Publications by authors named "Florin Sasarman"

29 Publications

  • Page 1 of 1

Biosynthesis of glycosaminoglycans: associated disorders and biochemical tests.

J Inherit Metab Dis 2016 Mar 21;39(2):173-88. Epub 2015 Dec 21.

Division of Medical Genetics, Department of Pediatrics, Université de Montréal and CHU Sainte-Justine, 3175 Côte Sainte-Catherine, Montreal, QC, H3T 1C5, Canada.

Glycosaminoglycans (GAG) are long, unbranched heteropolymers with repeating disaccharide units that make up the carbohydrate moiety of proteoglycans. Six distinct classes of GAGs are recognized. Their synthesis follows one of three biosynthetic pathways, depending on the type of oligosaccharide linker they contain. Chondroitin sulfate, dermatan sulfate, heparan sulfate, and heparin sulfate contain a common tetrasaccharide linker that is O-linked to specific serine residues in core proteins. Keratan sulfate can contain three different linkers, either N-linked to asparagine or O-linked to serine/threonine residues in core proteins. Finally, hyaluronic acid does not contain a linker and is not covalently attached to a core protein. Most inborn errors of GAG biosynthesis are reported in small numbers of patients. To date, in 20 diseases, convincing evidence for pathogenicity has been presented for mutations in a total of 16 genes encoding glycosyltransferases, sulfotransferases, epimerases or transporters. GAG synthesis defects should be suspected in patients with a combination of characteristic clinical features in more than one connective tissue compartment: bone and cartilage (short long bones with or without scoliosis), ligaments (joint laxity/dislocations), and subepithelial (skin, sclerae). Some produce distinct clinical syndromes. The commonest laboratory tests used for this group of diseases are analysis of GAGs, enzyme assays, and molecular testing. In principle, GAG analysis has potential as a general first-line diagnostic test for GAG biosynthesis disorders.
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http://dx.doi.org/10.1007/s10545-015-9903-zDOI Listing
March 2016

Mitochondrial Diseases and Cardiomyopathies.

Can J Cardiol 2015 Nov 28;31(11):1360-76. Epub 2015 Aug 28.

Medical Genetics Division, Department of Pediatrics, CHU Sainte-Justine, Montreal, Quebec, Canada.

Mitochondrial cardiomyopathies are clinically and genetically heterogeneous. An integrative approach encompassing clinical, biochemical, and molecular investigations is required to reach a specific diagnosis. In this review we summarize the clinical and genetic aspects of mitochondrial disorders associated with cardiomyopathy, including disorders of oxidative phosphorylation. It also describes groups of disorders that, although not usually classified as mitochondrial disorders, stem from defects in mitochondrial function (eg, disorders of β-oxidation and the carnitine cycle), are associated with secondary mitochondrial impairment (eg, organic acidurias), and are important diagnostically because they are treatable. Current biochemical and molecular techniques for the diagnosis of mitochondrial cardiomyopathies are described, and a diagnostic algorithm is proposed, to help clinicians in their approach to cardiomyopathies in the context of mitochondrial diseases.
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http://dx.doi.org/10.1016/j.cjca.2015.08.017DOI Listing
November 2015

An N-terminal formyl methionine on COX 1 is required for the assembly of cytochrome c oxidase.

Hum Mol Genet 2015 Jul 24;24(14):4103-13. Epub 2015 Apr 24.

Department of Human Genetics and Montreal Neurological Institute, McGill University, Montreal, QC., Canada,

Protein synthesis in mitochondria is initiated by formylmethionyl-tRNA(Met) (fMet-tRNA(Met)), which requires the activity of the enzyme MTFMT to formylate the methionyl group. We investigated the molecular consequences of mutations in MTFMT in patients with Leigh syndrome or cardiomyopathy. All patients studied were compound heterozygotes. Levels of MTFMT in patient fibroblasts were almost undetectable by immunoblot analysis, and BN-PAGE analysis showed a combined oxidative phosphorylation (OXPHOS) assembly defect involving complexes I, IV and V. The synthesis of only a subset of mitochondrial polypeptides (ND5, ND4, ND1, COXII) was decreased, whereas all others were translated at normal or even increased rates. Expression of the wild-type cDNA rescued the biochemical phenotype when MTFMT was expressed near control levels, but overexpression produced a dominant-negative phenotype, completely abrogating assembly of the OXPHOS complexes, suggesting that MTFMT activity must be tightly regulated. fMet-tRNA(Met) was almost undetectable in control cells and absent in patient cells by high-resolution northern blot analysis, but accumulated in cells overexpressing MTFMT. Newly synthesized COXI was under-represented in complex IV immunoprecipitates from patient fibroblasts, and two-dimensional BN-PAGE analysis of newly synthesized mitochondrial translation products showed an accumulation of free COXI. Quantitative mass spectrophotometry of an N-terminal COXI peptide showed that the ratio of formylated to unmodified N-termini in the assembled complex IV was ∼350:1 in controls and 4:1 in patient cells. These results show that mitochondrial protein synthesis can occur with inefficient formylation of methionyl-tRNA(Met), but that assembly of complex IV is impaired if the COXI N-terminus is not formylated.
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http://dx.doi.org/10.1093/hmg/ddv149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4476453PMC
July 2015

The 3' addition of CCA to mitochondrial tRNASer(AGY) is specifically impaired in patients with mutations in the tRNA nucleotidyl transferase TRNT1.

Hum Mol Genet 2015 May 4;24(10):2841-7. Epub 2015 Feb 4.

