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To access, purchase, authenticate, or subscribe to the full-text of this article, please visit this link: http://dx.doi.org/10.1016/j.ajhg.2017.07.005 Byline: Nicole J. Lake (1,2,23), Bryn D. Webb (3,4,5,23), David A. Stroud (6,23), Tara R. Richman (7,23), Benedetta Ruzzenente (8), Alison G. Compton (1,2), Hayley S. Mountford (1,2,9), Juliette Pulman (8), Coralie Zangarelli (8), Marlene Rio (10), Nathalie Boddaert (11), Zahra Assouline (10), Mingma D. Sherpa (3,5), Eric E. Schadt (3,5), Sander M. Houten (3,5), James Byrnes (12), Elizabeth M. McCormick (12), Zarazuela Zolkipli-Cunningham (12,13), Katrina Haude (14), Zhancheng Zhang (14), Kyle Retterer (14), Renkui Bai (14), Sarah E. Calvo (15,16,17), Vamsi K. Mootha (15,16,17), John Christodoulou (1,2), Agnes Rotig (8), Aleksandra Filipovska (7,18), Ingrid Cristian (19,20), Marni J. Falk (12,21,24), Metodi D. Metodiev (8,24), David R. Thorburn [david.thorburn@mcri.edu.au] (1,2,22,24,*) Keywords mitochondrial diseases; Leigh syndrome; mitochondrial ribosome; mitochondrial translation; MRPS34; respiratory chain; whole-exome sequencing; quantitative proteomics; ribosome profiling The synthesis of all 13 mitochondrial DNA (mtDNA)-encoded protein subunits of the human oxidative phosphorylation (OXPHOS) system is carried out by mitochondrial ribosomes (mitoribosomes). Defects in the stability of mitoribosomal proteins or mitoribosome assembly impair mitochondrial protein translation, causing combined OXPHOS enzyme deficiency and clinical disease. Here we report four autosomal-recessive pathogenic mutations in the gene encoding the small mitoribosomal subunit protein, MRPS34, in six subjects from four unrelated families with Leigh syndrome and combined OXPHOS defects. Whole-exome sequencing was used to independently identify all variants. Two splice-site mutations were identified, including homozygous c.321+1G>T in a subject of Italian ancestry and homozygous c.322-10G>A in affected sibling pairs from two unrelated families of Puerto Rican descent. In addition, compound heterozygous MRPS34 mutations were identified in a proband of French ancestry; a missense (c.37G>A [p.Glu13Lys]) and a nonsense (c.94C>T [p.Gln32.sup.*]) variant. We demonstrated that these mutations reduce MRPS34 protein levels and the synthesis of OXPHOS subunits encoded by mtDNA. Examination of the mitoribosome profile and quantitative proteomics showed that the mitochondrial translation defect was caused by destabilization of the small mitoribosomal subunit and impaired monosome assembly. Lentiviral-mediated expression of wild-type MRPS34 rescued the defect in mitochondrial translation observed in skin fibroblasts from affected subjects, confirming the pathogenicity of MRPS34 mutations. Our data establish that MRPS34 is required for normal function of the mitoribosome in humans and furthermore demonstrate the power of quantitative proteomic analysis to identify signatures of defects in specific cellular pathways in fibroblasts from subjects with inherited disease. Author Affiliation: (1) Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, VIC 3052, Australia (2) Department of Paediatrics, University of Melbourne, Melbourne, VIC 3052, Australia (3) Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA (4) Department of Pediatrics, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA (5) Icahn Institute for Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA (6) Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Clayton Campus, Melbourne, VIC 3800, Australia (7) Harry Perkins Institute of Medical Research and Centre for Medical Research, University of Western Australia, Nedlands, WA 6009, Australia (8) INSERM U1163, Universite Paris Descartes - Sorbonne Paris Cite, Institut Imagine, 75015 Paris, France (9) Department of Biological and Medical Sciences, Faculty of Health Sciences, Oxford Brookes University, Oxford OX3 0BP, UK (10) Departments of Pediatric, Neurology and Genetics, Hopital Necker-Enfants-Malades, 75015 Paris, France (11) Pediatric Radiology Department, Hopital Necker Enfants Malades, AP-HP, University Rene Descartes, PRES Sorbonne Paris Cite, INSERM U1000 and UMR 1163, Institut Imagine, 75015 Paris, France (12) Division of Human Genetics, Department of Pediatrics, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA (13) Division of Neurology, Department of Pediatrics, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA (14) GeneDx, Gaithersburg, MD 20877, USA (15) Howard Hughes Medical Institute, Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA (16) Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA (17) Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA (18) School of Molecular Sciences, University of Western Australia, Crawley, WA 6009, Australia (19) Nemours Children's Hospital, Orlando, FL 32827, USA (20) Division of Genetics, Arnold Palmer Hospital for Children, Orlando, FL 32806, USA (21) University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA (22) Victorian Clinical Genetic Services, Royal Children's Hospital, Melbourne, VIC 3052, Australia * Corresponding author Article History: Received 16 May 2017; Accepted 9 July 2017 (miscellaneous) Published: August 3, 2017; corrected online: March 19, 2018 (footnote)23 These authors contributed equally to this work (footnote)24 These authors contributed equally to this work