title: Single-nucleotide substitution T to A in the polypyrimidine stretch at the splice acceptor site of intron 9 causes exon 10 skipping in theACAT1gene.
abstract: BACKGROUND
  β-ketothiolase (T2, gene symbolACAT1) 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 ofACAT1. 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.
  
  
  CONCLUSIONS
  We showed that c.941-9T>A induces aberrant splicing in theACAT1gene. 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.
authors.affiliation: Department of Pediatrics Graduate School of Medicine Gifu University Gifu Japan.
authors.forename: Hideo
authors.identifier: 0000-0001-7704-6259
authors.initials: H
authors.lastname: Sasai
authors.affiliation: Department of PediatricsGraduate School of MedicineGifu UniversityGifuJapan; Department of Biomedical SciencesCollege of Life and Health SciencesChubu UniversityKasugaiJapan.
authors.forename: Yuka
authors.initials: Y
authors.lastname: Aoyama
authors.affiliation: Department of Pediatrics Graduate School of Medicine Gifu University Gifu Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Otsuka
authors.affiliation: Department of PediatricsGraduate School of MedicineGifu UniversityGifuJapan; Department of PediatricsFaculty of MedicineSohag UniversitySohagEgypt.
authors.forename: Elsayed
authors.initials: E
authors.lastname: Abdelkreem
authors.affiliation: Division of Clinical Genetics Gifu University Hospital Gifu Japan.
authors.forename: Mina
authors.initials: M
authors.lastname: Nakama
authors.affiliation: Department of Pediatrics Graduate School of Medicine Gifu University Gifu Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Hori
authors.affiliation: Department of Pediatrics Graduate School of Medicine Gifu University Gifu Japan.
authors.forename: Hidenori
authors.initials: H
authors.lastname: Ohnishi
authors.affiliation: Discipline of Genetics Memorial University of Newfoundland St John's NF Canada.
authors.forename: Lesley
authors.initials: L
authors.lastname: Turner
authors.affiliation: Department of PediatricsGraduate School of MedicineGifu UniversityGifuJapan; Division of Clinical GeneticsGifu University HospitalGifuJapan.
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Fukao
keywords: ACAT1; T2 deficiency; exon skipping; mitochondrial acetoacetyl‐CoA thiolase deficiency; polypyrimidine stretch; splice acceptor site