RESUMO
Holocarboxylase synthetase (HLCS) is an enzyme that catalyzes the incorporation of biotin into apo-carboxylases, and its deficiency causes biotin-responsive multiple carboxylase deficiency. The reported sequences of cDNA for human HLCS from liver, lymphocyte, and KG-1 myeloid cell lines differ at their 5' regions. To elucidate variations of the human HLCS mRNA and longer 5' cDNA ends, we performed screening of the human liver cDNA library and rapid amplification of the cDNA ends (RACE). Our results suggest the existence of three types of HLCS mRNA that start at different exons. The first type starts at exon 1, and the second type starts at exon 3, and both are found in various human tissues. The third type, corresponding to the cDNA from the KG-1 cell, starts at exon 2 of the HLCS gene. Various splicing patterns from exons 3-6 were also observed. None of the variations of cDNA found created a new initiation codon. Mutation screening from exons 6-14, therefore, was sufficient to detect amino acid changes in HLCS in patients. Our direct sequencing strategy for screening mutations in the HLCS gene revealed mutations in five Japanese patients and seven non-Japanese patients. Our analyses involving 12 Japanese and 13 non-Japanese patients and studies by others indicate that (1) there is no panethnically prevalent mutation; (2) the Arg508Trp, Gly581Ser, and Val550Met mutations are found in both Japanese and non-Japanese populations; (3) the IVS10+5G-->A mutation is predominant and probably a founder mutation in European patients; (4) the 655-656insA, Leu237Pro, and 780delG mutations are unique in Japanese patients; (5) the spectrum of the mutations in the HLCS gene may vary substantially among different ethnic groups.
Assuntos
Carbono-Nitrogênio Ligases/genética , Mutação , Sequência de Bases , Carbono-Nitrogênio Ligases/deficiência , Linhagem Celular Transformada , Cromossomos Humanos Par 21 , Primers do DNA , DNA Complementar , Etnicidade , Feminino , Humanos , Masculino , RNA Mensageiro/genéticaRESUMO
The glycine cleavage system (GCS) is a mitochondrial multienzyme system consisting of four individual proteins, three specific components (P-, T-, and H-proteins) and one house-keeping enzyme, dihydrolipoamide dehydrogenase. Inherited deficiency of the GCS causes nonketotic hyperglycinemia (NKH), an inborn error of glycine metabolism. NKH is characterized by massive accumulation of glycine in serum and cerebrospinal fluids and severe neuronal dysfunction in neonates. To elucidate the neuropathogenesis of NKH, we cloned cDNAs encoding three specific components of the GCS and studied the gene expression in rat central nervous system. P-, T-, and H-protein cDNAs encoded 1024, 403, and 170 amino acids, respectively. In situ hybridization analysis revealed that P-protein mRNA was expressed mainly in glial-like cells, including Bergmann glias in the cerebellum, while T- and H-protein mRNAs were detected in both glial-like cells and neurons. T- and H-protein mRNAs, but not P-protein mRNA, were expressed in the spinal cord. Primary astrocyte cultures established from cerebral cortex had higher GCS activities than hepatocytes whereas those from spinal cord expressed only H-protein mRNA and had no enzymatic activity. An important role of glycine as inhibitory neurotransmitter has been established in the brainstem and spinal cord and another role of glycine as an excitation modulator of N-methyl-D-aspartate receptor is suggested in the hippocampus, cerebral cortex, olfactory bulbus, and cerebellum. Our results suggest that the GCS plays a major role in the forebrain and cerebellum rather than in the spinal cord, and that N-methyl-D-aspartate receptor may participate in neuropathogenesis of NKH.
