title: Exon 10 skipping in ACAT1 caused by a novel c.949G>A mutation located at an exonic splice enhancer site.
abstract: Beta-ketothiolase deficiency, also known as mitochondrial acetoacetyl-CoA thiolase (T2) deficiency, is an autosomal recessive disease caused by mutations in the acetyl‑CoA acetyltransferase 1 (ACAT1) gene. A German T2‑deficient patient that developed a severe ketoacidotic episode at the age of 11 months, was revealed to be a compound heterozygote of a previously reported null mutation, c.472A>G (p.N158D) and a novel mutation, c.949G>A (p.D317N), in ACAT1. The c.949G>A mutation was suspected to cause aberrant splicing as it is located within an exonic splicing enhancer sequence (c. 947CTGACGC) that is a potential binding site for serine/arginine‑rich splicing factor 1. A mutation in this sequence, c.951C>T, results in exon 10 skipping. A minigene construct was synthesized that included exon 9‑truncated intron 9‑exon 10‑truncated intron 10‑exon 11, and the splicing of this minigene revealed that the c.949G>A mutant construct caused exon 10 skipping in a proportion of the transcripts. Furthermore, additional substitution of G for C at the first nucleotide of exon 10 (c.941G>C) abolished the effect of the c.949G>A mutation. Transient expression analysis of the c.949G>A mutant cDNA revealed no residual T2 activity in the mutated D317N enzyme. Therefore, c.949G>A (D317N) is a pathogenic missense mutation, and diminishes the effect of an exonic splicing enhancer and causes exon 10 skipping. The present study demonstrates that a missense mutation, or even a synonymous substitution, may disrupt enzyme function by interference with splicing.
authors.affiliation: Department of Pediatrics, Graduate School of Medicine, Gifu University, Gifu 501‑1194, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Otsuka
authors.affiliation: Department of Pediatrics, Graduate School of Medicine, Gifu University, Gifu 501‑1194, Japan.
authors.forename: Hideo
authors.initials: H
authors.lastname: Sasai
authors.affiliation: Division of Clinical Genetics, Gifu University Hospital, Gifu 501‑1194, Japan.
authors.forename: Mina
authors.initials: M
authors.lastname: Nakama
authors.affiliation: Department of Biomedical Sciences, College of Life and Health Sciences, Education and Training Center of Medical Technology, Chubu University, Kasugai 487‑8501, Japan.
authors.forename: Yuka
authors.initials: Y
authors.lastname: Aoyama
authors.affiliation: Department of Pediatrics, Graduate School of Medicine, Gifu University, Gifu 501‑1194, Japan.
authors.forename: Elsayed
authors.initials: E
authors.lastname: Abdelkreem
authors.affiliation: Department of Pediatrics, Graduate School of Medicine, Gifu University, Gifu 501‑1194, Japan.
authors.forename: Hidenori
authors.initials: H
authors.lastname: Ohnishi
authors.affiliation: Department of Pediatrics, Medical University of Vienna, Vienna A‑1090, Austria.
authors.forename: Vassiliki
authors.initials: V
authors.lastname: Konstantopoulou
authors.affiliation: Bioanalytics and Biochemistry, Department of Natural Sciences, University of Applied Sciences, D‑53359 Rheinbach, Germany.
authors.forename: Jörn Oliver
authors.initials: JO
authors.lastname: Sass
authors.affiliation: Department of Pediatrics, Graduate School of Medicine, Gifu University, Gifu 501‑1194, Japan.
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Fukao
mesh_terms: D000101:Acetyl-CoA C-Acetyltransferase / Q000235:genetics*; D000102:Acetyl-CoA C-Acyltransferase / Q000172:deficiency / Q000235:genetics; D017398:Alternative Splicing; D000592:Amino Acid Metabolism, Inborn Errors / Q000175:diagnosis / Q000235:genetics; D001483:Base Sequence; D004742:Enhancer Elements, Genetic; D004789:Enzyme Activation; D005091:Exons; D005260:Female; D015870:Gene Expression; D006801:Humans; D007223:Infant; D009154:Mutation; D020125:Mutation, Missense; D022821:RNA Splice Sites
chemical_list: D022821:RNA Splice Sites; D000102:Acetyl-CoA C-Acyltransferase; C506639:ACAT1 protein, human; D000101:Acetyl-CoA C-Acetyltransferase