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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.
Mol Genet Genomic Med 2017; 5(2):177-184MG

Abstract

BACKGROUND

β-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.

CONCLUSION

We 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.

Authors+Show Affiliations

Department of Pediatrics Graduate School of Medicine Gifu University Gifu Japan.Department of PediatricsGraduate School of MedicineGifu UniversityGifuJapan; Department of Biomedical SciencesCollege of Life and Health SciencesChubu UniversityKasugaiJapan.Department of Pediatrics Graduate School of Medicine Gifu University Gifu Japan.Department of PediatricsGraduate School of MedicineGifu UniversityGifuJapan; Department of PediatricsFaculty of MedicineSohag UniversitySohagEgypt.Division of Clinical Genetics Gifu University Hospital Gifu Japan.Department of Pediatrics Graduate School of Medicine Gifu University Gifu Japan.Department of Pediatrics Graduate School of Medicine Gifu University Gifu Japan.Discipline of Genetics Memorial University of Newfoundland St John's NF Canada.Department of PediatricsGraduate School of MedicineGifu UniversityGifuJapan; Division of Clinical GeneticsGifu University HospitalGifuJapan.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

28361105

Citation

Sasai, Hideo, et al. "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." Molecular Genetics & Genomic Medicine, vol. 5, no. 2, 2017, pp. 177-184.
Sasai H, Aoyama Y, Otsuka H, et al. 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. Mol Genet Genomic Med. 2017;5(2):177-184.
Sasai, H., Aoyama, Y., Otsuka, H., Abdelkreem, E., Nakama, M., Hori, T., ... Fukao, T. (2017). 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. Molecular Genetics & Genomic Medicine, 5(2), pp. 177-184. doi:10.1002/mgg3.275.
Sasai H, et al. 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. Mol Genet Genomic Med. 2017;5(2):177-184. PubMed PMID: 28361105.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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. AU - Sasai,Hideo, AU - Aoyama,Yuka, AU - Otsuka,Hiroki, AU - Abdelkreem,Elsayed, AU - Nakama,Mina, AU - Hori,Tomohiro, AU - Ohnishi,Hidenori, AU - Turner,Lesley, AU - Fukao,Toshiyuki, Y1 - 2017/02/08/ PY - 2016/11/25/received PY - 2017/01/03/revised PY - 2016/12/20/accepted PY - 2017/4/1/entrez PY - 2017/4/1/pubmed PY - 2017/4/1/medline KW - ACAT1 KW - T2 deficiency KW - exon skipping KW - mitochondrial acetoacetyl‐CoA thiolase deficiency KW - polypyrimidine stretch KW - splice acceptor site SP - 177 EP - 184 JF - Molecular genetics & genomic medicine JO - Mol Genet Genomic Med VL - 5 IS - 2 N2 - BACKGROUND: β-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. CONCLUSION: We 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. SN - 2324-9269 UR - https://www.unboundmedicine.com/medline/citation/28361105/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_ L2 - https://doi.org/10.1002/mgg3.275 DB - PRIME DP - Unbound Medicine ER -