European Journal of Human Genetics (2011) 19, 769–775 & 2011 Macmillan Publishers Limited All rights reserved 1018-4813/11 www.nature.com/ejhg ARTICLE Respiratory chain complex I deficiency caused by mitochondrial DNA mutations Helen Swalwell1, Denise M Kirby2, Emma L Blakely1, Anna Mitchell1, Renato Salemi2,6, Canny Sugiana2,3,7, Alison G Compton2, Elena J Tucker2,3, Bi-Xia Ke2, Phillipa J Lamont4, Douglass M Turnbull1, Robert McFarland1, Robert W Taylor1 and David R Thorburn*,2,3,5 Defects of the mitochondrial respiratory chain are associated with a diverse spectrum of clinical phenotypes, and may be caused by mutations in either the nuclear or the mitochondrial genome (mitochondrial DNA (mtDNA)). Isolated complex I deficiency is the most common enzyme defect in mitochondrial disorders, particularly in children in whom family history is often consistent with sporadic or autosomal recessive inheritance, implicating a nuclear genetic cause. In contrast, although a number of recurrent, pathogenic mtDNA mutations have been described, historically, these have been perceived as rare causes of paediatric complex I deficiency. We reviewed the clinical and genetic findings in a large cohort of 109 paediatric patients with isolated complex I deficiency from 101 families. Pathogenic mtDNA mutations were found in 29 of 101 probands (29%), 21 in MTND subunit genes and 8 in mtDNA tRNA genes. Nuclear gene defects were inferred in 38 of 101 (38%) probands based on cell hybrid studies, mtDNA sequencing or mutation analysis (nuclear gene mutations were identified in 22 probands). Leigh or Leigh-like disease was the most common clinical presentation in both mtDNA and nuclear genetic defects. The median age at onset was higher in mtDNA patients (12 months) than in patients with a nuclear gene defect (3 months). However, considerable overlap existed, with onset varying from 0 to 460 months in both groups. Our findings confirm that pathogenic mtDNA mutations are a significant cause of complex I deficiency in children. In the absence of parental consanguinity, we recommend whole mitochondrial genome sequencing as a key approach to elucidate the underlying molecular genetic abnormality. European Journal of Human Genetics (2011) 19, 769–775; doi:10.1038/ejhg.2011.18; published online 2 March 2011 Keywords: respiratory chain; complex I; mitochondrial DNA; mutation; genetic counselling INTRODUCTION NDUFS3, NDUFS4, NDUFS6, NDUFS7, NDUFS8, NDUFV1, Isolated complex I deficiency is the most frequently observed mito- NDUFV2, NDUFA2 and NDUFA11), 1 of the 2 chromosome X chondrial respiratory chain disorder, and is associated with a wide genes encoding complex I subunits (NDUFA1) and 9 autosomal range of clinical presentations, including marked and often fatal lactic genes encoding proteins involved in complex I assembly, namely acidosis, cardiomyopathy, leukoencephalopathy, pure myopathy and NDUFAF1 (CIA30), NDUFAF2 (B17.2L), NDUFAF3, NDUFAF4, hepatopathy with tubulopathy.1–3 Complex I deficiency is one of the C8orf38, C20orf7, NUBPL, FOXRED1 and ACAD9.10–16 Mutations in most common mitochondrial disorders of childhood; onset is usually nuclear genes involved in mtDNA maintenance, in particular POLG, early and death often occurs within the first year of life.4–7 which encodes the catalytic subunit of the mitochondrial polymerase Defining the molecular basis of complex I deficiency is a difficult g, may also cause isolated complex I deficiency in patients with Alpers’ task, not least because of the dual genetic control governing the correct syndrome.10 Equally, mutations have been identified in all seven of the assembly and function of complex I, but also as the largest complex in mtDNA genes encoding subunits of complex I,17 and also in mtDNA the mitochondrial respiratory chain, it is also the most intricate. tRNA (mt-tRNA) genes, particularly mitochondrial tRNALeu(UUR).2 Complex I is composed of 45 subunits,8 7 of which are encoded by The importance of mtDNA mutations as a potential molecular mitochondrial DNA (mtDNA), and the remaining 38 are nuclear aetiology for complex I deficiency was therefore not in doubt, but encoded. Moreover, a number of proteins are involved in the correct thus far their prevalence remained unclear. Indeed, it was previously processing and assembly of complex I, the total number of which as believed that complex I deficiency due to mtDNA mutations yet remains unknown.9 accounted for only 5–10% of paediatric complex I deficiency To date, pathogenic mutations in structural complex I proteins, cases.18,19 Recent screening of mitochondrial complex I subunit leading to complex I deficiency and associated with a disease pheno- genes has shown mutations in mtDNA accounting for a much higher type, have been identified in 21 