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Inherited ataxias are heterogeneous disorders affecting both children and adults. The primary cause can be identified in about half of the patients and only very few can receive causative therapy.The authors performed sequencing of known Coenzyme Q10 (CoQ10) deficiency genes in 22 patients with unexplained recessive or sporadic ataxia.CABC1/ADCK3 mutations were detected in four patients and two siblings presenting with cerebellar ataxia, epilepsy and muscle symptoms. Spasticity, dystonia, tremor and migraine were variably present; cognitive impairment was severe in early childhood cases, but was absent in adults. In contrast to previous reports, two of the patients had a later-onset, very mild phenotype and remained ambulatory in their late forties. Muscle biopsy revealed lipid accumulation, mitochondrial proliferation and cytochrome c oxidase-deficient fibres, but no typical ragged red fibres. Respiratory-chain enzyme activities and CoQ10 were decreased in severely affected patients but remained normal in a mildly affected patient at 46 years of age.These observations highlight the importance of screening for a potentially treatable cause, CABC1/ADCK3 mutations, not only in severe childhood-onset ataxia, but also in patients with mild cerebellar ataxia in adult life.

Disorders of mitochondrial DNA (mtDNA) maintenance leading to multiple mtDNA deletions are a significant cause of inherited neurologic disease in adults, but the underlying nuclear gene defects remain elusive in many patients. Following the recent description of a truncating mutation in the RRM2B gene—encoding the small subunit, p53R2, of the p53-inducible ribonucleotide reductase protein—in 2 families with autosomal-dominant progressive external ophthalmoplegia (adPEO),1 we determined the frequency of RRM2B mutations in a large cohort of patients with chronic PEO and multiple mtDNA deletions in muscle in whom mutations in all known candidate genes (e.g., POLG , POLG2 , SLC25A4 , and PEO1 ) had been excluded.2 ### Methods. We studied 75 unrelated probands with PEO, a mosaic defect of cytochrome c oxidase (COX) activity, and multiple mtDNA deletions in skeletal muscle who had been referred to Mitochondrial Diagnostic Centers at Newcastle, Oxford, or Munich for clinical assessment and histologic/molecular genetic analysis. The entire coding region, including intron–exon boundaries, of the RRM2B gene was determined as previously described.1 RRM2B exon copy number (exons 1–8) was assessed by MLPA (MRC-Holland kit P089-A1) in patients with single, heterozygous missense mutations. #### Standard protocol approvals, registrations, and patient consents. This study was approved and performed under the ethical guidelines issued by each institution for clinical studies, with written informed consent obtained from all subjects. ### Results. We identified 10 different …

Neutral lipid storage disease is caused by mutations in the CGI-58 or the PNPLA2 genes. Lipid storage can be detected in various cell types including blood granulocytes. While CGI-58 mutations are associated with Chanarin–Dorfman syndrome, a condition characterized by lipid storage and skin involvement (ichthyosis), mutations in the patatin-like phospholipase domain-containing protein 2 gene (PNPLA2) were reported with skeletal and cardiac muscle disease only. We describe clinical, myopathological, magnetic resonance imaging (MRI), and genetic findings of six patients carrying different recessive PNPLA2 mutations. Pulse-chase labeling of control and patient cells with supplementation of clenbuterol, salmeterol, and dexamethasone was performed in vitro. The patients share a recognizable phenotype with prominent shoulder girdle weakness and mild pelvic girdle and distal muscle weakness, with highly elevated creatine kinase (CK) and cardiomyopathy developing at later stages. Muscle histology invariably reveals massive accumulation of lipid droplets. New muscle or whole-body MRI techniques may assist diagnosis and may become a useful tool to quantify intramuscular lipid storage. Four novel and two previously reported mutations were detected, affecting different parts of the PNPLA2 gene. Activation of hormone-sensitive lipase by beta-adrenergic substances such as clenbuterol appears to bypass the enzymatic block in PNPLA2-deficient patient cells in vitro. PNPLA2 deficiency is a slowly progressive myopathy with onset around the third decade. Cardiac involvement is relatively common at a later stage. Muscle MRI may detect increased lipid in a characteristic distribution, which could be used for monitoring disease progression. Beta-adrenergic agents may be beneficial in improving triacylglycerol breakdown in patients with PNPLA2 mutations.

Mitochondrial disorders are a group of clinically heterogeneous diseases, commonly defined by lack of cellular energy due to genetic defects of oxidative phosphorylation (OXPHOS). Ocular involvement is a prominent clinical feature of mitochondrial disease. This can manifest as optic nerve dysfunction specifically involving retinal ganglion cells as typified by Leber hereditary optic neuropathy (LHON), or progressive external ophthalmoplegia (PEO) and ptosis involving the extraocular muscles which is commonly associated with either primary mitochondrial DNA (mtDNA) mutations or acquired mtDNA defects secondary to a nuclear genetic disorder of mtDNA maintenance. In this short review, we will outline the unique characteristics of mitochondrial genetic disease and its investigation with reference to the clinical features and molecular genetic abnormalities underlying mitochondrial ophthalmological disease.

