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Case Reports
. 2014 Dec 4;95(6):708-20.
doi: 10.1016/j.ajhg.2014.10.017. Epub 2014 Nov 26.

Mutations in GTPBP3 cause a mitochondrial translation defect associated with hypertrophic cardiomyopathy, lactic acidosis, and encephalopathy

Robert Kopajtich  1 , Thomas J Nicholls  2 , Joanna Rorbach  2 , Metodi D Metodiev  3 , Peter Freisinger  4 , Hanna Mandel  5 , Arnaud Vanlander  6 , Daniele Ghezzi  7 , Rosalba Carrozzo  8 , Robert W Taylor  9 , Klaus Marquard  10 , Kei Murayama  11 , Thomas Wieland  12 , Thomas Schwarzmayr  12 , Johannes A Mayr  13 , Sarah F Pearce  2 , Christopher A Powell  2 , Ann Saada  14 , Akira Ohtake  15 , Federica Invernizzi  7 , Eleonora Lamantea  7 , Ewen W Sommerville  9 , Angela Pyle  16 , Patrick F Chinnery  16 , Ellen Crushell  17 , Yasushi Okazaki  18 , Masakazu Kohda  19 , Yoshihito Kishita  20 , Yoshimi Tokuzawa  20 , Zahra Assouline  21 , Marlène Rio  21 , François Feillet  22 , Bénédict Mousson de Camaret  23 , Dominique Chretien  3 , Arnold Munnich  24 , Björn Menten  25 , Tom Sante  25 , Joél Smet  6 , Luc Régal  26 , Abraham Lorber  27 , Asaad Khoury  27 , Massimo Zeviani  28 , Tim M Strom  12 , Thomas Meitinger  29 , Enrico S Bertini  8 , Rudy Van Coster  6 , Thomas Klopstock  30 , Agnès Rötig  3 , Tobias B Haack  12 , Michal Minczuk  31 , Holger Prokisch  32
Affiliations
Case Reports

Mutations in GTPBP3 cause a mitochondrial translation defect associated with hypertrophic cardiomyopathy, lactic acidosis, and encephalopathy

Robert Kopajtich et al. Am J Hum Genet. .

Abstract

Respiratory chain deficiencies exhibit a wide variety of clinical phenotypes resulting from defective mitochondrial energy production through oxidative phosphorylation. These defects can be caused by either mutations in the mtDNA or mutations in nuclear genes coding for mitochondrial proteins. The underlying pathomechanisms can affect numerous pathways involved in mitochondrial physiology. By whole-exome and candidate gene sequencing, we identified 11 individuals from 9 families carrying compound heterozygous or homozygous mutations in GTPBP3, encoding the mitochondrial GTP-binding protein 3. Affected individuals from eight out of nine families presented with combined respiratory chain complex deficiencies in skeletal muscle. Mutations in GTPBP3 are associated with a severe mitochondrial translation defect, consistent with the predicted function of the protein in catalyzing the formation of 5-taurinomethyluridine (τm(5)U) in the anticodon wobble position of five mitochondrial tRNAs. All case subjects presented with lactic acidosis and nine developed hypertrophic cardiomyopathy. In contrast to individuals with mutations in MTO1, the protein product of which is predicted to participate in the generation of the same modification, most individuals with GTPBP3 mutations developed neurological symptoms and MRI involvement of thalamus, putamen, and brainstem resembling Leigh syndrome. Our study of a mitochondrial translation disorder points toward the importance of posttranscriptional modification of mitochondrial tRNAs for proper mitochondrial function.

