Large-scale mtDNA Deletions Research Articles

Abstract 15785: Long-term Cardiac Prognosis and Risk Stratification in 260 Adults Presenting With Mitochondrial Diseases

Introduction: The long-term cardiac prognosis of adults with mitochondrial diseases is unknown. Methods and Results: Between January 2000 and May 2014, we retrospectively included in this study 260 consecutive patients (60% women) ≥18 years of age, (interquartile range [IQR]; 31 to 54), with genetically proven mitochondrial diseases, including 109 with mitochondrial DNA (mtDNA) single large-scale deletions, 64 with the m.3243A>G mutation in MT-TL1 , 51 with other mtDNA point mutations, and 36 patients with nuclear genes mutations. Cardiac involvement was present at baseline in 81 patients (30%). Single and multiple variable analyses were performed in search of predictors of major adverse cardiac event (MACE), and hazard ratios (HR) and 95% confidence intervals (CI) were calculated. Over a median follow-up of 7 years [3.6 to 11.7], 27 patients (10%) suffered a MACE, defined as sudden death, death due to heart failure (HF), resuscitated cardiac arrest, 3 rd degree atrioventricular block, sinus node dysfunction, cardiac transplantation, or hospitalization for management of HF. Patients with single large-scale mtDNA deletions or m.3243A>G mutations had the highest incidence of MACE. By multiple variable analysis, intraventricular conduction block (HR = 16.9; 95% CI: 7.2 to 39.4), diabetes (HR = 7.0; 95% CI: 2.9 to 16.7), premature ventricular complexes (HR = 3.6; 95% CI: 1.4 to 9.2) and left ventricular (LV) hypertrophy (HR = 2.5; 95% CI: 1.1 to 5.8) were independent predictors of MACE. In patients with 0, 1, and ≥2 risk factors, the incidence of MACE was 1.7, 15 and 42% respectively. Conclusions: Patients with mitochondrial diseases are at high risk of MACE, independently predicted by intraventricular conduction block, diabetes, ventricular prematurity and LV hypertrophy.

Long-term cardiac prognosis and risk stratification in 260 adults presenting with mitochondrial diseases.

The aim of this study is to assess the long-term cardiac prognosis of adults with mitochondrial diseases. Between January 2000 and May 2014, we retrospectively included in this study 260 consecutive patients (60% women) ≥18 years (interquartile range 31-54), with genetically proven mitochondrial diseases, including 109 with mitochondrial DNA (mtDNA) single large-scale deletions, 64 with the m.3243A>G mutation in MT-TL1, 51 with other mtDNA point mutations, and 36 patients with nuclear gene mutations. Cardiac involvement was present at baseline in 81 patients (30%). Single and multiple variable analyses were performed in search of predictors of major adverse cardiac events (MACEs), and hazard ratios (HRs) and 95% confidence intervals (CI) were calculated. Over a median follow-up of 7 years (3.6-11.7), 27 patients (10%) suffered a MACE, defined as sudden death, death due to heart failure (HF), resuscitated cardiac arrest, third-degree atrioventricular block, sinus node dysfunction, cardiac transplantation, or hospitalization for management of HF. Patients with single large-scale mtDNA deletions or m.3243A>G mutations had the highest incidence of MACE. By multiple variable analysis, intraventricular conduction block (HR = 16.9; 95% CI: 7.2-39.4), diabetes (HR = 7.0; 95% CI: 2.9-16.7), premature ventricular complexes (HR = 3.6; 95% CI: 1.4-9.2), and left ventricular (LV) hypertrophy (HR = 2.5; 95% CI: 1.1-5.8) were independent predictors of MACEs. In patients with zero, one, and two or more risk factors, the incidences of MACE were 1.7, 15 and 42%, respectively. Patients with mitochondrial diseases are at high risk of MACE, independently predicted by intraventricular conduction block, diabetes, ventricular prematurity, and LV hypertrophy.

OP46 – 2969: Novel phenotypes of childhood encephalomyopathies with mitochondrial DNA depletion or deletions

