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The mammalian mitochondrial genome (mtDNA) is multicopy and its copy number (mtCN) varies widely across tissues, in development and in disease. Here, we systematically catalog this variation by assaying mtCN in 52 human tissues across 952 donors (10,499 samples from the Genotype-Tissue Expression project) and 20 murine tissues using qPCR, capturing 50-and 200-fold variation, respectively. We also estimate per cell mtCN across 173 human cell lines from the Cancer Cell Line Encyclopedia using whole-genome sequencing data and observe >50-fold variation. We then leverage the vast amount of genomics data available for these repositories to credential our resource and uncover mtDNA-related biology. Using already existing proteomics data, we show that variation in mtCN can be predicted by variation in TFAM, histone, and mitochondrial ribosome protein abundance. We also integrate mtCN estimates with the CRISPR gene dependency measurements to find that cell lines with high mtCN are resistant to loss of GPX4, a glutathione phospholipid hydroperoxidase. Our resource captures variation in mtCN across mammalian tissues and should be broadly useful to the research community. [ABSTRACT FROM AUTHOR]

Renal oncocytomas are benign tumors characterized by a marked accumulation of mitochondria. We report a combined exome, transcriptome, and metabolome analysis of these tumors. Joint analysis of the nuclear and mitochondrial (mtDNA) genomes reveals loss-of-function mtDNA mutations occurring at high variant allele fractions, consistent with positive selection, in genes encoding complex I as the most frequent genetic events. A subset of these tumors also exhibits chromosome 1 loss and/or cyclin D1 overexpression, suggesting they follow complex I loss. Transcriptome data revealed that many pathways previously reported to be altered in renal oncocytoma were simply differentially expressed in the tumor's cell of origin, the distal nephron, compared with other nephron segments. Using a heuristic approach to account for cell-of-origin bias we uncovered strong expression alterations in the gamma-glutamyl cycle, including glutathione synthesis (increased GCLC) and glutathione degradation. Moreover, the most striking changes in metabolite profiling were elevations in oxidized and reduced glutathione as well as γ-glutamyl-cysteine and cysteinyl-glycine, dipeptide intermediates in glutathione biosynthesis, and recycling, respectively. Biosynthesis of glutathione appears adaptive as blockade of GCLC impairs viability in cells cultured with a complex I inhibitor. Our data suggest that loss-of-function mutations in complex I are a candidate driver event in renal oncocytoma that is followed by frequent loss of chromosome 1, cyclin D1 overexpression, and adaptive up-regulation of glutathione biosynthesis. [ABSTRACT FROM AUTHOR]

Known disease mechanisms in mitochondrial DNA (mtDNA) maintenance disorders alter either the mitochondrial replication machinery (POLG, POLG2 and C10orf2) or the biosynthesis pathways of deoxyribonucleoside 5?-triphosphates for mtDNA synthesis. However, in many of these disorders, the underlying genetic defect has yet to be discovered. Here, we identify homozygous nonsense and missense mutations in the orphan gene C20orf72 in three families with a mitochondrial syndrome characterized by external ophthalmoplegia, emaciation and respiratory failure. Muscle biopsies showed mtDNA depletion and multiple mtDNA deletions. C20orf72, hereafter MGME1 (mitochondrial genome maintenance exonuclease 1), encodes a mitochondrial RecB-type exonuclease belonging to the PD-(D/E)XK nuclease superfamily. We show that MGME1 cleaves single-stranded DNA and processes DNA flap substrates. Fibroblasts from affected individuals do not repopulate after chemically induced mtDNA depletion. They also accumulate intermediates of stalled replication and show increased levels of 7S DNA, as do MGME1-depleted cells. Thus, we show that MGME1-mediated mtDNA processing is essential for mitochondrial genome maintenance. [ABSTRACT FROM AUTHOR]

The molecular diagnosis of mitochondrial disorders still remains elusive in a large proportion of patients, but advances in next generation sequencing are significantly improving our chances to detect mutations even in sporadic patients. Syndromes associated with mitochondrial DNA multiple deletions are caused by different molecular defects resulting in a wide spectrum of predominantly adult-onset clinical presentations, ranging from progressive external ophthalmoplegia to multi-systemic disorders of variable severity. The mutations underlying these conditions remain undisclosed in half of the affected subjects. We applied next-generation sequencing of known mitochondrial targets (MitoExome) to probands presenting with adult-onset mitochondrial myopathy and harbouring mitochondrial DNA multiple deletions in skeletal muscle. We identified autosomal recessive mutations in the DGUOK gene (encoding mitochondrial deoxyguanosine kinase), which has previously been associated with an infantile hepatocerebral form of mitochondrial DNA depletion. Mutations in DGUOK occurred in five independent subjects, representing 5.6% of our cohort of patients with mitochondrial DNA multiple deletions, and impaired both muscle DGUOK activity and protein stability. Clinical presentations were variable, including mitochondrial myopathy with or without progressive external ophthalmoplegia, recurrent rhabdomyolysis in a young female who had received a liver transplant at 9 months of age and adult-onset lower motor neuron syndrome with mild cognitive impairment. These findings reinforce the concept that mutations in genes involved in deoxyribonucleotide metabolism can cause diverse clinical phenotypes and suggest that DGUOK should be screened in patients harbouring mitochondrial DNA deletions in skeletal muscle. [ABSTRACT FROM PUBLISHER]