Purpose Mitochondrial disease is the most common neuromuscular disease and has a profound impact upon daily life, disease and longevity. Exercise therapy has been shown to improve mitochondrial function in patients with mitochondrial disease. However, no information exists about the level of habitual physical activity of people with mitochondrial disease and its relationship with clinical phenotype. Methods Habitual physical activity, genotype and clinical presentations were assessed in 100 patients with mitochondrial disease. Comparisons were made with a control group individually matched by age, gender and BMI. Results Patients with mitochondrial disease had significantly lower levels of physical activity in comparison to matched people without mitochondrial disease (steps/day; 6883±3944 vs. 9924±4088, p = 0.001). 78% of the mitochondrial disease cohort did not achieve 10,000 steps per day and 48%were classified as overweight or obese. Mitochondrial disease was associated with less breaks in sedentary activity (Sedentary to Active Transitions, % per day; 13±0.03 vs. 14±0.03, p = 0.001) and an increase in sedentary bout duration (bout lengths / fraction of total sedentary time; 0.206±0.044 vs. 0.187±0.026, p = 0.001). After adjusting for covariates, higher physical activity was moderately associated with lower clinical disease burden (steps / day; rs = −0.49; 95% CI −0.33, −0.63, P

Background: Mutations in the Twinkle (PEO1) gene are a recognized cause of autosomal dominant progressive external ophthalmoplegia (adPEO), resulting in the accumulation of multiple mitochondrial DNA (mtDNA) deletions and cytochrome c oxidase (COX)-deficient fibers in skeletal muscle secondary to a disorder of mtDNA maintenance. Patients typically present with isolated extraocular muscle involvement, with little apparent evidence of the clinical heterogeneity documented in other mtDNA maintenance disorders, in particular POLG-related disease. Methods: We reviewed the clinical, histochemical, and molecular genetics analysis of 33 unreported patients from 26 families together with all previous cases described in the literature to define the clinical phenotype associated with PEO1 mutations. Results: Ptosis and ophthalmoparesis were almost universal clinical features among this cohort, with 52% (17/33) reporting fatigue and 33% (11/33) having mild proximal myopathy. Features consistent with CNS involvement were rarely described; however, in 24% (8/33) of the patients, cardiac abnormalities were reported. Mitochondrial histochemical changes observed in muscle showed remarkable variability, as did the secondary mtDNA deletions, which in some patients were only detected by PCR-based assays and not Southern blotting. Moreover, we report 7 novel PEO1 variants. Conclusions: Our data suggest a shared clinical phenotype with variable mild multiorgan involvement, and that the contribution of PEO1 mutations as a cause of adPEO may well be underestimated. Direct sequencing of the PEO1 gene should be considered in adPEO patients prior to muscle biopsy.

Purpose: Single large-scale mitochondrial DNA deletions account for a quarter of mitochondrial disease cases and occur sporadically with unknown risk factors. Mitochondrial DNA deletions accumulate with age in many tissues. Primordial germ cells, the precursors of oocytes are made by our grandmothers, therefore we wanted to determine whether age of maternal grandmother is a risk factor for sporadic mitochondrial DNA deletions. Methods: Twenty-nine patients with sporadic single mitochondrial DNA deletions from the Newcastle UK cohort provided dates of birth for mothers and maternal grandmothers plus father and paternal grandmother (healthy controls). Results: Mean age for grandmothers at birth of a mother of an affected patient was 28.5 years (SD ± 6.9) for single mitochondrial DNA deletions maternal grandmothers and 28.2 years (SD ± 6.1) for healthy control paternal grandmothers. Conclusion: Maternal grandmother age is not a risk factor for sporadic mitochondrial DNA deletions, an important observation in a population where many women are delaying reproduction.

