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Introduction Neurofascin, encoded by NFASC, is a transmembrane protein that plays an essential role in nervous system development and node of Ranvier function. Anti-Neurofascin autoantibodies cause a specific type of chronic inflammatory demyelinating polyneuropathy (CIDP) often characterized by cerebellar ataxia and tremor. Recently, homozygous NFASC mutations were recently associated with a neurodevelopmental disorder in two families. Methods A combined approach of linkage analysis and whole-exome sequencing was performed to find the genetic cause of early-onset cerebellar ataxia and demyelinating neuropathy in two siblings from a consanguineous Italian family. Functional studies were conducted on neurons from induced pluripotent stem cells (iPSCs) generated from the patients. Results Genetic analysis revealed a homozygous p.V1122E mutation in NFASC. This mutation, affecting a highly conserved hydrophobic transmembrane domain residue, led to significant loss of Neurofascin protein in the iPSC-derived neurons of affected siblings. Conclusions The identification of NFASC mutations paves the way for genetic research in the developing field of nodopathies, an emerging pathological entity involving the nodes of Ranvier, which are associated for the first time with a hereditary ataxia syndrome with neuropathy.

Glycogen disease type III (GSDIII), a rare incurable autosomal recessive disorder due to glycogen debranching enzyme deficiency, presents with liver, heart and skeletal muscle impairment, hepatomegaly and ketotic hypoglycemia. Muscle weakness usually worsens to fixed myopathy and cardiac involvement may present in about half of the patients during disease. Management relies on careful follow-up of symptoms and diet. No common agreement was reached on sugar restriction and treatment in adulthood. We administered two dietary regimens differing in their protein and carbohydrate content, high-protein (HPD) and high-protein/glucose-free (GFD), to our mouse model of GSDIII, starting at one month of age. Mice were monitored, either by histological, biochemical and molecular analysis and motor functional tests, until 10 months of age. GFD ameliorated muscle performance up to 10 months of age, while HPD showed little improvement only in young mice. In GFD mice, a decreased muscle glycogen content and fiber vacuolization was observed, even in aged animals indicating a protective role of proteins against skeletal muscle degeneration, at least in some districts. Hepatomegaly was reduced by about 20%. Moreover, the long-term administration of GFD did not worsen serum parameters even after eight months of high-protein diet. A decreased phosphofructokinase and pyruvate kinase activities and an increased expression of Krebs cycle and gluconeogenesis genes were seen in the liver of GFD fed mice. Our data show that the concurrent use of proteins and a strictly controlled glucose supply could reduce muscle wasting, and indicate a better metabolic control in mice with a glucose-free/high-protein diet., Highlights • GSDIII is a rare incurable disease due to lacking of glycogen debrancher enzyme. • Essential features are liver, heart and skeletal muscle impairment. • Two diets differing in protein and sugar amount were tested in Agl-mouse model. • Glucose-free/high-protein diet decreased glycogen storage and hepatomegaly. • Improved muscle performance and better metabolic compensation were achieved.

Biallelic mutations in ECHS1, encoding the mitochondrial enoyl‐CoA hydratase, have been associated with mitochondrial encephalopathies with basal ganglia involvement. Here, we describe a novel clinical presentation consisting of dystonia‐ataxia syndrome with hearing loss and a peculiar torsional nystagmus observed in two adult siblings. The presence of a 0.9‐ppm peak at MR spectroscopy analysis suggested the accumulation of branched‐chain amino acids. Exome sequencing in index probands identified two ECHS1 mutations, one of which was novel (p.V82L). ECHS1 protein levels and residual activities were reduced in patients’ fibroblasts. This paper expands the phenotypic spectrum observed in patients with impaired valine catabolism.

Allgrove syndrome (AS) is a rare disease with broad neurological involvement. Neurodegeneration can affect spinal motor neurons, Purkinje cells, striatal neurons and the autonomic system. The mechanisms that lead to neuronal loss are still unclear. Recessive mutations in the AAAS gene affect the encoded protein Aladin, which would normally localize to the cytoplasmic face of the nuclear membrane as part of the nuclear pore complex (NPC). While the NPC is known to be a key factor for nucleocytoplasmic transport, the precise role of Aladin has not been elucidated yet. Here, we explored the consequences of the homozygous AAAS mutation c.464G>A (p.R155H) in central nervous system tissues and fibroblasts of a novel AS patient presenting motor neuron disease, cerebellar ataxia and autonomic dysfunction. Neuropathological analyses showed severe loss of motor neurons and Purkinje cells, with significant reduction in the perinuclear expression of Aladin. A reduced amount of protein was detected in the nuclear membrane fraction of the patient’s brain. RNA analysis revealed a significant reduction of the transcript AAAS-1, while the AAAS-2 transcript was upregulated in fibroblasts. To our knowledge, this is the first study to demonstrate the effects of AAAS mutations in the human central nervous system.

Aims Pompe disease is an autosomal recessive lysosomal storage disorder resulting from deficiency of acid α-glucosidase (GAA) enzyme. Histopathological hallmarks in skeletal muscle tissue are fiber vacuolization and autophagy. Since 2006, ERT is the only approved treatment with human recombinant GAA alglucosidase alfa. We designed a study to examine ERT-related skeletal muscle changes in 18 modestly to moderately affected LOPD patients along with the relationship between morphological/biochemical changes and clinical outcomes. Treatment duration was short-to-long term. Methods We examined muscle biopsies from 18 LOPD patients at both histopathological and biochemical level. All patients underwent two muscle biopsies, before and after ERT administration respectively. The study is partially retrospective because the first biopsies were taken before the study was designed whereas the second biopsy was always performed after at least six months of ERT administration. Results After ERT, 15 out of 18 patients showed improved 6MWT (p=0.0007) and most of them achieved respiratory stabilization. Pre-treatment muscle biopsies disclosed marked histopathological variability, ranging from an almost normal pattern to a severe vacuolar myopathy. After treatment, we detected morphological improvement in 15 patients and worsening in 3 patients. Post-ERT GAA enzymatic activity was mildly increased compared with pre-treatment levels in all patients. Protein levels of the mature enzyme increased in 14 of the 18 patients (mean increase = +35%; p

Summary Multiple system atrophy (MSA) is a progressive neurodegenerative disease that affects several areas of the CNS, whose pathogenesis is still widely unclear and for which an effective treatment is lacking. We have generated induced pluripotent stem cell-derived dopaminergic neurons from four MSA patients and four healthy controls and from two monozygotic twins discordant for the disease. In this model, we have demonstrated an aberrant autophagic flow and a mitochondrial dysregulation involving respiratory chain activity, mitochondrial content, and CoQ10 biosynthesis. These defective mechanisms may contribute to the onset of the disease, representing potential therapeutic targets., Highlights • An iPSC-based neuronal model of MSA is described • Mitochondria are dysfunctional in MSA neurons • Autophagic machinery is impaired in MSA neurons, Monzio Compagnoni et al. present an iPSC-based neuronal in vitro model of multiple system atrophy. Patients' dopaminergic neurons display a dysregulation of mitochondrial functioning and autophagy, suggesting new hints for the comprehension of the pathogenesis of the disease.

