Exon 10 skipping in ACAT1 caused by a novel c.949G>A mutation located at an exonic splice enhancer site

Otsuka,Hiroki; Sasai,Hideo; Nakama,Mina; Aoyama,Yuka; Abdelkreem,Elsayed; Ohnishi,Hidenori; Konstantopoulou,Vassiliki; Sass,Jörn  Oliver; Fukao,Toshiyuki

doi:10.3892/mmr.2016.5819

Exon 10 skipping in ACAT1 caused by a novel c.949G>A mutation located at an exonic splice enhancer site

Authors:
- Hiroki Otsuka
- Hideo Sasai
- Mina Nakama
- Yuka Aoyama
- Elsayed Abdelkreem
- Hidenori Ohnishi
- Vassiliki Konstantopoulou
- Jörn Oliver Sass
- Toshiyuki Fukao
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Affiliations: Department of Pediatrics, Graduate School of Medicine, Gifu University, Gifu 501‑1194, Japan, Division of Clinical Genetics, Gifu University Hospital, Gifu 501‑1194, Japan, Department of Biomedical Sciences, College of Life and Health Sciences, Education and Training Center of Medical Technology, Chubu University, Kasugai 487‑8501, Japan, Department of Pediatrics, Medical University of Vienna, Vienna A‑1090, Austria, Bioanalytics and Biochemistry, Department of Natural Sciences, University of Applied Sciences, D‑53359 Rheinbach, Germany
Published online on: October 10, 2016 https://doi.org/10.3892/mmr.2016.5819
Pages: 4906-4910

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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.