International audience; Additional neurological features have recently been described in seven families transmitting pathogenic mutations in OPA1, the most common cause of autosomal dominant optic atrophy. However, the frequency of these syndromal ‘dominant optic atrophy plus’ variants and the extent of neurological involvement have not been established. In this large multi-centre study of 104 patients from 45 independent families, including 60 new cases, we show that extra-ocular neurological complications are common in OPA1 disease, and affect up to 20% of all mutational carriers. Bilateral sensorineural deafness beginning in late childhood and early adulthood was a prominent manifestation, followed by a combination of ataxia, myopathy, peripheral neuropathy and progressive external ophthalmoplegia from the third decade of life onwards. We also identified novel clinical presentations with spastic paraparesis mimicking hereditary spastic paraplegia, and a multiple sclerosis-like illness. In contrast to initial reports, multi-system neurological disease was associated with all mutational subtypes, although there was an increased risk with missense mutations [odds ratio = 3.06, 95% confidence interval = 1.44–6.49; P = 0.0027], and mutations located within the guanosine triphosphate-ase region (odds ratio = 2.29, 95% confidence interval = 1.08–4.82; P = 0.0271). Histochemical and molecular characterization of skeletal muscle biopsies revealed the presence of cytochrome c oxidase-deficient fibres and multiple mitochondrial DNA deletions in the majority of patients harbouring OPA1 mutations, even in those with isolated optic nerve involvement. However, the cytochrome c oxidase-deficient load was over four times higher in the dominant optic atrophy + group compared to the pure optic neuropathy group, implicating a causal role for these secondary mitochondrial DNA defects in disease pathophysiology. Individuals with dominant optic atrophy plus phenotypes also had significantly worse visual outcomes, and careful surveillance is therefore mandatory to optimize the detection and management of neurological disability in a group of patients who already have significant visual impairment.

A 59-year-old, right-handed woman presented with a 7-day history of insidious onset of progressive blurring of vision of the right eye. She had undergone orthotopic liver transplantation 15 months previously due to non-A non-B fulminant hepatic failure. Her postoperative period was complicated by 2 episodes of acute rejection which responded to methylprednisolone, daclizumab, and change of immunosuppressive therapy from tacrolimus to cyclosporine. Three months prior she had developed nonspecific symptoms of weight loss, malaise, and anorexia. On examination she was alert and oriented. Visual acuity was 6/60 on the right and 6/36 on the left. Color vision was impaired bilaterally as tested with the Ishihara pseudoisochromatic plates. There was enlargement of both blind spots and severe constriction of fields with relative preservation of inferior nasal fields only. Ophthalmoscopic examination and fluorescein angiography showed bilateral pale swollen optic discs with peripapillary hemorrhages and venous engorgement (figure). The remaining neurologic examination was normal. Figure Photographs of the optic disc of the right and left eyes, showing bilateral pale swollen discs with flame-shaped hemorrhages and venous engorgement of the vessels ### Questions for consideration: 1. What are the possible differential diagnoses? 2. Which initial investigations would you recommend? The initial differential diagnosis for optic neuropathies with disc swelling included raised intracranial pressure, malignant hypertension, bilateral compressive thyroid ophthalmopathy, bilateral optic nerve tumors, bilateral simultaneous anterior ischemic optic neuropathy, idiopathic intracranial hypertension, and infective and inflammatory conditions. Twenty-four–hour ambulatory blood pressure monitoring was normal. Free thyroxin and thyroid stimulating hormone levels were normal. Thyroid antibodies were negative. Inflammatory markers were normal. Gadolinium-enhanced MRI of brain, orbits, pituitary, and optic nerves revealed increased signal uptake in the left optic nerve only, excluding …

A 58-year-old woman presented to a neuro-ophthalmologist with a 5-year history of progressively blurred vision, diplopia, and longstanding bilateral ptosis. She described occasional choking episodes after eating as well as fatigue and shortness of breath after minimal exertion. Her older sibling had received corrective eyelid surgery for ptosis and two nieces had ptosis and proximal myopathy and were being investigated in another center. Direct and consensual pupillary light reflexes were normal with no rapid alternating pupillary defect. Visual acuity was 20/20 on the right and 20/30 on the left. She had bilateral symmetric ptosis obscuring two-thirds of the pupil and restriction of eye movements below 60% of normal in all directions of gaze. Lower limb examination revealed symmetric proximal limb weakness (Medical Research Council grade 4+) with reduced reflexes and flexor plantars. Tandem gait was hesitant. ### Questions for consideration: 1. What are the possible diagnoses? 2. What initial investigations would you recommend? The initial differential diagnosis included Graves thyroid eye disease, a neuromuscular junction disorder (myasthenia gravis or botulism), oculopharyngeal muscular dystrophy, Miller Fisher variant of Guillain-Barre syndrome, and progressive muscular dystrophy. Free thyroxin and thyroid stimulating hormone levels were normal. Thyroid antibodies were negative. Acetylcholine receptor antibody assay was negative and repetitive nerve stimulation to exclude a neuromuscular junction disorder was normal. Electromyography of proximal upper limb muscles revealed an increased number of short duration motor units consistent with borderline myopathy. Nerve conduction velocities were …

Essential tremor (ET) is one of the most common movement disorders seen in routine neurological practice, but other diseases may mimic this condition. Here we describe a 62-year-old right-handed man with an action (kinetic) tremor fulfilling diagnostic criteria for ET which was the presenting feature of the fragile X associated tremor/ataxia syndrome. His sister’s grandchildren were the clue to the diagnosis, illustrating the importance of a detailed medical history in descendents as well as ancestors in the context of late onset neurological disorders. ET, typically a symmetric bilateral postural tremor with fluctuating amplitude, can be asymmetric and varies both in severity and functional impact. It classically begins in the fourth and fifth decades of life, is frequently associated with a family history and affects 0.8/1000 (95% confidence interval 0.5 to 1) of the general population, with a prevalence that increases with age.1 ET is one of the most common movement disorders seen in routine neurological practice, and the diagnosis is usually straightforward. The …