Multiple System Atrophy is a severe neurodegenerative disorder which is characterized by a variable clinical presentation and a broad neuropathological spectrum. The pathogenic mechanisms are almost completely unknown. In the present study, we established a cellular model of MSA by using fibroblasts' primary cultures and performed several experiments to investigate the causative mechanisms of the disease, with a particular focus on mitochondrial functioning. Fibroblasts' analyses (7 MSA-P, 7 MSA-C and 6 healthy controls) displayed several anomalies in patients: an impairment of respiratory chain activity, in particular for succinate Coenzyme Q reductase (p

Polyglucosan bodies (PBs) are deposits of amylopectin-like polysaccharides, detected in muscles of patients affected with glycogenoses-like branching enzyme (GBE) and phosphofructokinase deficiencies.1 Recently, mutations in RBCK1 have been associated with a skeletal PBs myopathy,2 as well as mutations in GYG1 , encoding for glycogenyn-1.3 All these conditions have autosomal recessive inheritance. Only seven unrelated cases with mutations in GYG1 have been reported, characterised by variable age at onset (from childhood to 7th decade), mainly proximal weakness, normal creatine kinases (CKs) levels and myopathic electromyography (EMG).3 We have been, for the past 35 years, following two affected sisters now aged 71 and 64 years (P.IV-5 and P.IV-10) (see online supplementary figure S1A). Their parents were both healthy first cousins from a little village in the Italian Alps. Both sisters had normal motor development. At the age of 30 years, P.IV-5 showed weakness in arm abduction, more prominent on the right side. The disease course was slowly progressive over the decades, with early involvement of proximal limb muscles. Waddling gait with hyperlordosis was first observed when she was in her late 40s. She started to use a wheelchair outdoors at the age of 59 years. In the following years she became completely a wheelchair user and dependent for her daily activities. Her last examination, at the age of 71 years, showed facial weakness, which was initially absent, with muscle atrophy and hypotonia, absent deep tendon reflexes (DTR) and severely compromised muscle power (Medical Research Council (MRC) score: neck flexors 3, neck extensors 2, shoulder abductors 0, forearm extensors/flexors 0, wrist extensors 0, wrist flexors and hand grip 2, hip flexors/extensors 0, thigh extensors/flexors 2, foot plantar flexors and …

Riboflavin is essential in numerous cellular oxidation/reduction reactions but is not synthesized by mammalian cells. Riboflavin absorption occurs through the human riboflavin transporters RFVT1 and RFVT3 in the intestine and RFVT2 in the brain. Mutations in these genes are causative for the Brown–Vialetto–Van Laere (BVVL), childhood-onset syndrome characterized by a variety of cranial nerve palsies as well as by spinal cord motor neuron (MN) degeneration. Why mutations in RFVTs result in a neural cell–selective disorder is unclear. As a novel tool to gain insights into the pathomechanisms underlying the disease, we generated MNs from induced pluripotent stem cells (iPSCs) derived from BVVL patients as an in vitro disease model. BVVL-MNs explained a reduction in axon elongation, partially improved by riboflavin supplementation. RNA sequencing profiles and protein studies of the cytoskeletal structures showed a perturbation in the neurofilament composition in BVVL-MNs. Furthermore, exploring the autophagy–lysosome pathway, we observed a reduced autophagic/mitophagic flux in patient MNs. These features represent emerging pathogenetic mechanisms in BVVL-associated neurodegeneration, partially rescued by riboflavin supplementation. Our data showed that this therapeutic strategy could have some limits in rescuing all of the disease features, suggesting the need to develop complementary novel therapeutic strategies.

Spinocerebellar ataxias (SCAs) are a genetically heterogeneous group of cerebellar degenerative disorders, characterized by progressive gait unsteadiness, hand incoordination, and dysarthria. Ataxia type 1 (SCA1) is caused by the expansion of a CAG trinucleotide repeat in the SCA1 gene resulting in the atypical extension of a polyglutamine (polyQ) tract within the ataxin-1 protein. Our main objective was to investigate the mitochondrial oxidative metabolism in the cerebellum of transgenic SCA1 mice. SCA1 transgenic mice develop clinical features in the early life stages (around 5 weeks of age) presenting pathological cerebellar signs with concomitant progressive Purkinje neuron atrophy and relatively little cell loss; this evidence suggests that the SCA1 phenotype is not the result of cell death per se, but a possible effect of cellular dysfunction that occurs before neuronal demise. We studied the mitochondrial oxidative metabolism in cerebellar cells from both homozygous and heterozygous transgenic SCA1 mice, aged 2 and 6 months. Histochemical examination showed a cytochrome-c-oxidase (COX) deficiency in the Purkinje cells (PCs) of both heterozygous and homozygous mice, the oxidative defect being more prominent in older mice, in which the percentage of COX-deficient PC was up to 30%. Using a laser-microdissector, we evaluated the mitochondrial DNA (mtDNA) content on selectively isolated COX-competent and COX-deficient PC by quantitative Polymerase Chain Reaction and we found mtDNA depletion in those with oxidative dysfunction. In conclusion, the selective oxidative metabolism defect observed in neuronal PC expressing mutant ataxin occurs as early as 8 weeks of age thus representing an early step in the PC degeneration process in SCA1 disease.

Glycogen storage disease type III is an autosomal recessive disease characterized by a deficiency in the glycogen debranching enzyme, encoded by AGL. Essential features of this disease are hepatomegaly, hypoglycemia, hyperlipidemia, and growth retardation. Progressive skeletal myopathy, neuropathy, and/or cardiomyopathy become prominent in adults. Currently, there is no available cure. We generated an Agl knockout mouse model by deletion of the carboxy terminus of the protein, including the carboxy end of the glucosidase domain and the glycogen-binding domain. Agl knockout mice presented serious hepatomegaly, but we did not observe signs of cirrhosis or adenomas. In affected tissues, glycogen storage was higher than in wild-type mice, even in the central nervous system which has never been tested in GSDIII patients. The biochemical findings were in accordance with histological data, which clearly documented tissue impairment due to glycogen accumulation. Indeed, electron microscopy revealed the disruption of contractile units due to glycogen infiltrations. Furthermore, adult Agl knockout animals appeared less prompt to move, and they exhibited kyphosis. Three-mo-old Agl knockout mice could not run, and adult mice showed exercise intolerance. In addition, older affected animals exhibited an accelerated respiratory rate even at basal conditions. This observation was correlated with severe glycogen accumulation in the diaphragm. Diffuse glycogen deposition was observed in the tongues of affected mice. Our results demonstrate that this Agl knockout mouse is a reliable model for human glycogenosis type III, as it recapitulates the essential phenotypic features of the disease.

