title: 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.
abstract: Most mutation-related aberrant splicing occurs in the conserved splice-acceptor and -donor sites and some exonic mutations also affect splicing. We identified and characterized a point mutation (380C>T) in a Spanish patient (GK25) with mitochondrial acetoacetyl-CoA thiolase (T2) deficiency. GK25 is a homozygote of 380C>T, which activates a cryptic splice-acceptor site 5 bases downstream from 380C>T within exon 5, causing aberrant splicing in 94% of transcripts. The aberrant splicing results in a 17-amino acids deletion, including the active-site 126Cys. The 380C>T mutation also results in A127V mutation in 6% of transcripts. Transient expression analysis showed that the A127V mutation did not retain T2 activity, indicating that 380C>T was a null mutation. Although this cryptic splice site has a higher Shapiro and Senapathy's score (86) in even a normal sequence than the authentic splice-acceptor site of intron 4 (78), it is not used in normal controls. While the 380C>T mutation increases the score slightly (90), the cryptic splice site is used in almost all transcripts in GK25 fibroblasts. This is an example in which a point mutation activates a cryptic splice-acceptor site motif that is used preferentially over the upstream authentic splice site.
authors.affiliation: Department of Pediatrics, Gifu University School of Medicine, Gifu, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Nakamura
authors.forename: T
authors.initials: T
authors.lastname: Fukao
authors.forename: C
authors.initials: C
authors.lastname: Perez-Cerda
authors.forename: C
authors.initials: C
authors.lastname: Luque
authors.forename: X Q
authors.initials: XQ
authors.lastname: Song
authors.forename: Y
authors.initials: Y
authors.lastname: Naiki
authors.forename: Y
authors.initials: Y
authors.lastname: Kohno
authors.forename: M
authors.initials: M
authors.lastname: Ugarte
authors.forename: N
authors.initials: N
authors.lastname: Kondo
mesh_terms: D000102:Acetyl-CoA C-Acyltransferase / Q000172:deficiency* / Q000235:genetics*; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry; D004252:DNA Mutational Analysis; D005091:Exons; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D017353:Gene Deletion; D006801:Humans; D015151:Immunoblotting; D007438:Introns; D008661:Metabolism, Inborn Errors / Q000235:genetics; D008928:Mitochondria / Q000201:enzymology*; D008957:Models, Genetic; D009154:Mutation; D017354:Point Mutation; D022821:RNA Splice Sites; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
chemical_list: D022821:RNA Splice Sites; D012333:RNA, Messenger; D000102:Acetyl-CoA C-Acyltransferase; D003545:Cysteine