Spinocerebellar ataxia type 6 (SCA6) is a calcium channelopathy due to a pathological CAG repeat expansion in CACNL1A4. Patients frequently describe paroxysmal vertigo early in the disease course, but it is not clear whether this is central or labyrinthine in origin. To address this issue we studied 21 SCA6 patients. Symptoms of vertigo were defined using a structured questionnaire. Signs were recorded during a standardised bed-side vestibular examination that included systematic positional testing with Frenzel goggles.Brief, recurrent attacks of vertigo occurred in 13 patients, usually preceding the onset of ataxia. Nystagmus was observed behind Frenzel goggles in 14 patients, and was induced either during positional testing, or head shaking in 20 patients. Only one patient had findings typical of benign paroxysmal positional vertigo (BPPV). Combined downbeat and horizontal gaze-evoked nystagmus ("side-pocket") was the most common form, occurring most commonly in supine and head-hanging positions, and following horizontal head-shaking. Nystagmus beating away from the ground (apogeotropic) occurred in 9 patients as they lay on their side.In conclusion, vertigo and abnormalities on bedside vestibular examination are common in SCA6, with forms of nystagmus typical of cerebellar, rather than labyrinthine, disease. These findings demonstrate phenotypic overlap between SCA6 and episodic ataxia type 2, which are both due to mutations in CACNL1A4.

In their comprehensive Review, Jennifer Fugate and Alejandro Rabinstein discuss how posterior reversible encephalopathy syndrome (PRES) is sometimes inappropriately regarded as a radiological diagnosis and rightly emphasise the importance of correlating clinical history in the diagnostic process. However, we believe that an important diff erential diagnosis has been omitted. Mitochondrial disorders are among the most common forms of inherited neurological disease and present with a frequency of 1:4300 in the adult population. The acute neurological symptoms associated with PRES are frequently non-specifi c, including seizures, encephalopathy, headache, and visual disturbances. These symptoms also characterise stroke-like episodes associated with mitochondrial disease, often recognised in the context of the mitochondrial DNA mutation 3243A→G or mutations in the nuclear POLG gene. In our clinical experience of a cohort of 37 patients harbouring the 3243A→G mutation and presenting with stroke-like episodes, PRES was frequently the initial working diagnosis before consideration of a mitochondrial disorder. Although hypertension frequently accompanies PRES, studies suggest that 15–20% of patients with PRES are in fact normotensive or even hypotensive. Moreover, PRES is suggested to be recurrent in 5–10% of cases. Particularly in the absence of the known predisposing conditions for PRES, such as an autoimmune disorder, immunosuppressive or cytotoxic drug treatment, or eclampsia, both normotension and recurrent episodes together with neurological and radiological features suggestive of PRES warrant consideration of a possible underlying mitochondrial disorder. Importantly, the brain MRI abnormalities associated with PRES can be very similar to those encountered in patients with mitochondrial stroke-like episodes. We wish to point out that in patients presenting acutely with any combination of seizures, encephalopathy, headache, and visual disturbances, particularly when brain MRI reveals features suggestive of PRES, the possibility of a mitochondrial disorder should be considered. Useful clinical pointers to discern mitochondrial disease from PRES include: the presence of seemingly unrelated multisystem diseases such as bilateral sensorineural hearing loss, diabetes mellitus, cardiomyopathy, and gut dysmotility, which are common in 3243A→G mutation carriers; the presence of cerebellar ataxia or axonal polyneuropathy, which are suggestive of POLG-associated mitochondrial disease; and in the case of mitochondrial disease caused by mutations in mitochondrial DNA, a detailed family history might reveal features compatible with a maternal inheritance pattern. Genetic testing for the 3243A→G mutation and the common POLG mutations are at present noninvasive, inexpensive, and readily available. Moreover, establishment of a diagnosis of mitochondrial disease mimicking PRES has important practical implications, not only from a prognostic perspective, but also in relation to therapeutic strategies (such as avoidance of sodium valproate in POLG-related mitochondrial disease due to the associated risk of fulminant hepatic failure) and the need for detailed family tracing of at-risk individuals.

BackgroundMitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome is an acute and severe neurological presentation most commonly associated with a point mutation in the mitochondrial tRNA Leu (UUR)gene, m.3243A>G. The onset of stroke-like episodes (SLE) is often subtle and the diagnosis is not considered until progressive encephalopathy and refractory focal seizures ensue.ObjectiveTo review the acute management of SLE in patients with m.3243A>G related MELAS syndrome and implement revision of current centre guidelines.MethodThe medical records of adult patients (over 16 years) with m.3243A>G-related MELAS syndrome were reviewed over a 20 year period (1995 to 2014). Clinical presentation, brain imaging, EEG findings and seizure management were evaluated.ResultsWe identified 47 SLEs occurring in 22 m.3243A>G MELAS patients (mean age 33 years). Seizures of occipital origin were most common. Time to hospital admission and investigation was highly variable. Recognition and treatment of seizure activity was often delayed.ConclusionThis audit is the first to highlight the need for early recognition and prompt management of SLE in MELAS and the role of seizure control to improve clinical outcome.

Background Heterogeneity of clinical phenotype associated with the m.3243A>G mutation is a significant diagnostic and prognostic challenge to clinicians. Current literature regarding disease progression in relation to this mutation focuses on individuals who are moderately or severely affected by the disease. Here we present two sudden unexpected deaths in individuals who were largely asymptomatic. Method Multiple tissues were investigated including muscle, brain and heart. The degree of respiratory chain deficiency was determined using histochemical techniques and m.3243A>G mutation load by pyrosequencing. Result A high level of m. 3243A>G mutation load was detected in cardiomyocytes, brain and skeletal muscle. Mild left ventricular hypertrophy was identified in one case. There were 15–60% of COX deficient cardiomyocytes in both cases. Interestingly, the extensive respiratory chain deficiency observed in brain tissues from both of these individuals is similar to a patient severely affected by MELAS (Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-like Episodes). Conclusion We propose that sudden unexpected death in asymptomatic individuals with high tissue heteroplasmy represents a new, but rare, phenotype. The exact mechanism of death is undetermined although cardiac cause is a strong possibility. This adds a further challenge to the increasingly complex counselling of asymptomatic carriers of the m.3243A>G mutation.

Mitochondrial diseases are increasingly recognised as common genetic disorders exhibiting prodigious genotypic and phenotypic heterogeneity. Given that all mammalian cells contain mitochondrial DNA except mature red blood cells, it is not surprising that manifestations of the disorder are frequently multi systemic. Whilst brain, skeletal muscle, and cardiac symptoms are recognised prominent manifestations of mitochondrial disorders, other non-mitotic tissues maybe similarly affected. MLASA is a rare but distinct clinical entity characterised by myopathy, lactic acidosis and sideroblastic anaemia; originally attributed to mutations in the PUS1 (pseudouridylate synthase 1) gene. Mutations in the YAR2 (tyrosyl-tRNA synthethase 2; mitochondrial) gene, have recently been implicated in eight patients with MLASA (7 Lebanese and one of French ethnic origin). We present three unrelated adults, with novel mutations in YAR2 gene that challenge the ‘Lebanese founder effect’ and acronym, synonymous with this form of mitochondrial disease.

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.

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.

OPA1 codes for a mitochondrial fusion protein found on the inner mitochondrial membrane. Mutations in OPA1 are the most common cause of autosomal dominant optic atrophy (DOA), and until recently, this was thought to be a pure ocular disorder. By studying 104 patients from 45 independent families we have defined the clinical spectrum of this new multisystem mitochondrial disease, which we have called DOA+. We show that extra-ocular neurological complications affect ∼20%, and include sensorineural deafness in childhood, followed by ataxia, myopathy, peripheral neuropathy and progressive external ophthalmoplegia in the very late stages. We have also identified novel clinical presentations mimicking hereditary spastic paraplegia, and a multiple sclerosis-like illness resembling “Harding9s disease”, first described in patients with Leber hereditary optic neuropathy. We show that the risk of DOA+ is greatest in patients with mis-sense mutations, particularly those affecting the GTPase region of the protein, and that secondary mitochondrial DNA defects are likely to be responsible for the extra-ocular features. Individuals with DOA+ phenotypes also had significantly worse visual outcomes, and careful surveillance is therefore mandatory to optimise the detection and management of neurological disability in a group of patients with already significant visual impairment.

Progressive encephalomyelitis with rigidity and myoclonus (PERM) is a rare disorder of subacute onset presenting as limb and truncal rigidity, muscle spasms, brainstem signs, and hyperekplexia.1 Life-threatening, it is part of the spectrum of stiff-person syndrome (SPS) with anti-glutamic acid decarboxylase (GAD) antibodies in up to 80% of patients.1 It may also be paraneoplastic with anti-amphiphysin antibodies.2 Mutations in the α1 subunit of the glycine receptor gene GLRA1 have been identified in hereditary hyperekplexia.3 Glycine receptors are concentrated in the caudal pontine brainstem and spinal cord; in PERM, hyperekplexia (brainstem myoclonus or excessive startle)4 implies disruption of the normal inhibitory glycinergic mechanism in the caudal pons where the nucleus reticularis pontis caudalis mediates the startle reflex. We report a patient with typical severe PERM associated with a novel anti-glycine receptor antibody. ### Clinical summary. A 54-year-old man presented in January 2001 with 3 weeks of left flank tingling and 2 weeks of worsening brief frequent violent jerks, spontaneous and triggered by sensory and auditory stimuli; his upper limbs would abduct and flex and his trunk and legs extend. Neurologic examination was otherwise normal; he was intubated and ventilated. The CSF contained 60 lymphocytes × 109/L, the CSF IgG index was 0.72 (n < 0.7), oligoclonal bands negative. Cranial and spinal MRI scans with gadolinium were normal. After …

