An in vitro system for expression analysis of mutations of the b-globin gene: validation and application to two mutations in the 5 0 UTR P. J OY H O,J ACKIE S LOANE -S TANLEY,AGLAIA ATHANASSIADOU,* WILLIAM G. WOOD AND S WEE L AY T HEIN MRC Molecular Haematology Unit, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, and *Laboratory of Biology, Department of Basic Sciences I, Medical Faculty, University of Patras, Greece Received 29 April 1999; accepted for publication 30 June 1999 Summary. We describe the setting up of an in vitro expression system for the analysis of mutations of the b-globin gene. The system is based on the stable transfection of a normal or mutated b-globin gene into mouse erythroleukaemia (MEL) cells. The expression construct contains an A g gene as an internal control and both globin genes are under the control of the HS2 element of the b LCR. The system enables analysis of transcription, RNA processing and transport, as well as mRNA stability. With non-mutant genes, high-level expres- sion of both b and A g genes is seen and both mRNAs are stable. The system was validated by comparing the expression of the b 654 thalassaemia splicing 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 faithfully reproduced. The system was used to examine the mechanism by which two mutations in the b-globin 5 0 untranslated region (5 0 UTR) result in b thalassaemia. Surprisingly, the mechanism appeared to differ in the two cases, with the C-G substitution at position 33 affecting transcription, whereas the T deletion at position 10 resulted in a translational defect. The stably transfected MEL cells, with an internal control and an endogenous enhancer, appear to be a valid and realistic experimental model, superior to transient expression studies. This system should ®nd wide application in the analysis of the effects and mechanisms of gene inactivation in mutations affecting the b-globin as well as other genes. Keywords: b-globin gene, 5 0 UTR mutations, in vitro expression. b thalassaemia is characterized by the reduced production of b-globin chains; the majority are caused by mutations of the b-globin gene (Thein, 1998). In the past the effects of these mutations were mainly assessed by clinical phenotype and/or in transient expression studies (Treisman et al, 1983; Cheng et al, 1984; Orkin et al, 1984a, b; Atweh et al, 1987). Mechanisms of down-regulation of gene expression were frequently deduced from the base change or its position rather than directly demonstrated, such that correlations between different mechanisms of gene inactivation and the severity of phenotype were dif®cult to establish. For mutations found only in cis with other b thalassaemia alleles such as the C-T substitution at position 20 in the b-globin 5 0 UTR (found in cis with the C-G mutation in bIVS2 position 745) (Gonzalez-Redondo et al, 1989), it was often not possible to examine their effects in isolation in the natural mutants. Here we report the setting up of an in vitro expression system to examine the effects and mechanisms of action of mutations of the b-globin gene. The system was based on the transfection of a construct bearing a normal or mutated b-globin gene and an A g globin gene which serves as internal control, under the control of the hypersensitive site 2 (HS2) enhancer element from the b locus control region (b LCR). A simple model of gene expression was devised in which transcription, post-transcriptional RNA processing and RNA stability were considered possible `targets' for the mutations. Our aim was to elucidate the predominant mechanism by measuring the quantity and stability of b-globin mRNA. We ®rst examined expression of the normal genes before validating the system with the b 654 thalassaemia splicing mutation that has been well characterized previously in vivo (Ho et al, 1998b). Analysis of two mutations in the 5 0 British Journal of Haematology , 1999, 106, 938±947 938 q 1999 Blackwell Science Ltd Correspondence: Dr P. J. Ho, Royal Prince Alfred Hospital, Missenden Road, Camperdown, Sydney, N.S.W. 2050, Australia. e-mail: j.ho@centenary.usyd.edu.au.