Objective: To investigate the molecular defect underlying a large Italian kindred with progressive adult-onset respiratory failure, proximal weakness of the upper limbs, and evidence of lower motor neuron degeneration. Methods: We describe the clinical features of 5 patients presenting with prominent respiratory insufficiency, proximal weakness of the upper limbs, and no signs of frontotemporal lobar degeneration or semantic dementia. Molecular analysis was performed combining linkage and exome sequencing analyses. Further investigations included transcript analysis and immunocytochemical and protein studies on established cell models. Results: Genome-wide linkage analysis showed an association with chromosome 17q21. Exome analysis disclosed a missense change in MAPT segregating dominantly with the disease and resulting in D348G-mutated tau protein. Motor neuron cell lines overexpressing mutated D348G tau isoforms displayed a consistent reduction in neurite length and arborization. The mutation does not seem to modify tau interactions with microtubules. Neuropathologic studies were performed in one affected subject, which exhibited α-motoneuron loss and atrophy of the spinal anterior horns with accumulation of phosphorylated tau within the surviving motor neurons. Staining for 3R- and 4R-tau revealed pathology similar to that observed in familial cases harboring MAPT mutations. Conclusion: Our study broadens the phenotype of tauopathies to include lower motor neuron disease and implicate tau degradation pathway defects in motor neuron degeneration.

Syndromes associated with multiple mtDNA deletions are due to different molecular defects that can result in a wide spectrum of predominantly adult-onset clinical presentations, ranging from progressive external ophthalmoplegia (PEO) to multisystemic disorders of variable severity. The autosomal-dominant form of PEO is genetically heterogeneous. Recently, causative mutations have been reported in several nuclear genes that encode proteins of the mtDNA replisome machinery (POLG, POLG2, and C10orf2) or that are involved in pathways for the synthesis of deoxyribonuclotides (ANT1 and RRM2B). Despite these findings, putative mutations remain unknown in half of the subjects with PEO. We report the identification, by exome sequencing, of mutations in DNA2 in adult-onset individuals with a form of mitochondrial myopathy featuring instability of muscle mtDNA. DNA2 encodes a helicase/nuclease family member that is most likely involved in mtDNA replication, as well as in the long-patch base-excision repair (LP-BER) pathway. In vitro biochemical analysis of purified mutant proteins revealed a severe impairment of nuclease, helicase, and ATPase activities. These results implicate human DNA2 and the LP-BER pathway in the pathogenesis of adult-onset disorders of mtDNA maintenance.

Patient 1 A 56-year-old male patient was investigated for progressive limb weakness over the previous 3 years. Past medical and family history was unremarkable. Neurologic examination revealed a proximal quite symmetric weakness [Medical Research Council (MRC) score 3–4/5], without sensory disturbances or pyramidal signs. Because no etiologies were found to explain Dear Sir, Late-onset Pompe disease (LOPD) is an autosomal recessive lysosomal storage disease due to glucosidase alpha acid (GAA) deficiency leading to intracytoplasmic glycogen accumulation [1] . Prevalence of LOPD varies in populations at between 1: 40,000 and 1: 146,000 [2] . Hydromyelia is a dilatation of the spinal central canal which communicates with the intracranial cerebrospinal fluid compartment [3, 4] . It can lead to neurologic deficits including in some cases muscle weakness and atrophy [3, 5] . Hydromyelia is considered a rare condition [6] , but its exact incidence seems unknown. Syringomyelia arises primarily outside the central canal and secondary to Chi ari malformation, tumor, traumatism or bleeding [4, 5] . Its incidence is estimated at around 8.4 new cases/year/100,000 people [7] . In some cases, hydromyelia is considered a ‘pre-syringomyelic’ stage or a predisposition to syringomyelia [8, 9] . We report for the first time, 2 cases of association between hydromyelic cavity Received: January 21, 2013 Accepted: March 20, 2013 Published online: July 9, 2013

Neurotoxicity due to the accumulation of mutant proteins is thought to drive pathogenesis in neurodegenerative diseases. Mutations in superoxide dismutase 1 (SOD1) are linked to familial amyotrophic lateral sclerosis (fALS); these mutations result in progressive motor neuron death through one or more acquired toxicities. Interestingly, SOD1 is not only responsible for fALS but may also play a significant role in sporadic ALS; therefore, SOD1 represents a promising therapeutic target. Here, we report slowed disease progression, improved neuromuscular function, and increased survival in an in vivo ALS model following therapeutic delivery of morpholino oligonucleotides (MOs) designed to reduce the synthesis of human SOD1. Neuropathological analysis demonstrated increased motor neuron and axon numbers and a remarkable reduction in astrogliosis and microgliosis. To test this strategy in a human model, we treated human fALS induced pluripotent stem cell (iPSC)-derived motor neurons with MOs; these cells exhibited increased survival and reduced expression of apoptotic markers. Our data demonstrated the efficacy of MO-mediated therapy in mouse and human ALS models, setting the stage for human clinical trials.

Charcot-Marie-Tooth 2A (CMT2A) is an inherited peripheral neuropathy caused by mutations in MFN2, which encodes a mitochondrial membrane protein involved in mitochondrial network homeostasis. Because MFN2 is expressed ubiquitously, the reason for selective motor neuron (MN) involvement in CMT2A is unclear. To address this question, we generated MNs from induced pluripotent stem cells (iPSCs) obtained from CMT2A patients as an in vitro disease model. CMT2A iPSC-derived MNs (CMT2A-MNs) exhibited a global reduction in mitochondrial content and altered mitochondrial positioning without significant differences in survival and axon elongation. RNA sequencing profiles and protein studies of key components of the apoptotic executioner program (i.e., p53, BAX, caspase 8, cleaved caspase 3, and the anti-apoptotic marker Bcl2) demonstrated that CMT2A-MNs are more resistant to apoptosis than wild-type MNs. Exploring the balance between mitochondrial biogenesis and the regulation of autophagy-lysosome transcription, we observed an increased autophagic flux in CMT2A-MNs that was associated with increased expression of PINK1, PARK2, BNIP3, and a splice variant of BECN1 that was recently demonstrated to be a trigger for mitochondrial autophagic removal. Taken together, these data suggest that the striking reduction in mitochondria in MNs expressing mutant MFN2 is not the result of impaired biogenesis, but more likely the consequence of enhanced mitophagy. Thus, these pathways represent possible novel molecular therapeutic targets for the development of an effective cure for this disease.

