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Transport And Golgi Organization protein 2 (TANGO2) deficiency has recently been identified as a rare metabolic disorder with a distinct clinical and biochemical phenotype of recurrent metabolic crises, hypoglycemia, lactic acidosis, rhabdomyolysis, arrhythmias, and encephalopathy with cognitive decline. We report nine subjects from seven independent families, and we studied muscle histology, respiratory chain enzyme activities in skeletal muscle and proteomic signature of fibroblasts. All nine subjects carried autosomal recessive TANGO2 mutations. Two carried the reported deletion of exons 3 to 9, one homozygous, one heterozygous with a 22q11.21 microdeletion inherited in trans. The other subjects carried three novel homozygous (c.262C>T/p.Arg88*; c.220A>C/p.Thr74Pro; c.380+1G>A), and two further novel heterozygous (c.6_9del/p.Phe6del); c.11‐13delTCT/p.Phe5del mutations. Immunoblot analysis detected a significant decrease of TANGO2 protein. Muscle histology showed mild variation of fiber diameter, no ragged‐red/cytochrome c oxidase‐negative fibers and a defect of multiple respiratory chain enzymes and coenzyme Q10 (CoQ10) in two cases, suggesting a possible secondary defect of oxidative phosphorylation. Proteomic analysis in fibroblasts revealed significant changes in components of the mitochondrial fatty acid oxidation, plasma membrane, endoplasmic reticulum‐Golgi network and secretory pathways. Clinical presentation of TANGO2 mutations is homogeneous and clinically recognizable. The hemizygous mutations in two patients suggest that some mutations leading to allele loss are difficult to detect. A combined defect of the respiratory chain enzymes and CoQ10 with altered levels of several membrane proteins provides molecular insights into the underlying pathophysiology and may guide rational new therapeutic interventions., Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR), Grant/Award Number: 2014: SGR 393; Association Française contre les Myopathies (FR), Grant/Award Number: 21644; CERCA Programme/ Generalitat de Catalunya, the Hesperia Foundation, the Secretariat for Universities and Research of the Ministry of Business and Knowledge of the Government of Catalonia, Grant/Award Number: [2017SGR1206]; European Research Council, Grant/Award Number: 309548; FEuropean Union Seventh Framework Programme, Grant/Award Number: (FP7/2007‐2013); Instituto de la Marató de TV3, Grant/Award Number: 345/C/2014rm Care (CA); Instituto de Salud Carlos III, Grant/Award Number: grants PI17‐01286, PI17/00109, PI16/00579, PI16/01048, PI14/00581 and CP09/00011; Medical Research Council, Grant/Award Number: MR/N025431/1; Mitochondrial Disease Patient Cohort (UK) , Grant/Award Number: (G0800674); National Institute for Health Research (NIHR) doctoral fellowship, Grant/Award Number: NIHR‐HCS‐D12‐03‐04; Newton Fund, Grant/Award Number: MR/N027302/1; Wellcome Centre for Mitochondrial Research, Grant/Award Number: (203105/Z/16/Z); Wellcome Investigator fund, Grant/Award Number: 109915/Z/15/Z; Wellcome Trust Pathfinder Scheme, Grant/Award Number: 201064/Z/16/Z

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.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.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.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.).

Reversible infantile respiratory chain deficiency (RIRCD) is a rare mitochondrial myopathy leading to severe metabolic disturbances in infants, which recover spontaneously after 6‐months of age. RIRCD is associated with the homoplasmic m.14674T>C mitochondrial DNA mutation; however, only ~ 1/100 carriers develop the disease. We studied 27 affected and 15 unaffected individuals from 19 families and found additional heterozygous mutations in nuclear genes interacting with mt‐tRNAGlu including EARS2 and TRMU in the majority of affected individuals, but not in healthy carriers of m.14674T>C, supporting a digenic inheritance. Our transcriptomic and proteomic analysis of patient muscle suggests a stepwise mechanism where first, the integrated stress response associated with increased FGF21 and GDF15 expression enhances the metabolism modulated by serine biosynthesis, one carbon metabolism, TCA lipid oxidation and amino acid availability, while in the second step mTOR activation leads to increased mitochondrial biogenesis. Our data suggest that the spontaneous recovery in infants with digenic mutations may be modulated by the above described changes. Similar mechanisms may explain the variable penetrance and tissue specificity of other mtDNA mutations and highlight the potential role of amino acids in improving mitochondrial disease., Heterozygous mutations in nuclear genes interacting with mt‐tRNAGlu induce the integrated stress response and metabolic rearrangements, reducing penetrance and promoting spontaneous recovery in a rare mitochondrial myopathy.