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1	Fukao T, Mitchell G, Sass JO, Hori T, Orii K and Aoyama Y: Ketone body metabolism and its defects. J Inherit Metab Dis. 37:541–551. 2014. View Article : Google Scholar : PubMed/NCBI
2	Daum RS, Lamm PH, Mamer OA and Scriver CR: A ‘new’ disorder of isoleucine catabolism. Lancet. 2:1289–1290. 1971. View Article : Google Scholar : PubMed/NCBI
3	Fukao T, Scriver CR and Kondo N: t2 Collaborative Working Group: The clinical phenotype and outcome of mitochondrial acetoacetyl-CoA thiolase deficiency (beta-ketothiolase or T2 deficiency) in 26 enzymatically proved and mutation-defined patients. Mol Genet Metab. 72:109–114. 2001. View Article : Google Scholar : PubMed/NCBI
4	Sass JO: Inborn errors of ketogenesis and ketone body utilization. J Inherit Metab Dis. 35:23–28. 2012. View Article : Google Scholar : PubMed/NCBI
5	Hori T, Yamaguchi S, Shinkaku H, Horikawa R, Shigematsu Y, Takayanagi M and Fukao T: Inborn errors of ketone body utilization. Pediatr Int. 57:41–48. 2015. View Article : Google Scholar : PubMed/NCBI
6	Abdelkreem E, Otsuka H, Sasai H, Aoyama Y, Hori T, Abd El Aal M, Mahmoud S and Fukao T: Beta-ketothiolase deficiency: Resolving challenges in diagnosis. Journal of Inborn Errors of Metabolism & Screening. 4:2016. View Article : Google Scholar
7	Kano M, Fukao T, Yamaguchi S, Orii T, Osumi T and Hashimoto T: Structure and expression of the human mitochondrial acetoacetyl-CoA thiolase-encoding gene. Gene. 109:285–290. 1991. View Article : Google Scholar : PubMed/NCBI
8	Masuno M, Kano M, Fukao T, Yamaguchi S, Osumi T, Hashimoto T, Takahashi E, Hori T and Orii T: Chromosome mapping of the human mitochondrial acetoacetyl-coenzyme A thiolase gene to 11q22.3-q23.1 by fluorescence in situ hybridization. Cytogenet Cell Genet. 60:121–122. 1992. View Article : Google Scholar : PubMed/NCBI
9	Fukao T, Yamaguchi S, Kano M, Orii T, Fujiki Y, Osumi T and Hashimoto T: Molecular cloning and sequence of the complementary DNA encoding human mitochondrial acetoacetyl-coenzyme A thiolase and study of the variant enzymes in cultured fibroblasts from patients with 3-ketothiolase deficiency. J Clin Invest. 86:2086–2092. 1990. View Article : Google Scholar : PubMed/NCBI
10	Fukao T, Maruyama S, Ohura T, Hasegawa Y, Toyoshima M, Haapalainen AM, Kuwada N, Imamura M, Yuasa I, Wierenga RK, et al: Three Japanese patients with beta-ketothiolase deficiency who share a mutation, c.431A>C (H144P) in ACAT1: Subtle abnormality in urinary organic acid analysis and blood acylcarnitine analysis using tandem mass spectrometry. JIMD Rep. 3:107–115. 2012. View Article : Google Scholar : PubMed/NCBI
11	Fukao T, Yamaguchi S, Orii T, Osumi T and Hashimoto T: Molecular basis of 3-ketothiolase deficiency: Identification of an AG to AC substitution at the splice acceptor site of intron 10 causing exon 11 skipping. Biochim Biophys Acta. 1139:184–188. 1992. View Article : Google Scholar : PubMed/NCBI
12	Fukao T, Yamaguchi S, Orii T, Schutgens RB, Osumi T and Hashimoto T: Identification of three mutant alleles of the gene for mitochondrial acetoacetyl-coenzyme A thiolase. A complete analysis of two generations of a family with 3-ketothiolase deficiency. J Clin Invest. 89:474–479. 1992. View Article : Google Scholar : PubMed/NCBI
13	Fukao T, Song XQ, Yamaguchi S, Kondo N, Orii T, Matthieu JM, Bachmann C and Hashimoto T: Identification of three novel frameshift mutations (83delAT, 754insCT, and 435 + 1G to A) of mitochondrial acetoacetyl-coenzyme A thiolase gene in two Swiss patients with CRM-negative beta-ketothiolase deficiency. Hum Mutat. 9:277–279. 1997. View Article : Google Scholar : PubMed/NCBI
14	Thummler S, Dupont D, Acquaviva C, Fukao T and de Ricaud D: Different clinical presentation in siblings with mitochondrial acetoacetyl-CoA thiolase deficiency and identification of two novel mutations. Tohoku J Exp Med. 220:27–31. 2010. View Article : Google Scholar : PubMed/NCBI
15	Law CY, Lam CW, Ching CK, Yau KC, Ho TW, Lai CK and Mak CM: NMR-based urinalysis for beta-ketothiolase deficiency. Clin Chim Acta. 438:222–225. 2015. View Article : Google Scholar : PubMed/NCBI