Objective To study the clinical manifestations and occurrence of mtDNA depletion and deletions in paediatric patients with neuromuscular diseases, in order to estimate the role of mtDNA rearrangements in pathogenesis of these diseases and to identify novel clinical phenotypes associated with mtDNA depletion or deletions. Methods Muscle DNA samples from patients presenting with undefined encephalomyopathies or myopathies were analysed for mtDNA content by quantitative real-time PCR and for deletions by long-range PCR. Direct sequencing of mtDNA maintenance genes and whole-exome sequencing were used to study the genetic aetiologies of diseases. Clinical and laboratory findings were collected. Results Muscle samples were obtained from 104 paediatric patients with neuromuscular diseases. MtDNA depletion was found in three patients with severe early-onset encephalomyopathy or myopathy Two of these patients presented with novel types of mitochondrial DNA depletion syndromes associated with increased serum creatine kinase and multiorgan disease without mutations in any of the known mtDNA maintenance genes; muscle electron micrograph of one patient revealed disordered myofibrillar structure, pathological endoplasmic reticulum membranes and accumulation of glycogen and extracellular collagen fibres. The third patient with mtDNA depletion and severe myopathy was diagnosed with merosine-deficient muscular dystrophy caused by a homozygous mutation in the LAMA2 gene. Two patients with an early-onset Kearns-Sayre/Pearson-like phenotype harboured a large-scale mtDNA deletion, minor multiple deletions and high mtDNA content (4.9 and 6.9-fold increase relative to the median of age-matched controls). Conclusion We describe two novel early-onset phenotypes associated with mtDNA depletion, one of them presenting with pathologic endoplasmic reticulum membranes. Furthermore, mtDNA depletion can be a secondary finding in hereditary muscular dystrophy. We suggest that a large-scale mtDNA deletion, minor multiple deletions and high mtDNA content associated with Kearns-Sayre/Pearson syndromes may be secondary changes caused by mutations in an unknown nuclear gene.

0370 : Long-term cardiac prognosis and risk stratification in 260 adults presenting with mitochondrial diseases

The long-term cardiac prognosis of adults with mitochondrial diseases is unknown. Between January 2000 and May 2014, we retrospectively included in this study 260 consecutive patients (60% women) ε18 years of age, [interquartile range (IQR) = 31 to 54], with genetically proven mitochondrial diseases, including 109 with mtDNA single large-scale deletions, 64 with the m.3243A>G mutation in MT-TL1, 51 with other mtDNA point mutations, and 36 patients with nuclear genes mutations. Cardiac involvement was present at baseline in 81 patients (30%). Single and multiple variable analyses were performed in search of predictors of major adverse cardiac event (MACE), and hazard ratios (HR) and 95% confidence intervals (CI) were calculated. Over a median follow-up of 7 years (IQR = 3.6 to 11.7), 27 patients (10%) experienced a MACE, defined as sudden death, death due to heart failure (HF), resuscitated cardiac arrest, 3rd degree atrioventricular block, sinus node dysfunction, cardiac transplantation, or hospitalization for management of HF. Patients with single large-scale mtDNA deletions or m.3243A>G mutations had the highest incidence of MACE. By multiple variable analysis, intraventricular conduction block (HR = 16.9; 95% CI: 7.2 to 39.4), diabetes (HR = 7.0; 95% CI: 2.9 to 16.7), premature ventricular complexes (HR = 3.6; 95% CI: 1.4 to 9.2) and left ventricular hypertrophy (HR=2.5; 95% CI: 1.1 to 5.8) were independent predictors of MACE. In patients with 0, 1, and ε2 risk factors, the incidence of MACE was 1.7, 15 and 42% respectively. Patients with mitochondrial diseases are at high risk of MACE, independently predicted by intraventricular conduction block, diabetes, ventricular prematurity and left ventricular hypertrophy.

Mitochondrial DNA Depletion and Deletions in Paediatric Patients with Neuromuscular Diseases: Novel Phenotypes.

To study the clinical manifestations and occurrence of mtDNA depletion and deletions in paediatric patients with neuromuscular diseases and to identify novel clinical phenotypes associated with mtDNA depletion or deletions. Muscle DNA samples from patients presenting with undefined encephalomyopathies or myopathies were analysed for mtDNA content by quantitative real-time PCR and for deletions by long-range PCR. Direct sequencing of mtDNA maintenance genes and whole-exome sequencing were used to study the genetic aetiologies of the diseases. Clinical and laboratory findings were collected. Muscle samples were obtained from 104 paediatric patients with neuromuscular diseases. mtDNA depletion was found in three patients with severe early-onset encephalomyopathy or myopathy. Two of these patients presented with novel types of mitochondrial DNA depletion syndromes associated with increased serum creatine kinase (CK) and multiorgan disease without mutations in any of the known mtDNA maintenance genes; one patient had pathologic endoplasmic reticulum (ER) membranes in muscle. The third patient with mtDNA depletion was diagnosed with merosine-deficient muscular dystrophy caused by a homozygous mutation in the LAMA2 gene. Two patients with an early-onset Kearns-Sayre/Pearson-like phenotype harboured a large-scale mtDNA deletion, minor multiple deletions and high mtDNA content. Novel encephalomyopathic mtDNA depletion syndrome with structural alterations in muscle ER was identified. mtDNA depletion may also refer to secondary mitochondrial changes related to muscular dystrophy. We suggest that a large-scale mtDNA deletion, minor multiple deletions and high mtDNA content associated with Kearns-Sayre/Pearson syndromes may be secondary changes caused by mutations in an unknown nuclear gene.