Cell-free mitochondrial DNA (cfmtDNA) is detectable in almost all human body fluids and has been associated with the onset and progression of several complex traits. In-life assessments indicate that reduced cfmtDNA is a feature of neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease and multiple sclerosis. However, whether this feature is conserved across all neurodegenerative diseases and how it relates to the neurodegenerative processes remains unclear. In this study, we assessed the levels of ventricular cerebrospinal fluid-cfmtDNA (vCSF-cfmtDNA) in a diverse group of neurodegenerative diseases (NDDs) to determine if the in-life observations of reduced cfmtDNA seen in lumbar CSF translated to the post-mortem ventricular CSF. To investigate further, we compared vCSF-cfmtDNA levels to known protein markers of neurodegeneration, synaptic vesicles and mitochondrial integrity. Our data indicate that reduced vCSF-cfmtDNA is a feature specific to Parkinson’s and appears consistent throughout the disease course. Interestingly, we observed increased vCSF-cfmtDNA in the more neuropathologically severe NDD cases, but no association to protein markers of neurodegeneration, suggesting that vCSF-cfmtDNA release is more complex than mere cellular debris produced following neuronal death. We conclude that vCSF-cfmtDNA is reduced in PD, but not other NDDs, and appears to correlate to pathology. Although its utility as a prognostic biomarker is limited, our data indicate that higher levels of vCSF-cfmtDNA is associated with more severe clinical presentations; suggesting that it is associated with the neurodegenerative process. However, as vCSF-cfmtDNA does not appear to correlate to established indicators of neurodegeneration or indeed indicators of mitochondrial mass, further work to elucidate its exact role is needed.

Mitochondrial DNA copy number (mtDNA CN) exhibits interindividual and intercellular variation, but few genome-wide association studies (GWAS) of directly assayed mtDNA CN exist.We undertook a GWAS of qPCR-assayed mtDNA CN in the Avon Longitudinal Study of Parents and Children (ALSPAC), and the UK Blood Service (UKBS) cohort. After validating and harmonising data, 5461 ALSPAC mothers (16-43 years at mtDNA CN assay), and 1338 UKBS females (17-69 years) were included in a meta-analysis. Sensitivity analyses restricted to females with white cell-extracted DNA, and adjusted for estimated or assayed cell proportions. Associations were also explored in ALSPAC children, and UKBS males.A neutrophil-associated locus approached genome-wide significance (rs709591 [MED24], β[SE] −0.084 [0.016], p=1.54e-07) in the main meta-analysis of adult females. This association was concordant in magnitude and direction in UKBS males and ALSPAC neonates. SNPs in and around ABHD8 were associated with mtDNA CN in ALSPAC neonates (rs10424198, β[SE] 0.262 [0.034], p=1.40e-14), but not other study groups. In a meta-analysis of unrelated individuals (N=11253), we replicated a published association in TFAM β[SE] 0.046 [0.017], p=0.006), with an effect size much smaller than that observed in the replication analysis of a previous in silico GWAS.In a hypothesis-generating GWAS, we confirm an association between TFAM and mtDNA CN, and present putative loci requiring replication in much larger samples. We discuss the limitations of our work, in terms of measurement error and cellular heterogeneity, and highlight the need for larger studies to better understand nuclear genomic control of mtDNA copy number.

Deletions in mitochondrial DNA (mtDNA) are an important cause of human disease and their accumulation has been implicated in the ageing process. As mtDNA is a high copy number genome, the coexistence of deleted and wild-type mtDNA molecules within a single cell defines heteroplasmy. When deleted mtDNA molecules, driven by intracellular clonal expansion, reach a sufficiently high level, a biochemical defect emerges, contributing to the appearance and progression of clinical pathology. Consequently, it is relevant to determine the heteroplasmy levels within individual cells to understand the mechanism of clonal expansion. Heteroplasmy is reflected in a mosaic distribution of cytochrome c oxidase (COX)-deficient muscle fibers. We applied droplet digital PCR (ddPCR) to single muscle fibers collected by laser-capture microdissection (LCM) from muscle biopsies of patients with different paradigms of mitochondrial disease, characterized by the accumulation of single or multiple mtDNA deletions. By combining these two sensitive approaches, ddPCR and LCM, we document different models of clonal expansion in patients with single and multiple mtDNA deletions, implicating different mechanisms and time points for the development of COX deficiency in these molecularly distinct mitochondrial cytopathies.

Recent studies have linked cell-free mitochondrial DNA (ccf-mtDNA) to neurodegeneration in both Alzheimer's and Parkinson's disease, raising the possibility that the same phenomenon could be seen in other diseases which manifest a neurodegenerative component. Here, we assessed the role of circulating cell-free mitochondrial DNA (ccf-mtDNA) in end-stage progressive multiple sclerosis (PMS), where neurodegeneration is evident, contrasting both ventricular cerebral spinal fluid ccf-mtDNA abundance and integrity between PMS cases and controls, and correlating ccf-mtDNA levels to known protein markers of neurodegeneration and PMS. Our data indicate that reduced ccf-mtDNA is a component of PMS, concluding that it may indeed be a hallmark of broader neurodegeneration.

