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s, 7 Annual UK Neuromuscular Translational Research Conference, 2014 /Neuromuscular Disorders 24S1 (2014) S7–S27 S15 to the age-dependent manifestation or spontaneous recovery of infantile reversible COX deficiency myopathy. We performed immunohistochemistry and immunoblotting with antibodies against COXVI and COXVII isoforms in mice, in human skeletal muscle of controls and patients in different ages (0–6 months, >1 year, adults) and in human muscle cells. Both in mice and humans, the liver type isoforms gradually decreased, and the heart/muscle-type isoforms increased through development in the first weeks of life, confirming an age-dependent isoform switch. In skeletal muscle of a patient with reversible COX deficiency myopathy we proved the existence of the COXVI and COXVII isoform switch, although we could not confirm an association with the clinical recovery. However, understanding developmental changes of the COX isoforms may have implications for other mitochondrial diseases. P31 Genotypic and phenotypic heterogeneity in adult-onset progressive external ophthalmoplegia (PEO) with mitochondrial DNA instability: a systematic review E.W. Sommerville, P.F. Chinnery, G.S. Gorman, R.W. Taylor. Wellcome Trust Centre for Mitochondrial Research, Cookson Building, Framlington Place, Newcastle University, Newcastle upon Tyne, Tyne and Wear, NE2 4HH, UK; Institute of Genetic Medicine, International Centre for Life, Newcastle University, Central Parkway, Newcastle upon Tyne NE1 3BZ, UK Background: Progressive external ophthalmoplegia (PEO) is an eye movement disorder characterised by extraocular muscle paresis and muscle restricted mitochondrial DNA (mtDNA) deletions. Patient classification is difficult due to overlapping clinical phenotypes and poor genotype-phenotype correlates. This is compounded by the fact that approximately half of PEO patients do not have a genetic diagnosis. Aims: To review the phenotypic and genotypic manifestations of adult-onset PEO and to identify possible novel candidate genes. Patients: Patients were identified in the literature using electronic searches from Scopus, Medline via PubMed and Genetics Abstracts databases (1 January 1970 to 8 November 2013). Adult patients presenting PEO (≥16 years) presenting with PEO and mtDNA instability and with a confirmed genetic diagnosis were selected. The criterion was extended when searching candidate novel genes. Results: We identified 575 PEO patients, harbouring 12 known nuclear encoded genes (TYMP, SLC25A4, POLG, C10ORF2, OPA1, POLG2, RRM2B, TK2, DGUOK , MPV17, MGME1, and DNA2). Additional novel candidate genes (twenty in total), including several encoding proteins not predicted to localise to mitochondria, were also identified. Conclusion: We propose to use the findings of this systematic review coupled to whole exome and targeted next-generation sequencing technology, to help direct the investigation of a large cohort of clinically well-defined, genetically undetermined adult patients with PEO and mtDNA instability. P32 Mutations in SPG7 cause chronic progressive external ophthalmoplegia through disordered mtDNA maintenance G.S. Gorman, G. Pfeffer, H. Griffin, M. Kurzawa-Akanbi, E.L. Blakely, I. Wilson, K. Sitarz, D. Moore, J.L. Murphy, C.L. Alston, A. Pyle, J. Coxhead, B. Payne, G.H. Gorrie, C. Longman, M. Hadjivassiliou, J. McConville, D. Dick, I. Imam, D. Hilton, F. Norwood, M.R. Baker, S.R. Jaiser, P. Yu-Wai-Man, M. Farrell, A. McCarthy, T. Lynch, R. McFarland, A.M. Schaefer, D.M. Turnbull, R. Horvath, R.W. Taylor, P.F. Chinnery. Wellcome Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK Background: Despite being a canonical presenting feature of mitochondrial (mt) disease, the genetic basis of progressive external ophthalmoplegia (PEO) remains unknown in a large proportion of patients. Aims: To identify the causative gene in patients with genetically undetermined mtDNA maintenance disorders. Methods: Whole exome sequencing, targeted Sanger sequencing and MLPA analysis were used to study 68 adult patients with PEO either with or without multiple mtDNA deletions in skeletal muscle. Functional studies included transcript analysis, proteomics, mitochondrial network analysis, single fibre mtDNA analysis and deep re-sequencing of mtDNA. Results: Nine patients (eight probands) were found to carry compound heterozygous SPG7 mutations, including three novel mutations: c.2221G>A; p.(Glu741Lys), c.2224G>A; p.(Asp742Asn), and c.861dupT; p.Asn288*, and seven previously reported mutations. We identified a further six patients with single heterozygous mutations in SPG7, including two further novel mutations: c.184–3C>T (predicted to remove a splice site before exon 2) and c.1067C>T; p.