Molecular studies on beta-ketothiolase deficiency

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3-Ketothiolase deficiency is an inherited metabolic disorder of organic acids and shows an autosomal recessive inheritance. Many patients present severe ketoacidosis reccurrently. It is caused by a defect in biosynthesis of mitochondrial acetoacetyl-CoA thiolase (T2) among four thiolases identified in mammals known up to now. For the study of the disease at the protein and gene levels, we analyzed fibroblasts from 12 patients with the disease, using immunochemical procedures, Northern blotting, or PCR method, and cloned the human T2 cDNA and T2 gene. The results were as follows :(1) In the pulse-chase experiments, the pattern of defects of T2 biosynthesis observed in 12 patients appeared to be separated into at least six groups in respect to the molecular size, the amount, or the stability of the cross reactive protein to T2. (2) It was confirmed that enzyme assay and immunoblotting of T2 can be performed using peripheral lymphcytes and rectal mucosa that are obtainable less-invasively. (3) We cloned human T2cDNA and T2 gene. Human T2 cDNA was found to have a 1281-base open reading frame encoding 427 amnio acids. Human T2 gene was found to span approximately 27 kilobases, including twelve exons. (4) Heterogeneity in mRNA expression was also revealed by Northern blot analysis using T2 cDNA as a probe. (5) Molecular analysis of a patient using a PCR method revealed that he is a compound heterozygote, consisting of a single nucleotide replacement causing an amino acid substitution in one allele, and a skip of exon 8 in another allele. The exon 8 skipping was found to be caused by a single nucleotide replacement at the 5' splicing site of intron 8. These studies should contribute elucidation in the molecular basis of this disease.