-globin gene cluster haplotypes has been associated with differing risks for major organ failure, and more recently with response to hydroxyurea treatment. Early identification of β-globin haplotypes in individuals with sickle-cell anemia may be a clinically useful prognostic factor for severity of disease expression. This report describes the use of whole-blood spots on filter papers from newborn hemoglobinopathy screening for β-globin gene cluster haplotyping by the polymerase chain reaction. Am. J. Hematol. 54:76–78, 1997. © 1997 Wiley-Liss, Inc.     

Codon 4 ACT→ACA,codon 5 CCT→TCT,and codon 6 GAG→TAG mutations in cis position: A form of thalassemia trait

A female of Uttar Pradesh, of Indian origin, who had a transfusion-dependent child, carried codon 4 ACT→ACA, codon 5 CCT→TCT, and codon 6 GAG→TAG mutations at the cis position. The mutation was detected through sequencing of the amplified β-globin gene. Heterozygosity is expressed as a thalassemia trait with moderate anemia, low MCV (57 fl), raised HbA₂ (6.7%), and normal fetal hemoglobin (1.4%). Am. J. Hematol. 56:187–188, 1997. © 1997 Wiley-Liss, Inc.     

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.     

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.     

An Individual with Hb-Lepore-Baltimore- δβ-Thalassaemia in a Yugoslavian Family

The clinical, haematological and biochemical findings in a person with δβ-thalassaemia and Hb-Lepore are described. The patient was a 24-year-old student who suffered from anaemia of intermediate seventy with late onset of the clinical manifestations, had minor bone and facial deformities, but had no necessity for regular transfusions. Haemoglobins A and A₂ were absent in this individual, and the Hb-Lepore has been identified as Lepore-Baltimore. Heterogeneity of γ chain of the Hb-F follows the expected pattern. The study provides further evidence that neither β nor δ chains are synthesized in cis to δβ-thalassaemia or Hb-Lepore.