Objective: To better clarify the pathogenesis of linezolid-induced lactic acidosis. Design: Case report. Setting: ICU. Patient: A 64-year-old man who died with linezolid-induced lactic acidosis. Interventions: Skeletal muscle was sampled at autopsy to study mitochondrial function. Measurements and Main Results: Lactic acidosis developed during continuous infusion of linezolid while oxygen consumption and oxygen extraction were diminishing from 172 to 52 mL/min/m 2 and from 0.27 to 0.10, respectively. Activities of skeletal muscle respiratory chain complexes I, III, and IV, encoded by nuclear and mitochondrial DNA, were abnormally low, whereas activity of complex II, entirely encoded by nuclear DNA, was not. Protein studies confirmed stoichiometric imbalance between mitochondrial (cytochrome c oxidase subunits 1 and 2) and nuclear (succinate dehydrogenase A) DNA-encoded respiratory chain subunits. These findings were not explained by defects in mitochondrial DNA or transcription. There were no compensatory mitochondrial biogenesis (no induction of nuclear respiratory factor 1 and mitochondrial transcript factor A) or adaptive unfolded protein response (reduced concentration of heat shock proteins 60 and 70). Conclusions: Linezolid-induced lactic acidosis is associated with diminished global oxygen consumption and extraction. These changes reflect selective inhibition of mitochondrial protein synthesis (probably translation) with secondary mitonuclear imbalance. One novel aspect of linezolid toxicity that needs to be confirmed is blunting of reactive mitochondrial biogenesis and unfolded protein response.

Leigh syndrome (LS) is an incurable, nearly always fatal, neurodegenerative, pediatric disorder that results from respiratory chain failure. The most common mitochondrial DNA (mtDNA) mutations that result in LS are m.8993T→C/G and m.9176T→C/G, which were previously found in several patients with early-onset Leigh syndrome. Here, we describe clinical and molecular features of a novel pedigree, where LS developed in two siblings. The proband was a young woman with an unusual adult-onset LS. She harbored a homoplasmic m.9176T→C mutation, based on analysis of a muscle biopsy. In contrast, the brother died at a young age. This novel case report and literature review highlights the variability of phenotypic expression of the m.9176T→C mutation.

Duchenne and Becker muscular dystrophy (DMD and BMD, respectively) are allelic disorders with different clinical presentations and severity determined by mutations in the gene DMD, which encodes the sarcolemmal protein dystrophin. Diagnosis is based on clinical aspects and muscle protein analysis, followed by molecular confirmation. We revised the main aspects of the natural history of dystrophinopathies to define genotype-phenotype correlations in large patient cohorts with extended follow-up. We also specifically explored subjects carrying nucleotide substitutions in the DMD gene, a comparatively less investigated DMD/BMD subgroup. We studied 320 dystrophinopathic patients (205 DMD and 115 BMD), defining muscular, cardiac, respiratory, and cognitive involvement. We also subdivided patients according to the kind of molecular defect (deletions, duplications, nucleotide substitutions or other microrearrangements) and the mutation sites (proximal/distal to exon 45), studying phenotype-genotype correlations for each group. In DMD, mutation type did not influence clinical evolution; mutations located in distal regions (irrespective of their nature) are more likely to be associated with lower IQ levels (p = 0.005). BMD carrying proximal deletions showed a higher degree of cardiac impairment than BMD with distal deletions (p = 0.0046). In the BMD population, there was a strong correlation between the entity of muscle dystrophin deficiency and clinical course (p = 0.002). An accurate knowledge of natural history may help in the clinical management of patients. Furthermore, several clinical trials are ongoing or are currently planned, some of which aim to target specific DMD mutations: a robust natural history is therefore essential to correctly design these experimental trials.

Mitochondrial disorders are often associated with mutations in mitochondrial tRNA. Independent observation of the same molecular defect in unrelated subjects is a generally required proof of pathogenicity. A sporadic case of chronic external ophthalmoplegia (cPEO) with ragged red fibres (RRFs) has been previously related to an m.12316G>A substitution in tRNA(Leu(CUN)). Sequencing muscle-derived mtDNA, we found the m.12316G>A substitution in an adult woman with mitochondrial myopathy and respiratory impairment. Her muscle biopsy presented several cytochrome c oxidase-negative (COX-) fibres, and RRFs as signs of mitochondrial proliferation. Restriction-fragment length polymorphism (RFLP) analysis of the mutation in isolated muscle fibres showed a threshold of at least 60% of mutated mtDNA to determine a COX deficiency phenotype. This second report of the m.12316G>A mutation in a sporadic patient consolidates its pathogenic nature and provides further elements for genetic counselling.

A disulfide relay system (DRS) was recently identified in the yeast mitochondrial intermembrane space (IMS) that consists of two essential components: the sulfhydryl oxidase Erv1 and the redox-regulated import receptor Mia40. The DRS drives the import of cysteine-rich proteins into the IMS via an oxidative folding mechanism. Erv1p is reoxidized within this system, transferring its electrons to molecular oxygen through interactions with cytochrome c and cytochrome c oxidase (COX), thereby linking the DRS to the respiratory chain. The role of the human Erv1 ortholog, GFER, in the DRS has been poorly explored. Using homozygosity mapping, we discovered that a mutation in the GFER gene causes an infantile mitochondrial disorder. Three children born to healthy consanguineous parents presented with progressive myopathy and partial combined respiratory-chain deficiency, congenital cataract, sensorineural hearing loss, and developmental delay. The consequences of the mutation at the level of the patient's muscle tissue and fibroblasts were 1) a reduction in complex I, II, and IV activity; 2) a lower cysteine-rich protein content; 3) abnormal ultrastructural morphology of the mitochondria, with enlargement of the IMS space; and 4) accelerated time-dependent accumulation of multiple mtDNA deletions. Moreover, the Saccharomyces cerevisiae erv1(R182H) mutant strain reproduced the complex IV activity defect and exhibited genetic instability of the mtDNA and mitochondrial morphological defects. These findings shed light on the mechanisms of mitochondrial biogenesis, establish the role of GFER in the human DRS, and promote an understanding of the pathogenesis of a new mitochondrial disease.

Ever increasing evidence has been provided on the accumulation of mutations in the mitochondrial DNA (mtDNA) during the aging process. However, the lack of direct functional consequences of the mutant mtDNA load on the mitochondria-dependent cell metabolism has raised many questions on the physiological importance of the age-related mtDNA variations. In the present work, we have analyzed the bioenergetic properties associated with the age-related T414G mutation of the mtDNA control region in transmitochondrial cybrids. The results show that the T414G mutation does not cause per se any detectable bioenergetic change. Moreover, three mtDNA mutations clustered in the 16S ribosomal RNA gene cosegregated together with the T414G in the same cybrid cell line. Two of them, namely T1843C and A1940G, are novel and associate with a negative bioenergetic phenotype. The results are discussed in the more general context of the complex heterogeneity and the dramatic instability of the mitochondrial genome during cell culture of transmitochondrial cybrids.

