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Distinct Clinical Phenotypes Associated with a Mutation in the Mitochondrial Translation Elongation Factor EFTs

2006; Elsevier BV; Volume: 79; Issue: 5 Linguagem: Inglês

10.1086/508434

1537-6605

Jan Smeitink, Orly Elpeleg, Hana Antonická, Heleen Diepstra, Ann Saada, P. Smits, Florin Sasarman, Gert Vriend, Jasmine Jacob‐Hirsch, Avraham Shaag, Gideon Rechavi, Brigitte Welling, Jürgen Horst, Richard J. Rodenburg, Bert van den Heuvel, Eric A. Shoubridge,

Metabolism and Genetic Disorders

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. 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. Defects in mitochondrial oxidative phosphorylation (OXPHOS) are estimated to occur in ∼1 in 5,000 live births.1Thorburn DR Mitochondrial disorders: prevalence, myths and advances.J Inherit Metab Dis. 2004; 27: 349-362Crossref PubMed Scopus (168) Google Scholar They can be classified biochemically as either isolated or combined deficiencies of any of the five OXPHOS complexes.2Smeitink J van den Heuvel L DiMauro S The genetics and pathology of oxidative phosphorylation.Nat Rev Genet. 2001; 2: 342-352Crossref PubMed Scopus (513) Google Scholar Genetic defects leading to combined deficiencies of complexes I, III, IV, and V can be caused by mutations in mtDNA or in the components of the mitochondrial translation apparatus encoded by nuclear genes.3Jacobs HT Disorders of mitochondrial protein synthesis.Hum Mol Genet. 2003; 12: R293-R301Crossref PubMed Scopus (84) Google Scholar Synthesis of the 13 mitochondrial-encoded proteins occurs on a dedicated mitochondrial translation apparatus similar to that found in prokaryotes and requires, in addition to the tRNAs and ribosomal RNAs (rRNAs) encoded in mtDNA, the concerted action of several translation factors and a large number of mitochondrial ribosomal (mitoribosome) proteins, all of which are encoded by nuclear genes. Two mammalian initiation factors, IF2 and IF3; four elongation factors, EFTu, EFTs, EFG1, and EFG2 (reviewed by Spremulli et al.4Spremulli LL Coursey A Navratil T Hunter SE Initiation and elongation factors in mammalian mitochondrial protein biosynthesis.Prog Nucleic Acid Res Mol Biol. 2004; 77: 211-261Crossref PubMed Scopus (61) Google Scholar); one release factor, RF1; and one ribosomal recycling factor, RRF,5Zhang Y Spremulli LL Identification and cloning of human mitochondrial translational release factor 1 and the ribosome recycling factor.Biochim Biophys Acta. 1998; 1443: 245-250Crossref PubMed Scopus (60) Google Scholar have been cloned and sequenced. Mitoribosomes are distinct from both prokaryotic and eukaryotic cytosolic ribosomes; they are 55S particles composed of a small (28S) and a large (39S) subunit and contain a much higher protein:RNA ratio than that of the bacterial 70S ribosome.6O'Brien TW Evolution of a protein-rich mitochondrial ribosome: implications for human genetic disease.Gene. 2002; 286: 73-79Crossref PubMed Scopus (93) Google Scholar Although the vast majority of components of the mitochondrial translation system are nuclear encoded, most mutations associated with mitochondrial translation defects have been reported in mtDNA-encoded tRNAs and rRNAs.7DiMauro S Schon EA Mitochondrial respiratory-chain diseases.N Engl J Med. 2003; 348: 2656-2668Crossref PubMed Scopus (1191) Google Scholar, 8Taylor RW Turnbull DM Mitochondrial DNA mutations in human disease.Nat Rev Genet. 2005; 6: 389-402Crossref PubMed Scopus (1167) Google Scholar However, we recently reported mutations in the nuclear-encoded translation elongation factor EFG19Coenen MJ Antonicka H Ugalde C Sasarman F Rossi R Heister JG Newbold RF Trijbels FJ van den Heuvel LP Shoubridge EA Smeitink JA Mutant mitochondrial elongation factor G1 and combined oxidative phosphorylation deficiency.N Engl J Med. 2004; 351: 2080-2086Crossref PubMed Scopus (152) Google Scholar, 10Antonicka H Sasarman F Kennaway NG Shoubridge EA 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; 15: 1835-1846Crossref PubMed Scopus (104) Google Scholar and in MRPS16, a protein of the small ribosomal subunit,11Miller C Saada A Shaul N Shabtai N Ben-Shalom E Shaag A Hershkovitz E Elpeleg O Defective mitochondrial translation caused by a ribosomal protein (MRPS16) mutation.Ann Neurol. 