Wolfram syndrome (WS) is an ultra-rare progressive neurodegenerative disorder defined by early-onset diabetes mellitus and optic atrophy. The majority of patients harbour recessive mutations in the WFS1 gene, which encodes for Wolframin, a transmembrane endoplasmic reticulum protein. There is limited availability of human ocular and brain tissues, and there are few animal models for WS that replicate the neuropathology and clinical phenotype seen in this disorder. We, therefore, characterised two wfs1 zebrafish knockout models harbouring nonsense wfs1a and wfs1b mutations. Both homozygous mutant wfs1a −/− and wfs1 b −/− embryos showed significant morphological abnormalities in early development. The wfs1 b −/− zebrafish exhibited a more pronounced neurodegenerative phenotype with delayed neuronal development, progressive loss of retinal ganglion cells and clear evidence of visual dysfunction on functional testing. At 12 months of age, wfs1b −/− zebrafish had a significantly lower RGC density per 100 μm 2 (mean ± standard deviation; 19 ± 1.7) compared with wild-type (WT) zebrafish (25 ± 2.3, p < 0.001). The optokinetic response for wfs1b −/− zebrafish was significantly reduced at 8 and 16 rpm testing speeds at both 4 and 12 months of age compared with WT zebrafish. An upregulation of the unfolded protein response was observed in mutant zebrafish indicative of increased endoplasmic reticulum stress. Mutant wfs1 b −/− zebrafish exhibit some of the key features seen in patients with WS, providing a versatile and cost-effective in vivo model that can be used to further investigate the underlying pathophysiology of WS and potential therapeutic interventions.

BACKGROUND:Autophagy is the major intracellular degradation route in mammalian cells. Systemic ablation of core autophagy-related (ATG) genes in mice leads to embryonic or perinatal lethality, and conditional models show neurodegeneration. Impaired autophagy has been associated with a range of complex human diseases, yet congenital autophagy disorders are rare.METHODS:We performed a genetic, clinical, and neuroimaging analysis involving five families. Mechanistic investigations were conducted with the use of patient-derived fibroblasts, skeletal muscle-biopsy specimens, mouse embryonic fibroblasts, and yeast.RESULTS:We found deleterious, recessive variants in human ATG7, a core autophagy-related gene encoding a protein that is indispensable to classical degradative autophagy. Twelve patients from five families with distinct ATG7 variants had complex neurodevelopmental disorders with brain, muscle, and endocrine involvement. Patients had abnormalities of the cerebellum and corpus callosum and various degrees of facial dysmorphism. These patients have survived with impaired autophagic flux arising from a diminishment or absence of ATG7 protein. Although autophagic sequestration was markedly reduced, evidence of basal autophagy was readily identified in fibroblasts and skeletal muscle with loss of ATG7. Complementation of different model systems by deleterious ATG7 variants resulted in poor or absent autophagic function as compared with the reintroduction of wild-type ATG7.CONCLUSIONS:We identified several patients with a neurodevelopmental disorder who have survived with a severe loss or complete absence of ATG7, an essential effector enzyme for autophagy without a known functional paralogue. (Funded by the Wellcome Centre for Mitochondrial Research and others.).

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Objective: To study the prevalence, molecular cause, and clinical presentation of hereditary motor neuropathies in a large cohort of patients from the North of England.Methods: Detailed neurologic and electrophysiologic assessments and next-generation panel testing or whole exome sequencing were performed in 105 patients with clinical symptoms of distal hereditary motor neuropathy (dHMN, 64 patients), axonal motor neuropathy (motor Charcot-Marie-Tooth disease [CMT2], 16 patients), or complex neurologic disease predominantly affecting the motor nerves (hereditary motor neuropathy plus, 25 patients).Results: The prevalence of dHMN is 2.14 affected individuals per 100,000 inhabitants (95% confidence interval 1.62-2.66) in the North of England. Causative mutations were identified in 26 out of 73 index patients (35.6%). The diagnostic rate in the dHMN subgroup was 32.5%, which is higher than previously reported (20%). We detected a significant defect of neuromuscular transmission in 7 cases and identified potentially causative mutations in 4 patients with multifocal demyelinating motor neuropathy.Conclusions: Many of the genes were shared between dHMN and motor CMT2, indicating identical disease mechanisms; therefore, we suggest changing the classification and including dHMN also as a subcategory of Charcot-Marie-Tooth disease. Abnormal neuromuscular transmission in some genetic forms provides a treatable target to develop therapies.

Dementia with Lewy bodies (DLB) is the second most common form of degenerative dementia. Siblings of affected individuals are at greater risk of developing DLB, but little is known about the underlying genetic basis of the disease. We set out to determine whether mutations in known highly penetrant neurodegenerative disease genes are found in patients with DLB. Whole-exome sequencing was performed on 91 neuropathologically confirmed cases of DLB, supplemented by independent APOE genotyping. Genetic variants were classified using established criteria, and additional neuropathological examination was performed for putative mutation carriers. Likely pathogenic variants previously described as causing monogenic forms of neurodegenerative disease were found in 4.4% of patients with DLB. The APOE ɛ4 allele increased the risk of disease (P=0.0001), conferred a shorter disease duration (P=0.043) and earlier age of death (P=0.0015). In conclusion, although known pathogenic mutations in neurodegenerative disease genes are uncommon in DLB, known genetic risk factors are present in >60% of cases. APOE ɛ4 not only modifies disease risk, but also modulates the rate of disease progression. The reduced penetrance of reported pathogenic alleles explains the lack of a family history in most patients, and the presence of variants previously described as causing frontotemporal dementia suggests a mechanistic overlap between DLB and other neurodegenerative diseases.