nuclear genes: 11 of the 36 autosomal number of cases,20–27 and therefore, its screening is an essential step genes encoding complex I subunits (namely NDUFS1, NDUFS2, in the genetic diagnosis of complex I deficiency. Previous studies 1Mitochondrial Research Group, Institute for Ageing and Health, The Medical School, Newcastle University, Newcastle upon Tyne, UK; 2Murdoch Childrens Research Institute, Royal Children’s Hospital, Melbourne, Victoria, Australia; 3Department of Paediatrics, University of Melbourne, Melbourne, Victoria, Australia; 4Neurogenetic Unit, Department of Neurology, Royal Perth Hospital, Perth, Western Australia, Australia; 5Genetic Health Services Victoria, Royal Children’s Hospital, Melbourne, Victoria, Australia *Correspondence: Dr D Thorburn, Murdoch Childrens Research Institute, Royal Children’s Hospital, Flemington Road, Parkville, Victoria 3052, Australia. Tel: +613 8341 6235; Fax: +613 8341 6212; E-mail: [email protected] 6Current address: Peter MacCallum Cancer Centre, Melbourne, Australia. 7Current address: The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia. Received 13 October 2010; revised 12 January 2011; accepted 14 January 2011; published online 2 March 2011 mtDNA mutations causing complex I deficiency HSwalwellet al 770 assessing the prevalence of nuclear gene mutations in complex I- NDUFS8 and NDUFV1 genes by denaturing HPLC, as described elsewhere.34 deficient paediatric cases have found the prevalence of these to also be Next-generation sequencing was also used to screen patients from 94 families for high. Three studies have reported mutations in nuclear genes encoding mutations in 103 genes. These included the 45 subunits, the 15 mt-tRNA genes subunits, assembly factors or related proteins in 20–25% of children flanking the complex I subunit genes, known assembly factors and genes that with complex I deficiency.15,28,29 shared an evolutionary relationship with complex I. The approach involved a discovery screen that sequenced PCR-amplified targets in pools of DNA obtained In this study, we report a detailed analysis of a large cohort of 109 from 20 patients, with each pool analysed in a single lane of an Illumina Genome patients, representing all patients diagnosed with childhood-onset Analyzer (Illumina, San Diego, CA, USA). Sequence variants were prioritised by complex I deficiency at a large national referral centre. We present bioinformatic analyses and assigned to individual patients by Sequenom geno- the identification of the underlying molecular basis of the enzyme typing (Sequenom, San Diego, CA, USA), followed by experimental studies to defects in a number of these patients, and have used these data to determine pathogenicity. The inclusion of 43 patients with known pathogenic estimate the prevalence of such mutations in these disorders and to mutations allowed us to determine a detection sensitivity of 78% for causal present data surveying the overall clinical findings in these patients. nuclear DNA variants and 28% for causal mtDNA variants; the latter figure was low because of highly variable amplification of mtDNA from different patients in PATIENTS AND METHODS the whole-genome amplification step before pooling.15 Patient cohort The cohort of 109 patients (from 101 families) represents all patients diagnosed RESULTS in Melbourne between 1992 and 2007, with definite childhood-onset mito- mtDNA mutations chondrial disease and an enzyme diagnosis of complex I deficiency. The Melbourne laboratory is referred almost all children from Australia and A total of 30 patients (from 29 families) were found to harbour a New Zealand who are biopsied for investigation of mitochondrial disease. pathogenic mutation in mtDNA (29% of probands), either in MTND Before biopsy, children had been referred to a geneticist or paediatrician subunit genes (21 probands from 22 families) or in MTTL1 or MTTW specialising in inborn errors of metabolism. Testing for metabolic or genetic tRNA genes (8 probands) (Table 1). Each of the mtDNA mutations conditions with phenotypes similar to mitochondrial disease would typically has been reported to be pathogenic in previous studies that provided have been performed (eg, organic acids, acylcarnitines, chromosome analysis), evidence to support pathogenicity such as sequence conservation, the but the extent of this testing varied. Histological and enzyme histochemical presence of lower levels of heteroplasmy in healthy maternal relatives analyses were routinely performed on muscle biopsies, but usually produced and either cybrid analysis or single-fibre PCR used to confirm an normal or equivocal results, as reported previously for patients with complex I association between heteroplasmic mutant load and mitochondrial deficiency.2 The criteria used