2004; 56: 734-738Crossref PubMed Scopus (173) Google Scholar in patients with autosomal recessive mitochondrial translation defects. Encouraged by these findings, we investigated a cohort of 16 patients with combined OXPHOS enzyme defects expressed in both muscle and fibroblasts in which mtDNA sequencing and complex II activity–measurement results were normal. We describe the first mutation in the mitochondrial elongation factor EFTs (encoded by TSFM [MIM 604723]) associated with a fatal mitochondrial encephalomyopathy in one pedigree and with fatal hypertrophic cardiomyopathy in another, and we demonstrate the molecular basis for the defect. Patient 1, a boy, was born at term as the second child of consanguineous Turkish parents by cesarean section performed on maternal indication. Birth weight (3,140 g), length (50 cm), and head circumference (36 cm) were age appropriate. Muscular hypotonia, sucking weakness, and a severe lactic acidosis (pH 6.8; base excess −26; lactic acid 42 mM [control <2.1 mM]) were the initial clinical signs and symptoms, followed by rhabdomyolysis with creatine kinase values up to 7,252 U/liter (control <248 U/liter). The initial lactate:pyruvate (L:P) ratio was 92 (control 12–15). During life, lactic acid dropped to values between 4 and 5.5 mM, with L:P ratios ∼25. Artificial ventilation was needed because of persistent dyspnea. On the 3rd day of life, generalized convulsions became obvious. The electroencephalogram showed a discontinuous pattern with bifrontal symmetrical sharper spikes. Repeated ultrasound imaging of the brain showed reduced gyri on day 4, plexus bleeding with enlarged ventricles on day 6, and abnormal signal intensity of the thalami on day 9. Echocardiographic examination on day 6 revealed a persistent ductus arteriosus Botalli, with left-to-right shunting, and a persistent foramen ovale. Septum thickness and contractility were age appropriate. On the basis of the suspicion of a mitochondrial disorder, a fibroblast culture was established and a skeletal muscle (m. vastus lateralis dextra) biopsy sample was obtained for measurements of the OXPHOS enzymes. Despite intensive treatment, the patient died of progressive encephalomyopathy and respiratory failure at age 7 wk. Patient 2, a girl, was the third child of second cousins once removed whose parents are of Kurdish Jewish origin. Two older children were healthy. The mother reported a paucity of fetal movements throughout the pregnancy. Delivery was vaginal, and birth weight was 2,600 g. Initial examination results were normal, and the patient was breast-fed. At age 36 h, apathy, irregular breathing, and severe muscular hypotonia were noted. Laboratory investigation revealed severe metabolic acidosis (pH 6.93; HCO3 −4.6; base excess −26). Serum lactate level was increased (17.6 mM; control <2.4 mM), as were blood ketone levels (3-OH-butyrate 4,803 μM; acetoacetate 257 μM; control values <200 μM and <100 μM, respectively) and serum ammonia levels (268 μM; control 60), two proteins with known function in mitochondrial translation were identified within these regions—the mitochondrial ribosomal protein S35 and the mitochondrial translation elongation factor EFTs (encoded by TSFM). Sequence analysis of TSFM revealed the same homozygous C997T mutation in exon 7 in both patients (fig. 2). A BLAST search showed that the C997T mutation was not present in any reported human EST, and the mutation was not found in the other 14 patients tested or in 135 controls. The mutation was heterozygous in the parents in both families and in the two healthy sibs of patient 2 (data not shown). A sib of patient 1 was homozygous wild type (data not shown). Analysis of microsatellite markers on chromosome 12 demonstrated that the mutation did not arise on a common haplotype in the two patients (fig. 2D). Human TSFM appears to be alternatively spliced, since exon 5 was not present in any of the patient or control cDNAs analyzed. Exon 5 is present in a single EST sequence in the human database, derived form bone marrow of a patient with acute myelogenous leukemia. The mutation predicts an Arg333Trp substitution in the C-terminal domain of EFTs, and sequence alignment of this protein from several species shows that the mutated amino acid is highly conserved from human to bacteria (fig. 2B). This Arg residue makes favorable hydrophobic and electrostatic interactions within the helical part of subdomain C (fig. 3). Mutational analysis using the crystal structure of the bovine EFTu•EFTs complex shows that it is not possible to introduce a Trp at this position without serious clashes between the Trp side chain and the backbone atoms in the immediate surroundings. The close-up of the region of Ts that harbors the mutation shows three preferred rotamers of Trp, representing three different energy minima (fig. 3, lower panel). The rotamer that is predicted to be the most favorable in the absence of a surrounding bumps into the helical domain immediately beneath it, and the other two rotamers that normally are energetically