In Parkinson disease (PD), mitochondrial dysfunction associates with nigral dopaminergic neuronal loss. Cholinergic neuronal loss co-occurs, particularly within a brainstem structure, the pedunculopontine nucleus (PPN). We isolated single cholinergic neurons from postmortem PPNs of aged controls and PD patients. Mitochondrial DNA (mtDNA) copy number and mtDNA deletions were increased significantly in PD patients compared to controls. Furthermore, compared to controls the PD patients had significantly more PPN cholinergic neurons containing mtDNA deletion levels exceeding 60%, a level associated with deleterious effects on oxidative phosphorylation. The current results differ from studies reporting mtDNA depletion in nigral dopaminergic neurons of PD patients. Ann Neurol 2017;82:1016-1021.

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.

Despite extensive efforts, half of patients with rare movement disorders such as hereditary spastic paraplegias and cerebellar ataxias remain genetically unexplained, implicating novel genes and unrecognized mutations in known genes. Non-coding DNA variants are suspected to account for a substantial part of undiscovered causes of rare diseases. Here we identified mutations located deep in introns of POLR3A to be a frequent cause of hereditary spastic paraplegia and cerebellar ataxia. First, whole-exome sequencing findings in a recessive spastic ataxia family turned our attention to intronic variants in POLR3A, a gene previously associated with hypomyelinating leukodystrophy type 7. Next, we screened a cohort of hereditary spastic paraplegia and cerebellar ataxia cases (n = 618) for mutations in POLR3A and identified compound heterozygous POLR3A mutations in ∼3.1% of index cases. Interestingly, >80% of POLR3A mutation carriers presented the same deep-intronic mutation (c.1909+22G>A), which activates a cryptic splice site in a tissue and stage of development-specific manner and leads to a novel distinct and uniform phenotype. The phenotype is characterized by adolescent-onset progressive spastic ataxia with frequent occurrence of tremor, involvement of the central sensory tracts and dental problems (hypodontia, early onset of severe and aggressive periodontal disease). Instead of the typical hypomyelination magnetic resonance imaging pattern associated with classical POLR3A mutations, cases carrying c.1909+22G>A demonstrated hyperintensities along the superior cerebellar peduncles. These hyperintensities may represent the structural correlate to the cerebellar symptoms observed in these patients. The associated c.1909+22G>A variant was significantly enriched in 1139 cases with spastic ataxia-related phenotypes as compared to unrelated neurological and non-neurological phenotypes and healthy controls (P = 1.3 × 10−4). In this study we demonstrate that (i) autosomal-recessive mutations in POLR3A are a frequent cause of hereditary spastic ataxias, accounting for about 3% of hitherto genetically unclassified autosomal recessive and sporadic cases; and (ii) hypomyelination is frequently absent in POLR3A-related syndromes, especially when intronic mutations are present, and thus can no longer be considered as the unifying feature of POLR3A disease. Furthermore, our results demonstrate that substantial progress in revealing the causes of Mendelian diseases can be made by exploring the non-coding sequences of the human genome.

The molecular diagnosis of adult-onset autosomal recessive cerebellar ataxias remains challenging because of genetic heterogeneity. However, recently developed molecular genetic techniques will potentially revolutionize the diagnostic approach. Here we set out to define the genetic basis of the ataxia in two brothers with no molecular diagnosis. Clinical evaluation was followed by whole-exome second-generation sequencing and comparative genomic hybridization to determine the diagnosis. Whole-exome sequencing identified a hemizygous novel spastic ataxia of Charlevoix-Saguenay (SACS) stop-codon mutation in both brothers (c.13048G→T, p.E4350*) that was present in the mother, but not the father. Comparative genomic hybridization revealed a 0.7-Mb deletion on chromosome 13q12.12 in both brothers, which included SACS and was heterozygous in the asymptomatic father. The milder phenotype, caused by a whole-gene deletion and a stop-codon mutation in SACS, indicates a loss-of-function mechanism in late-onset autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS), and illustrates the importance of chromosomal rearrangements in the investigation of adult-onset ataxia.

BACKGROUND: Neurodegeneration with brain iron accumulation is clinically and genetically heterogeneous because of mutations in at least 7 nuclear genes. METHODS: We performed homozygosity mapping and whole-exome sequencing in 2 brothers with brain iron accumulation from a consanguineous family. RESULTS: We identified a homozygous missense mutation in both brothers in the very recently identified chromosome 19 open-reading frame 12 gene. The disease presented before age 10 with slowly progressive tremor, dystonia, and spasticity. Additional features were optic atrophy, peripheral neuropathy, and learning difficulties. A raised serum creatine kinase indicated neuromuscular involvement, and compensatory mitochondrial proliferation implicated mitochondrial dysfunction as a pathological mechanism. CONCLUSIONS: Further studies are needed to explore the function of the chromosome 19 open-reading frame 12 gene, and extended genetic analysis on larger patient cohorts will provide more information about the presentation and frequency of this disease.

