In vitro expression of β-thalassaemia gene (IVS1-1G>C) reveals complete inactivation of the normal 5' splice site and alternative aberrant RNA splicing

We previously reported a case of heterozygous β-thalassaemia with IVS1-1G>C substitution in the β-globin gene and a non-detectable level of mutant mRNA in the patient's reticulocytes. The purpose of this study was to determine whether the transcription and RNA splicing and processing of the mutant gene occurred. We analysed the expression of the mRNA encoded by the cloned mutant gene in COS-1 cells by reverse transcription-polymerase chain reaction followed by agarose gel electrophoresis and nucleotide sequencing. The G>C mutation completely inactivated the normal 5' splice site and resulted in the activation of two cryptic 5' splice sites, located 16 and 38 nt upstream of the normal site. The usage of these two cryptic sites accords with the findings of reports on IVS1-1G>A or IVS1-1G>C substitution of exon 1 of the β-globin gene. Additional experiments that involved transfection of equal amounts of both normal and mutant vectors into COS-1 cells indicated the presence of mutant mRNAs. In conclusion, the β-thalassaemia gene (IVS1-1G>C) was expressed in transfected cells, but showed aberrant RNA splicing. Further studies will be required to clarify the molecular mechanism that results in severe reduction in the mutant mRNA level in vivo.