(Thr356Met). The clinical phenotype typically developed in mid adult life with either PEO/ptosis and spastic ataxia, or a progressive ataxic disorder. Functional studies revealed increased mitochondrial biogenesis in patient muscle, and mitochondrial fusion in patient fibroblasts associated with the clonal expansion of mtDNA mutations. Conclusion: The SPG7 gene should be screened in patients in whom a disorder of mtDNA maintenance is suspected when spastic ataxia is prominent. The complex neurological phenotype is likely due to the clonal expansion of secondary mtDNA mutations modulating the phenotype, driven by compensatory mitochondrial biogenesis. a Joint first authors. P33 Do modulators of mitophagy select pathogenic mtDNA mutations? A. Hinks-Roberts, E. Dombi, A. Diot, C. Liao, K. Morten, J. Carver, T. Lodge, H. Mortiboys, J. Poulton. Nuffield Department of Obstetrics and Gynaecology, University of Oxford, UK; Sheffield Institute for Translational Neuroscience, University of Sheffield, UK Mitochondrial diseases that result from maternally transmitted mitochondrial DNA (mtDNA) mutations occur in 1/400 individuals. In heteroplasmic diseases, the balance between co-existing mutant and wild type mtDNA usually underlies disease progression. Cellular mechanisms for maintaining mitochondrial quality include mitophagy, and this could be a critical determinant of disease severity. Previous investigators showed that mitophagy was increased by the drug phenanthroline, a metallopeptidase inhibitor. We reasoned that activating mitophagy with phenanthroline might potentially reduce the load of pathogenic mutant mtDNA in tissue culture cells. We used a previously developed high throughput imaging for quantifying mitophagy in cultured primary fibroblasts bearing the common pathogenic A3243G mtDNA mutation, associated with the mitochondrial encephalomyopathy, lactic acidosis, and strokelike episode syndrome (MELAS) and with diabetes mellitus and deafness. We showed that phenanthroline significantly increased mitophagy in fibroblasts and reduced both the mitochondrial volume and mtDNA content. We conclude that phenanthroline activates mitophagy. However, there was little evidence that it reduced the load of mutant mtDNA. This suggests that both wild type and mutant mtDNA are turned over during mitophagy activated by phenanthroline. We conclude that phenanthroline is a poorly selective activator of mitophagy. Modulators of mitophagy need to target mitochondria enriched for mutant mtDNA if they are to benefit patients with heteroplasmic mtDNA disease.

L eber hereditary optic neuropathy (LHON, MIM 535000) is a mitochondrial genetic disease that causes blindness in young adults, with an estimated prevalence of 1 in 25 000 in the north east of England.1 It classically presents as bilateral subacute loss of central vision owing to the focal neurodegeneration of the retinal ganglion cell layer. There is marked cell body and axonal degeneration, with associated demyelination and atrophy observed from the optic nerves to the lateral geniculate bodies.2,3 Over 95% of cases are principally the result of one of three “primary” mtDNA point mutations, G3460A, G11778A, and T14484C, which all involve genes encoding complex I subunits of the mitochondrial respiratory chain.4 However, only ~50% of male and ~10% of female LHON carriers will develop the optic neuropathy.5 This marked incomplete penetrance and gender bias clearly indicates that additional factors are required for the phenotypic expression of the pathogenic mtDNA mutations in LHON. Segregation analysis initially suggested that a nuclear encoded modifier locus on the X chromosome could account for these intriguing features.6,7 However, attempts to map this X linked susceptibility locus using standard linkage analysis have so far not been successful.8–10 Furthermore, the Bu and Rotter segregation model could not be replicated in every LHON population.11 It is therefore likely that the phenotypic expression of the primary mtDNA LHON mutations is dependent upon the complex interaction of multiple susceptibility factors, which may include nuclear modifier genes and environmental agents.12 There is a strong association between APOE genotype and a number of common neurodegenerative conditions, including Alzheimer disease and dementia with Lewy bodies. The APOE e4 allele is associated with an increased lifetime risk and an earlier age of onset for these …

Genetic factors are important in the aetiology of the two most common neurodegenerative diseases: Alzheimer’s disease (AD) and dementia with Lewy bodies (DLB).1,2 Individuals with AD are more likely to have a similarly affected mother than a similarly affected father,3 raising the possibility of a maternally transmitted susceptibility factor. Patients with AD accumulate cytochrome c oxidase deficient neurones at a faster rate than age matched controls,4 and although contentious, abnormal mitochondrial function has been documented in the brains of patients with AD. Cybrid studies suggest that the biochemical abnormality is due to a defect of the mitochondrial genome (mtDNA).5 Different mtDNA sequence variants have been associated with AD and DLB, but there have been no consistent findings, and a maternally transmitted susceptibility factor remains elusive. The 16.5 kb mtDNA molecule codes for 13 essential respiratory chain subunits and the 24 RNAs required for intramitochondrial protein synthesis. Transcription and translation of mtDNA is controlled by the short 1 kb non-coding D-loop. In the wild-type genome, a thymidine residue at nucleotide position (np) 16189 interrupts a tract of cytosine residues between np 16184 and 16193, close to the origin of heavy strand replication (OH).6 The presence of a cytosine residue at np 16189 generates a homopolymeric C tract which appears to be unstable. It is thought …