Carnitine palmitoyltransferase II (CPT II) deficiency is the most common inherited disorder of lipid metabolism characterized in its adult form by attacks of myalgia and myoglobinuria. We analyzed a cohort of 22 CPT II-deficient patients (representing 20 independent probands) to correlate clinical presentation and molecular data. The common p.Ser113Leu mutation was detected with an allelic frequency of 67.5% (27/40), in association with mild adult-onset phenotype. In addition to the p.Ser113Leu mutation, other 10 disease-causing mutations were identified, 5 of which were novel. They are a micro-insertion within exon 5, three aminoacid substitutions within the coding region, namely p.Arg151Trp, p.Asp576Gly, p.Arg247Trp and a truncating stop codon mutation (p.Arg554Ter). Our data expand the spectrum of CPT II mutations and help to evaluate possible correlations between genotypes and phenotypes.

Limb girdle muscular dystrophies (LGMD) are characterized by genetic and clinical heterogeneity: seven autosomal dominant and 12 autosomal recessive loci have so far been identified. Aims of this study were to evaluate the relative proportion of the different types of LGMD in 181 predominantly Italian LGMD patients (representing 155 independent families), to describe the clinical pattern of the different forms, and to identify possible correlations between genotype, phenotype, and protein expression levels, as prognostic factors. Based on protein data, the majority of probands (n=72) presented calpain-3 deficiency; other defects were as follows: dysferlin (n=31), sarcoglycans (n=32), α-dystroglycan (n=4), and caveolin-3 (n=2). Genetic analysis identified 111 different mutations, including 47 novel ones. LGMD relative frequency was as follows: LGMD1C (caveolin-3) 1.3%; LGMD2A (calpain-3) 28.4%; LGMD2B (dysferlin) 18.7%; LGMD2C (γ-sarcoglycan) 4.5%; LGMD2D (α-sarcoglycan) 8.4%; LGMD2E (β-sarcoglycan) 4.5%; LGMD2F (δ-sarcoglycan) 0.7%; LGMD2I (Fukutin-related protein) 6.4%; and undetermined 27.1%. Compared to Northern European populations, Italian patients are less likely to be affected with LGMD2I. The order of decreasing clinical severity was: sarcoglycanopathy, calpainopathy, dysferlinopathy, and caveolinopathy. LGMD2I patients showed both infantile noncongenital and mild late-onset presentations. Age at disease onset correlated with variability of genotype and protein levels in LGMD2B. Truncating mutations determined earlier onset than missense substitutions (20±5.1 years vs. 36.7±11.1 years; P=0.0037). Similarly, dysferlin absence was associated with an earlier onset when compared to partial deficiency (20.2±standard deviation [SD] 5.2 years vs. 28.4±SD 11.2 years; P=0.014). Hum Mutat 29(2), 258–266, 2008. © 2007 Wiley-Liss, Inc.

The important depletion of mitochondrial DNA (mtDNA) and the general depression of mitochondrial respiratory chain complex levels (including complex II) have been confirmed, implying an increasing paucity of mitochondria in the muscle from patients with types I, II, and III spinal muscular atrophy (SMA-I, -II, and -III, respectively).To investigate mitochondrial dysfunction in a large series of muscle biopsy samples from patients with SMA.We studied quadriceps muscle samples from 24 patients with genetically documented SMA and paraspinal muscle samples from 3 patients with SMA-II undergoing surgery for scoliosis correction. Postmortem muscle samples were obtained from 1 additional patient. Age-matched controls consisted of muscle biopsy specimens from healthy children aged 1 to 3 years who had undergone analysis for suspected myopathy. Analyses were performed at the Neuromuscular Unit, Istituto di Ricovero e Cura a Carattere Scientifico Foundation Ca' Granda Ospedale Maggiore Policlinico-Milano, from April 2011 through January 2015.We used histochemical, biochemical, and molecular techniques to examine the muscle samples.Respiratory chain activity and mitochondrial content.Results of histochemical analysis revealed that cytochrome-c oxidase (COX) deficiency was more evident in muscle samples from patients with SMA-I and SMA-II. Residual activities for complexes I, II, and IV in muscles from patients with SMA-I were 41%, 27%, and 30%, respectively, compared with control samples (P .005). Muscle mtDNA content and cytrate synthase activity were also reduced in all 3 SMA types (P .05). We linked these alterations to downregulation of peroxisome proliferator-activated receptor coactivator 1α, the transcriptional activators nuclear respiratory factor 1 and nuclear respiratory factor 2, mitochondrial transcription factor A, and their downstream targets, implying depression of the entire mitochondrial biogenesis. Results of Western blot analysis confirmed the reduced levels of the respiratory chain subunits that included mitochondrially encoded COX1 (47.5%; P = .004), COX2 (32.4%; P .001), COX4 (26.6%; P .001), and succinate dehydrogenase complex subunit A (65.8%; P = .03) as well as the structural outer membrane mitochondrial porin (33.1%; P .001). Conversely, the levels of expression of 3 myogenic regulatory factors-muscle-specific myogenic factor 5, myoblast determination 1, and myogenin-were higher in muscles from patients with SMA compared with muscles from age-matched controls (P .05).Our results strongly support the conclusion that an altered regulation of myogenesis and a downregulated mitochondrial biogenesis contribute to pathologic change in the muscle of patients with SMA. Therapeutic strategies should aim at counteracting these changes.

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is an infantile autosomal-recessive motor neuron disease caused by mutations in the immunoglobulin micro-binding protein 2. We investigated the potential of a spinal cord neural stem cell population isolated on the basis of aldehyde dehydrogenase (ALDH) activity to modify disease progression of nmd mice, an animal model of SMARD1. ALDH(hi)SSC(lo) stem cells are self-renewing and multipotent and when intrathecally transplanted in nmd mice generate motor neurons properly localized in the spinal cord ventral horns. Transplanted nmd animals presented delayed disease progression, sparing of motor neurons and ventral root axons and increased lifespan. To further investigate the molecular events responsible for these differences, microarray and real-time reverse transcription-polymerase chain reaction analyses of wild-type, mutated and transplanted nmd spinal cord were undertaken. We demonstrated a down-regulation of genes involved in excitatory amino acid toxicity and oxidative stress handling, as well as an up-regulation of genes related to the chromatin organization in nmd compared with wild-type mice, suggesting that they may play a role in SMARD1 pathogenesis. Spinal cord of nmd-transplanted mice expressed high transcript levels for genes related to neurogenesis such as doublecortin (DCX), LIS1 and drebrin. The presence of DCX-expressing cells in adult nmd spinal cord suggests that both exogenous and endogenous neurogeneses may contribute to the observed nmd phenotype amelioration.

