1 Department of Clinical Medicine - Research Unit for Molecular Medicine, Department of Clinical Medicine, Health, Aarhus University2 Aarhus BSS Administrative Centre - Efter- og videreuddannelsesområdet, BSS, Aarhus BSS Administrative Centre, Aarhus BSS, Aarhus University3 Institut for Biokemi og Molekylær Biologi4 Neurobiologisk Forskning5 Department of Molecular Biology, Faculty of Science, Aarhus University, Aarhus University6 unknown7 Department of Human Genetics, Faculty of Health Sciences, Aarhus University, Aarhus University8 Department of Clinical Medicine - Research Unit for Molecular Medicine, Department of Clinical Medicine, Health, Aarhus University9 Aarhus BSS Administrative Centre - Efter- og videreuddannelsesområdet, BSS, Aarhus BSS Administrative Centre, Aarhus BSS, Aarhus University
Multiple acyl-CoA dehydrogenation deficiency is a disorder of fatty acid and amino acid oxidation caused by defects of electron transfer flavoprotein (ETF) or its dehydrogenase (ETFDH). A clear relationship between genotype and phenotype makes genotyping of patients important not only diagnostically but also for prognosis and for assessment of treatment. In the present study, we show that a predicted benign ETFDH missense variation (c.158A>G/p.Lys53Arg) in exon 2 causes exon skipping and degradation of ETFDH protein in patient samples. Using splicing reporter minigenes and RNA pull-down of nuclear proteins, we show that the c.158A>G variation increases the strength of a preexisting exonic splicing silencer (ESS) motif UAGGGA. This ESS motif binds splice inhibitory hnRNP A1, hnRNP A2/B1, and hnRNP H proteins. Binding of these inhibitory proteins prevents binding of the positive splicing regulatory SRSF1 and SRSF5 proteins to nearby and overlapping exonic splicing enhancer elements and this causes exon skipping. We further suggest that binding of hnRNP proteins to UAGGGA is increased by triggering synergistic hnRNP H binding to GGG triplets located upstream and downsteam of the UAGGGA motif. A number of disease-causing exonic elements that induce exon skipping in other genes have a similar architecture as the one in ETFDH exon 2.