We have recently identified mutations in the translation activator of cytochrome c oxidase 1 (TACO1) gene, leading to cytochrome c oxidase (COX) deficiency. Here, we report the clinical and neuroimaging findings of five members of a big consanguinous family homozygous for c.472insC in TACO1. All 5 patients had an uneventful early childhood and a subtle onset, slowly progressive cognitive dysfunction, dystonia or visual impairment between ages 4 and 16 years. Affected girls had a milder phenotype and preserved ambulation into the late twenties. Brain MRI revealed bilateral, symmetric lesions of the basal ganglia in all affected family members, but less prominent in girls. TACO1 analysis showed no mutations in 17 patients with juvenile-onset Leigh syndrome and isolated COX or combined respiratory chain deficiency, indicating that TACO1 mutations are a rare cause of Leigh syndrome.

Contains fulltext : 52562.pdf (Publisher’s version ) (Closed access) Leber hereditary optic neuropathy (LHON) is due primarily to one of three common point mutations of mitochondrial DNA (mtDNA), but the incomplete penetrance implicates additional genetic or environmental factors in the pathophysiology of the disorder. Both the 11778G-->A and 14484T-->C LHON mutations are preferentially found on a specific mtDNA genetic background, but 3460G-->A is not. However, there is no clear evidence that any background influences clinical penetrance in any of these mutations. By studying 3,613 subjects from 159 LHON-affected pedigrees, we show that the risk of visual failure is greater when the 11778G-->A or 14484T-->C mutations are present in specific subgroups of haplogroup J (J2 for 11778G-->A and J1 for 14484T-->C) and when the 3460G-->A mutation is present in haplogroup K. By contrast, the risk of visual failure is significantly less when 11778G-->A occurs in haplogroup H. Substitutions on MTCYB provide an explanation for these findings, which demonstrate that common genetic variants have a marked effect on the expression of an ostensibly monogenic mtDNA disorder.

The identification of cell-free circulating mitochondrial DNA (ccf-mtDNA) in early-stage Alzheimer's disease (AD) raised the possibility that the same neurodegenerative effect could be observed in Parkinson's disease (PD). Here, and for the first time, we investigated the role of ccf-mtDNA in PD, identifying a significant reduction of ccf-mtDNA in PD patient cerebrospinal fluid (CSF) when compared to controls. Our data demonstrates that CSF ccf-mtDNA is not only a powerful biomarker for PD, but, given that the effect is also observed in AD, is likely a biomarker for neurodegeneration.

Sir, Erin Sandford and colleagues raise three main concerns that they believe undermine the likely causal nature of some of the genetic variants we detected by exome sequencing undiagnosed patients with ataxia (Pyle et al. , 2015): 1. Some of our proposed pathogenic variants have a minor allele frequency (MAF) of >0.01 in the population. Although useful as an initial guide, the 1% MAF is an arbitrary cut-off designed to rapidly filter-out common polymorphic genetic variants from a large exome data set. However, this must be done with care for several reasons. First, allele frequencies vary from database to database, and throughout the world. To choose their example, c.709C>T p.Pro237Ser in Patients P15 and P16 has a MAF = 0.0153 in the US-based ESP6500 (their Table 1), which contrasts with our local population (MAF = 0.0052), and the UK-based 1000 Genomes project (MAF = 0.0064). To our mind it would be ridiculous to reject this variant as non-pathogenic because one database documents a MAF > 0.01. Surely the important point here is …

Whole exome sequencing (WES) is a recently developed technique in genetics research that attempts to identify causative mutations in complex, undiagnosed genetic conditions. Causative mutations are usually identified after filtering the hundreds of variants on WES from an individual’s DNA selected by the phenotype. We investigated a patient with a slowly progressive chronic axonal distal motor neuropathy and extrapyramidal syndrome using WES, in whom common genetic mutations had been excluded. Variant filtering identified potentially deleterious mutations in three known disease genes: DCTN1, KIF5A and NEFH, which have been all associated with similar clinical presentations of amyotrophic lateral sclerosis, Parkinsonism and/or hereditary spastic paraplegia. Predicting the functional effect of the mutations were analysed in parallel with detailed clinical investigations. This case highlights the difficulties and pitfalls of applying WES in patients with complex neurological diseases and serves as an instructive tale.

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 photoreceptors 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.

Summary Background Human genome evolution has been shaped by infectious disease. Although most genetic studies have focused on the immune system, recovery after sepsis is directly related to physiological reserve that is critically dependent on mitochondrial function. We investigated whether haplogroup H, the most common type of mitochondrial DNA (mtDNA) in Europe, contributes to the subtle genetic variation in survival after sepsis. Methods In a prospective study, we included 150 individuals who were sequentially admitted to the intensive care unit in a hospital in Newcastle upon Tyne, UK. After clinical data were obtained, patients underwent mtDNA haplotyping by analysis with PCR and restriction fragment length polymorphism. As endpoints, we used death during the 6-month period or survival at 6 months. Findings Follow-up was complete for all study participants, although the haplotype of two patients could not be reliably determined. On admission to the intensive care unit, the frequency of mtDNA haplogroup H in study patients did not differ between study patients admitted with severe sepsis and 542 age-matched controls from the northeast of England. MtDNA haplogroup H was a strong independent predictor of outcome during severe sepsis, conferring a 2·12-fold (95% CI 1·02–4·43) increased chance of survival at 180 days compared with individuals without the haplogroup H. Interpretation Although haplogroup H is the most recent addition to the group of European mtDNA, paradoxically it is also the most common. Increased survival after sepsis provides one explanation for this observation. MtDNA haplotyping offers a new means of risk stratification of patients with severe infections, which suggests new avenues for therapeutic intervention.