Summary: The metazoan mitochondrial translation machinery is unusual in having a single tRNAMet that fulfills the dual role of the initiator and elongator tRNAMet. A portion of the Met-tRNAMet pool is formylated by mitochondrial methionyl-tRNA formyltransferase (MTFMT) to generate N-formylmethionine-tRNAMet (fMet-tRNAmet), 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-tRNAMet 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-tRNAMet formylation. [Copyright &y& Elsevier]

Disorders of oxidative phosphorylation (OXPHOS) have a birth prevalence of ∼1/5,000 and are the most common inborn errors of metabolism. The most common OXPHOS disorder is complex I deficiency. Patients with complex I deficiency present with variable symptoms, such as muscle weakness, cardiomyopathy, developmental delay or regression, blindness, seizures, failure to thrive, liver dysfunction or ataxia. Molecular diagnosis of patients with complex I deficiency is a challenging task due to the clinical heterogeneity of patients and the large number of candidate disease genes, both nuclear-encoded and mitochondrial DNA (mtDNA)-encoded. In this review, we have thoroughly surveyed the literature to identify 149 patients described with both isolated complex I deficiency and pathogenic mutations within nuclear genes. In total, 115 different pathogenic mutations have been reported in 22 different nuclear genes encoding complex I subunits or assembly factors, highlighting the allelic and locus heterogeneity of this disorder. Missense mutations predominate in genes encoding core subunits and some assembly factors while null-type mutations are common in the genes encoding supernumerary subunits and other assembly factors. Despite developments in molecular technology, many patients do not receive molecular diagnosis and no gene has yet been identified that accounts for more than 5% of cases, suggesting that there are likely many disease genes that await discovery.© 2011 IUBMB Life, 63(9): 669-677, 2011 [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

For nearly three decades, the sequence of the human mitochondrial genome (mtDNA) has provided a molecular framework for understanding maternally inherited diseases. However, the vast majority of human mitochondrial disorders are caused by nuclear genome defects, which is not surprising since the mtDNA encodes only 13 proteins. Advances in genomics, mass spectrometry, and computation have only recently made it possible to systematically identify the complement of over 1,000 proteins that comprise the mammalian mitochondrial proteome. Here, we review recent progress in characterizing the mitochondrial proteome and highlight insights into its complexity, tissue heterogeneity, evolutionary origins, and biochemical versatility. We then discuss how this proteome is being used to discover the genetic basis of respiratory chain disorders as well as to expand our definition of mitochondrial disease. Finally, we explore future prospects and challenges for using the mitochondrial proteome as a foundation for systems analysis of the organelle. [ABSTRACT FROM AUTHOR]

Complex I (NADH:ubiquinone oxidoreductase) is the first and largest multimeric complex of the mitochondrial respiratory chain. Human complex I comprises seven subunits encoded by mitochondrial DNA and 38 nuclear-encoded subunits that are assembled together in a process that is only partially understood. To date, mutations causing complex I deficiency have been described in all 14 core subunits, five supernumerary subunits, and four assembly factors. We describe complex I deficiency caused by mutation of the putative complex 1 assembly factor C20orf7. A candidate region for a lethal neonatal form of complex I deficiency was identified by homozygosity mapping of an Egyptian family with one affected child and two affected pregnancies predicted by enzyme-based prenatal diagnosis. The region was confirmed by microcell-mediated chromosome transfer, and 11 candidate genes encoding potential mitochondrial proteins were sequenced. A homozygous missense mutation in C20orf7 segregated with disease in the family. We show that C20orf7 is peripherally associated with the matrix face of the mitochondrial inner membrane and that silencing its expression with RNAi decreases complex I activity. C20orf7 patient fibroblasts showed an almost complete absence of complex I holoenzyme and were defective at an early stage of complex I assembly, but in a manner distinct from the assembly defects caused by mutations in the assembly factor NDUFAF1. Our results indicate that C20orf7 is crucial in the assembly of complex I and that mutations in C20orf7 cause mitochoridrial disease. [ABSTRACT FROM AUTHOR]