Assuntos
Aminoácido Oxirredutases/genética , Encéfalo/enzimologia , Proteínas de Transporte/genética , Glicina/metabolismo , Mitocôndrias/enzimologia , Fatores Etários , Aminoácido Oxirredutases/metabolismo , Sequência de Aminoácidos , Animais , Astrócitos/citologia , Astrócitos/fisiologia , Sequência de Bases , Encéfalo/citologia , Proteínas de Transporte/metabolismo , Células Cultivadas , Clonagem Molecular , DNA Complementar , Expressão Gênica/fisiologia , Proteína H do Complexo Glicina Descarboxilase , Glicina Desidrogenase (Descarboxilante) , Hiperglicinemia não Cetótica/genética , Hiperglicinemia não Cetótica/metabolismo , Hibridização In Situ , Fígado/enzimologia , Dados de Sequência Molecular , RNA Mensageiro/análise , Ratos , Ratos Wistar , Receptores de Glicina/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
Serum phenylalanine concentrations decreased in 4 patients with hyperphenylalaninemia after loading with tetrahydrobiopterin. There were no abnormalities in urinary pteridine excretion or in dihydropteridine reductase activity. However, mutations were detected in the phenylalanine hydroxylase gene, suggesting a novel subtype of phenylalanine hydroxylase deficiency that may respond to treatment with cofactor supplementation.
Assuntos
Antioxidantes/uso terapêutico , Biopterinas/análogos & derivados , Fenilalanina Hidroxilase/deficiência , Fenilcetonúrias/diagnóstico , Fenilcetonúrias/tratamento farmacológico , Administração Oral , Adolescente , Biopterinas/uso terapêutico , Criança , Análise Mutacional de DNA , Di-Hidropteridina Redutase/urina , Monitoramento de Medicamentos , Feminino , Humanos , Masculino , Mutação/genética , Triagem Neonatal , Fenilalanina/sangue , Fenilalanina Hidroxilase/genética , Fenilcetonúrias/genética , Fenilcetonúrias/metabolismoRESUMO
Carnitine palmitoyltransferase II (CPT II) deficiency manifests as two different clinical phenotypes: a muscular form and a hepatic form. We have investigated three nonconsanguineous Japanese patients with CPT II deficiency. Molecular analysis revealed two missense mutations, a glutamate (174)-to-lysine substitution (E174K) and a phenylalanine (383)-to-tyrosine substitution (F383Y) in the CPT II cDNA. Transfection experiments in COS-1 cells demonstrated that the two mutations markedly decreased the catalytic activity of mutant CPT II. Case 1 (hepatic form) was homozygous for the F383Y mutation, whereas case 3 (muscular form) was homozygous for the E174K mutation. Case 2 and her brother, who were compound heterozygotes for E174K and F383Y, exhibited the hepatic phenotype. We also identified a novel polymorphism in the CPT2 gene, a phenylalanine (352)-to-cysteine substitution (F352C), which did not alter CPT II activity in transfected cells. It was present in 21 out of 100 normal alleles in the Japanese population, but absent in Caucasian populations. Genotyping with the F352C polymorphism and the two previously reported polymorphisms, V368I and M647V, allowed normal Japanese alleles to be classified into five haplotypes. In all three families with CPT II deficiency, the E174K mutation resided only on the F1V1M1 allele, whereas the F383Y mutation was observed on the F2V2M1 allele, suggesting a single origin for each mutation.
Assuntos
Carnitina O-Palmitoiltransferase/deficiência , Carnitina O-Palmitoiltransferase/genética , Erros Inatos do Metabolismo Lipídico/genética , Mutação , Adulto , Alelos , Animais , Células COS , Criança , DNA Complementar , Feminino , Haplótipos , Heterozigoto , Humanos , Lactente , Japão , Masculino , Linhagem , Fenótipo , Reação em Cadeia da Polimerase , Polimorfismo GenéticoRESUMO
Mutations in the dihydropteridine reductase (DHPR) gene result in hyperphenylalaninaemia and deficiency of various neurotransmitters in the central nervous system, causing severe neurological symptoms. We studied two Japanese patients with DHPR deficiency and identified a missense and a splicing error mutation, respectively. A homozygous missense mutation (tryptophan36-to-arginine) was detected in patient 1. The mutation abolished DHPR activity according to in vitro expression studies. The DHPR mRNA in patient 2 was markedly decreased. Reverse transcription-polymerase chain reaction of the mRNA generated a cDNA fragment with a 152-bp insertion. The inserted sequence contained a termination codon, which was likely to affect the stability of the mRNA. Analysis of genomic DNA showed that the insertion was derived from putative intron 3 of the DHPR gene, and an intronic A-to-G substitution was present adjacent to the 3'-end of the inserted sequence. The nucleotide change generated a sequence similar to an RNA splice donor site and probably activated an upstream cryptic acceptor site, thus producing an abnormal extra exon.