Recent reports have questioned the accepted dogma that mammalian mitochondrial DNA (mtDNA) is strictly maternally inherited. In humans, the argument hinges on detecting a signature of inter-molecular recombination in mtDNA sequences sampled at the population level, inferring a paternal source for the mixed haplotypes. However, interpreting these data is fraught with difficulty, and direct experimental evidence is lacking. Using extreme-high depth mtDNA re-sequencing up to ~1.2 million-fold coverage, we find no evidence that paternal mtDNA haplotypes are transmitted to offspring in humans, thus excluding a simple dilution mechanism for uniparental transmission of mtDNA present in all healthy individuals. Our findings indicate that an active mechanism eliminates paternal mtDNA which likely acts at the molecular level.

International audience; Respiratory chain deficiencies exhibit a wide variety of clinical phenotypes resulting from defective mitochondrial energy production through oxidative phosphorylation. These defects can be caused by either mutations in the mtDNA or mutations in nuclear genes coding for mitochondrial proteins. The underlying pathomechanisms can affect numerous pathways involved in mitochondrial physiology. By whole-exome and candidate gene sequencing, we identified 11 individuals from 9 families carrying compound heterozygous or homozygous mutations in GTPBP3, encoding the mitochondrial GTP-binding protein 3. Affected individuals from eight out of nine families presented with combined respiratory chain complex deficiencies in skeletal muscle. Mutations in GTPBP3 are associated with a severe mitochondrial translation defect, consistent with the predicted function of the protein in catalyzing the formation of 5-taurinomethyluridine (τm(5)U) in the anticodon wobble position of five mitochondrial tRNAs. All case subjects presented with lactic acidosis and nine developed hypertrophic cardiomyopathy. In contrast to individuals with mutations in MTO1, the protein product of which is predicted to participate in the generation of the same modification, most individuals with GTPBP3 mutations developed neurological symptoms and MRI involvement of thalamus, putamen, and brainstem resembling Leigh syndrome. Our study of a mitochondrial translation disorder points toward the importance of posttranscriptional modification of mitochondrial tRNAs for proper mitochondrial function.

Synaptotagmin 2 is a synaptic vesicle protein that functions as a calcium sensor for neurotransmission but has not been previously associated with human disease. Via whole-exome sequencing, we identified heterozygous missense mutations in the C2B calcium-binding domain of the gene encoding Synaptotagmin 2 in two multigenerational families presenting with peripheral motor neuron syndromes. An essential calcium-binding aspartate residue, Asp307Ala, was disrupted by a c.920A>C change in one family that presented with an autosomal-dominant presynaptic neuromuscular junction disorder resembling Lambert-Eaton myasthenic syndrome. A c.923C>T variant affecting an adjacent residue (p.Pro308Leu) produced a presynaptic neuromuscular junction defect and a dominant hereditary motor neuropathy in a second family. Characterization of the mutation homologous to the human c.920A>C variant in Drosophila Synaptotagmin revealed a dominant disruption of synaptic vesicle exocytosis using this transgenic model. These findings indicate that Synaptotagmin 2 regulates neurotransmitter release at human peripheral motor nerve terminals. In addition, mutations in the Synaptotagmin 2 C2B domain represent an important cause of presynaptic congenital myasthenic syndromes and link them with hereditary motor axonopathies., National Institutes of Health (U.S.) (NIH Grant NS40296), Picower Institute for Learning and Memory (Picower Neurological Disease Research Fund), JPB Foundation

IMPORTANCE: Mitochondrial disorders have emerged as a common cause of inherited disease, but their diagnosis remains challenging. Patients with multiple respiratory chain complex defects are particularly difficult to diagnose at the molecular level because of the massive number of nuclear genes potentially involved in intra-mitochondrial protein synthesis, with many not yet linked to human disease. OBJECTIVE: To determine the molecular basis of multiple respiratory chain complex deficiencies. DESIGN: We studied 53 patients referred to 2 national centers in the United Kingdom and Germany between 2005-2012. All had biochemical evidence of multiple respiratory chain complex defects, and no primary pathogenic mitochondrial DNA mutation. Whole exome sequencing was performed using 62Mb exome enrichment, followed by variant prioritization using bioinformatic prediction tools, variant validation by Sanger sequencing, and segregation of the variant with the disease phenotype in the family. PARTICIPANTS: 53 patients with biochemical evidence of multiple respiratory chain complex defects but no primary pathogenic mitochondrial DNA mutation. RESULTS: Presumptive causal variants were identified in 28 patients (53%, 95% CI=39-67), and possible causal variants were identified in 4 (8%, 95% CI=2-18). Together these account for 32 patients (60%, 95% CI=46-74), and involved 18 different genes. These included recurrent mutations in RMND1, AARS2 and MTO1, each on a haplotype background consistent with a shared founder allele, and potential novel mutations in 4 possible mitochondrial disease genes (VARS2, GARS, FLAD1 and PTCD1). Distinguishing clinical features included deafness and renal involvement associated with RMND1, and cardiomyopathy with AARS2, and MTO1. However, “classical” features were absent in some patients, including normal liver function and Leigh syndrome (subacute necrotizing encephalomyelopathy) seen in association with TRMU mutations, and no cardiomyopathy with founder SCO2 mutations. It was not possible to confidently identify the underlying genetic basis in 21 patients (40%, 95% CI=26-54). CONCLUSIONS AND RELEVANCE: Exome sequencing enhances the ability to identify potential nuclear gene mutations in patients with biochemically-defined defects affecting multiple mitochondrial respiratory chain complexes. Additional study will be required in independent patient populations to determine the utility of this approach in comparison to traditional diagnostic methods.