Objective: To identify the cause of isolated distal weakness in a family with both neuropathic and myopathic features on EMG and muscle histology. Methods: Case study with exome sequencing in 2 affected individuals, bioinformatic prioritization of genetic variants, and segregation analysis of the likely causal mutation. Functional studies included Western blot analysis of the candidate protein before and after heat shock treatment of primary skin fibroblasts. Results: A novel HSPB1 variant (c.387C>G, p.Asp129Glu) segregated with the phenotype and was predicted to alter the conserved α-crystallin domain common to small heat shock proteins. At baseline, there was no difference in HSPB1 protein levels nor its binding partner αB-crystallin. Heat shock treatment increased HSPB1 protein levels in both patient-derived and control fibroblasts, but the associated increase in αB-crystallin expression was greater in patient-derived than control fibroblasts. Conclusions: The HSPB1 variant (c.387C>G, p.Asp129Glu) is the likely cause of distal neuromyopathy in this pedigree with pathogenic effects mediated through binding to its partner heat shock protein αB-crystallin. Mutations in HSBP1 classically cause a motor axonopathy, but this family shows that the distal weakness can be both myopathic and neuropathic. The traditional clinical classification of distal weakness into “myopathic” or “neuropathic” forms may be misleading in some instances, and future treatments need to address the pathology in both tissues.

Mitochondrial disorders are clinically and genetically diverse, with mutations in mitochondrial or nuclear genes able to cause defects in mitochondrial gene expression. Recently, mutations in several genes encoding factors involved in mt-tRNA processing have been identified to cause mitochondrial disease. Using whole-exome sequencing, we identified mutations in TRMT10C (encoding the mitochondrial RNase P protein 1 [MRPP1]) in two unrelated individuals who presented at birth with lactic acidosis, hypotonia, feeding difficulties, and deafness. Both individuals died at 5 months after respiratory failure. MRPP1, along with MRPP2 and MRPP3, form the mitochondrial ribonuclease P (mt-RNase P) complex that cleaves the 50 ends of mt-tRNAs from polycistronic precursor transcripts. Additionally, a stable complex of MRPP1 and MRPP2 has m(1)R9 methyltransferase activity, which methylates mt-tRNAs at position 9 and is vital for folding mt-tRNAs into their correct tertiary structures. Analyses of fibroblasts from affected individuals harboring TRMT10C missense variants revealed decreased protein levels of MRPP1 and an increase in mt-RNA precursors indicative of impaired mt-RNA processing and defective mitochondrial protein synthesis. The pathogenicity of the detected variants-compound heterozygous c.542G>T (p.Arg181Leu) and c.814A>G (p.Thr272Ala) changes in subject 1 and a homozygous c.542G>T (p.Arg181Leu) variant in subject 2-was validated by the functional rescue of mt-RNA processing and mitochondrial protein synthesis defects after lentiviral transduction of wild-type TRMT10C. Our study suggests that these variants affect MRPP1 protein stability and mt-tRNA processing without affecting m(1)R9 methyltransferase activity, identifying mutations in TRMT10C as a cause of mitochondrial disease and highlighting the importance of RNA processing for correct mitochondrial function.

Mutations in STXBP1 have recently been identified as a cause of infantile epileptic encephalopathy. The underlying mechanism of the disorder remains unclear and, recently, several case reports have described broad and progressive neurological phenotypes in addition to early-onset epilepsy. Herein, we describe a patient with early-onset epilepsy who subsequently developed a progressive neurological phenotype including parkinsonism in her early teens. A de novo mutation in STXBP1 (c.416C>T, p.(Pro139Leu)) was detected with exome sequencing together with profound impairment of complex I of the mitochondrial respiratory chain on muscle biopsy. These findings implicate a secondary impairment of mitochondrial function in the progressive nature of the disease phenotype.

Blindness due to retinal degeneration affects millions of people worldwide, but many disease-causing mutations remain unknown. PNPLA6 encodes the patatin-like phospholipase domain containing protein 6, also known as neuropathy target esterase (NTE), which is the target of toxic organophosphates that induce human paralysis due to severe axonopathy of large neurons. Mutations in PNPLA6 also cause human spastic paraplegia characterized by motor neuron degeneration. Here we identify PNPLA6 mutations in childhood blindness in seven families with retinal degeneration, including Leber congenital amaurosis and Oliver McFarlane syndrome. PNPLA6 localizes mostly at the inner segment plasma membrane in photo-receptors and mutations in Drosophila PNPLA6 lead to photoreceptor cell death. We also report that lysophosphatidylcholine and lysophosphatidic acid levels are elevated in mutant Drosophila. These findings show a role for PNPLA6 in photoreceptor survival and identify phospholipid metabolism as a potential therapeutic target for some forms of blindness.