Montreal Neurological Institute and Department of Human Genetics, McGill University, Montreal, QC, Canada,

Addition of the trinucleotide cytosine/cytosine/adenine (CCA) to the 3' end of transfer RNAs (tRNAs) is essential for translation and is catalyzed by the enzyme TRNT1 (tRNA nucleotidyl transferase), which functions in both the cytoplasm and mitochondria. Exome sequencing revealed TRNT1 mutations in two unrelated subjects with different clinical features. The first presented with acute lactic acidosis at 3 weeks of age and developed severe developmental delay, hypotonia, microcephaly, seizures, progressive cortical atrophy, neurosensorial deafness, sideroblastic anemia and renal Fanconi syndrome, dying at 21 months. The second presented at 3.5 years with gait ataxia, dysarthria, gross motor regression, hypotonia, ptosis and ophthalmoplegia and had abnormal signals in brainstem and dentate nucleus. In subject 1, muscle biopsy showed combined oxidative phosphorylation (OXPHOS) defects, but there was no OXPHOS deficiency in fibroblasts from either subject, despite a 10-fold-reduction in TRNT1 protein levels in fibroblasts of the first subject. Furthermore, in normal controls, TRNT1 protein levels are 10-fold lower in muscle than in fibroblasts. High resolution northern blots of subject fibroblast RNA suggested incomplete CCA addition to the non-canonical mitochondrial tRNA(Ser(AGY)), but no obvious qualitative differences in other mitochondrial or cytoplasmic tRNAs. Complete knockdown of TRNT1 in patient fibroblasts rendered mitochondrial tRNA(Ser(AGY)) undetectable, and markedly reduced mitochondrial translation, except polypeptides lacking Ser(AGY) codons. These data suggest that the clinical phenotypes associated with TRNT1 mutations are largely due to impaired mitochondrial translation, resulting from defective CCA addition to mitochondrial tRNA(Ser(AGY)), and that the severity of this biochemical phenotype determines the severity and tissue distribution of clinical features.
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http://dx.doi.org/10.1093/hmg/ddv044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4406295PMC
May 2015

RMND1 deficiency associated with neonatal lactic acidosis, infantile onset renal failure, deafness, and multiorgan involvement.

Eur J Hum Genet 2015 Oct 21;23(10):1301-7. Epub 2015 Jan 21.

Department of Human Genetics, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada.

RMND1 is an integral inner membrane mitochondrial protein that assembles into a large 240 kDa complex to support translation of the 13 polypeptides encoded on mtDNA, all of which are essential subunits of the oxidative phosphorylation (OXPHOS) complexes. Variants in RMND1 produce global defects in mitochondrial translation and were first reported in patients with severe neurological phenotypes leading to mortality in the first months of life. Using whole-exome sequencing, we identified compound heterozygous RMND1 variants in a 4-year-old patient with congenital lactic acidosis, severe myopathy, hearing loss, renal failure, and dysautonomia. The levels of mitochondrial ribosome proteins were reduced in patient fibroblasts, causing a translation defect, which was rescued by expression of the wild-type cDNA. RMND1 was almost undetectable by immunoblot analysis in patient muscle and fibroblasts. BN-PAGE analysis showed a severe combined OXPHOS assembly defect that was more prominent in patient muscle than in fibroblasts. Immunofluorescence experiments showed that RMND1 localizes to discrete foci in the mitochondrial network, juxtaposed to RNA granules where the primary mitochondrial transcripts are processed. RMND1 foci were not detected in patient fibroblasts. We hypothesize that RMND1 acts to anchor or stabilize the mitochondrial ribosome near the sites where the mRNAs are matured, spatially coupling post-transcriptional handling mRNAs with their translation, and that loss of function variants in RMND1 are associated with a unique constellation of clinical phenotypes that vary with the severity of the mitochondrial translation defect.
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http://dx.doi.org/10.1038/ejhg.2014.293DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4592087PMC
October 2015

Tissue-specific responses to the LRPPRC founder mutation in French Canadian Leigh Syndrome.

Hum Mol Genet 2015 Jan 11;24(2):480-91. Epub 2014 Sep 11.

Montreal Neurological Institute and Department of Human Genetics, McGill University, Montreal, QC H3A 2B4, Canada

French Canadian Leigh Syndrome (LSFC) is an early-onset, progressive neurodegenerative disorder with a distinct pattern of tissue involvement. Most cases are caused by a founder missense mutation in LRPPRC. LRPPRC forms a ribonucleoprotein complex with SLIRP, another RNA-binding protein, and this stabilizes polyadenylated mitochondrial mRNAs. LSFC fibroblasts have reduced levels of LRPPRC and a specific complex IV assembly defect; however, further depletion of mutant LRPPRC results in a complete failure to assemble a functional oxidative phosphorylation system, suggesting that LRPPRC levels determine the nature of the biochemical phenotype. We tested this hypothesis in cultured muscle cells and tissues from LSFC patients. LRPPRC levels were reduced in LSFC muscle cells, resulting in combined complex I and IV deficiencies. A similar combined deficiency was observed in skeletal muscle. Complex IV was only moderately reduced in LSFC heart, but was almost undetectable in liver. Both of these tissues showed elevated levels of complexes I and III. Despite the marked biochemical differences, the steady-state levels of LRPPRC and mitochondrial mRNAs were extremely low, LRPPRC was largely detergent-insoluble, and SLIRP was undetectable in all LSFC tissues. The level of the LRPPRC/SLIRP complex appeared much reduced in control tissues by the first dimension blue-native polyacrylamide gel electrophoresis (BN-PAGE) analysis compared with fibroblasts, and even by second dimension analysis it was virtually undetectable in control heart. These results point to tissue-specific pathways for the post-transcriptional handling of mitochondrial mRNAs and suggest that the biochemical defects in LSFC reflect the differential ability of tissues to adapt to the mutation.
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http://dx.doi.org/10.1093/hmg/ddu468DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4275074PMC
January 2015

Mutation in the nuclear-encoded mitochondrial isoleucyl-tRNA synthetase IARS2 in patients with cataracts, growth hormone deficiency with short stature, partial sensorineural deafness, and peripheral neuropathy or with Leigh syndrome.

Hum Mutat 2014 Nov 18;35(11):1285-9. Epub 2014 Oct 18.

McGill University and Genome Quebec Innovation Centre, Montreal, Quebec, Canada.

