An in vitro system for expression analysis of mutations of the beta-globingene: validation and application to two mutations in the 5 ' UTR

We describe the setting up of an in vitro expression system for the analysi s of mutations of the beta-globin gene, The system is based on the stable t ransfection of a normal or mutated beta-globin gene into mouse erythroleuka emia (MEL) cells. The expression construct contains an (A)gamma gene as an internal control and both globin genes are under the control of the HS2 ele ment of the beta LCR. The system enables analysis of transcription, RNA processing and transport, as well as mRNA stability. With non-mutant genes, high-level expression of both beta and (A)gamma genes is seen and both mRNAs are stable. The system was validated by comparing the expression of the beta(654) thalassaemia sp licing mutation in MEL cells with its well-characterized expression in vivo . The level of the initial transcript, the proportion of abnormally spliced mRNA and its instability during erythroid cell maturation were all faithfu lly reproduced, The system was used to examine the mechanism by which two m utations in the beta-globin 5' untranslated region (5' UTR) result in beta thalassaemia. Surprisingly, the mechanism appeared to differ in the two cas es, with the C-G substitution at position +33 affecting transcription, wher eas the -T deletion at position +10 resulted in a translational defect, The stably transfected MEL cells, with an internal control and an endogenous e nhancer, appear to be a valid and realistic experimental model, superior to transient expression studies. This system should find wide application in the analysis of the effects and mechanisms of gene inactivation in mutation s affecting the beta-globin as well as other genes.