Mutations in the cytochrome c1 subunit of respiratory chain complex III cause insulin-responsive hyperglycemia and recurrent ketoacidosis. J. Christodoulou1,2,3,4, P. Gaignard5, M. Menezes3,4, M. Schiff5,6,7, A. Bayot6,7, M. Rak6,7, H. Ogier de Baulny8, C.-H. Su9, M. Gilleron10,11, A. Lombes10,11, H. Abida9, A. Tzagoloff9, L. Riley4, S. T. Cooper3,12, K. Mina13, M. R. Davis14, R. J. N. Allcock15,16, N. Kresoje15, N. G. Laing17, D. R. Thorburn18,19, A. Slama5, P. Rustin6,7 1) Western Sydney Genetics Program, Children's Hospital at Westmead, Westmead, NSW, Australia; 2) Discipline of Genetic Medicine, Sydney Medical School, University of Sydney, Australia; 3) Discipline of Paediatrics and Child Health, Sydney Medical School, University of Sydney, Australia; 4) Genetic Metabolic Disorders Research Unit, Research Institute, Childrens Hospital at Westmead, Sydney, NSW, Australia; 5) Laboratoire de Biochimie, APHP Hôpital de Bicêtre, 94275 Le Kremlin Bicêtre cedex, France; 6) INSERM UMR 676, Hôpital Robert Debré, 75019, Paris, France; 7) Université Paris 7, Faculté de Médecine Denis Diderot, Paris, France; 8) Reference Center for Inherited Metabolic Diseases, Hôpital Robert Debré, APHP, 75019, Paris, France; 9) Department of Biological Sciences, Columbia University, New York, NY 10027, USA; 10) INSERM, UMRS 1016, Institut Cochin, F-75014 Paris, France; 11) AP-HP, Hôpital de La Salpêtrière, Service de Biochimie Métabolique et Centre de Génétique Moléculaire et Chromosomique, 75651 Paris, France; 12) Institute for Neuroscience and Muscle Research, Childrens Hospital at Westmead, Sydney, NSW, Australia,; 13) Department of Molecular Genetics, PathWest and School of Pathology and Laboratory Medicine, The University of Western Australia, Nedlands, Perth, Western Australia; 14) Neurogenetic Laboratory, Department of Anatomical Pathology, Pathwest Laboratory Medicine WA, Royal Perth Hospital, Perth, Western Australia,; 15) Lotterywest State Biomedical Facility Genomics and School of Pathology and Laboratory Medicine, University of Western Australia, Nedlands, Perth, Western Australia; 16) Department of Clinical Immunology, Pathwest Laboratory Medicine WA, Royal Perth Hospital, Perth, Western Australia; 17) Centre for Medical Research, University of Western Australia & Western Australian Institute for Medical Research, QEII Medical Centre, Nedlands, Perth, Western Australia; 18) Murdoch Childrens Research Institute and Victorian Clinical Genetics Services, Royal Childrens Hospital, Flemington Road, Parkville, Melbourne Melbourne, VIC, Australia; 19) Department of Paediatrics, University of Melbourne, Melbourne, VIC, Australia.

   Primary complex III (CIII) deficiency is an uncommon and clinically highly variable mitochondrial respiratory chain disorder. Using a combination of whole exome and candidate gene sequencing, we have identified mutations in the CYC1 gene (c.643C>T; p.Leu215Phe and c.288G>T; p.Trp96Cys) encoding the cytochrome c1 subunit of CIII, in two unrelated patients (Sri Lankan and Lebanese, both the offspring of consanguineous unions), presenting with recurrent episodes of severe keto- and lactic acidosis and insulin-responsive hyperglycemia. A functional deficiency of CIII in skeletal muscle was associated with reduced levels of CYC1 protein in skeletal muscle and fibroblasts from the patients, suggesting that the mutation make the CYC1 protein unstable. In addition, there were reduced levels of assembly-dependent CIII subunits (core 2), and of assembly-dependent subunits of CI (subunit NDUFB8) and CIV (subunit1), suggesting that CYC1 mutations affect the in vivo stability or assembly of the respirasome. We then examined the effect of the two CYC1 mutations on mitochondrial function in Saccharomyces cerevisiae CYT1. Transformants harboring either mutant gene in single copy failed to grow on non-fermentable carbon sources, and had only 6% of NADH-cytochrome c reductase activity compared to wild-type, confirming that the two mutations severely affect respiratory chain function. Finally, lentiviral rescue experiments in patient fibroblasts showed that wild-type CYC1 corrected complex III activity (patient 1), and resulted in significant increases in protein levels of CYC1, CIII subunits (core 2) and assembly-dependent subunits of CI and CIV (patient 2). Together, our findings demonstrate that the mutations we have identified have a deleterious effect on cytochrome c1 stability and complex III assembly, and highlight that whole exome sequencing has matured to the point where it should be considered as a first line molecular screening when one of multiple genes could be causative for a particular phenotype.

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