Identification of three mutant alleles of the gene for mitochondrial acetoacetyl-coenzyme A thiolase. A complete analysis of two generations of a family with 3-ketothiolase deficiency.

Fukao, Toshiyuki; Yamaguchi, Seiji; Orii, Tadao; Schutgens, R. B. H.; Osumi, Takashi; Hashimoto, Takashi

doi:10.1172/jci115608

Cited by 41 publications

(28 citation statements)

References 39 publications

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“…Similar findings were noted in another family (Fukao et al , 1992aWajner et al 1992). Pulse labeling of the parents' fibroblasts revealed that the mutant T2 was inherited from the mother.…”

Section: Discussionsupporting

confidence: 83%

“…Subsequently, we reported data on four other patients and noted that a heterogeneity in defects of T2 biosynthesis at the protein and mRNA expression levels (Nagasawa et al 1989;Fukao et al 1990; Yamaguchi et al 1990). We cloned and sequenced the human T2 cDNA and human T2 gene ; Kano et al 1991), and began studies on 3KTD patients at the gene level (Fukao et al ,1992a. Our findings to date show that the gene mutation in 3KTD is likely heterogeneous.…”

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confidence: 95%

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Further Analysis of Mutant Thiolase Protein in Fibroblasts from a Japanese Boy with 3-Ketothiolase Deficiency.

Yamaguchi

Fukao

Kano

et al. 1992

Tohoku J. Exp. Med.

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We examined the mutant protein of mitochondrial acetoacetyl-CoA thiolase (mutant T2) in fibroblasts from a Japanese boy with 3-ketothiolase deficiency. The molecular size of the mutant T2 protein, determined by pulse labeling and SDS/PAGE, was intermediate between the mature subunit and the precursor of T2. To characterize the mutant T2 protein, pulselabeling and rhodamine 6G inhibition of mitochondrial transport in fibroblasts, cell-free translation experiments, and family studies by thiolase assay, immunoblotting, and pulse-labeling were carried out. The mutant T2 was detectable as early as a 10-min pulse. The probable precursor of the mutant T2 was not detectable in either the rhodamine 6G inhibition or cell-free translation experiments. In the parents, the K+ ion dependency of acetoacetyl-CoA thiolase activity was low and the T2 bands in immunoblots were faint. It would thus appear that the parents are heterozygotes of this disease. In pulse-labeling, only a band for the mutant T2 was detected in the patient and a single band for the normal mature subunit of T2 in the father ; both bands were detected in the mother. These findings suggested that the mutant T2 in the patient was inherited from the mother, and that the expression of another mutant allele of the father may be either abolished or scanty.3-ktothiolase deficiency ; inherited metabolic disorder ; organic acidemia ; mitochondrial acetoacetyl-CoA thiolase ; immunochemical study 3-Ketothiolase deficiency (3KTD) is an inherited metabolic disorder of organic acids, showing autosomal recessive traits. It is characterized by hyperexcretion of 2-methyl-3-hydroxybutyrate, 2-methylacetoacetate and tiglylglycine in

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“…Similar findings were noted in another family (Fukao et al , 1992aWajner et al 1992). Pulse labeling of the parents' fibroblasts revealed that the mutant T2 was inherited from the mother.…”

Section: Discussionsupporting

confidence: 83%

mentioning

confidence: 95%

Further Analysis of Mutant Thiolase Protein in Fibroblasts from a Japanese Boy with 3-Ketothiolase Deficiency.

Yamaguchi

Fukao

Kano

et al. 1992

Tohoku J. Exp. Med.

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“…C.52-53insC causes a reading frame shift and produces a premature termination. IVS8ϩ1gϾt causes exon 8 skipping and was proved to produce no T2 protein in the previous study (9). To confirm the other mutations as being causative mutations, we performed a transient expression analysis of mutant cDNAs.…”

Section: Resultsmentioning

confidence: 97%

“…Genomic DNA was prepared using Sepa gene kits (Sanko Junyaku, Tokyo, Japan) from fibroblasts. The oligonucleotide primers for cDNA synthesis and PCR amplification of T2 cDNA were described previously (9). The PCR fragments were extracted, subcloned, and sequenced as described (9).…”

Section: Patientsmentioning

confidence: 99%

Mitochondrial Acetoacetyl-CoA Thiolase (T2) Deficiency: T2-Deficient Patients with “Mild” Mutation(s) Were Previously Misinterpreted as Normal by the Coupled Assay with Tiglyl-CoA

et al. 2004

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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...

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“…We previously identified several mutations, which resulted in exon 8 skipping in the T2 gene. Among them, IVS8+1g>t caused exon 8 skipping in almost all transcripts (Fukao et al 1992) and c.816C>T (Q272X) caused exon 8 skipping in some transcripts (Fukao et al 1994). In both cases, no aberrant splicing variants using any other cryptic splice sites were identified.…”

Section: Discussionmentioning

confidence: 96%

Different Clinical Presentation in Siblings with Mitochondrial Acetoacetyl-CoA Thiolase Deficiency and Identification of Two Novel Mutations

Thümmler

Dupont

Acquaviva

et al. 2010

Tohoku J. Exp. Med.

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Identification of three mutant alleles of the gene for mitochondrial acetoacetyl-coenzyme A thiolase. A complete analysis of two generations of a family with 3-ketothiolase deficiency.

Cited by 41 publications

References 39 publications

Further Analysis of Mutant Thiolase Protein in Fibroblasts from a Japanese Boy with 3-Ketothiolase Deficiency.

Further Analysis of Mutant Thiolase Protein in Fibroblasts from a Japanese Boy with 3-Ketothiolase Deficiency.

Mitochondrial Acetoacetyl-CoA Thiolase (T2) Deficiency: T2-Deficient Patients with “Mild” Mutation(s) Were Previously Misinterpreted as Normal by the Coupled Assay with Tiglyl-CoA

Different Clinical Presentation in Siblings with Mitochondrial Acetoacetyl-CoA Thiolase Deficiency and Identification of Two Novel Mutations

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