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Abstract: Mitochondrial acetoacetyl-CoA thiolase deficiency is an autosomal recessive disorder, characterized by intermittent ketoacidosis. We developed a multiplex ligation-dependent probe amplification method for mutation detection in the ACAT1 gene, which encodes this enzyme, and validated it using DNAs from two previously reported patients having partial deletion and duplication in this gene. Using this method, we identified a heterozygous deletion including exons 3–4 in a third patient, likely due to Alu-mediated non-equal homologous recombination between Alu sequences. [Copyright &y& Elsevier]

Abstract: Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency is an inherited disorder affecting isoleucine catabolism and ketone body metabolism. A Japanese female developed a severe ketoacidotic attack at the age of 7months. Urinary organic acid analysis showed elevated excretion of 2-methyl-3-hydroxybutyrate but not tiglylglycine. She was diagnosed as having T2 deficiency by enzyme assay using fibroblasts. Mutation analysis revealed a compound heterozygote of c.556G>T(D186Y) and c.951C>T(D317D). Since c.951C>T does not cause amino acid change, we performed cDNA analysis and found that exon 10 skipping had occurred in the c.951C>T allele. A computer search using an ESE finder showed that an exonic splicing enhancer sequence, SF2/ASF, was located in CTGA 951CGC. We hypothesized that the exonic splicing enhancer is necessary for accurate splicing since the first nucleotide of exon 10 is C, which weakens the splice acceptor site of intron 9. We made a mini gene construct including exon 9-truncated intron 9-exon 10-truncated intron 10-exon 11 for a splicing experiment. We also made three mutant constructs which alter the SF2/ASF site (947C>T, 951C>T, 952G>A). An min-gene splicing experiment clearly showed that exon 10 skipping was induced in all three mutant constructs. Moreover, additional substitution of G for C at the first nucleotide of exon 10 resulted in normal splicing in these three mutants. These results confirmed that c.951C>T diminished the effect of the exonic splicing enhancer and caused exon 10 skipping. [Copyright &y& Elsevier]

Abstract: Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency is an inborn error of metabolism affecting isoleucine catabolism and ketone body utilization. This disorder is clinically characterized by intermittent ketoacidotic episodes with no clinical symptoms between episodes. In general, T2 gene mutations are heterogenous. No common mutations have been identified and more than 70 mutations have been identified in 70 patients with T2 deficiency (including unpublished data). We herein identified a common mutation, R208X, in Vietnamese patients. We identified R208X homozygously in six patients and heterozygously in two patients among eight Vietnamese patients. This R208X mutation was also identified heterozygously in two Dutch patients, however, R208X mutant alleles in the Vietnamese have a different haplotype from that in the Dutch, when analyzed using Msp I and Taq I polymorphisms in the T2 gene. The R208X mutant allele was not so frequent in the Vietnamese since we could not find that mutant allele in 400 healthy Vietnamese controls using the Nla III restriction enzyme assay. DNA diagnosis of T2 deficiency may be applicable to the Vietnamese population. [Copyright &y& Elsevier]

Abstract: Most mutations related to aberrant splicing occur in conserved splice acceptor and donor sites. Some exonic mutations also affect splicing. We identified and characterized a point mutation (c.1124A>G) in an Australian patient (GK43) with mitochondrial acetoacetyl-CoA thiolase (T2) deficiency. GK43 is a homozygote of c.1124A>G, which activates a cryptic splice donor site 5 bases upstream from c.1124A>G within exon 11, causing aberrant splicing in most transcripts. The aberrant splicing results in c.1120–1163 (44-base) deletion, causing a frameshift in T2 mRNA. A mini-gene splicing experiment confirmed that the c.1124A>G substitution was responsible for this aberrant splicing. This cryptic splice site has a Shapiro and Senapathy score (70.0) in a normal sequence but if mutated, the score (84.3) becomes higher than the one in the authentic splice donor site of intron 11 (81.4). This is an example in which a point mutation activates a cryptic splice donor site motif that is used preferentially over a downstream authentic splice site. [Copyright &y& Elsevier]

Abstract: Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency is a rare inherited metabolic disorder affecting isoleucine catabolism and ketone body metabolism. So far, more than 39 different mutations have been identified in 60 T2-deficient patients. However, no large deletions have been reported. We herein report the first case of a large T2 gene deletion from intron 1 to intron 4 in a T2-deficient patient (GK41). cDNA analysis revealed that an aberrant cDNA with exons 2–5 skipping was a major transcript, associated with a minor transcript of exons 2–4 skipping with a 94-bp insertion composed of an intron 1 sequence. Genomic analysis indicated an absence of PCR amplification of exons 2–4 and gene deletion was revealed by Southern blot analysis. Cloning and sequencing long range PCR products revealed a 6.4kb deletion. Alu element-mediated unequal homologous recombination between an Alu-Sx in intron 1 and another Alu-Y in intron 4 appears to be responsible for this deletion. [Copyright &y& Elsevier]