A 45-year-old male patient had an episode of acute renal failure with myoglobinuria, myalgias, weakness, and markedly increased serum CK levels. Similar episodes had occurred in the past. Carnitine palmitoyl-transferase II (CPT II) deficiency was documented both biochemically and genetically. Interestingly, muscle biopsy also showed some ragged red fibers (RRF) and complete mitochondrial DNA (mtDNA) sequence disclosed a homoplasmic T3394C point mutation. This mutation is described in Leber's hereditary optic neuropathy (LHON) or in patients with diabetes mellitus.

Extremely variable clinic and genetic features characterize mitochondrial encephalomyopathy (MEM). Pathogenic mitochondrial DNA (mtDNA) defects can be divided into large-scale rearrangements and single point mutations. Clinical manifestations become evident when a threshold percentage of the total mtDNA is mutated. In some MEM, the "mutant load" in an affected tissue is directly related to the severity of the phenotype. However, the clinical phenotype is not simply a direct consequence of the relative abundance of mutated mtDNA. Other factors, such as nuclear background, can contribute to the disease process, resulting in a wide range of phenotypes caused by the same mutation. Using Affymetrix oligonucleotide cDNA microarrays (HG-U133A), we studied the gene expression profile of muscle tissue biopsies obtained from 12 MEM patients [4 common 4977 bp deleted mtDNA and 8 A3243G: 4 progressive external ophthalmoplegia (PEO) and 4 mitochondrial myopathy, encephalopathy, lactic cidosis, and stroke-like episodes syndrome (MELAS) phenotypes] compared with age-matched healthy individuals. We found several differentially expressed genes: 35 were markedly up-regulated in the mtDNA macro-deletion group (vs. the control group) and 4 decreased; 56 genes were dysregulated in A3243G-related disorders (53 down-regulated in PEO and 3 up-regulated in MELAS). Finally, 12 genes were similarly regulated in the majority of the MEM patients under study. Amongst these, we identified an increased expression of genes related to the metabolism of the amino groups, as well as of several genes involved in genetic information processing. Moreover, few genes were similarly decreased in MEM patients vs. the control group. Real-time PCR demonstrated excellent reproducibility of the microarray-based findings. The observed expression changes are likely to represent a molecular signature for mitochondrial disorders. Furthermore, the differential expression profile of MELAS(A3243G) vs. PEO(A3243G) may support a role of nuclear background in contributing to these different clinical phenotypes. MEM microarray data are available from GEO database (http://www.ncbi.nlm.nih.gov/geo/) with the accession number: GSE1462.

Objective: To better understand the still unknown pathologic mechanism involved in the accumulation of multiple mtDNA deletions in stable tissues. Methods: A large-scale screening of mtDNA molecules from skeletal muscle was performed in 14 patients with progressive external ophthalmoplegia (PEO) and 2 patients with mitochondrial neurogastrointestinal encephalomyopathy carrying mutations on ANT1 , C10ORF2 or POLG1 , and TP genes. Results: Patients with at least one mutation in the exonuclease domain of POLG1 showed the highest frequency of individually rare point mutations only in the mtDNA control region; in addition, high levels, in terms of frequency and heteroplasmy, of recurrent mutations (A189G, T408A, and T414G) and alterations affecting the (HT)D310 region were detectable in many of the patients. Two homozygous POLG1 mutations, within the exonuclease domain, were able to induce an increased mutational burden also in fibroblasts from patients with PEO. Conclusions: Specific POLG1 mutations directly affect the integrity of the mtDNA by reducing its proof-reading exonuclease activity, resulting in the accumulation of heteroplasmic levels of both randomly rare and recurrent point mutations in the skeletal muscle tissue and fibroblasts.

The ageing process is associated with the accumulation of somatic mutations of mitochondrial DNA (mtDNA). The aged human skeletal muscle tissue presents a mosaic of fibers when stained histochemically for cytochrome c oxidase (COX) activity with a proportion of COX negative fibers. Given the potential relevance of any alteration in the mtDNA control region for replication, we analysed the correlation between the presence of mutations and their degree of heteroplasmy and the COX phenotype in individual muscle fibers of aged healthy donors. A region of the mtDNA D-loop was cloned from single fiber-derived DNA and multiple clones were analysed. This strategy showed that a high level of mutational burden is present in all fibers and that several types of mtDNA rearrangements are detectable: recurrent (A189G, T408A and T414G) and rare point mutations, length variations affecting the homopolymeric tract and the (CA)n repeat and macrodeletions. The aggregate mutational load in the D-loop region correlated with the single fiber COX phenotype, suggesting that the cumulative burden of multiple, individually rare, mtDNA alterations might functionally impair the mitochondrial genetic machinery.

The progressive accumulation of mitochondrial DNA (mtDNA) alterations, ranging from single mutations to large-scale deletions, in both the normal ageing process and pathological conditions is a relevant phenomenon in terms of frequency and heteroplasmic degree. Recently, two point mutations (A189G and T408A) within the Displacement loop (D-loop) region, the control region for mtDNA replication, were shown to occur in skeletal muscles from aged individuals. We evaluated the presence and the heteroplasmy levels of these two mutations in muscle biopsies from 91 unrelated individuals of different ages (21 healthy subjects and 70 patients affected by mitochondrial encephalomyopathies). Overall, both mutations significantly accumulate with age. However, a different relationship was discovered among the different subgroups of patients: a higher number of A189G positive subjects younger than 53 years was detected in the subgroup of multiple-deleted patients; furthermore, a trend towards an increased risk for the mutations was evidenced among patients carrying multiple deletions when compared to healthy controls. These findings support the idea that a common biological mechanism determines the accumulation of somatic point mutations in the D-loop region, both in healthy subjects and in mitochondrial myopathy patients. At the same time, it appears that disorders caused by mutations of nuclear genes controlling mtDNA replication (the "mtDNA multiple deletions" syndromes) present a temporal advantage to mutate in the D-loop region. This observation may be relevant to the definition of the molecular pathogenesis of these latter syndromes.

Autosomal dominant progressive external ophthalmoplegia (adPEO) is caused by mutations in at least three different genes: ANT1 (chromosome 4q34-35), TWINKLE, and POLG. The ANT1 gene encodes the adenine nucleotide translocator-1 (ANT1). We identified a heterozygous T293C mutation of the ANT1 gene in a Greek family with adPEO. The resulting leucine to proline substitution likely modifies the secondary structure of the ANT1 protein. ANT1 gene mutations may account for adPEO in families with different ethnic backgrounds.