Mutations in the nuclear-encoded mitochondrial aminoacyl-tRNA synthetases are associated with a range of clinical phenotypes. Here, we report a novel disorder in three adult patients with a phenotype including cataracts, short-stature secondary to growth hormone deficiency, sensorineural hearing deficit, peripheral sensory neuropathy, and skeletal dysplasia. Using SNP genotyping and whole-exome sequencing, we identified a single likely causal variant, a missense mutation in a conserved residue of the nuclear gene IARS2, encoding mitochondrial isoleucyl-tRNA synthetase. The mutation is homozygous in the affected patients, heterozygous in carriers, and absent in control chromosomes. IARS2 protein level was reduced in skin cells cultured from one of the patients, consistent with a pathogenic effect of the mutation. Compound heterozygous mutations in IARS2 were independently identified in a previously unreported patient with a more severe mitochondrial phenotype diagnosed as Leigh syndrome. This is the first report of clinical findings associated with IARS2 mutations.
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http://dx.doi.org/10.1002/humu.22629DOI Listing
November 2014

The arginine methyltransferase NDUFAF7 is essential for complex I assembly and early vertebrate embryogenesis.

Hum Mol Genet 2014 Oct 16;23(19):5159-70. Epub 2014 May 16.

Montreal Neurological Institute and Department of Human Genetics, McGill University, Montreal, QC, Canada H3A 2B4

Complex I of the mitochondrial respiratory chain is a large multisubunit enzyme that assembles from nuclear and mtDNA-encoded components. Several complex I assembly factors have been identified, but their precise functions are not well understood. Here, we have investigated the function of one of these, NDUFAF7, a soluble matrix protein comprised of a DUF185 domain that harbors a methyltransferase motif. Knockdown of NDUFAF7 by siRNA in human fibroblasts produced a specific complex I assembly defect, as did morpholino-mediated knockdown of the zebrafish ortholog. Germline disruption of the murine ortholog was an early embryonic lethal. The complex I assembly defect was characterized by rapid, AFG3L2-dependent, turnover of newly synthesized ND1, the subunit that seeds the assembly pathway, and by decreased steady-state levels of several other structural subunits including NDUFS2, NDUFS1 and NDUFA9. Expression of an NDUFAF7 mutant (G124V), predicted to disrupt methyltransferase activity, impaired complex I assembly, suggesting an assembly factor or structural subunit as a substrate for methylation. To identify the NDUFAF7 substrate, we used an anti-ND1 antibody to immunoprecipitate complex I and its associated assembly factors, followed by mass spectrometry to detect posttranslational protein modifications. Analysis of an NDUFAF7 methyltransferase mutant showed a 10-fold reduction in an NDUFS2 peptide containing dimethylated Arg85, but a 5-fold reduction in three other NDUFS2 peptides. These results show that NDUFAF7 functions to methylate NDUFS2 after it assembles into a complex I, stabilizing an early intermediate in the assembly pathway, and that this function is essential for normal vertebrate development.
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http://dx.doi.org/10.1093/hmg/ddu239DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4159157PMC
October 2014

Novel mutations in SCO1 as a cause of fatal infantile encephalopathy and lactic acidosis.

Hum Mutat 2013 Oct 12;34(10):1366-70. Epub 2013 Aug 12.

Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada.

Isolated cytochrome c oxidase (COX) deficiency is a common cause of mitochondrial disease, yet its genetic basis remains unresolved in many patients. Here, we identified novel compound heterozygous mutations in SCO1 (p.M294V, p.Val93*) in one such patient with fatal encephalopathy. The patient lacked the severe hepatopathy (p.P174L) or hypertrophic cardiomyopathy (p.G132S) observed in previously reported SCO1 cases, so we investigated whether allele-specific defects in SCO1 function might underlie the genotype-phenotype relationships. Fibroblasts expressing p.M294V had a relatively modest decrease in COX activity compared with those expressing p.P174L, whereas both SCO1 lines had marked copper deficiencies. Overexpression of known pathogenic variants in SCO1 fibroblasts showed that p.G132S exacerbated the COX deficiency, whereas COX activity was partially or fully restored by p.P174L and p.M294V, respectively. These data suggest that the clinical phenotypes in SCO1 patients might reflect the residual capacity of the pathogenic alleles to perform one or both functions of SCO1.
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http://dx.doi.org/10.1002/humu.22385DOI Listing
October 2013

The mitochondrial RNA-binding protein GRSF1 localizes to RNA granules and is required for posttranscriptional mitochondrial gene expression.

Cell Metab 2013 Mar;17(3):386-98

Montreal Neurological Institute and Department of Human Genetics, McGill University, Montreal, QC H3A 2B4, Canada.

RNA-binding proteins are at the heart of posttranscriptional gene regulation, coordinating the processing, storage, and handling of cellular RNAs. We show here that GRSF1, previously implicated in the binding and selective translation of influenza mRNAs, is targeted to mitochondria where it forms granules that colocalize with foci of newly synthesized mtRNA next to mitochondrial nucleoids. GRSF1 preferentially binds RNAs transcribed from three contiguous genes on the light strand of mtDNA, the ND6 mRNA, and the long noncoding RNAs for cytb and ND5, each of which contains multiple consensus binding sequences. RNAi-mediated knockdown of GRSF1 leads to alterations in mitochondrial RNA stability, abnormal loading of mRNAs and lncRNAs on the mitochondrial ribosome, and impaired ribosome assembly. This results in a specific protein synthesis defect and a failure to assemble normal amounts of the oxidative phosphorylation complexes. These data implicate GRSF1 as a key regulator of posttranscriptional mitochondrial gene expression.
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http://dx.doi.org/10.1016/j.cmet.2013.02.006DOI Listing
March 2013

MITRAC links mitochondrial protein translocation to respiratory-chain assembly and translational regulation.

Cell 2012 Dec;151(7):1528-41

Department for Biochemistry II, University of Göttingen, D-37073 Göttingen, Germany.

Mitochondrial respiratory-chain complexes assemble from subunits of dual genetic origin assisted by specialized assembly factors. Whereas core subunits are translated on mitochondrial ribosomes, others are imported after cytosolic translation. How imported subunits are ushered to assembly intermediates containing mitochondria-encoded subunits is unresolved. Here, we report a comprehensive dissection of early cytochrome c oxidase assembly intermediates containing proteins required for normal mitochondrial translation and reveal assembly factors promoting biogenesis of human respiratory-chain complexes. We find that TIM21, a subunit of the inner-membrane presequence translocase, is also present in the major assembly intermediates containing newly mitochondria-synthesized and imported respiratory-chain subunits, which we term MITRAC complexes. Human TIM21 is dispensable for protein import but required for integration of early-assembling, presequence-containing subunits into respiratory-chain intermediates. We establish an unexpected molecular link between the TIM23 transport machinery and assembly of respiratory-chain complexes that regulate mitochondrial protein synthesis in response to their assembly state.
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http://dx.doi.org/10.1016/j.cell.2012.11.053DOI Listing
December 2012

The conserved interaction of C7orf30 with MRPL14 promotes biogenesis of the mitochondrial large ribosomal subunit and mitochondrial translation.

