A novel mutation (c.121-13T>A) in the polypyrimidine tract of the splice acceptor site of intron 2 causes exon 3 skipping in Mitochondrial acetoacetyl-CoA thiolase gene

Mitochondrial acetoacetyl-CoA thiolase (T2) (gene symbol: ACAT1) deficiency is an autosomal recessive disorder affecting isoleucine catabolism and ketone body utilization. In this study, mutational analysis of an Indian T2-deficient patient revealed a homozygous mutation (c.121-13T>A) located at the polypyrimidine tract of the splice acceptor site of intron 2, and exon 3 skipping was identified by cDNA analysis using cycloheximide. We made three mutant constructs (c.121-13T>A, T>C, and T>G substitutions) followed by making a wild-type minigene construct that included an ACAT1 segment from exon 2 to 4 for a splicing experiment. The minigene splicing experiment demonstrated that exon 3 skipping was induced not only by c.121-13T>A mutation, but also by the other two substitutions. It was difficult to predict the effect of these mutations on splicing using in silico tools, as predictions of different tools were inconsistent with each other. The minigene splicing experiment remains the most reliable method to unravel splicing abnormalities.