Calpainopathy (LGMD2A) is the most common type of autosomal recessive limb-girdle muscular dystrophy. We performed a systematic clinical evaluation in 13 calpainopathy patients from 11 families, with particular attention to the pattern of muscle involvement. Eleven patients had a muscle biopsy with deficiency of calpain 3 on western blotting. The other two patients were not biopsied as they were siblings from the same families. Confirmatory CAPN3 mutations were detected in seven patients. The age at presentation was 2–45 years, wider than previously reported. We confirm the highly characteristic and recognisable phenotype of predominant muscular atrophy with early pelvic girdle involvement, relative sparing of the hip abductors, scapular winging and abdominal laxity. Early primary contractures were also a prominent feature in this group, expanding the breadth of the phenotype. Recognition of the clinical pattern of calpainopathy is of diagnosic significance. It is important, especially in sporadic cases, in targeting and interpreting laboratory investigations in order to provide accurate diagnostic and prognostic information.

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 5' 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.542GT (p.Arg181Leu) and c.814AG (p.Thr272Ala) changes in subject 1 and a homozygous c.542GT (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.

OBJECTIVE To determine the genetic basis of an unexplained multisystem neurological disorder affecting 2 siblings. DESIGN Case reports and whole-exome DNA sequencing. SETTING Neurogenetics clinic, Institute of Genetic Medicine, Newcastle upon Tyne, England. PATIENTS Two adult siblings with a sensorimotor neuropathy, ataxia, and spasticity. MAIN OUTCOME MEASURES Clinical, neurophysiological, imaging, and genetic data. RESULTS Novel compound heterozygous frameshift mutations were detected in the SACS gene of both siblings, predicted to drastically truncate the sacsin protein. CONCLUSIONS Whole-exome sequencing rapidly defined the genetic cause of the disorder, expanding the clinical phenotype associated with SACS mutations to include a severe sensorimotor neuropathy.

OPA1 mutations cause autosomal dominant optic atrophy (DOA), a debilitating mitochondrial optic neuropathy characterized by irreversible loss of retinal ganglion cells (RGCs) and progressive visual failure starting in early childhood.1 Interestingly, ∼20% of OPA1 carriers will develop a more severe form of the disease, DOA+, where the optic atrophy is compounded by additional neurologic features.1 OPA1 is a multifunctional mitochondrial inner membrane protein, and rather unexpectedly, high levels of cytochrome c oxidase (COX)-negative muscle fibers were recently identified in biopsy specimens from patients manifesting both the pure and syndromal phenotypes.2,3 These COX-negative muscle fibers harbored high levels of somatically acquired mitochondrial DNA (mtDNA) deletions and marked mtDNA proliferation was also observed, clearly revealing OPA1 to be a novel disorder of mtDNA maintenance.4 To further investigate this key pathologic mechanism, mtDNA copy number was quantified in blood leukocytes from 3 independent patient cohorts with molecularly confirmed OPA1 mutations.

ARTICLE Sir, We read with great interest the report of a Tunisian family by Rouzier et al . (2011) describing the neurological disorder linked to a novel heterozygous missense mutation in MFN2 (1p36.2) (Rouzier et al ., 2011). MFN2 mutations typically cause autosomal dominant axonal Charcot–Marie–Tooth disease (CMT2A, OMIM 609260), with peripheral nerve degeneration occasionally associated with visual failure and optic atrophy (Zuchner et al ., 2004, 2006). Interestingly, the clinical manifestations among mutational carriers in this Tunisian family were even more variable, ranging from asymptomatic subclinical disease to an axonal sensorimotor neuropathy complicated by optic atrophy, deafness, cerebellar ataxia and proximal myopathy. Furthermore, the intriguing finding of cytochrome c oxidase (COX)-deficient fibres and multiple mitochondrial DNA deletions in skeletal muscle biopsies suggest that MFN2 mutations can result in disturbed mitochondrial DNA maintenance and an overt respiratory chain defect, in addition to marked fragmentation of the mitochondrial network. These deleterious consequences are strikingly reminiscent of the pathological features recently highlighted in Brain for autosomal dominant optic atrophy due to OPA1 mutations (Amati-Bonneau et al ., 2008; Hudson et al ., 2008; Yu-Wai-Man et al ., 2010 a ). Here, we provide additional evidence that MFN2 …

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.