Mol Biol Cell 2013 Feb 21;24(3):184-93. Epub 2012 Nov 21.

Montreal Neurological Institute and Department of Human Genetics, McGill University, Montreal, QC H3A 2B4, Canada.

Mammalian mitochondria harbor a dedicated translation apparatus that is required for the synthesis of 13 mitochondrial DNA (mtDNA)-encoded polypeptides, all of which are essential components of the oxidative phosphorylation (OXPHOS) complexes. Little is known about the mechanism of assembly of the mitoribosomes that catalyze this process. Here we show that C7orf30, a member of the large family of DUF143 proteins, associates with the mitochondrial large ribosomal subunit (mt-LSU). Knockdown of C7orf30 by short hairpin RNA (shRNA) does not alter the sedimentation profile of the mt-LSU, but results in the depletion of several mt-LSU proteins and decreased monosome formation. This leads to a mitochondrial translation defect, involving the majority of mitochondrial polypeptides, and a severe OXPHOS assembly defect. Immunoprecipitation and mass spectrometry analyses identified mitochondrial ribosomal protein (MRP)L14 as the specific interacting protein partner of C7orf30 in the mt-LSU. Reciprocal experiments in which MRPL14 was depleted by small interfering RNA (siRNA) phenocopied the C7orf30 knockdown. Members of the DUF143 family have been suggested to be universally conserved ribosomal silencing factors, acting by sterically inhibiting the association of the small and large ribosomal subunits. Our results demonstrate that, although the interaction between C7orf30 and MRPL14 has been evolutionarily conserved, human C7orf30 is, on the contrary, essential for mitochondrial ribosome biogenesis and mitochondrial translation.
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http://dx.doi.org/10.1091/mbc.E12-09-0651DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3564531PMC
February 2013

An RMND1 Mutation causes encephalopathy associated with multiple oxidative phosphorylation complex deficiencies and a mitochondrial translation defect.

Am J Hum Genet 2012 Oct 27;91(4):737-43. Epub 2012 Sep 27.

Department of Human Genetics, McGill University, 1205 Penfield Avenue, Montréal, QC H3A 1B1, Canada.

Mutations in the genes composing the mitochondrial translation apparatus are an important cause of a heterogeneous group of oxidative phosphorylation (OXPHOS) disorders. We studied the index case in a consanguineous family in which two children presented with severe encephalopathy, lactic acidosis, and intractable seizures leading to an early fatal outcome. Blue native polyacrylamide gel electrophoretic (BN-PAGE) analysis showed assembly defects in all of the OXPHOS complexes with mtDNA-encoded structural subunits, and these defects were associated with a severe deficiency in mitochondrial translation. Immunoblot analysis showed reductions in the steady-state levels of several structural subunits of the mitochondrial ribosome. Whole-exome sequencing identified a homozygous missense mutation (c.1250G>A) in an uncharacterized gene, RMND1 (required for meiotic nuclear division 1). RMND1 localizes to mitochondria and behaves as an integral membrane protein. Retroviral expression of the wild-type RMND1 cDNA rescued the biochemical phenotype in subject cells, and siRNA-mediated knockdown of the protein recapitulated the defect. BN-PAGE, gel filtration, and mass spectrometry analyses showed that RMND1 forms a high-molecular-weight and most likely homopolymeric complex (∼240 kDa) that does not assemble in subject fibroblasts but that is rescued by expression of RMND1 cDNA. The p.Arg417Gln substitution, predicted to be in a coiled-coil domain, which is juxtaposed to a transmembrane domain at the extreme C terminus of the protein, does not alter the steady-state level of RMND1 but might prevent protein-protein interactions in this complex. Our results demonstrate that the RMND1 complex is necessary for mitochondrial translation, possibly by coordinating the assembly or maintenance of the mitochondrial ribosome.
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http://dx.doi.org/10.1016/j.ajhg.2012.08.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3484649PMC
October 2012

A novel mutation in YARS2 causes myopathy with lactic acidosis and sideroblastic anemia.

Hum Mutat 2012 Aug 7;33(8):1201-6. Epub 2012 May 7.

Montreal Neurological Institute, McGill University, Montreal, Canada.

Mutations in the mitochondrial aminoacyl-tRNA synthetases (ARSs) are associated with a strikingly broad range of clinical phenotypes, the molecular basis for which remains obscure. Here, we report a novel missense mutation (c.137G>A, p.Gly46Asp) in the catalytic domain of YARS2, which codes for the mitochondrial tyrosyl-tRNA synthetase, in a subject with myopathy, lactic acidosis, and sideroblastic anemia (MLASA). YARS2 was undetectable by immunoblot analysis in subject myoblasts, resulting in a generalized mitochondrial translation defect. Retroviral expression of a wild-type YARS2 complementary DNA completely rescued the translation defect. We previously demonstrated that the respiratory chain defect in this subject was only present in fully differentiated muscle, and we show here that this likely reflects an increased requirement for YARS2 as muscle cells differentiate. An additional, heterozygous mutation was detected in TRMU/MTU1, a gene encoding the mitochondrial 2-thiouridylase. Although subject myoblasts and myotubes contained half the normal levels of TRMU, thiolation of mitochondrial tRNAs was normal. YARS2 eluted as part of high-molecular-weight complexes of ∼250 kDa and 1 MDa by gel filtration. This study confirms mutations in YARS2 as a cause of MLASA and shows that, like some of the cytoplasmic ARSs, mitochondrial ARSs occur in high-molecular-weight complexes.
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http://dx.doi.org/10.1002/humu.22098DOI Listing
August 2012

Mutations in the mitochondrial methionyl-tRNA synthetase cause a neurodegenerative phenotype in flies and a recessive ataxia (ARSAL) in humans.

