Isolated complex I deficiency is a common biochemical phenotype observed in pediatric mitochondrial disease and often arises as a consequence of pathogenic variants affecting one of the ∼65 genes encoding the complex I structural subunits or assembly factors. Such genetic heterogeneity means that application of next-generation sequencing technologies to undiagnosed cohorts has been a catalyst for genetic diagnosis and gene-disease associations. We describe the clinical and molecular genetic investigations of four unrelated children who presented with neuroradiological findings and/or elevated lactate levels, highly suggestive of an underlying mitochondrial diagnosis. Next-generation sequencing identified bi-allelic variants in NDUFA6, encoding a 15 kDa LYR-motif-containing complex I subunit that forms part of the Q-module. Functional investigations using subjects' fibroblast cell lines demonstrated complex I assembly defects, which were characterized in detail by mass-spectrometry-based complexome profiling. This confirmed a marked reduction in incorporated NDUFA6 and a concomitant reduction in other Q-module subunits, including NDUFAB1, NDUFA7, and NDUFA12. Lentiviral transduction of subjects' fibroblasts showed normalization of complex I. These data also support supercomplex formation, whereby the ∼830 kDa complex I intermediate (consisting of the P- and Q-modules) is in complex with assembled complex III and IV holoenzymes despite lacking the N-module. Interestingly, RNA-sequencing data provided evidence that the consensus RefSeq accession number does not correspond to the predominant transcript in clinically relevant tissues, prompting revision of the NDUFA6 RefSeq transcript and highlighting not only the importance of thorough variant interpretation but also the assessment of appropriate transcripts for analysis.

Cambridge University Hospitals NHS Foundation Trust Cambridge Biomedical Campus Cambridge CB2 0QQ UK

Cologne Center for Genomics University of Cologne 50931 Cologne Germany

Department of Biochemistry and Molecular Biology Monash Biomedicine Discovery Institute Monash University 3800 Melbourne Australia

Department of Inherited Metabolic Disease Guy's and St Thomas' NHS Foundation Trusts Evelina London Children's Hospital London SE1 7EH UK

Department of Medical Genetics Cambridge Institute for Medical Research University of Cambridge Cambridge CB2 0XY UK

Department of Medical Genetics Cambridge Institute for Medical Research University of Cambridge Cambridge CB2 0XY UK; Cambridge University Hospitals NHS Foundation Trust Cambridge Biomedical Campus Cambridge CB2 0QQ UK; NIHR BioResource Rare Diseases Cambridge University Hospitals NHS Foundation Trust Cambridge Biomedical Campus Cambridge CB2 0QQ UK

Department of Medical Genetics Cambridge Institute for Medical Research University of Cambridge Cambridge CB2 0XY UK; NIHR BioResource Rare Diseases Cambridge University Hospitals NHS Foundation Trust Cambridge Biomedical Campus Cambridge CB2 0QQ UK

Department of Neuroradiology Oxford University Hospitals NHS Foundation Trust Oxford OX3 9DU UK

Department of Pediatrics and Adolescent Medicine Division of Pediatric Neurology University Medical Center Göttingen 37075 Göttingen Germany

Department of Pediatrics Drammen Sykehus 3004 Drammen Norway

Department of Pediatrics Salzburger Landeskliniken and Paracelsus Medical University 5020 Salzburg Austria

Functional Proteomics SFB 815 Core Unit Faculty of Medicine Goethe University 60590 Frankfurt am Main Germany

Functional Proteomics SFB 815 Core Unit Faculty of Medicine Goethe University 60590 Frankfurt am Main Germany; German Center of Cardiovascular Research Partner Site Rhein Main 60590 Frankfurt am Main Germany; Cluster of Excellence Macromolecular Complexes Goethe Universität 60590 Frankfurt am Main Germany

Institute of Human Genetics Technische Universität München 81675 Munich Germany; Institute of Human Genetics Helmholtz Zentrum München 85764 Neuherberg Germany

