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.

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.