PLoS Biol 2012 20;10(3):e1001288. Epub 2012 Mar 20.

Program in Developmental Biology, Baylor College of Medicine, Houston, Texas, United States of America.

An increasing number of genes required for mitochondrial biogenesis, dynamics, or function have been found to be mutated in metabolic disorders and neurological diseases such as Leigh Syndrome. In a forward genetic screen to identify genes required for neuronal function and survival in Drosophila photoreceptor neurons, we have identified mutations in the mitochondrial methionyl-tRNA synthetase, Aats-met, the homologue of human MARS2. The fly mutants exhibit age-dependent degeneration of photoreceptors, shortened lifespan, and reduced cell proliferation in epithelial tissues. We further observed that these mutants display defects in oxidative phosphorylation, increased Reactive Oxygen Species (ROS), and an upregulated mitochondrial Unfolded Protein Response. With the aid of this knowledge, we identified MARS2 to be mutated in Autosomal Recessive Spastic Ataxia with Leukoencephalopathy (ARSAL) patients. We uncovered complex rearrangements in the MARS2 gene in all ARSAL patients. Analysis of patient cells revealed decreased levels of MARS2 protein and a reduced rate of mitochondrial protein synthesis. Patient cells also exhibited reduced Complex I activity, increased ROS, and a slower cell proliferation rate, similar to Drosophila Aats-met mutants.
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http://dx.doi.org/10.1371/journal.pbio.1001288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3308940PMC
July 2012

Mutations in C12orf62, a factor that couples COX I synthesis with cytochrome c oxidase assembly, cause fatal neonatal lactic acidosis.

Am J Hum Genet 2012 Jan;90(1):142-51

Department of Human Genetics and Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada.

We investigated a family in which the index subject presented with severe congenital lactic acidosis and dysmorphic features associated with a cytochrome c oxidase (COX)-assembly defect and a specific decrease in the synthesis of COX I, the subunit that nucleates COX assembly. Using a combination of microcell-mediated chromosome transfer, homozygosity mapping, and transcript profiling, we mapped the gene defect to chromosome 12 and identified a homozygous missense mutation (c.88G>A) in C12orf62. C12orf62 was not detectable by immunoblot analysis in subject fibroblasts, and retroviral expression of the wild-type C12orf62 cDNA rescued the biochemical phenotype. Furthermore, siRNA-mediated knockdown of C12orf 62 recapitulated the biochemical defect in control cells and exacerbated it in subject cells. C12orf62 is apparently restricted to the vertebrate lineage. It codes for a very small (6 kDa), uncharacterized, single-transmembrane protein that localizes to mitochondria and elutes in a complex of ∼110 kDa by gel filtration. COX I, II, and IV coimmunoprecipated with an epitope-tagged version of C12orf62, and 2D blue-native-polyacrylamide-gel-electrophoresis analysis of newly synthesized mitochondrial COX subunits in subject fibroblasts showed that COX assembly was impaired and that the nascent enzyme complex was unstable. We conclude that C12orf62 is required for coordination of the early steps of COX assembly with the synthesis of COX I.
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http://dx.doi.org/10.1016/j.ajhg.2011.11.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3257963PMC
January 2012

Radioactive labeling of mitochondrial translation products in cultured cells.

Methods Mol Biol 2012 ;837:207-17

Montreal Neurological Institute and Department of Human Genetics, McGill University, Montreal, QC, Canada.

The mammalian mitochondrial genome contains 37 genes, 13 of which encode polypeptide subunits in the enzyme complexes of the oxidative phosphorylation system. The other genes encode the rRNAs and tRNAs necessary for their translation. The mitochondrial translation machinery is located in the mitochondrial matrix, and is exclusively dedicated to the synthesis of these 13 enzyme subunits. Mitochondrial disease in humans is often associated with defects in mitochondrial translation. This can manifest as a global decrease in the rate of mitochondrial protein synthesis, a decrease in the synthesis of specific polypeptides, the synthesis of abnormal polypeptides, or in altered stability of specific translation products. All of these changes in the normal pattern of mitochondrial translation can be assessed by a straightforward technique that takes advantage of the insensitivity of the mitochondrial translation machinery to antibiotics that completely inhibit cytoplasmic translation. Thus, specific radioactive labeling of the mitochondrial translation products can be achieved in cultured cells, and the results can be visualized on gradient gels. The analysis of mitochondrial translation in cells cultured from patient biopsies is useful in the study of disease-causing mutations in both the mitochondrial and the nuclear genomes.
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http://dx.doi.org/10.1007/978-1-61779-504-6_14DOI Listing
April 2012

The 2-thiouridylase function of the human MTU1 (TRMU) enzyme is dispensable for mitochondrial translation.

Hum Mol Genet 2011 Dec 1;20(23):4634-43. Epub 2011 Sep 1.

Montreal Neurological Institute, McGill University, Montreal H3A 2B4, Canada.

MTU1 (TRMU) is a mitochondrial enzyme responsible for the 2-thiolation of the wobble U in tRNA(Lys), tRNA(Glu) and tRNA(Gln), a post-transcriptional modification believed to be important for accurate and efficient synthesis of the 13 respiratory chain subunits encoded by mtDNA. Mutations in MTU1 are associated with acute infantile liver failure, and this has been ascribed to a transient lack of cysteine, the sulfur donor for the thiouridylation reaction, resulting in a mitochondrial translation defect during early development. A mutation in tRNA(Lys) that causes myoclonic epilepsy with ragged-red fibers (MERRF) is also reported to prevent modification of the wobble U. Here we show that mitochondrial translation is unaffected in fibroblasts from an MTU1 patient, in which MTU1 is undetectable by immunoblotting, despite the severe reduction in the 2-thiolation of mitochondrial tRNA(Lys), tRNA(Glu) and tRNA(Gln). The only respiratory chain abnormality that we could observe in these cells was an accumulation of a Complex II assembly intermediate, which, however, did not affect the level of the fully assembled enzyme. The identical phenotype was observed by siRNA-mediated knockdown of MTU1 in HEK 293 cells. Further, the mitochondrial translation deficiencies present in myoblasts from mitochondrial encephalomyopathy, lactic acidosis and stroke-like episode and MERRF patients, which are associated with defects in post-transcriptional modification of mitochondrial tRNAs, did not worsen following knockdown of MTU1 in these cells. This study demonstrates that MTU1 is not required for mitochondrial translation at normal steady-state levels of tRNAs, and that it may possess an as yet uncharacterized function in another sulfur-trafficking pathway.
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http://dx.doi.org/10.1093/hmg/ddr397DOI Listing
December 2011

Mutations in C12orf65 in patients with encephalomyopathy and a mitochondrial translation defect.