Institute of Physiology Czech Academy of Sciences 142 20 Prague Czech Republic

National Institute for Health Research Oxford Biomedical Research Centre Wellcome Centre for Human Genetics University of Oxford Oxford OX3 7BN UK

Nuffield Department of Women's and Reproductive Health University of Oxford Oxford OX3 9DU UK

Oxford Medical Genetics Laboratories Oxford University Hospitals NHS Foundation Trust Churchill Hospital Oxford OX3 7LE UK

Trevor Mann Baby Unit Brighton and Sussex University Hospitals NHS Trust Brighton BN2 5BE UK

Wellcome Centre for Mitochondrial Research Institute of Neuroscience Medical School Newcastle University Newcastle upon Tyne NE2 4HH UK

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Formosa L.E., Dibley M.G., Stroud D.A., Ryan M.T. Building a complex complex: Assembly of mitochondrial respiratory chain complex I. Semin. Cell Dev. Biol. 2018;76:154–162. PubMed

Calvo S.E., Clauser K.R., Mootha V.K. MitoCarta2.0: An updated inventory of mammalian mitochondrial proteins. Nucleic Acids Res. 2016;44(D1):D1251–D1257. PubMed PMC

Frazier A.E., Thorburn D.R., Compton A.G. Mitochondrial energy generation disorders: Genes, mechanisms and clues to pathology. J. Biol. Chem. 2017 Published online December 12, 2017. PubMed PMC

Piekutowska-Abramczuk D., Assouline Z., Mataković L., Feichtinger R.G., Koňařiková E., Jurkiewicz E., Stawiński P., Gusic M., Koller A., Pollak A. NDUFB8 mutations cause mitochondrial complex I deficiency in individuals with Leigh-like encephalomyopathy. Am. J. Hum. Genet. 2018;102:460–467. PubMed PMC

Haack T.B., Kopajtich R., Freisinger P., Wieland T., Rorbach J., Nicholls T.J., Baruffini E., Walther A., Danhauser K., Zimmermann F.A. ELAC2 mutations cause a mitochondrial RNA processing defect associated with hypertrophic cardiomyopathy. Am. J. Hum. Genet. 2013;93:211–223. PubMed PMC

Freisinger P., Haack T., Kopajtich R., Johannes M., Ahting U., Sperl W., Plecko B., Wilichowski E., Meitinger T., Prokisch H. Mitochondriopathy due to mutations in MTFMT: A predominant neurologic phenotype. Neuropediatrics. 2013;44 FV12_03.

Ohtake A., Murayama K., Mori M., Harashima H., Yamazaki T., Tamaru S., Yamashita Y., Kishita Y., Nakachi Y., Kohda M. Diagnosis and molecular basis of mitochondrial respiratory chain disorders: Exome sequencing for disease gene identification. Biochim. Biophys. Acta. 2014;1840:1355–1359. PubMed

Sobreira N., Schiettecatte F., Valle D., Hamosh A. GeneMatcher: A matching tool for connecting investigators with an interest in the same gene. Hum. Mutat. 2015;36:928–930. PubMed PMC

Alston C.L., Howard C., Oláhová M., Hardy S.A., He L., Murray P.G., O’Sullivan S., Doherty G., Shield J.P., Hargreaves I.P. A recurrent mitochondrial p.Trp22Arg NDUFB3 variant causes a distinctive facial appearance, short stature and a mild biochemical and clinical phenotype. J. Med. Genet. 2016;53:634–641. PubMed PMC

Ploski R., Pollak A., Müller S., Franaszczyk M., Michalak E., Kosinska J., Stawinski P., Spiewak M., Seggewiss H., Bilinska Z.T. Does p.Q247X in TRIM63 cause human hypertrophic cardiomyopathy? Circ. Res. 2014;114:e2–e5. PubMed

Lunter G., Goodson M. Stampy: A statistical algorithm for sensitive and fast mapping of Illumina sequence reads. Genome Res. 2011;21:936–939. PubMed PMC

Rimmer A., Phan H., Mathieson I., Iqbal Z., Twigg S.R.F., Wilkie A.O.M., McVean G., Lunter G., WGS500 Consortium Integrating mapping-, assembly- and haplotype-based approaches for calling variants in clinical sequencing applications. Nat. Genet. 2014;46:912–918. PubMed PMC

