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Cell Metab ; 14(3): 428-34, 2011 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-21907147


The metazoan mitochondrial translation machinery is unusual in having a single tRNA(Met) that fulfills the dual role of the initiator and elongator tRNA(Met). A portion of the Met-tRNA(Met) pool is formylated by mitochondrial methionyl-tRNA formyltransferase (MTFMT) to generate N-formylmethionine-tRNA(Met) (fMet-tRNA(met)), which is used for translation initiation; however, the requirement of formylation for initiation in human mitochondria is still under debate. Using targeted sequencing of the mtDNA and nuclear exons encoding the mitochondrial proteome (MitoExome), we identified compound heterozygous mutations in MTFMT in two unrelated children presenting with Leigh syndrome and combined OXPHOS deficiency. Patient fibroblasts exhibit severe defects in mitochondrial translation that can be rescued by exogenous expression of MTFMT. Furthermore, patient fibroblasts have dramatically reduced fMet-tRNA(Met) levels and an abnormal formylation profile of mitochondrially translated COX1. Our findings demonstrate that MTFMT is critical for efficient human mitochondrial translation and reveal a human disorder of Met-tRNA(Met) formylation.

Ciclo-Oxigenase 1/metabolismo , DNA Mitocondrial/química , Fibroblastos/metabolismo , Doença de Leigh/genética , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Biossíntese de Proteínas , RNA de Transferência de Metionina/metabolismo , Células Cultivadas , Criança , Ciclo-Oxigenase 1/genética , DNA Mitocondrial/genética , Fibroblastos/patologia , Heterozigoto , Humanos , Hidroximetil e Formil Transferases , Immunoblotting , Doença de Leigh/metabolismo , Doença de Leigh/patologia , Lentivirus , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Mutação , Biossíntese de Proteínas/genética , Análise de Sequência de DNA , Transdução Genética , Vírion
Nat Genet ; 42(10): 851-8, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20818383


Discovering the molecular basis of mitochondrial respiratory chain disease is challenging given the large number of both mitochondrial and nuclear genes that are involved. We report a strategy of focused candidate gene prediction, high-throughput sequencing and experimental validation to uncover the molecular basis of mitochondrial complex I disorders. We created seven pools of DNA from a cohort of 103 cases and 42 healthy controls and then performed deep sequencing of 103 candidate genes to identify 151 rare variants that were predicted to affect protein function. We established genetic diagnoses in 13 of 60 previously unsolved cases using confirmatory experiments, including cDNA complementation to show that mutations in NUBPL and FOXRED1 can cause complex I deficiency. Our study illustrates how large-scale sequencing, coupled with functional prediction and experimental validation, can be used to identify causal mutations in individual cases.

Complexo I de Transporte de Elétrons/genética , Estudos de Associação Genética , Doenças Mitocondriais/genética , Proteínas Mitocondriais/genética , Mutação/genética , Western Blotting , Estudos de Casos e Controles , Dosagem de Genes , Humanos , Proteínas Mitocondriais/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA
Nat Genet ; 38(5): 576-82, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16582907


The majority of inherited mitochondrial disorders are due to mutations not in the mitochondrial genome (mtDNA) but rather in the nuclear genes encoding proteins targeted to this organelle. Elucidation of the molecular basis for these disorders is limited because only half of the estimated 1,500 mitochondrial proteins have been identified. To systematically expand this catalog, we experimentally and computationally generated eight genome-scale data sets, each designed to provide clues as to mitochondrial localization: targeting sequence prediction, protein domain enrichment, presence of cis-regulatory motifs, yeast homology, ancestry, tandem-mass spectrometry, coexpression and transcriptional induction during mitochondrial biogenesis. Through an integrated analysis we expand the collection to 1,080 genes, which includes 368 novel predictions with a 10% estimated false prediction rate. By combining this expanded inventory with genetic intervals linked to disease, we have identified candidate genes for eight mitochondrial disorders, leading to the discovery of mutations in MPV17 that result in hepatic mtDNA depletion syndrome. The integrative approach promises to better define the role of mitochondria in both rare and common human diseases.

Genômica , Doenças Mitocondriais/genética , Sequência de Bases , Primers do DNA , Ligação Genética , Humanos , Espectrometria de Massas/métodos