16	Fukao T, Yamaguchi S, Wakazono A, Orii T, Hoganson G and Hashimoto T: Identification of a novel exonic mutation at −13 from 5′ splice site causing exon skipping in a girl with mitochondrial acetoacetyl-coenzyme A thiolase deficiency. J Clin Invest. 93:1035–1041. 1994. View Article : Google Scholar : PubMed/NCBI
17	Fukao T, Horikawa R, Naiki Y, Tanaka T, Takayanagi M, Yamaguchi S and Kondo N: A novel mutation (c.951C>T) in an exonic splicing enhancer results in exon 10 skipping in the human mitochondrial acetoacetyl-CoA thiolase gene. Mol Genet Metab. 100:339–344. 2010. View Article : Google Scholar : PubMed/NCBI
18	Fukao T, Nakamura H, Song XQ, Nakamura K, Orii KE, Kohno Y, Kano M, Yamaguchi S, Hashimoto T, Orii T and Kondo N: Characterization of N93S, I312T, and A333P missense mutations in two Japanese families with mitochondrial acetoacetyl-CoA thiolase deficiency. Hum Mutat. 12:245–254. 1998. View Article : Google Scholar : PubMed/NCBI
19	Fukao T, Boneh A, Aoki Y and Kondo N: A novel single-base substitution (c.1124A>G) that activates a 5-base upstream cryptic splice donor site within exon 11 in the human mitochondrial acetoacetyl-CoA thiolase gene. Mol Genet Metab. 94:417–421. 2008. View Article : Google Scholar : PubMed/NCBI
20	Watanabe H, Orii KE, Fukao T, Song XQ, Aoyama T, IJlst L, Ruiter J, Wanders RJ and Kondo N: Molecular basis of very long chain acyl-CoA dehydrogenase deficiency in three Israeli patients: Identification of a complex mutant allele with P65L and K247Q mutations, the former being an exonic mutation causing exon 3 skipping. Hum Mutat. 15:430–438. 2000. View Article : Google Scholar : PubMed/NCBI
21	Niwa H, Yamamura K and Miyazaki J: Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene. 108:193–199. 1991. View Article : Google Scholar : PubMed/NCBI
22	Zhang GX, Fukao T, Rolland MO, Zabot MT, Renom G, Touma E, Kondo M, Matsuo N and Kondo N: Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency: T2-deficient patients with ‘mild’ mutation were previously misinterpreted as normal by the coupled assay with tiglyl-CoA. Pediatr Res. 56:60–64. 2004. View Article : Google Scholar : PubMed/NCBI
23	Goldstrohm AC, Greenleaf AL and Garcia-Blanco MA: Co-transcriptional splicing of pre-messenger RNAs: Considerations for the mechanism of alternative splicing. Gene. 277:31–47. 2001. View Article : Google Scholar : PubMed/NCBI
24	Cooper TA and Mattox W: The regulation of splice-site selection, and its role in human disease. Am J Hum Genet. 61:259–266. 1997. View Article : Google Scholar : PubMed/NCBI
25	Robberson BL, Cote GJ and Berget SM: Exon definition may facilitate splice site selection in RNAs with multiple exons. Mol Cell Biol. 10:84–94. 1990. View Article : Google Scholar : PubMed/NCBI
26	Shapiro MB and Senapathy P: RNA splice junctions of different classes of eukaryotes: Sequence statistics and functional implications in gene expression. Nucleic Acids Res. 15:7155–7174. 1987. View Article : Google Scholar : PubMed/NCBI
27	Lin S and Fu XD: SR proteins and related factors in alternative splicing. Adv Exp Med Biol. 623:107–122. 2007. View Article : Google Scholar : PubMed/NCBI
28	Caceres EF and Hurst LD: The evolution, impact and properties of exonic splice enhancers. Genome Biol. 14:R1432013. View Article : Google Scholar : PubMed/NCBI
29	Cartegni L, Hastings ML, Calarco JA, de Stanchina E and Krainer AR: Determinants of exon 7 splicing in the spinal muscular atrophy genes, SMN1 and SMN2. Am J Hum Genet. 78:63–77. 2006. View Article : Google Scholar : PubMed/NCBI
30	Singh NN, Androphy EJ and Singh RN: An extended inhibitory context causes skipping of exon 7 of SMN2 in spinal muscular atrophy. Biochem Biophys Res Commun. 315:381–388. 2004. View Article : Google Scholar : PubMed/NCBI
31	Nakamura K, Fukao T, Perez-Cerda C, Luque C, Song XQ, Naiki Y, Kohno Y, Ugarte M and Kondo N: A novel single-base substitution (380C>T) that activates a 5-base downstream cryptic splice-acceptor site within exon 5 in almost all transcripts in the human mitochondrial acetoacetyl-CoA thiolase gene. Mol Genet Metab. 72:115–121. 2001. View Article : Google Scholar : PubMed/NCBI