Carss K.J., Arno G., Erwood M., Stephens J., Sanchis-Juan A., Hull S., Megy K., Grozeva D., Dewhurst E., Malka S., NIHR-BioResource Rare Diseases Consortium Comprehensive rare variant analysis via whole-genome sequencing to determine the molecular pathology of inherited retinal disease. Am. J. Hum. Genet. 2017;100:75–90. PubMed PMC

Calabrese C., Simone D., Diroma M.A., Santorsola M., Guttà C., Gasparre G., Picardi E., Pesole G., Attimonelli M. MToolBox: A highly automated pipeline for heteroplasmy annotation and prioritization analysis of human mitochondrial variants in high-throughput sequencing. Bioinformatics. 2014;30:3115–3117. PubMed PMC

Richards S., Aziz N., Bale S., Bick D., Das S., Gastier-Foster J., Grody W.W., Hegde M., Lyon E., Spector E., ACMG Laboratory Quality Assurance Committee Standards and guidelines for the interpretation of sequence variants: A joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet. Med. 2015;17:405–424. PubMed PMC

Kircher M., Witten D.M., Jain P., O’Roak B.J., Cooper G.M., Shendure J. A general framework for estimating the relative pathogenicity of human genetic variants. Nat. Genet. 2014;46:310–315. PubMed PMC

Lee C., Kalmar L., Xue B., Tompa P., Daughdrill G.W., Uversky V.N., Han K.H. Contribution of proline to the pre-structuring tendency of transient helical secondary structure elements in intrinsically disordered proteins. Biochim. Biophys. Acta. 2014;1840:993–1003. PubMed

Thermann R., Neu-Yilik G., Deters A., Frede U., Wehr K., Hagemeier C., Hentze M.W., Kulozik A.E. Binary specification of nonsense codons by splicing and cytoplasmic translation. EMBO J. 1998;17:3484–3494. PubMed PMC

Kremer L.S., Bader D.M., Mertes C., Kopajtich R., Pichler G., Iuso A., Haack T.B., Graf E., Schwarzmayr T., Terrile C. Genetic diagnosis of Mendelian disorders via RNA sequencing. Nat. Commun. 2017;8:15824. PubMed PMC

Pesta D., Gnaiger E. High-resolution respirometry: OXPHOS protocols for human cells and permeabilized fibers from small biopsies of human muscle. Methods Mol. Biol. 2012;810:25–58. PubMed

Oláhová M., Hardy S.A., Hall J., Yarham J.W., Haack T.B., Wilson W.C., Alston C.L., He L., Aznauryan E., Brown R.M. LRPPRC mutations cause early-onset multisystem mitochondrial disease outside of the French-Canadian population. Brain. 2015;138:3503–3519. PubMed PMC

Zerbetto E., Vergani L., Dabbeni-Sala F. Quantification of muscle mitochondrial oxidative phosphorylation enzymes via histochemical staining of blue native polyacrylamide gels. Electrophoresis. 1997;18:2059–2064. PubMed

Neuhoff V., Arold N., Taube D., Ehrhardt W. Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis. 1988;9:255–262. PubMed

Stroud D.A., Maher M.J., Lindau C., Vögtle F.N., Frazier A.E., Surgenor E., Mountford H., Singh A.P., Bonas M., Oeljeklaus S. COA6 is a mitochondrial complex IV assembly factor critical for biogenesis of mtDNA-encoded COX2. Hum. Mol. Genet. 2015;24:5404–5415. PubMed

Wirth C., Brandt U., Hunte C., Zickermann V. Structure and function of mitochondrial complex I. Biochim. Biophys. Acta. 2016;1857:902–914. PubMed

Fuhrmann D.C., Wittig I., Dröse S., Schmid T., Dehne N., Brüne B. Degradation of the mitochondrial complex I assembly factor TMEM126B under chronic hypoxia. Cell. Mol. Life Sci. 2018;75:3051–3067. PubMed PMC