Am J Hum Genet 2010 Jul;87(1):115-22

Montreal Neurological Institute and Department of Human Genetics, McGill University, Montreal H3A 2B4, Quebec, Canada.

We investigated the genetic basis for a global and uniform decrease in mitochondrial translation in fibroblasts from patients in two unrelated pedigrees who developed Leigh syndrome, optic atrophy, and ophthalmoplegia. Analysis of the assembly of the oxidative phosphorylation complexes showed severe decreases of complexes I, IV, and V and a smaller decrease in complex III. The steady-state levels of mitochondrial mRNAs, tRNAs, and rRNAs were not reduced, nor were those of the mitochondrial translation elongation factors or the protein components of the mitochondrial ribosome. Using homozygosity mapping, we identified a 1 bp deletion in C12orf65 in one patient, and DNA sequence analysis showed a different 1 bp deletion in the second patient. Both mutations predict the same premature stop codon. C12orf65 belongs to a family of four mitochondrial class I peptide release factors, which also includes mtRF1a, mtRF1, and Ict1, all characterized by the presence of a GGQ motif at the active site. However, C12orf65 does not exhibit peptidyl-tRNA hydrolase activity in an in vitro assay with bacterial ribosomes. We suggest that it might play a role in recycling abortive peptidyl-tRNA species, released from the ribosome during the elongation phase of translation.
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http://dx.doi.org/10.1016/j.ajhg.2010.06.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2896764PMC
July 2010

LRPPRC and SLIRP interact in a ribonucleoprotein complex that regulates posttranscriptional gene expression in mitochondria.

Mol Biol Cell 2010 Apr 3;21(8):1315-23. Epub 2010 Mar 3.

Department of Human Genetics, Montreal Neurological Institute, McGill University, Montreal, QC, H3A 2B4, Canada.

Mutations in LRPPRC are responsible for the French Canadian variant of Leigh syndrome (LSFC), a neurodegenerative disorder caused by a tissue-specific deficiency in cytochrome c oxidase (COX). To investigate the pathogenic mechanism of disease, we studied LRPPRC function in LSFC and control fibroblasts. The level of mutated LRPPRC is reduced in LSFC cells, and this results in decreased steady-state levels of most mitochondrial mRNAs, but not rRNAs or tRNAs, a phenotype that can be reproduced by siRNA-mediated knockdown of LRPPRC in control cells. Processing of the primary transcripts appears normal. The resultant defect in mitochondrial protein synthesis in LSFC cells disproportionately affects the COX subunits, leading to an isolated COX assembly defect. Further knockdown of LRPPRC produces a generalized assembly defect in all oxidative phosphorylation complexes containing mtDNA-encoded subunits, due to a severe decrease in all mitochondrial mRNAs. LRPPRC exists in a high-molecular-weight complex, and it coimmunoprecipitates with SLIRP, a stem-loop RNA-binding protein. Although this interaction does not depend on mitochondrial mRNA, both proteins show reduced stability in its absence. These results implicate LRPPRC in posttranscriptional mitochondrial gene expression as part of a ribonucleoprotein complex that regulates the stability and handling of mature mRNAs.
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http://dx.doi.org/10.1091/mbc.e10-01-0047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2854090PMC
April 2010

Oxidative phosphorylation: synthesis of mitochondrially encoded proteins and assembly of individual structural subunits into functional holoenzyme complexes.

Methods Mol Biol 2009 ;554:143-62

Montreal Neurological Institute and Department of Human Genetics, McGill University, Montreal, QC, Canada.

The bulk of ATP consumed by various cellular processes in higher eukaryotes is normally produced by five multimeric protein complexes (I-V) embedded within the inner mitochondrial membrane, in a process known as oxidative phosphorylation (OXPHOS). Maintenance of energy homeostasis under most physiological conditions is therefore contingent upon the ability of OXPHOS to meet cellular changes in bioenergetic demand, with a chronic failure to do so being a frequent cause of human disease. With the exception of Complex II, the structural subunits of OXPHOS complexes are encoded by both the nuclear and the mitochondrial genomes. The physical separation of the two genomes necessitates that the expression of the 13 mitochondrially encoded polypeptides be co-ordinated with that of relevant nuclear-encoded partners in order to assemble functional holoenzyme complexes. Complex biogenesis is a highly ordered process, and several nuclear-encoded factors that function at distinct stages in the assembly of individual OXPHOS complexes have been identified.
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http://dx.doi.org/10.1007/978-1-59745-521-3_10DOI Listing
October 2009

Mutation in TACO1, encoding a translational activator of COX I, results in cytochrome c oxidase deficiency and late-onset Leigh syndrome.

Nat Genet 2009 Jul 7;41(7):833-7. Epub 2009 Jun 7.

Department of Human Genetics, McGill University, Montreal, Quebec, Canada.

Defects in mitochondrial translation are among the most common causes of mitochondrial disease, but the mechanisms that regulate mitochondrial translation remain largely unknown. In the yeast Saccharomyces cerevisiae, all mitochondrial mRNAs require specific translational activators, which recognize sequences in 5' UTRs and mediate translation. As mammalian mitochondrial mRNAs do not have significant 5' UTRs, alternate mechanisms must exist to promote translation. We identified a specific defect in the synthesis of the mitochondrial DNA (mtDNA)-encoded COX I subunit in a pedigree segregating late-onset Leigh syndrome and cytochrome c oxidase (COX) deficiency. We mapped the defect to chromosome 17q by functional complementation and identified a homozygous single-base-pair insertion in CCDC44, encoding a member of a large family of hypothetical proteins containing a conserved DUF28 domain. CCDC44, renamed TACO1 for translational activator of COX I, shares a notable degree of structural similarity with bacterial homologs, and our findings suggest that it is one of a family of specific mammalian mitochondrial translational activators.
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http://dx.doi.org/10.1038/ng.390DOI Listing
July 2009

Human SCO2 is required for the synthesis of CO II and as a thiol-disulphide oxidoreductase for SCO1.