Mutations in several mitochondrial DNA and nuclear genes involved in mitochondrial protein synthesis have recently been reported in combined respiratory chain deficiency, indicating a generalized defect in mitochondrial translation. However, the number of patients with pathogenic mutations is small, implying that nuclear defects of mitochondrial translation are either underdiagnosed or intrauterine lethal. No comprehensive studies have been reported on large cohorts of patients with combined respiratory chain deficiency addressing the role of nuclear genes affecting mitochondrial protein synthesis to date. We investigated a cohort of 52 patients with combined respiratory chain deficiency without causative mitochondrial DNA mutations, rearrangements or depletion, to determine whether a defect in mitochondrial translation defines the pathomechanism of their clinical disease. We followed a combined approach of sequencing known nuclear genes involved in mitochondrial protein synthesis (EFG1, EFTu, EFTs, MRPS16, TRMU), as well as performing in vitro functional studies in 22 patient cell lines. The majority of our patients were children (

Disorders of mitochondrial DNA (mtDNA) maintenance have emerged as an important cause of human genetic disease, but demonstrating the functional consequences of de novo mutations remains a major challenge. We studied the rate of depletion and repopulation of mtDNA in human fibroblasts exposed to ethidium bromide in patients with heterozygous POLG mutations, POLG2 and TK2 mutations. Ethidium bromide induced mtDNA depletion occurred at the same rate in human fibroblasts from patients and healthy controls. By contrast, the restoration of mtDNA levels was markedly delayed in fibroblasts from patients with compound heterozygous POLG mutations. Specific POLG2 and TK2 mutations did not delay mtDNA repopulation rates. These observations are consistent with the hypothesis that mutations in POLG impair mtDNA repopulation within intact cells, and provide a potential method of demonstrating the functional consequences of putative pathogenic alleles causing a defect of mtDNA synthesis.

Loss of mitochondrial DNA (mtDNA) has been described in whole blood samples from a small number of patients with sepsis, but the underlying mechanism and clinical implications of this observation are not clear. We have investigated the cellular basis of the mtDNA depletion in sepsis, and determined clinical correlates with mtDNA depletion.Whole blood samples were obtained from 147 consecutive patients with severe sepsis admitted to a General Critical Care Unit in a University Hospital and 83 healthy controls. In a separate study of 13 patients with severe sepsis, blood was obtained for immediate cell sorting by flow cytometry. MtDNA content was determined in whole blood DNA by PCR methods, and subsequently in the 13 samples where white cell subtypes were separated.The mtDNA content of peripheral blood in human subjects was lower in patients with sepsis than controls (P0.0001). By studying leukocyte subsets in a subgroup of 13 patients, we showed that this was largely due to an increase in the proportion of circulating neutrophils, which contained approximately 3-fold less mtDNA than mononuclear leukocytes. However, isolated monocytes (P = 0.041) and lymphocytes (P = 0.021) from septic patients showed clear evidence of mtDNA depletion, which correlated with the APACHE II score (P = 0.015).In severe sepsis much of the apparent whole blood mtDNA depletion is due to a change in the differential leukocyte count. However mtDNA depletion in mononuclear cells occurs in patients with sepsis and correlates with disease severity.

Background: Insulin resistance and hyperglycaemia are common in severe sepsis. Mitochondrial uncoupling protein 2 (UCP2) plays a role in insulin release and sensitivity. Objectives: To determine if a common, functional polymorphism in the UCP2 gene promoter region (the -866 G/A polymorphism) contributes to the risk of hyperglycaemia in severe sepsis. Results: In the prospective group 120 non-diabetic patients who were carriers of the G allele had significantly higher maximum blood glucose recordings than non-carriers (AA 8.5±2.2 mmol/l; GA 8.5+2.4 mmol/l, GG 10.1+3.1 mmol/l; p=0.0042) and required significantly more insulin to maintain target blood glucose (p = 0.0007). In the retrospective study 103 non-diabetic patients showed a similar relationship between maximum glucose and UCP genotype (AA 6.8±2.3 mmol/l; GA 7.8+2.2 mmol/l, GG 9.2+2.9 mmol/l; p = 0.0078). Conclusions: A common, functional polymorphism in the promoter region of the UCP2 gene is associated with hyperglycaemia and insulin resistance in severe sepsis. This has implications for our understanding of the genetic pathophysiology of sepsis and is of use in the stratification of patients for more intensive management.