favorable bump into other local residues, as indicated in the legend of figure 3. The mutation is predicted to disrupt the interaction between the long helix and two strands in subdomain C, moving the domain that holds the long helix away from domain III of EFTu 19Jeppesen MG Navratil T Spremulli LL Nyborg J Crystal structure of the bovine mitochondrial elongation factor Tu.Ts complex.J Biol Chem. 2005; 280: 5071-5081Crossref PubMed Scopus (38) Google Scholar and preventing it from contributing to EFTu•EFTs binding (fig. 3). To directly test whether the lack of a functional EFTs protein was the cause of the defective mitochondrial translation, we transduced patient fibroblasts with a retroviral vector expressing the wild-type TSFM cDNA. Since EFTs and EFTu form a dimer in human cells, we also tested whether overexpression of wild-type EFTu could suppress the translation defect. Overexpression of either of these constructs (two-to-threefold for EFTu and three-to-fourfold for EFTs; fig. 4) had a dominant negative effect on mitochondrial translation in control cells. In independent experiments with different control lines, global mitochondrial translation was 61% ± 18% (five independent transductions in two control lines) of control in cells overexpressing EFTu and 74% ± 14% of control in cells overexpressing EFTs (four independent transductions in two control lines). In the results shown in figure 1, global mitochondrial translation is 68% and 75% of the control in cells overexpressing EFTu and EFTs, respectively. A relatively modest increase in global mitochondrial protein synthesis was observed in patient cells overexpressing EFTs compared with the appropriate control; however, there was a significant increase in the rate of synthesis of several polypeptides of complex I and complex IV, and the rate of synthesis of ATP6 and ATP8 was significantly reduced relative to that observed in untransduced patient fibroblasts (table 2 and fig. 1). The results were generally similar in patient cells overexpressing EFTu. Thus, overexpression of either translation elongation factor could partially suppress the translation defect in the patient cells. To investigate the mechanism of suppression of the translation defect by EFTu, we analyzed the steady-state levels of the translation elongation factors by immunoblot analysis. EFTs and EFTu in patient fibroblasts were reduced to 25% and 40% of control levels, respectively (fig. 4). Overexpression of EFTu partially rescued EFTs levels, and EFTu levels were completely rescued by overexpression of EFTs. Overexpression of EFTs also slightly reduced EFTu levels in control cells. The levels of another mitochondrial translation elongation factor, EFG1, were unaltered in patient cells and were not changed in any of the cells overexpressing EFTs or EFTu. To investigate whether the increased mitochondrial protein synthesis in patient cells overexpressing EFTu or EFTs resulted in increased OXPHOS function, we examined the assembly of the OXPHOS complexes by blue-native PAGE analysis (fig. 5). Patient fibroblasts showed reduced levels of fully assembled complexes I, IV, and V but normal levels of complex II and III, consistent with the deficiencies demonstrated by enzyme activity assays (table 1). The assembly of all three affected complexes was restored to control levels (in comparison with control fibroblasts overexpressing the same construct) by overexpression of either elongation factor (fig. 5). Functional complementation of the biochemical defect in patient cells overexpressing the elongation factors was also demonstrated by immunoblot analysis, which showed similar steady-state levels of both nuclear- and mitochondrial-encoded subunits of the OXPHOS complexes in patient cells and controls (fig. 4). Interestingly, overexpression of both factors led to slightly higher steady-state levels of some complex I subunits (39 kDa, 49 kDa, and ND1) in the patient cell line compared with controls and a markedly decreased expression of MnSOD, the mitochondrial superoxide dismutase. This study firmly establishes mutations in the mitochondrial translation elongation factor EFTs as the cause of disease in two unrelated patients with mitochondrial dysfunction due to a combined deficiency of OXPHOS enzymes. Several pieces of evidence support this conclusion. First, pulse labeling of the mitochondrial translation products in patient fibroblasts identified a global translation defect in both patients, leading to a failure to assemble adequate amounts of three of the OXPHOS complexes containing subunits encoded in mtDNA. This phenotype could be rescued by retroviral expression of the wild-type cDNAs for eit