Deletions within the dystrophin gene (DMD) account for >70% of mutations leading to Duchenne and Becker muscular dystrophies (DMD and BMD). Deletion breakpoints were reported to be scattered within regions that also represent meiotic recombination hot spots. Recent studies indicates that deletion junctions arise from nonhomologous end joining (NHEJ), a major pathway for repairing DNA double-strand breaks (DSBs) in mammals. Here we show that a region in intron 47 (i.e., a major deletion hot spot in the DMD gene) generates DSBs during meiosis in yeast and harbors a cluster of previously sequenced deletion breaks. Mapping of breakpoints in 26 BMD/DMD patients indicated that the frequency of breakpoint occurrence around this region is 3-fold higher than expected by chance. These findings suggest that DSBs mediate deletion formation in intron 47 and possibly account for the high frequency of meiotic recombination in the region. Statistical analysis indicated the presence of at least one other breakpoint cluster in intron 47. Taken together, these results suggest that the primary events in deletion formation occur within discrete regions and that the scattered breakpoint distribution reflects both a variable degree of DSB end processing and the availability of a small (compared to the huge regions involved) deletion junction sample.

Glycogen storage disease type II (GSDII) is a lysosomal storage disorder caused by acid alpha-1,4-glucosidase deficiency and associated with recessive mutations in its coding gene GAA. Few studies have provided so far a detailed phenotypical characterization in late onset GSDII (LO-GSDII) patients. Genotype-phenotype correlation has been previously attempted with controversial results. We aim to provide an in-depth description of a cohort (n = 36) of LO-GSDII patients coming from the north of Italy and compare our population's findings to the literature. We performed a clinical record-based retrospective and prospective study of our patients. LO-GSDII in our cohort covers a large variability of phenotype including subtle clinical presentation and did not differ significantly from previous data. In all patients, molecular analysis disclosed GAA mutations, five of them being novel. To assess potential genotype-phenotype correlations we divided IVS1-32-13T>G heterozygous patients into two groups following the severity of the mutations on the second allele. Our patients harbouring "severe" mutations (n = 21) presented a strong tendency to have more severe phenotypes and more disability, more severe phenotypes and more disability, higher prevalence of assisted ventilation and a shorter time of evolution to show it. The determination of prognostic factors is mandatory in order to refine the accuracy of prognostic information, to develop follow-up strategy and, more importantly, to improve the decision algorithm for enzyme replacement therapy administration. The demonstration of genotype-phenotype correlations could help to reach this objective. Clinical assessment homogeneity is required to overcome limitations due to the lack of power of most studies.

Respiratory failure associated with diaphragmatic weakness is the first cause of death in late-onset type II glycogenosis (LO-GSDII). We aim to identify predictive factors of diaphragmatic weakness and investigate the pathophysiology of respiratory muscles impairment. Pulmonary function and chest wall volumes were measured in ten patients and eight controls (supine and seated). According to the change in forced vital capacity in supine (ΔFVC) we considered patients with (DW, ΔFVC25%) and without (noDW, ΔFVC25%) diaphragmatic weakness. Postural change made the supine abdominal contribution to tidal volume (%VAB) of DW to fall and the ribcage to increase and good correlation was found between %VAB and ΔFVC (R=0.776). Patients showed reduced chest wall and abdominal inspiratory capacity (ICCW and ICAB) (p0.001) and low abdominal expiratory reserve volume (p0.01). Passing to supine DW did not increase ICCW and ICAB. ΔFVC occurs in LO-GSDII due to weakened diaphragm and abdominal muscles while intercostals are preserved. %VAB represents a new reliable index to detect diaphragmatic weakness.

Mitochondrial (mt)DNA alterations cause cellular energy failure and respiratory chain dysfunction. Single large-scale rearrangements represent the most common mtDNA mutations and are responsible for very variable clinical manifestations. Here, we show an increased frequency of the A12308G substitution, a common polymorphism used to define the European mtDNA haplogroup U, in mitochondrial patients carrying mtDNA single macrodeletion. In this group of patients, A12308G substitution is associated with a higher relative risk of developing pigmentary retinal degeneration, short stature, dysphasia-dysarthria and cardiac conduction defects. MtDNA haplotype might modulate the clinical expression of mitochondrial encephalomyopathies due to mtDNA macrodeletions.

Background Brown-Vialetto-Van Laere (BVVL) syndrome is a rare disorder characterised by progressive pontobulbar palsy and sensorineural deafness. Causative mutations in genes encoding human riboflavin transporter 2 (hRFT2) and 3 (hRFT3) have been identified in BVVL patients. Methods and results We report the clinical and molecular features of a severe BVVL patient in whom screening of SLC52A3/hRFT2 was negative. Sequence analysis identified two novel compound heterozygous mutations in SLC52A2/hRFT3 , namely c.155C>T and c.1255G>A, leading to the amino acid changes p.S52F and p.G419S, respectively. Functional studies show that these defects impair the gene expression of the corresponding transporter, resulting in a significant reduction of riboflavin transport. Conclusions These findings support the pathogenetic role of SLC52A2/hRFT3 in BVVL with important clinical and therapeutic implications.

The molecular diagnosis of mitochondrial disorders still remains elusive in a large proportion of patients, but advances in next generation sequencing are significantly improving our chances to detect mutations even in sporadic patients. Syndromes associated with mitochondrial DNA multiple deletions are caused by different molecular defects resulting in a wide spectrum of predominantly adult-onset clinical presentations, ranging from progressive external ophthalmoplegia to multi-systemic disorders of variable severity. The mutations underlying these conditions remain undisclosed in half of the affected subjects. We applied next-generation sequencing of known mitochondrial targets (MitoExome) to probands presenting with adult-onset mitochondrial myopathy and harbouring mitochondrial DNA multiple deletions in skeletal muscle. We identified autosomal recessive mutations in the DGUOK gene (encoding mitochondrial deoxyguanosine kinase), which has previously been associated with an infantile hepatocerebral form of mitochondrial DNA depletion. Mutations in DGUOK occurred in five independent subjects, representing 5.6% of our cohort of patients with mitochondrial DNA multiple deletions, and impaired both muscle DGUOK activity and protein stability. Clinical presentations were variable, including mitochondrial myopathy with or without progressive external ophthalmoplegia, recurrent rhabdomyolysis in a young female who had received a liver transplant at 9 months of age and adult-onset lower motor neuron syndrome with mild cognitive impairment. These findings reinforce the concept that mutations in genes involved in deoxyribonucleotide metabolism can cause diverse clinical phenotypes and suggest that DGUOK should be screened in patients harbouring mitochondrial DNA deletions in skeletal muscle. © 2012 The Author (2012). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Limb-girdle muscular dystrophy (LGMD) 2L, caused by mutations in the anoctamin 5 (ANO5) gene, is the third most common LGMD in Northern and Central Europe, where the c.191dupA mutation causes the majority of cases. We evaluated data from 228 Italian LGMD patients to determine the prevalence of LGMD2L and the c.191dupA mutation, and to describe the clinical, muscle biopsy, and magnetic resonance imaging findings in these patients. Forty-three patients who lacked molecular diagnosis were studied for ANO5 mutations, and four novel mutations were found in three probands. Only one proband carried the c.191dupA mutation, which was compound heterozygous with c.2516T>G. Two probands were homozygous for the c.1627dupA and c.397A>T mutations, respectively, while a fourth proband had a compound heterozygous status (c.220C>T and c.1609T>C). Therefore occurrence and molecular epidemiology of LGMD2L in this Italian cohort differed from those observed in other European countries. ANO5 mutations accounted for ∼2% of our sample. Affected patients exhibited benign progression with variable onset and an absence of cardiac and respiratory impairment; muscle biopsy generally showed mild signs, except when performed on the quadriceps muscles; MRI showed predominant involvement of the posterior thigh. Overall these common clinical, morphological and imaging findings could be useful in differential diagnosis.