Background: The POLG1 gene encodes the catalytic subunit of DNA polymerase gamma, essential for mitochondrial DNA replication and repair. Mutations in POLG1 have been linked to a spectrum of clinical phenotypes, and may account for up to 25% of all adult presentations of mitochondrial disease. Methods and results: We present 14 patients, with characteristic features of mitochondrial disease including progressive external ophthalmoplegia (PEO) and Alpers– Huttenlocher syndrome and laboratory findings indicative of mitochondrial dysfunction, including cytochrome c oxidase (COX) deficiency and multiple deletions or depletion of the mitochondrial DNA. Four novel POLG1 missense substitutions (p.R597W, p.L605R, p.G746S, p.A862T), are described, together with the first adult patient with a recently described polymerase domain mutation (p.R1047W). All novel changes were rare in a control population and affected highly conserved amino acids. Conclusion: The addition of these substitutions— including the first report of a dinucleotide mutation (c.1814_1815TT.GC)—to the growing list of defects further confirms the importance of POLG1 mutations as the underlying abnormality in a range of neurological presentations.

Childhood-onset mitochondrial encephalomyopathies are usually severe, relentlessly progressive conditions that have a fatal outcome. However, a puzzling infantile disorder, long known as 'benign cytochrome c oxidase deficiency myopathy' is an exception because it shows spontaneous recovery if infants survive the first months of life. Current investigations cannot distinguish those with a good prognosis from those with terminal disease, making it very difficult to decide when to continue intensive supportive care. Here we define the principal molecular basis of the disorder by identifying a maternally inherited, homoplasmic m.14674T>C mt-tRNA(Glu) mutation in 17 patients from 12 families. Our results provide functional evidence for the pathogenicity of the mutation and show that tissue-specific mechanisms downstream of tRNA(Glu) may explain the spontaneous recovery. This study provides the rationale for a simple genetic test to identify infants with mitochondrial myopathy and good prognosis.

Background: The 3243A→G MTTL1 mutation is the most common heteroplasmic mitochondrial DNA (mtDNA) mutation associated with disease. Previous studies have shown that the percentage of mutated mtDNA decreases in blood as patients get older, but the mechanisms behind this remain unclear. Objectives and method: To understand the dynamics of the process and the underlying mechanisms, an accurate fluorescent assay was established for 3243A→G heteroplasmy and the amount of mtDNA in blood with real-time polymerase chain reaction was determined. The amount of mutated and wild-type mtDNA was measured at two time points in 11 subjects. Results: The percentage of mutated mtDNA decreases exponentially during life, and peripheral blood leucocytes in patients harbouring 3243A→G are profoundly depleted of mtDNA. Conclusions: A similar decrease in mtDNA has been seen in other mitochondrial disorders, and in 3243A→G cell lines in culture, indicating that depletion of mtDNA may be a common secondary phenomenon in several mitochondrial diseases. Depletion of mtDNA is not always due to mutation of a nuclear gene involved in mtDNA maintenance.

There is increasing evidence that genetic variants of mitochondrial DNA have an important role in the cause of idiopathic Parkinson's disease. We determined the mitochondrial DNA haplogroup of 455 Parkinson's disease cases, 185 Alzheimer's disease cases, and 447 healthy English control subjects. The UKJT haplogroup cluster was associated with a 22% reduction in population-attributable risk for Parkinson's disease. There was no association between individual haplogroups or the UKJT cluster and Alzheimer's disease, confirming that the association with Parkinson's disease was disease specific and not a general effect seen in all neurodegenerative diseases.

Nimantha de Alwis, Guruprasad Aithal, Elizabetta Bugianesi, Julian Leathart, Gavin Hudson, Angela Pyle, Catherine Mowbray, Elsbeth Henderson, Alistair D. Burt, Patrick F. Chinnery, Christopher P. Day

Sir, we read with great interest the report describing 12 patients presented with early onset severe neurological disease, characterized by unique MRI features of leukoencephalopathy involving the thalamus and brainstem with high lactate (LTBL; Steenweg et al., 2012). Clinically, the patients fell into two distinct groups. Four patients showed a severe progressive psychomotor regression soon after birth followed by complex irreversible neurological features, however, all patients were alive up to 7 years of age. Eight patients had mild spasticity, seizures and irritability between 6 and 12 months of life with improvement of both clinical and MRI signs in the second year. Stereotypical brain MRI appearances led to molecular diagnosis of mutations in the mitochondrial glutamyl-tRNA synthetase ( EARS2 ) gene. An increasing number of mutations have been identified recently in genes involved in mitochondrial protein synthesis (Smits et al., 2010; Chrzanowska-Lightowlers et al., 2011; Rotig, 2011). Most of these gene defects result in histological [cytochrome c oxidase (COX) deficient or ragged red fibres] and biochemical (multiple respiratory chain defect) abnormalities in affected organs. The clinical phenotypes are usually early-onset, severe, and often fatal diseases, implying the importance of mitochondrial translation from birth. Although some have distinguishing features (Chrzanowska-Lightowlers et al., 2011; Rotig, 2011), as a group they form an overlapping spectrum of disease. The MRI features described by Steenweg et al. (2012) in patients with EARS2 mutations add characteristic radiological features to …