Hum Mol Genet 2009 Jun 31;18(12):2230-40. Epub 2009 Mar 31.

Montreal Neurological Institute, McGill University, 3801 University Street, Montreal, Quebec, Canada H3A 2B4.

Human SCO1 and SCO2 code for essential metallochaperones with ill-defined functions in the biogenesis of the CuA site of cytochrome c oxidase subunit II (CO II). Here, we have used patient cell lines to investigate the specific roles of each SCO protein in this pathway. By pulse-labeling mitochondrial translation products, we demonstrate that the synthesis of CO II is reduced in SCO2, but not in SCO1, cells. Despite this biosynthetic defect, newly synthesized CO II is more stable in SCO2 cells than in control cells. RNAi-mediated knockdown of mutant SCO2 abolishes CO II labeling in the translation assay, whereas knockdown of mutant SCO1 does not affect CO II synthesis. These results indicate that SCO2 acts upstream of SCO1, and that it is indispensable for CO II synthesis. The subsequent maturation of CO II is contingent upon the formation of a complex that includes both SCO proteins, each with a functional CxxxC copper-coordinating motif. In control cells, the cysteines in this motif in SCO1 exist as a mixed population comprised of oxidized disulphides and reduced thiols; however, the relative ratio of oxidized to reduced cysteines in SCO1 is perturbed in cells from both SCO backgrounds. Overexpression of wild-type SCO2, or knockdown of mutant SCO2, in SCO2 cells alters the ratio of oxidized to reduced cysteines in SCO1, suggesting that SCO2 acts as a thiol-disulphide oxidoreductase to oxidize the copper-coordinating cysteines in SCO1 during CO II maturation. Based on these data we present a model in which each SCO protein fulfills distinct, stage-specific functions during CO II synthesis and CuA site maturation.
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http://dx.doi.org/10.1093/hmg/ddp158DOI Listing
June 2009

The A3243G tRNALeu(UUR) MELAS mutation causes amino acid misincorporation and a combined respiratory chain assembly defect partially suppressed by overexpression of EFTu and EFG2.

Hum Mol Genet 2008 Dec 27;17(23):3697-707. Epub 2008 Aug 27.

Montreal Neurological Institute, Montreal, QC, Canada.

The majority of patients with MELAS (mitochondrial encephalomyophathy, lactic acidosis, stroke-like episodes) carry a heteroplasmic A3243G mutation in the mitochondrial tRNA(Leu(UUR)). The mutation prevents modification of the wobble U base, impairing translation at UUA and UUG codons; however, whether this results in amino acid misincorporation in the mitochondrial translation products remains controversial. We tested this hypothesis in homoplasmic mutant myoblasts isolated from a MELAS patient and investigated whether overexpression of the mitochondrial translation elongation factors could suppress the translation defect. Blue-Native gel electrophoretic analysis demonstrated an almost complete lack of assembly of respiratory chain complexes I, IV and V in MELAS myoblasts. This phenotype could be partially suppressed by overexpression of EFTu or EFG2 but not EFTs or EFG1. Despite the severity of the assembly defect, overall mitochondrial protein synthesis was only moderately affected, but some anomalously migrating translation products were present. Pulse-chase labeling showed reduced stability of all mitochondrial translation products consistent with the assembly defect. Labeling patterns of the translation products were similar with [(3)H]-leucine or [(3)H]-phenylalanine, showing that loss of the wobble U modification did not permit decoding of UUY codons; however, endoproteinase fingerprint analysis showed clear evidence of amino acid misincorporation in three polypeptides: CO III, CO II and ATP6. Taken together, these data demonstrate that the A3243G mutation produces both loss- and gain-of-function phenotypes, explaining the apparent discrepancy between the severity of the translation and respiratory chain assembly defects, and suggest a function for EFG2 in quality control of translation elongation.
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http://dx.doi.org/10.1093/hmg/ddn265DOI Listing
December 2008

Distinct clinical phenotypes associated with a mutation in the mitochondrial translation elongation factor EFTs.

Am J Hum Genet 2006 Nov 15;79(5):869-77. Epub 2006 Sep 15.

Nijmegen Centre for Mitochondrial Disorders, Department of Pediatrics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.

The 13 polypeptides encoded in mitochondrial DNA (mtDNA) are synthesized in the mitochondrial matrix on a dedicated protein-translation apparatus that resembles that found in prokaryotes. Here, we have investigated the genetic basis for a mitochondrial protein-synthesis defect associated with a combined oxidative phosphorylation enzyme deficiency in two patients, one of whom presented with encephalomyopathy and the other with hypertrophic cardiomyopathy. Sequencing of candidate genes revealed the same homozygous mutation (C997T) in both patients in TSFM, a gene coding for the mitochondrial translation elongation factor EFTs. EFTs functions as a guanine nucleotide exchange factor for EFTu, another translation elongation factor that brings aminoacylated transfer RNAs to the ribosomal A site as a ternary complex with guanosine triphosphate. The mutation predicts an Arg333Trp substitution at an evolutionarily conserved site in a subdomain of EFTs that interacts with EFTu. Molecular modeling showed that the substitution disrupts local subdomain structure and the dimerization interface. The steady-state levels of EFTs and EFTu in patient fibroblasts were reduced by 75% and 60%, respectively, and the amounts of assembled complexes I, IV, and V were reduced by 35%-91% compared with the amounts in controls. These phenotypes and the translation defect were rescued by retroviral expression of either EFTs or EFTu. These data clearly establish mutant EFTs as the cause of disease in these patients. The fact that the same mutation is associated with distinct clinical phenotypes suggests the presence of genetic modifiers of the mitochondrial translation apparatus.
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http://dx.doi.org/10.1086/508434DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1698578PMC
November 2006

The molecular basis for tissue specificity of the oxidative phosphorylation deficiencies in patients with mutations in the mitochondrial translation factor EFG1.

