Validation of a Recombinant DNA Construct (μLCR and Full-Length β-Globin Gene) for Quantification of Human β-Globin Expression: Application to Mutations in the Promoter, Intronic, and 5′- and 3′-Untranslated Regions of the Human β-Globin Gene

β-Thalassemia is characterized by the reduced production of β-globin chains as a result of mutations in the β-globin gene (1). This reduction is predictable when mutations occur in the coding sequence, but not when they occur in the 5′- and 3′-untranslated regions (UTRs), the locus control region (LCR), the promoter, or the introns. Whether such mutations are involved in the reduction of the β-globin chain production or are simple polymorphisms cannot always be inferred from clinical data. Transient transfection studies with a β-globin promoter and an heterologous reporter gene have shown that promoter mutations can decrease transcription (2) and are then associated with the β-thalassemia phenotype, as illustrated by the −30T→A mutation (3). However, such studies have often failed to provide clear-cut data regarding the transcriptional effect of a mutation or a deletion occurring in a noncoding sequence (4), and quantitative data are lacking. To bypass these limitations and to mimic as closely as possible the regulatory mechanisms of β-human globin gene expression in vivo, we created a construct (pBLG), in which the entire human β-globin gene was cloned behind the β-μLCR. Whereas previous assays used constructs bearing HS2 as a single LCR enhancer element (5)(6), we used the entire β-μLCR because it has been shown that the other three HS elements play also a key role in β-globin transcription (7)(8)(9)(10)(11). Nucleotides changes in various untranscribed or untranslated parts of the β-globin gene representing thalassemic mutations or deletions were introduced in the construct. All the mutations assessed in our study were found in members of proband families presenting with β-thalassemia or were created by directed mutagenesis. In addition to the wild type, variant pBLG constructs carrying the following mutations were generated: −101C→T, +20C→T, IVS-I-108T→C, and IVS-I-110G→A mutations (12) …