Abstract
While point mutations affecting the promoter region of beta-globin gene are widely described, there are no well characterised reports of any point mutations currently found in the promoter of the alpha 2-globin (HBA2) gene. We present clinical and experimental data for three novel HBA2 gene core and proximal promoter mutations. Using an in vitro system designed to assess the impact of point mutations, the three novel [HBA2:c.-59C>T], [HBA2:c.-81C>A] and [HBA2:c.91G>A] promoter mutations identified in three unrelated patients were analysed for HBA2 gene transcriptional and translational activities. Following the generation and transfection of expression vectors carrying each mutation, the HBA2 transcription activity of the promoters from each mutant was analysed with quantitative real time-PCR (qReTi-PCR) technique. Immunofluorochemistry (IFC) was used to analyse HBA2 protein synthesis. The analyses showed that [HBA2:c.-59C>T and [HBA2:c.-91G>A] mutant constructs caused significant reduction in the HBA2 transcription levels by 53.7% (p=0.0008) and 36.2% (p = 0.004), respectively, resulting in markedly lower HBA2 protein labelling when compared to the wild type as shown with subsequent IFC analysis. Conversely, the [HBA2:c.-81C>A] construct showed no significant changes in either transcription (p = 0.089) or in protein labelling when compared to the wild type. The equal pAmp transcription levels found in each group confirmed that the observed labelling differences were not due to varying transfection efficiencies. This study emphasises the importance of in vitro studies to establish the impact of base substitutions on the level of gene expression, and the value of these studies in clinicopathological correlation so that appropriate advice can be given in genetic counselling.