[Show abstract][Hide abstract] ABSTRACT: 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 IVS8+1g>t) 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.
Pediatric Research 08/2004; 56(1):60-4. · 2.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Site of Immunogenesis of Autoantibodies Against GluRα2 in Patients with Chronic Progressive Epilepsia Partialis Continua of Childhood. *†† YukitoshiTakahashi, * NaomiSakaguchi, ‡ MasahikoWatanabe, ‡ HisashiMori, ‡ MasayoshiMishina, § HideoAiba, ∥ TomokoMiyajima, ¶ YoshiakiSaitou, ** YasushiUchida, † NaokiMatsuo, † SatomiHattori, and * NaomiKondo Department of Pediatrics, Gifu University School of Medicine, Gifu ; Department of Anatomy and Embryology, Hokkaido University Graduate School of Medicine, Hokkaido ; Department of Molecular Neurobiology and Pharmacology, School of Medicine, University of Tokyo, Tokyo ; Shizuoka Children's Hospital, Shizuoka ; Shiga Medical Center for Children, Shiga ; Department of Pediatrics, Tokyo Women's Medical University, Tokyo ; Department of Pediatrics, Nagahama Municipal Hospital, Nagahama ; and Gifu Prefectural Gifu Hospital, Gifu, Japan Purpose: Glutamate receptors (GluRs) in the central nervous system play an important role in the excitatory synapse and have been implicated in neurodegenerative diseases and symptomatic epilepsy. After Rogers et al. reported that autoantibodies against GluR3 were involved in some cases of Rasmussen encephalitis (Science 1994; 265:648–51), studies have focused on other neurologic diseases with a possible causal relation to autoantibodies against GluRs. To reveal the pathophysiologic mechanisms of severe epilepsia partialis continua (Y. Takahashi et al. ActaNeurolScand 1997; 96:345–52), we developed a detection system highly sensitive to autoantibodies against the N-methyl-d-aspartate (NMDA) GluRα2 subunit and found autoantibodies against GluRα2 in patients with chronic progressive epilepsia partialis continua of childhood (Y. Takahashi et al. AnnRepJpnEpilepsyResFound 1999; 11:45–52). Serum autoantibodies against GluRα2 in patients with chronic progressive epilepsia partialis continua of childhood have epitopes on the C-terminal of GluR2 molecules. In this study, we tried to reveal the site of immunogenesis of autoantibodies by comparing the epitopes in serum and cerebrospinal fluid (CSF). Methods: Six patients with immunoglobulin G (IgG) autoantibodies against the NMDA GluRα2 subunit in serum and CSF were examined. The age at onset of their disease ranged from 2 months to 12.5 years for epilepsy, and 7 months to 12.9 years for epilepsia partialis continua. Probable causative factors were aseptic meningitis, encephalopathy, IgA deficiency, influenza infection, and immunization. The patients had intractable epilepsia partialis continua and complex partial seizures. Neuropsychological outcomes were poor in three patients; quadriplegia developed in four patients, and hemiplegia, in one. Four sequences of GluRα2 cDNA (an N-terminal fragment, and three C-terminal fragments) were inserted into bacterial fusion protein vectors (pGEX and pMAL), and the production of those bacterial fusion proteins was induced by isopropyl thiogalactose (IPTG). Three C-terminal peptides existed after the fourth transmembrane region in the order of C1, C2, and C3. Western blots transferred bacterial fusion proteins composed of GluRα2 fragments to nitrocellulose membranes, and the membranes were incubated with the patients' serum or CSF and stained with IgG-type second antibodies coupled to alkaline phosphatase. Results: In five of the six patients, IgG autoantibodies against GluRα2 in CSF had all four epitopes, and in a patient with mild disease, without mental retardation or motor dysfunction, IgG autoantibodies against GluR2 in CSF had all epitopes except C1. In the five patients with all epitopes in CSF, one patient had one serum epitope, two patients had two serum epitopes, and two patients had three serum epitopes. Their sera showed epitope N in two patients, epitope C1 in two patients, epitope C2 in five patients, and epitope C3 in two patients. The patient with mild disease and three epitopes in CSF had only one epitope, C2, in serum. Conclusions: The diversity of epitopes of IgG autoantibodies against GluRα2 was broader in the CSF than in the sera of all six patients. It is, therefore, suggested that the immunogenesis of IgG autoantibodies against GluRα2 occurs mainly in the central nervous system of patients with chronic progressive epilepsia partialis continua of childhood.
[Show abstract][Hide abstract] ABSTRACT: 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.
Human Mutation 07/2003; 21(6):587-92. · 5.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency is an inborn error of metabolism affecting isoleucine and ketone bodies in the catabolic process. Mutation analysis and expression analysis of mutant cDNAs have facilitated the division of T2-deficient patients into two groups: those with null mutations in either allele (group 1) and those with mutation(s) retaining some residual T2 activity in at least one of two mutant alleles (group II). Among 5 Japanese T2-deficient patients, GK01 belonged to group I and the other patients (GK19, GK19B, GK30 and GK31) to group II. As we have suggested previously, the severity of ketoacidotic episodes in the group II patients was similar to that in the group I patient. However, the urinary organic acid and blood spot acylcarnitine profiles under stable conditions differed between the two groups. The group I patient had typical profiles for the T2 deficiency. In contrast, in all four patients in group II, tiglylglycine was not or was only faintly detected and the 2-methyl-3-hydroxybutyrate levels were less than the cutoff value. Their tiglylcarnitine levels were within the normal range and 2-methyl-3-hydroxy-, butyrylcarnitine was detected just around the cutoff value in our newborn screening pilot test. Hence, these analyses under stable conditions are not reliable for diagnosing the T2 deficiency in the group II patients. The T2 deficiency (group II) can be misdiagnosed as normal if these analyses are performed under nonepisodic conditions and possibly during the newborn screening for inborn errors of metabolism.
[Show abstract][Hide abstract] ABSTRACT: In ketone body metabolism, hepatocyte-specific silencing of the succinyl-CoA:3-ketoacid CoA transferase (SCOT) gene appears to be physiologically important to avoid a futile cycle in the liver, whereas the SCOT gene is expressed in extrahepatic tissues. It is not possible to explain hepatocyte-specific silencing by cis-elements in the 2.2-kb 5' flanking region. The molecular basis of this gene silencing is unknown thus far. In the present study, the methylation status of CpG islands around exon 1 in the SCOT gene was analyzed by sodium bisulfite treatment and by sequencing of genomic DNA from the HepG2, Chang liver and HeLa human cell lines, and also from mouse liver, heart and kidney cells. Most CpG dinucleotides in the CpG island of the human SCOT promoter region were not methylated in the DNA of HeLa and Chang cells, while HepG2 DNA was hypomethylated in this CpG island. CpG dinucleotides in the mouse SCOT CpG island were almost completely unmethylated in the liver DNA as well as in the heart and kidney DNA. CpG islands around the promoter region of the SCOT gene were hypomethylated in the DNA from both human HepG2 cells and mouse liver. Hence, methylation status does not contribute to hepatocyte-specific SCOT gene silencing.
Molecular Medicine Reports 3(2):355-9. · 1.17 Impact Factor