Hum Mol Genet 2006 Jun 21;15(11):1835-46. Epub 2006 Apr 21.

Montreal Neurological Institute, McGill University, 3801 University Street, Montreal, Quebec, Canada.

Defects in mitochondrial translation are associated with a remarkable, but unexplained diversity of clinical phenotypes. Here we have investigated the molecular basis for tissue specificity in patients with a fatal hepatopathy due to mutations in the mitochondrial translation elongation factor EFG1. Blue-native gel electrophoresis revealed unique, tissue-specific patterns in the nature and severity of the defect. Liver was the most severely affected tissue, with less than 10% residual assembly of complexes I and IV, and a 50% decrease in complex V. Skeletal muscle showed a 50% reduction in complex I, and complexes IV and V were 20% of control. In fibroblasts, complexes I and IV were 20% of control, and there was a 40-60% reduction in complexes III and V. In contrast, except for a 50% decrease in complex IV, all complexes were near normal in heart. The severity of the defect paralleled the steady-state level of the mutant EFG1 protein, which varied from 60% of control in heart to undetectable in liver. The ratio of translation elongation factors EFTu:EFTs increased from 1:6 to 1:2 in patient heart, whereas in liver it decreased from 1:1 to 1:4. Over-expression of either EFTu or EFTs in control and patient fibroblasts produced dominant negative effects, indicating that the relative abundance of these factors is an important determinant of translation efficiency. Our results demonstrate marked differences among tissues in the organization of the mitochondrial translation system and its response to dysfunction, and explain the severe hepatopathy, but normal cardiac function in EFG1 patients.
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http://dx.doi.org/10.1093/hmg/ddl106DOI Listing
June 2006

The P174L mutation in human Sco1 severely compromises Cox17-dependent metallation but does not impair copper binding.

J Biol Chem 2006 May 6;281(18):12270-6. Epub 2006 Mar 6.

Department of Medicine, University of Utah Health Sciences Center, Salt Lake City, Utah 84132, USA.

Sco1 is a metallochaperone that is required for copper delivery to the Cu(A) site in the CoxII subunit of cytochrome c oxidase. The only known missense mutation in human Sco1, a P174L substitution in the copper-binding domain, is associated with a fatal neonatal hepatopathy; however, the molecular basis for dysfunction of the protein is unknown. Immortalized fibroblasts from a SCO1 patient show a severe deficiency in cytochrome c oxidase activity that was partially rescued by overexpression of P174L Sco1. The mutant protein retained the ability to bind Cu(I) and Cu(II) normally when expressed in bacteria, but Cox17-mediated copper transfer was severely compromised both in vitro and in a yeast cytoplasmic assay. The corresponding P153L substitution in yeast Sco1 was impaired in suppressing the phenotype of cells harboring the weakly functional C57Y allele of Cox17; however, it was functional in sco1delta yeast when the wild-type COX17 gene was present. Pulse-chase labeling of mitochondrial translation products in SCO1 patient fibroblasts showed no change in the rate of CoxII translation, but there was a specific and rapid turnover of CoxII protein in the chase. These data indicate that the P174L mutation attenuates a transient interaction with Cox17 that is necessary for copper transfer. They further suggest that defective Cox17-mediated copper metallation of Sco1, as well as the subsequent failure of Cu(A) site maturation, is the basis for the inefficient assembly of the cytochrome c oxidase complex in SCO1 patients.
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http://dx.doi.org/10.1074/jbc.M600496200DOI Listing
May 2006

Mutant mitochondrial elongation factor G1 and combined oxidative phosphorylation deficiency.

N Engl J Med 2004 Nov;351(20):2080-6

Nijmegen Center for Mitochondrial Disorders, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands.

Although most components of the mitochondrial translation apparatus are encoded by nuclear genes, all known molecular defects associated with impaired mitochondrial translation are due to mutations in mitochondrial DNA. We investigated two siblings with a severe defect in mitochondrial translation, reduced levels of oxidative phosphorylation complexes containing mitochondrial DNA (mtDNA)-encoded subunits, and progressive hepatoencephalopathy. We mapped the defective gene to a region on chromosome 3q containing elongation factor G1 (EFG1), which encodes a mitochondrial translation factor. Sequencing of EFG1 revealed a mutation affecting a conserved residue of the guanosine triphosphate (GTP)-binding domain. These results define a new class of gene defects underlying disorders of oxidative phosphorylation.
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http://dx.doi.org/10.1056/NEJMoa041878DOI Listing
November 2004

Nuclear genetic control of mitochondrial translation in skeletal muscle revealed in patients with mitochondrial myopathy.

Hum Mol Genet 2002 Jul;11(14):1669-81

Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec, Canada, H3A 2B4.

Oxidative phosphorylation deficiencies can be caused by mutations in either the nuclear genome or the mitochondrial genome (mtDNA); however, most pathogenic mutations reported in adults occur in mtDNA. Such mutations often impair mitochondrial translation, and are associated with a characteristic muscle pathology consisting of a mosaic pattern of normal fibres interspersed with fibres that show mitochondrial proliferation (ragged-red fibres) and little or no complex IV (COX) activity. We investigated two adult patients with a severe mitochondrial myopathy in whom all muscle fibres showed mitochondrial proliferation with barely detectable COX activity - a pattern never before reported. Biochemical studies of the respiratory chain in muscle showed decreased activities of complexes I and IV (5% of control) and complex II+III (41% of control). Immunoblot analysis of nuclear and mitochondrial subunits of complexes I, III and IV showed a greater than 90% decrease in the steady-state level of these subunits in mature muscle, but no change in nuclear-encoded subunits of complexes II and V. A generalized mitochondrial translation defect was identified in pulse-label experiments in myotubes, but not in myoblasts cultured from both patients. This defect moved with the nucleus in patient cybrid cells. Myoblasts from one patient transplanted into the muscle bed of SCID mice differentiated into mature human muscle fibres that displayed a defect similar to that seen in the patient muscle. These results suggest a defect in a developmentally regulated nuclear factor important for mitochondrial translation in skeletal muscle.
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http://dx.doi.org/10.1093/hmg/11.14.1669DOI Listing
July 2002
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