Background Neuromuscular disease includes a spectrum of inflammatory and mitochondrial myopathies where dysfunction in the energy–producing mitochondria can cause muscle weakness, exercise intolerance and fatigue. Continuous endurance training has been shown to improve cardiorespiratory fitness but disease symptoms also limit many patients9 ability to perform such exercise. Aim To determine the effects of low volume, high intensity interval training (HIIT) on clinical symptoms and functional capacity in adults with sporadic Inclusion Body Myositis and mitochondrial myopathies. Methods Subjects with a clinical diagnosis of mitochondrial disease or sporadic Inclusion Body Myositis completed 16 weeks of the HIIT, where short bursts of high intensity exercise were followed by low intensity, recovery intervals. Exercise tolerance (i.e. peak oxygen consumption and work rate), muscle and whole body composition, electrical activity in muscle and disease burden were assessed before and after exercise intervention. Results Preliminary results from seven subjects (65±10 years of age) show an improvement in peak exercise VO2 (1.3l/min±0.3 to 1.4l/min±0.4, p=0.296) and a significant increase in peak work capacity following HIIT (95±28 to 107±34 watts, p=0.036). For two subjects, muscle electrical activity during voluntary contraction decreased by more than 50% after HIIT, however, there was no significant change in peak muscle strength or symptoms of fatigue. There was also no significant change in bone mineral density with HIIT although one subject with mitochondrial disease was found to have marked osteopenia prior to, and after exercise intervention. Conclusion Preliminary findings suggest that high intensity interval training improved aerobic work capacity in patients with mitochondrial myopathies and sporadic Inclusion Body Myositis without exacerbating symptoms of fatigue. However, Macro–EMG findings did identify possible denervation in one subject with sporadic Inclusion Body Myositis following HIIT. Further investigations and analyses will help to better characterise subjects9 disease burden and assess the wider safety implications and therapeutic potential of exercise in neuromuscular disease.

Introduction Mitochondrial diseases are a common group of genetic neurometabolic disorders characterised by heterogeneous clinical features including myopathy, auditory, visual, cardiac, and brain dysfunction. Mitochondrial dysfunction has been implicated in ageing as well as in a number of other neurodegenerative diseases. Thus, information gained from research in patients with mitochondrial disease may provide useful insights into other age–related and neurodegenerative diseases. Cognitive features are present in patients with mitochondrial disease, but have not been comprehensively explored. Aims This longitudinal study sought to compare estimated premorbid and current levels of general cognitive functioning and examine the cognitive profile of patients with mitochondrial disease. Methods Forty–nine adult patients harbouring either the m.3243A>G or m.8344A>G mtDNA point mutations and 49 age–and ability–matched controls were recruited. The Wechsler Test of Adult Reading (WTAR) was performed to estimate premorbid cognitive ability and the Wechsler Adult Intelligence Scale–IV (WAIS–IV) was used to assess current cognitive functioning. The Speed of Comprehension test and the Birt Memory & Information Processing Battery were employed to assess processing speed in addition to WAIS–IV tests. The Wechsler Memory Scale–IV (WMS–IV) was used to measure auditory memory. The Tower test, Verbal Fluency (phonetic and semantic), and the Self–Ordered Pointing task were employed to measure executive functions. The Addenbrooke9s Cognitive Examination–Revised (ACE–R) provided a clinical screening tool for cognitive impairment. Baseline, cross–sectional data of patients with mitochondrial disease are considered here. Results: Patients with mitochondrial disease performed significantly worse than the norm on the WTAR (p=.002), suggestive of developmental cognitive problems. Current cognitive functioning, measured by the WAIS–IV, was also significantly lower than the norm (p Conclusion These findings demonstrate widespread cognitive difficulties that can undermine independence and quality of life in patients with mitochondrial disease, and emphasise the need to incorporate cognitive testing into clinical practice, in order to ensure that appropriate care is provided. These findings serve as an informative guide for assessing the true nature and severity of cognitive disability in patients with mitochondrial disease and preface future studies assessing speed of progression and final cognitive outcome, whilst providing clues to the role of mitochondrial dysfunction in other neurodegenerative conditions and age–related cognitive decline.

