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

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.

Keywords: ACAT1; T2 deficiency; exon skipping; mitochondrial acetoacetyl‐CoA thiolase deficiency; polypyrimidine stretch; splice acceptor site.

Figure 1

Immunoblot analysis. The amount of fibroblast protein extract applied is indicated in each lane. T2 protein was obtained from GK03 fibroblasts and two healthy control fibroblasts. The first antibody was a mixture of an anti‐T2 antibody and an anti‐SCOT (succinyl‐CoA: 3‐ketoacid CoA transferase) antibody. The positions of the bands for T2 and SCOT are indicated by arrows.

Figure 2

cDNA analysis using CHX‐treated fibroblasts. Five percent polyacrylamide gel electrophoresis of amplified T2 cDNA fragments using RNAs extracted from cycloheximide (CHX)‐treated and ‐untreated fibroblasts from the patient and a control. Bands corresponding to normal transcripts and those with exon 10 skipping are depicted by arrows.

Figure 3

Minigene splicing experiment. (A) Schematic presentation of minigene splicing construct. pCAGGS expression vector was used for this construct. The minigene construct had a T2 gene fragment from c.842 of exon 9 and intron 9 (from +1 to a HindIII site, 476 bp) and intron 10 (from a PstI site to −1, 732 bp), and exon 11 (to c. 1122). In the cases of mutant constructs, the region around exon 10, highlighted in black, was replaced as a cassette. (B) Minigene splicing experiment. Detection of chimeric cDNAs derived from transfected minigenes. First‐strand cDNA was reverse‐transcribed using the glo2 primer. cDNA amplification was performed using Ex9 (Eco RI) and glo3 primers. Those fragments were electrophoresed on 5% polyacrylamide gel. Normal splicing and exon 10 skipping produced 309‐bp and 244‐bp PCR fragments, respectively.