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Figures

Figure 1
Figure 1
GTPBP3 Mutation Status and Gene Structure (A) Pedigrees of nine families with mutations in GTPBP3. (B) Gene structure of GTPBP3 with known protein domains of the gene product and localization and conservation of amino acid residues affected by mutations. Black and orange text indicate exonic and intronic variants. Intronic regions are not drawn to scale. Coloring in the sequence alignment represents the identity of amino acid residues.
Figure 2
Figure 2
Brain MRI of Affected Individuals #72425, #82790, and #75168 (A) MRI of individual #72425 shows small T2 hyperintensities in the anterior thalamus bilaterally (arrow). (B) In individual #82790, T2-weighted MRI shows bilateral hyperintensities in the putamen (arrowhead) and weakly also in the anterior thalamus (arrow). (C) T2-weighted MRI of individual #75168 shows pronounced bilateral hyperintensities affecting the whole thalamus (arrow, axial view at the left) and extending to the mesencephalon (arrowhead, sagittal view at the right).
Figure 3
Figure 3
Analysis of Respiration Defects and GTPBP3 Protein Levels in Fibroblast Cell Lines (A) Oxygen consumption rate (OCR) of fibroblast cell lines from five affected individuals and five control subjects cultured in high-glucose (Glc) medium. Each analysis was performed in more than 15 replicates. Control one (C1) was measured five time at different passage numbers (C1.1–1.5, NHDFneo, Lonza). OCR was expressed as percentage relative to the average of all controls. Cells from individuals #75191 and #76671 showed a significant reduction of oxygen consumption whereas cells from individuals #49665, #72425, and #66143 presented no defective respiration. Error bar indicates 1 SD; ∗∗∗p < 0.001. (B) Oxygen consumption rate of fibroblast cells cultured in galactose (Gal) growth medium. The average increase of OCR from five control cells cultured in galactose-containing medium compared to glucose-containing medium was 107%. Cell lines from individuals #49665, #72425, and #66143 show significant lower increase in OCR. Lentiviral expression of wtGTPBP3 in cells from individual #66143 significantly increases the change in OCR although it has only little effect in control cells (C6-T). Error bar indicates 1 SD; ∗∗∗p < 0.001. (C) Activities of respiratory chain complexes I and IV (expressed as ratio to CII activity) are decreased in individual #83904 cells transfected by electroporation with empty vector (pIRES2-EGFP) according to the manufacturer’s protocol (LONZA) but are improved upon expression of GTPBP3 cDNAs from the same plasmid. Measurements were performed as previously described. Error bar indicates 1 SD. Activity in controls was set as 100%. ∗∗p < 0.01, ∗∗p < 0.001. (D) Levels of GTPBP3 were reduced in cells from individuals #49665, #75191, and #66143 and elevated after transduction with wtGTPBP3 cDNA. MRPS18B and MRPL3 served as mitochondrial loading controls.
Figure 4
Figure 4
Analysis of Mitochondrial Protein Synthesis in Primary Fibroblasts and in HeLa Cells Treated with RNAi against GTPBP3 (A) [35S]methionine metabolic labeling of mitochondrial proteins in fibroblasts. Products of mitochondrial translation were labeled with [35S]methionine for 30 min, separated by a 4%–12% gradient SDS-PAGE, and visualized by autoradiography. To validate equal protein loading, a small section of the gel was stained with Coomassie (CGS). Fibroblasts from individuals #49665, #66143, and #75191 demonstrate significant inhibition of mitochondrial protein synthesis although translation in cells from individual #72425 is not affected. (B) Quantification of radiolabelled products of mitochondrial translation. Incorporation of [35S] as in (A) was quantified by ImageQuant software after exposure to a PhosphorImager screen from three independent experiments. Error bar indicates 1 SD. (C) Downregulation of GTPBP3 in HeLa cells via RNA interference. Immunoblot analysis of total HeLa cell lysate transfected with two different siRNA to GTPBP3 show decreased level of GTPBP3 upon RNAi treatment for 6 days. siRNA to GFP was used as transfection control. Asterisk indicates nonspecific band recognized by anti-GTPBP3 antibody in HeLa cells. β-actin serves as a loading control. Two different siRNA duplexes targeting GTPBP3 were used, 1 and 2. (D) Mitochondrial translation in HeLa cells upon GTPBP3 downregulation. HeLa cells were transfected for 6 days with siRNA against GTPBP3 and subjected to [35S]methionine metabolic labeling. Inactivation of GTPBP3 leads to the reduced efficiency of mitochondrial translation. Two different siRNA duplexes targeting GTPBP3 were used, 1 and 2.
Figure 5
Figure 5
Immunoblot Analysis of OXPHOS Proteins in Fibroblasts 10 μg of detergent-solubilized total cell extract was subjected to immunoblot analysis of OXPHOS components. Amounts of SDHA (complex II) and ATP5A (ATPase) were unchanged in all individuals. In cells from individuals #72425, #75191, and #76671, a reduction of COXIV (complex IV) was observed. Cells from individuals #49665, #72425, and #75191 showed decreased levels of NDUFB8 (complex I). Antibodies used: mouse antibodies against SDHA (ab14715), NDUFB8 (ab110242), ATP5A (ab14748), and rabbit antibodies against COXIV (ab16056) from Abcam and rabbit anti GTPBP3 (HPA042158) from SIGMA Aldrich.
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References

    1. Boczonadi V., Horvath R. Mitochondria: impaired mitochondrial translation in human disease. Int. J. Biochem. Cell Biol. 2014;48:77–84. - PMC - PubMed
    1. Osawa S. ASM Press; Washington, DC: 1995. Evolution of the Genetic Code.
    1. Suzuki T. Biosynthesis and function of tRNA wobble modifications. Top. Curr. Genet. 2005;12:23–69.
    1. Suzuki T., Suzuki T. A complete landscape of post-transcriptional modifications in mammalian mitochondrial tRNAs. Nucleic Acids Res. 2014;42:7346–7357. - PMC - PubMed
    1. Colby G., Wu M., Tzagoloff A. MTO1 codes for a mitochondrial protein required for respiration in paromomycin-resistant mutants of Saccharomyces cerevisiae. J. Biol. Chem. 1998;273:27945–27952. - PubMed

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