Background Cardiomyopathy is an independent predictor of morbidity and early mortality in mitochondrial DNA disease, and occurs in 20%–40% of adult patients harbouring the common m.3243A>G mutation, usually with a hypertrophic phenotype. Pathogenetic mechanisms are unclear yet no detailed study of myocardial structure, function and bioenergetics has been performed in m.3243A>G mutation carriers to identify early markers of cardiac involvement. Methods Cardiac MRI was performed in 20 adult patients (10 males, mean age 38.7±13.1 years) harbouring the m.3243A>G mutation, without clinical evidence of cardiac involvement on routine ECG and echocardiographic screening, and 20 age- and gender-matched healthy controls (10 males, 38.4±14.2 years): (1) phosphorus-31 magnetic resonance spectroscopy, (2) cine imaging (3), cardiac tagging, and (4) late gadolinium enhancement (LGE) imaging on a Philips Intera Achieva 3 Tesla scanner. Clinical disease burden was determined using: (1) the Newcastle Mitochondrial Disease Adult Scale (NMDAS), a 29-domain validated scoring system with a single cardiac domain, and (2) urinary mutation load, the best predictor of overall clinical outcome in m.3243A>G mutation carriers. Results Compared to control subjects and following Bonferroni correction for multiple comparisons, patients had increased left ventricular mass index (LVMI), LV mass to end-diastolic volume ratio (M/V ratio), LV radial wall thicknesses (all p Conclusions Concentric remodelling and subendocardial dysfunction are prevalent in m.3243A>G mutation carriers without clinical cardiac disease. Assessment of myocardial deformation may be useful in monitoring disease progression or response to early intervention. Patients with higher urinary mutation loads and disease burden may be at increased risk of cardiac involvement.

Introduction Rapid progress has been made in relation to our understanding of the molecular and genetic basis of many mitochondrial disorders. Yet despite this, avenues for therapeutic intervention are limited. Low levels of habitual physical activity have been recognised to have a strong negative relationship with muscle mitochondrial capacity, disease development and mortality. The aims of this study were to systematically assess habitual physical activity in a cohort of mitochondrial patients. Methods One hundred patients were enrolled in the study. Habitual physical activity was measured by a multisensor array and by completion of a self-report questionnaire. Disease severity was assessed using the Newcastle Mitochondrial Disability Adult Scale (NMDAS). Results Low levels of habitual physical activity were common. Seventy-eight per cent of the patients achieved less than nationally advised levels of physical activity (10 000 steps per day) and almost half had an average daily energy expenditure of less than 1.4 METS. Higher physical activity was associated with lower BMI and lower clinical disease burden. Conclusion Low levels of physical activity are prominent and constitute a significant and important modifiable risk factor in mitochondrial disease. These findings advocate the promotion of increased physical activity irrespective of genotype.

Introduction Mitochondrial diseases are a clinically multifarous group of genetic disorders with extensive phenotypic and disease burden variability. Health related quality of life (HRQoL) is increasingly recognised as a fundamental patient-based outcome measure in both clinical intervention and research. Generic HRQoL outcome measures have been extensively validated across populations and different disease states. Due to their inclusive construct not all relevant aspects of a specific illness may be captured. It is acknowledged that there is a need to develop disease-specific HRQoL measures that centre on symptoms characteristic of a specific disease or condition. This study presents the conceptualisation, development and assessment of a disease-specific HRQoL measure (Mito-QoL) for patients with mitochondrial cytopathies. Methods Domain and item content for the Mito-QoL was derived from semi-structured key-informant interviews and modified following piloting of the questionnaire by post. Items were eliminated with the use of standard psychometric criteria. Construct validity was assessed by comparing domain scores with similar Short Form 36 domains. Results Mito-QoL consists of 63 items within 16 unidimensional domains demonstrating excellent internal reliability (Cronbach9s α>0.74) and construct validity. Conclusion Mito-QoL is a valid and reliable disease-specific HRQoL measure which may be of use in clinical practice and research.

Two Irish women presented with difficulty in completion of hair straightening, limited by upper limb dysaesthesiae due to claudication or brachial plexus entrapment induced by sustained shoulder abduction beyond 90 degrees. The first described arm pain precipitated by elevation and sustained abduction above shoulder level, particularly while using her GHD hair-straightener. Elevated arm stress test was positive and a left cervical rib was seen on chest x ray. Neurogenic thoracic outlet syndrome (TOS) was diagnosed and the cervical rib was resected. The second described arm tingling and weakness when performing tasks involving shoulder abduction and elevation, limiting GHD use to 10 min before having to rest. Upper limb blood pressure and pulses were undetectable. Erythrocyte sedimentation rate was elevated and flow on radial Doppler disappeared on abduction and elevation of each arm. A CT pulmonary angiography demonstrated features of Takayasu's arteritis. Vascular TOS was diagnosed. Symptoms resolved with corticosteroids, followed by long-term immunosuppression and anti-coagulation.