Objective Axonal form of Charcot Marie Tooth disease (CMT2) is a clinicogenetically heterogeneous group of hereditary sensorimotor neuropathies. There is a phenotypic overlap between CMT2 and distal hereditary motor neuropathy (dHMN), a subgroup of rare inherited motor axonal neuropathies with no significant sensory involvement. Mutations were described in genes encoding axonal proteins. Aminoacyl-tRNA synthetases (ARS) are essential enzymes for translation the genetic code into proteins. There have been six ARS implicated in CMT2 and dHMN related axonal pathology. Majority of the mutations were described in glycyl-tRNA synthetase (GARS). The aim was to analyse the pheno-and genotypic characteristics of patients with GARS and alanyl-tRNA synthetase (AARS) mutations indentified in a cohort of CMT2/dHMN from the North East of England. Methods In a large cohort of 438 CMT patients, multi-gene panel assay and whole exome sequencing confirmed 4 clinically suspected GARS neuropathies and identified AARS mutation in 4 CMT2 patients. Results All patients carrying GARS mutations presented with early adulthood onset upper limb predominant axonal motor neuropathy associated with mild motor weakness in the lower limbs and minor sensory involvement. A characteristic wasting in the first dorsal interosseus hand muscles led to a split hand malformation. Identification of heterozygous novel mutations in the GARS gene supported the clinical diagnosis. The previously described c.986G>A recurrent heterozygous AARS mutation was present in all 4 CMT2 patients. All had a moderate predominantly motor axonal neuropathy with variable age onset. However hand involvement was present, the lower limbs were more affected with minor sensory changes. Conclusion Mutations in ARSs are implicated in dominant axonal CMT. Despite the large number of variants and multiple theories of underlying pathology, GARS related clinical phenotype has remained quite distinctive. The recurrent AARS variant is associated with a more heterogeneous phenotype due to a methylation mediated loss of function mechanism. Axonal form of Charcot Marie Tooth disease (CMT2) is a clinicogenetically heterogeneous group of hereditary sensorimotor neuropathies. There is a phenotypic overlap between CMT2 and distal hereditary motor neuropathy (dHMN), a subgroup of rare inherited motor axonal neuropathies with no significant sensory involvement. Mutations were described in genes encoding axonal proteins. Aminoacyl-tRNA synthetases (ARS) are essential enzymes for translation the genetic code into proteins. There have been six ARS implicated in CMT2 and dHMN related axonal pathology. Majority of the mutations were described in glycyl-tRNA synthetase (GARS). The aim was to analyse the pheno-and genotypic characteristics of patients with GARS and alanyl-tRNA synthetase (AARS) mutations indentified in a cohort of CMT2/dHMN from the North East of England. In a large cohort of 438 CMT patients, multi-gene panel assay and whole exome sequencing confirmed 4 clinically suspected GARS neuropathies and identified AARS mutation in 4 CMT2 patients. All patients carrying GARS mutations presented with early adulthood onset upper limb predominant axonal motor neuropathy associated with mild motor weakness in the lower limbs and minor sensory involvement. A characteristic wasting in the first dorsal interosseus hand muscles led to a split hand malformation. Identification of heterozygous novel mutations in the GARS gene supported the clinical diagnosis. The previously described c.986G>A recurrent heterozygous AARS mutation was present in all 4 CMT2 patients. All had a moderate predominantly motor axonal neuropathy with variable age onset. However hand involvement was present, the lower limbs were more affected with minor sensory changes. Mutations in ARSs are implicated in dominant axonal CMT. Despite the large number of variants and multiple theories of underlying pathology, GARS related clinical phenotype has remained quite distinctive. The recurrent AARS variant is associated with a more heterogeneous phenotype due to a methylation mediated loss of function mechanism.

Sporadic late onset cerebellar ataxia is a well-described clinical presentation with a broad differential diagnosis that adult neurologists should be familiar with. However, despite extensive clinical investigations, an acquired cause is identified in only a minority of cases. Thereafter, an underlying genetic basis is often considered, even in those without a family history. Here we apply whole exome sequencing to a cohort of 12 patients with late onset cerebellar ataxia. We show that 33% of 'idiopathic' cases harbor compound heterozygous mutations in known ataxia genes, including genes not included on multi-gene panels, or primarily associated with an ataxic presentation.

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.

Boczonadi V, Muller JS, Pyle A, Munkley J, Dor T, Quartararo J, Ferrero I, Karcagi V, Giunta M, Polvikoski T, Birchall D, Princzinger A, Cinnamon Y, Lutzkendorf S, Piko H, Reza M, Florez L, Santibanez-Koref M, Griffin H, Schuelke M, Elpeleg O, Kalaydjieva L, Lochmuller H, Elliott DJ, Chinnery PF, Edvardson S, Horvath R. EXOSC8 mutations alter mRNA metabolism and cause hypomyelination with spinal muscular atrophy and cerebellar hypoplasia. Nature Communications 2014, 5: 4287.