Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency is an inborn error of metabolism that affects the catabolism of isoleucine and ketone bodies. This disorder is characterized by intermittent ketoacidotic episodes. Recently, we diagnosed T2 deficiency in two patients (GK45 and GK47) by the absence of potassium ion-activated acetoacetyl-CoA thiolase activity, whereas these patients were previously misinterpreted as normal by a coupled assay with tiglyl-CoA as a substrate. This method has been widely used for the enzymatic diagnosis of the T2 deficiency in the United States and Europe. We hypothesized that some residual T2 activity showed normal results in the assay. To prove this hypothesis, we analyzed these two patients together with three typical T2-deficient patients (GK46, GK49, and GK50) at the DNA level. Expression analysis of mutant cDNAs clearly showed that GK45 and GK47 had "mild" mutations (A132G, D339-V340insD) that retained some residual T2 activity, at least one of two mutant alleles, whereas the other three patients had null mutations (c.52-53insC, G152A, H397D, and IVS8ϩ1gϾt) in either allele. These results raise the possibility that T2-deficient patients with mild mutations have been misinterpreted as normal by the coupled assay with tiglyl-CoA. Mitochondrial acetoacetyl-CoA thiolase (T2; EC 2.3.1.9) deficiency is an autosomal recessive disorder that affects the catabolism of isoleucine and ketone bodies (1,2). This disorder, commonly known as the ␤-ketothiolase deficiency (McKusick catalogue no. 203750), is characterized by intermittent ketoacidotic episodes. The urinary organic acid profiles of T2 deficiency are typically characterized by massive excretion of tiglylglycine, 2-methyl-3-hydroxybutyrate, and 2-methylacetoacetate in both ketoacidotic and stable conditions (2,3). We previously carried out mutation analysis on 26 patients and evaluated missense mutations by transient expression analysis of the mutant cDNAs (3). On the basis of the residual T2 activity in the expression analysis of mutant cDNAs, we divided T2-deficient patients into two groups: patients with null mutations in either allele (patients with "severe" mutations) and patients with mutation(s) that retain some residual T2 activity in at least one of two mutant alleles (patients with "mild" mutations). We found no apparent correlation between clinical severity and genotype (3). However, 7 of the 26 did not excrete any detectable amount of tiglylglycine even during ketoacidotic episodes, and, at that time, 6 of the 7 had mild mutations. We recently showed that urinary organic acid and acylcarnitine profiles during nonepisodic conditions were different between patients with mild mutations and those with severe mutations (4). Hence, there is a clear correlation between biochemical phenotype and genotype.We recently diagnosed T2 deficiency in two patients who were previously misinterpreted as normal by the coupled assay with tiglyl-CoA (5). We hypothesized that the presence of residual T2 activity in cells from these patients...

Initiator codon mutations are relatively uncommon and less well characterized compared to other types of mutations. We identified a novel initiator codon mutation (c.2T>C) heterozygously in a Japanese patient (Patient GK30) with mitochondrial acetoacetyl-CoA thiolase (T2) gene deficiency (ACAT1 deficiency); c.149delC was on the other allele. We examined translation efficiencies of nine mutant T2 cDNAs harboring one-base substitutions at the initiator methionine codon using in vivo transient expression analysis. We found that all the mutants produced wild-type T2 polypeptide, to various degrees (wild type (100%) > c.1A>C (66%) > c.2T>C, c.3G>C, c.3G>T (22%) > c3G>A, c.1A>G (11%) > c.2T>A, c.2T>G, c.1A>T (7.4%)). T2 mRNA expression levels in Patient GK08 (a homozygote of c.2T>A) and Patient GK30 fibroblasts, respectively, were almost the same as in control fibroblasts, when examined using semiquantitative PCR. This means that initiator codon mutations did not affect T2 mRNA levels. We propose that all one-base substitutions at the initiator methionine codon in the T2 gene could be mutations, which retain some residual T2 activity.

Bloom syndrome and ataxia-telangiectasia are autosomal recessive human disorders characterized by immunodeficiency, genome instability and predisposition to develop cancer. Recent data reveal that the products of these two genes, BLM and ATM, interact and function together in recognizing abnormal DNA structures. To investigate the function of these two molecules in DNA damage recognition, we generated double knockouts of ATM cells. There was no evidence for exacerbation of either phenotype; however, the more extreme radiosensitivity seen in ATM À/À and the elevated sister chromatid exchange seen in BLM À/À cells were retained in the double mutants. These results suggest that ATM and BLM have largely distinct roles in recognizing different forms of damage in DNA, but are also compatible with partially overlapping functions in recognizing breaks in radiation-damaged DNA.