Complementary DNAs encoding the precursor of human hepatic mitochondrial acetoacetyl-CoA thiolase (T2) (EC 2.3.1.9) were cloned and sequenced. The cDNA inserts in these clones were 1,518 bases in length when overlapped, and encoded the 427-amino acid precursor of this enzyme (45,199 mol wt). This amino acid sequence included a 33-residue leader peptide moiety and a 394-amino acid subunit of the mature enzyme (41,385 mol wt). The T2 gene expression in fibroblasts from four patients with 3-ketothiolase deficiency was analyzed by Northern blotting. The T2 mRNA in all four cell lines had the same 1.7 kb as that of the control. However, the amounts of T2 mRNA differed: the content was reduced in two cell lines (cases 1 and 3), whereas it was within a normal range in others (cases 2 and 4). Pulse labeling followed by subcellular fractionation revealed that the T2 proteins in the fibroblasts from these patients are present in the mitochondria. These results suggest that different mechanisms are involved in the enzyme defects in the four patients. (J. Clin.

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We describe mutations identified in stored skin fibroblast cell lines from two original probands (JB and JM), first reported with 2-methylacetoacetic aciduria, and shown later to have a deficiency of the K(+)-activated enzyme, mitochondrial acetoacetyl-coenzyme A thiolase (T2). JB is homozygous for a 4-base insertion (GCAG) which is derived mutation. The primary mutation is an AG/gt to AG/gc transition at the 5'-splice-junction site in intron 11. An alternative splice site 4 bp downstream (Ggcag/gt) is used which causes a frame shift and replaces 39 C-terminal residues by 70 nonfunctional residues. JM is homozygous for a mutation in the translation-initiation codon (ATG to AAG). By expression analyses the JB mutation (IVS11nt2) causes an unstable T2 polypeptide and the JM mutation (M1K) severely impairs T2 mRNA translation. The JB allele associates with Dutch ancestry (no consanguinity) and the JM allele with Chilean ancestry (distant consanguinity).

A plasmid clone of an 8.2-kb EcoRl genomic fragment of the gene for human mitochondrial acetoacetyl-CoA thiolase (ACAT; E.C.2.3.1.9), containing exon 9 to exon 12, was utilized in a fluorescence in situ hybridization study to assign the ACAT locus to chromosome 11q22.3→q23.1.