Heide H., Bleier L., Steger M., Ackermann J., Dröse S., Schwamb B., Zörnig M., Reichert A.S., Koch I., Wittig I., Brandt U. Complexome profiling identifies TMEM126B as a component of the mitochondrial complex I assembly complex. Cell Metab. 2012;16:538–549. PubMed

Angerer H., Radermacher M., Mańkowska M., Steger M., Zwicker K., Heide H., Wittig I., Brandt U., Zickermann V. The LYR protein subunit NB4M/NDUFA6 of mitochondrial complex I anchors an acyl carrier protein and is essential for catalytic activity. Proc. Natl. Acad. Sci. USA. 2014;111:5207–5212. PubMed PMC

Ogilvie I., Kennaway N.G., Shoubridge E.A. A molecular chaperone for mitochondrial complex I assembly is mutated in a progressive encephalopathy. J. Clin. Invest. 2005;115:2784–2792. PubMed PMC

Mckenzie M., Ryan M.T. Assembly factors of human mitochondrial complex I and their defects in disease. IUBMB Life. 2010;62:497–502. PubMed

Lazarou M., McKenzie M., Ohtake A., Thorburn D.R., Ryan M.T. Analysis of the assembly profiles for mitochondrial- and nuclear-DNA-encoded subunits into complex I. Mol. Cell. Biol. 2007;27:4228–4237. PubMed PMC

Stroud D.A., Surgenor E.E., Formosa L.E., Reljic B., Frazier A.E., Dibley M.G., Osellame L.D., Stait T., Beilharz T.H., Thorburn D.R. Accessory subunits are integral for assembly and function of human mitochondrial complex I. Nature. 2016;538:123–126. PubMed

Wu M., Gu J., Guo R., Huang Y., Yang M. Structure of mammalian respiratory supercomplex I1III2IV1. Cell. 2016;167:1598–1609.e10. PubMed

Alston C.L., Compton A.G., Formosa L.E., Strecker V., Oláhová M., Haack T.B., Smet J., Stouffs K., Diakumis P., Ciara E. Biallelic mutations in TMEM126B cause severe complex I deficiency with a variable clinical phenotype. Am. J. Hum. Genet. 2016;99:217–227. PubMed PMC

Sánchez-Caballero L., Ruzzenente B., Bianchi L., Assouline Z., Barcia G., Metodiev M.D., Rio M., Funalot B., van den Brand M.A., Guerrero-Castillo S. Mutations in complex I assembly factor TMEM126B result in muscle weakness and isolated complex I deficiency. Am. J. Hum. Genet. 2016;99:208–216. PubMed PMC

Punzi G., Porcelli V., Ruggiu M., Hossain M.F., Menga A., Scarcia P., Castegna A., Gorgoglione R., Pierri C.L., Laera L. SLC25A10 biallelic mutations in intractable epileptic encephalopathy with complex I deficiency. Hum. Mol. Genet. 2018;27:499–504. PubMed PMC

Schottmann G., Picker-Minh S., Schwarz J.M., Gill E., Rodenburg R.J.T., Stenzel W., Kaindl A.M., Schuelke M. Recessive mutation in EXOSC3 associates with mitochondrial dysfunction and pontocerebellar hypoplasia. Mitochondrion. 2017;37:46–54. PubMed

Haack T.B., Haberberger B., Frisch E.M., Wieland T., Iuso A., Gorza M., Strecker V., Graf E., Mayr J.A., Herberg U. Molecular diagnosis in mitochondrial complex I deficiency using exome sequencing. J. Med. Genet. 2012;49:277–283. PubMed

Swalwell H., Kirby D.M., Blakely E.L., Mitchell A., Salemi R., Sugiana C., Compton A.G., Tucker E.J., Ke B.X., Lamont P.J. Respiratory chain complex I deficiency caused by mitochondrial DNA mutations. Eur. J. Hum. Genet. 2011;19:769–775. PubMed PMC

Multisystem mitochondrial diseases due to mutations in mtDNA-encoded subunits of complex I