Although neuromuscular clinical features often dominate the clinical presentation of mitochondrial disease due to the m.3243A>G mitochondrial DNA (mtDNA) mutation, many patients develop cardiac failure, which is often overlooked until it reaches an advanced stage. We set out to determine whether cardiac complications are sufficiently common to warrant prospective screening in all mutation carriers. Routine clinical echocardiography and 3 Tesla cardiac MRI were performed on ten m.3243A>G mutation carriers and compared to age and gender matched controls, with contemporaneous quadriceps muscle biopsies to measure respiratory chain activity and mtDNA mutation levels. Despite normal echocardiography, all ten m.3243A>G mutation carriers had evidence of abnormal cardiac function on MRI. The degree of cardiac dysfunction correlated with the percentage level of mutant mtDNA in skeletal muscle. Sub-clinical cardiac dysfunction was a universal finding in this study, adding weight to the importance of screening for cardiac complications in patients with m.3243A>G. The early detection of cardiac dysfunction with MRI opens up opportunities to prevent heart failure in these patients through early intervention.

Coenzyme Q10 (CoQ10) deficiency is a clinically and genetically heterogeneous subtype of mitochondrial disease. We report two girls with ataxia and mitochondrial respiratory chain deficiency who were shown to have primary CoQ10 deficiency. Muscle histochemistry displayed signs of mitochondrial dysfunction—ragged red fibers, mitochondrial paracrystalline inclusions, and lipid deposits while biochemical analyses revealed complex II+III respiratory chain deficiencies. MRI brain demonstrated cerebral and cerebellar atrophy. Targeted molecular analysis identified a homozygous c.1015G>A, p.(Ala339Thr) COQ8A variant in subject 1, while subject 2 was found to harbor a single heterozygous c.1029_1030delinsCA variant predicting a p.Gln343_Val344delinsHisMet amino acid substitution. Subsequent investigations identified a large‐scale COQ8A deletion in trans to the c.1029_1030delinsCA allele. A skin biopsy facilitated cDNA studies that confirmed exon skipping in the fibroblast derived COQ8A mRNA transcript. This report expands the molecular genetic spectrum associated with COQ8A‐related mitochondrial disease and highlights the importance of thorough investigation of candidate pathogenic variants to establish phase. Rapid diagnosis is of the utmost importance as patients may benefit from therapeutic CoQ10 supplementation.

How mtDNA replication is terminated and the newly formed genomes are separated remain unknown. We here demonstrate that the mitochondrial isoform of topoisomerase 3alpha (Top3alpha) fulfills this function, acting independently of its nuclear role as a component of the Holliday junction-resolving BLM-Top3alpha-RMI1-RMI2 (BTR) complex. Our data indicate that mtDNA replication termination occurs via a hemicatenane formed at the origin of H-strand replication and that Top3alpha is essential for resolving this structure. Decatenation is a prerequisite for separation of the segregating unit of mtDNA, the nucleoid, within the mitochondrial network. The importance of this process is highlighted in a patient with mitochondrial disease caused by biallelic pathogenic variants in TOP3A, characterized by muscle-restricted mtDNA deletions and chronic progressive external ophthalmoplegia (CPEO) plus syndrome. Our work establishes Top3alpha as an essential component of the mtDNA replication machinery and as the first component of the mtDNA separation machinery.

Objectives: Patient preference studies are increasingly used to inform decision-making during the medical product lifecycle but are rarely used to inform early stages of drug development. The primary aim of this study is to quantify treatment preferences of patients with neuromuscular disorders, which represent serious and debilitating conditions with limited or no treatment options available. Methods: This quantitative patient preferences study was designed as an online survey, with a cross-over design. This study will target two different diseases from the neuromuscular disorders disease group, myotonic dystrophy type 1 (DM1) and mitochondrial myopathies (MM). Despite having different physio-pathological pathways both DM1 and MM manifest in a clinically similar manner and may benefit from similar treatment options. The sample will be stratified into three subgroups: two patient groups differentiated by age of symptom onset and one caregivers group. Each subgroup will be randomly assigned to complete two of three different preference elicitation methods at two different time points: Q-methodology survey, discrete choice experiment, and best-worst scaling type 2, allowing cross-comparisons of the results across each study time within participants and within elicitation methods. Additional variables such as sociodemographic, clinical and health literacy will be collected to enable analysis of potential heterogeneity. Ethics and Dissemination: This study protocol has undergone ethical review and approval by the Newcastle University R&D Ethics Committee (Ref: 15169/2018). All participants will be invited to give electronic informed consent to take part in the study prior accessing the online survey. All electronic data will be anonymised prior analysis. This study is part of the Patient Preferences in Benefit-Risk Assessments during the Drug Life Cycle (IMI-PREFER) project, a public-private collaborative research project aiming to develop expert and evidence-based recommendations on how and when patient preferences can be assessed and used to inform medical product decision making.