Single‐nucleotide substitution T to A in the polypyrimidine stretch at the splice acceptor site of intron 9 causes exon 10 skipping in the ACAT1 gene

Abstract

Backgroundβ‐ketothiolase (T2, gene symbol ACAT1) deficiency is an autosomal recessive disorder, affecting isoleucine and ketone body metabolism. We encountered a patient (GK03) with T2 deficiency whose T2 mRNA level was <10% of the control, but in whom a previous routine cDNA analysis had failed to find any mutations. Genomic PCR‐direct sequencing showed homozygosity for c.941‐9T>A in the polypyrimidine stretch at the splice acceptor site of intron 9 of ACAT1. Initially, we regarded this variant as not being disease‐causing by a method of predicting the effect of splicing using in silico tools. However, based on other findings of exon 10 splicing, we eventually hypothesized that this mutation causes exon 10 skipping.Methods cDNA analysis was performed using GK03's fibroblasts treated with/without cycloheximide (CHX), since exon 10 skipping caused a frameshift and nonsense‐mediated mRNA decay (NMD). Minigene splicing experiment was done to confirm aberrant splicing.Results cDNA analysis using fibroblasts cultured with cycloheximide indeed showed the occurrence of exon 10 skipping. A minigene splicing experiment clearly showed that the c.941‐9T>A mutant resulted in transcripts with exon 10 skipping. There are few reports describing that single‐nucleotide substitutions in polypyrimidine stretches of splice acceptor sites cause aberrant splicing.ConclusionWe showed that c.941‐9T>A induces aberrant splicing in the ACAT1 gene. Our ability to predict the effects of mutations on splicing using in silico tools is still limited. cDNA analysis and minigene splicing experiments remain useful alternatives to reveal splice defects.

Clinical and Mutation Analysis of Patients with Best Vitelliform Macular Dystrophy or Autosomal Recessive Bestrophinopathy in Chinese Population

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Mutation update on ACAT1 variants associated with mitochondrial acetoacetyl‐CoA thiolase (T2) deficiency

“…Nevertheless, two of these variants, c.121–13T>A and c.941–9T>A, are located at the polypyrimidine tract of the splice acceptor site. Although in silico tools failed to predict the pathogenic effect of the latter two variants on splicing, minigene splicing experiments recently proved that c.121–13T>A and c.941–9T>A variants induce skipping of exons 3 and 10, respectively, in greater than 90% of transcripts (Aoyama et al, ; Sasai et al, ).…”

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