Background POLG1 mutations have been associated with MELAS-like phenotypes. However given several clinical differences it is unknown whether POLG1 mutations are possible causes of MELAS or give raise to a distinct clinical and genetic entity, named POLG1-associated encephalopathy. Case presentation We describe a 74 years old man carrying POLG1 mutations presenting with strokes, myopathy and ragged red fibers with some atypical aspects for MELAS such as late onset, lack of cerebral calcification and presence of frontal and occipital MRI lesions better consistent with the POLG associated-encephalopathy spectrum. Conclusion The lack of available data hampers a definite diagnosis in our patient as well as makes it difficult to compare MELAS, which is a clearly defined clinical syndrome, with POLG1-associated encephalopathy, which is so far a purely molecularly defined syndrome with a quite heterogeneous clinical picture. However, the present report contributes to expand the phenotypic spectrum of POLG1 mutations underlining the importance of searching POLG1 mutations in patients with mitochondrial signs and MELAS like phenotypes but negative for common mtDNA mutations.

Although mutations in mitochondrial tRNAs constitute the most common mtDNA defect, the presence of pathological variants in mitochondrial tRNA(Asn) is extremely rare. We were able to identify a novel mtDNA tRNA(Asn) gene pathogenic mutation associated with a myopathic phenotype and a previously unreported respiratory impairment. Our proband is an adult woman with ophthalmoparesis and respiratory impairment. Her muscle biopsy presented several cytochrome c oxidase-negative (COX-) fibres and signs of mitochondrial proliferation (ragged red fibres). Sequence analysis of the muscle-derived mtDNA revealed an m.5709T>C substitution, affecting mitochondrial tRNA(Asn) gene. Restriction-fragment length polymorphism analysis of the mutation in isolated muscle fibres showed that a threshold of at least 91.9% mutated mtDNA results in the COX deficiency phenotype. The new phenotype further increases the clinical spectrum of mitochondrial diseases caused by mutations in the tRNA(Asn) gene.

Mitochondrial DNA host a wide number of molecular defects associated with a broad spectrum of human clinical presentations ranging from tissue-specific diseases such as isolated myopathy or Leber hereditary optic neuropathy to multisystem disorders. Mutations affecting mitochondrial protein synthesis (transfer and ribosomal RNA genes) are quite frequent while molecular defects affecting genes encoding respiratory chain subunits are relatively less common. Here we discuss clinical, histological and molecular features of a patient presenting isolated myopathy secondary to mitochondrial complex I deficiency. Muscle biopsy showed ragged-red fibers that stained intensely for cytochrome c oxidase activity (COX-positive RRF). Muscle respiratory chain biochemical analysis revealed a severe ( A mutation, resulting in a premature termination codon within ND1 subunit. Mutational load was assessed by PCR-RFLP analysis and found to be 50% in skeletal muscle. Conversely we did not detect the variant in proband’s blood and proliferating cells including fibroblasts and myoblasts. To our knowledge, only two other non sense mutations have been reported in ND1 gene: the m.3308T > G mutation (p. M1X) in three cases of infantile onset sudden death syndromes and the intragenic inversion of seven nucleotides, resulting, similar to our proband, in isolated mitochondrial myopathy.

Maintenance and replication of mitochondrial DNA require the concerted action of several factors encoded by nuclear genome. The mitochondrial helicase Twinkle is a key player of replisome machinery. Heterozygous mutations in its coding gene, PEO1, are associated with progressive external ophthalmoplegia (PEO) characterised by ptosis and ophthalmoparesis, with cytochrome c oxidase (COX)-deficient fibres, ragged-red fibres (RRF) and multiple mtDNA deletions in muscle. Here we describe clinical, histological and molecular features of two patients presenting with mitochondrial myopathy associated with PEO. PEO1 sequencing disclosed two novel mutations in exons 1 and 4 of the gene, respectively. Although mutations in PEO1 exon 1 have already been described, this is the first report of mutation occurring in exon 4.

Background Amyotrophic lateral sclerosis (ALS) is a major cause of neurological disability and its pathogenesis remains elusive despite a multitude of studies. Although defects of the mitochondrial respiratory chain have been described in several ALS patients, their pathogenic significance is unclear. Objective To review systematically the muscle biopsy specimens from patients with typical sporadic ALS to search for possible mitochondrial oxidative impairment. Design Retrospective histochemical, biochemical, and molecular studies of muscle specimens. Setting Tertiary care university. Subjects Fifty patients with typical sporadic ALS (mean age, 55 years). Main Outcome Measure Number of patients showing a clear muscle mitochondrial dysfunction assessed through histochemical and biochemical muscle analysis. Results Histochemical data showed cytochrome c oxidase (COX)–negative fibers in 46% patients. Based on COX histochemical activity, patients fell into 4 groups: 27 had normal COX activity; and 8 had mild (2-4 COX-negative fibers of 100 fibers), 8 had moderate (5-10 COX-negative fibers of 100), and 7 had severe (>10 COX-negative fibers of 100) COX deficiency. Spectrophotometric measurement of respiratory chain activities showed that 3 patients with severe histochemical COX deficiency also showed combined enzyme defects. In 1 patient, COX deficiency worsened in a second biopsy taken 9 months after the first. Among the patients with severe COX deficiency, one had a new mutation in the SOD1 gene, another a mutation in the TARDBP gene, and a third patient with biochemically confirmed COX deficiency had multiple mitochondrial DNA deletions detectable by Southern blot analysis. Conclusions Our data confirm that the histochemical finding of COX-negative fibers is common in skeletal muscle from patients with sporadic ALS. We did not find a correlation between severity of the oxidative defect and age of the patients or duration of the disease. However, the only patient who underwent a second muscle biopsy did show a correlation between severity of symptoms and worsening of the respiratory chain defect. In 7 patients, the oxidative defect was severe enough to support the hypothesis that mitochondrial dysfunction must play a role in the pathogenesis of the disease.