SANDO syndrome in a cohort of 107 patients with CPEO and mitochondrial DNA deletions

ObjectiveThe sensory ataxic neuropathy with dysarthria and ophthalmoparesis (SANDO) syndrome is a subgroup of mitochondrial chronic progressive external ophthalmoplegia (CPEO)-plus disorders associated with multiple mitochondrial DNA (mtDNA) deletions. There is...

Mitochondrial DNA Mutation Load

Mitochondrial DNA Mutation Load

Extraocular Muscle Atrophy and Central Nervous System Involvement in Chronic Progressive External Ophthalmoplegia

BackgroundChronic progressive external ophthalmoplegia (CPEO) is a classical mitochondrial ocular disorder characterised by bilateral progressive ptosis and ophthalmoplegia. These ocular features can develop either in isolation or in association with other prominent neurological deficits (CPEO+). Molecularly, CPEO can be classified into two distinct genetic subgroups depending on whether patients harbour single, large-scale mitochondrial DNA (mtDNA) deletions or multiple mtDNA deletions secondary to a nuclear mutation disrupting mtDNA replication or repair. The aim of this magnetic resonance imaging (MRI) study was to investigate whether the ophthalmoplegia in CPEO is primarily myopathic in origin or whether there is evidence of contributory supranuclear pathway dysfunction.MethodsTen age-matched normal controls and twenty patients with CPEO were recruited nine patients with single, large-scale mtDNA deletions and eleven patients with multiple mtDNA deletions secondary to mutations in POLG, PEO1, OPA1, and RRM2B. All subjects underwent a standardised brain and orbital MRI protocol, together with proton magnetic resonance spectroscopy in two voxels located within the parietal white matter and the brainstem.ResultsThere was evidence of significant extraocular muscle atrophy in patients with single or multiple mtDNA deletions compared with controls. There was no significant difference in metabolite concentrations between the patient and control groups in both the parietal white matter and brainstem voxels. Volumetric brain measurements revealed marked cortical and cerebellar atrophy among patients with CPEO+ phenotypes.ConclusionThe results of this study support a primary myopathic aetiology for the progressive limitation of eye movements that develops in CPEO.

Mitochondrial DNA deletions in muscle satellite cells: implications for therapies

Progressive myopathy is a major clinical feature of patients with mitochondrial DNA (mtDNA) disease. There is limited treatment available for these patients although exercise and other approaches to activate muscle stem cells (satellite cells) have been proposed. The majority of mtDNA defects are heteroplasmic (a mixture of mutated and wild-type mtDNA present within the muscle) with high levels of mutated mtDNA and low levels of wild-type mtDNA associated with more severe disease. The culture of satellite cell-derived myoblasts often reveals no evidence of the original mtDNA mutation although it is not known if this is lost by selection or simply not present in these cells. We have explored if the mtDNA mutation is present in the satellite cells in one of the commonest genotypes associated with mitochondrial myopathies (patients with single, large-scale mtDNA deletions). Analysis of satellite cells from eight patients showed that the level of mtDNA mutation in the satellite cells is the same as in the mature muscle but is most often subsequently lost during culture. We show that there are two periods of selection against the mutated form, one early on possibly during satellite cell activation and the other during the rapid replication phase of myoblast culture. Our data suggest that the mutations are also lost during rapid replication in vivo, implying that strategies to activate satellite cells remain a viable treatment for mitochondrial myopathies in specific patient groups.

Large scale deletions of the mitochondrial DNA in astheno, asthenoterato and oligoasthenoterato-spermic men

The purpose of this study was to investigate the association of large-scale deletions of mtDNA between idiopathic astheno, asthenoterato and oligoasthenoterato-spermic as patient group and normospermic as control group. Forty semen samples including: 10 asthenospermic (A), 10 asthenoteratospermic (AT), 10 oligoasthenoteratospermic (OAT) and 10 normospermic samples as control group, were collected from IVF center. Our analysis of long-range polymerase chain reaction were shown multiple deletions; 4977-bp, 7599-bp and 7491-bp of mtDNA in spermatozoa of patients (A, AT and OAT) and control groups. However, the frequency of multiple mtDNA deletions in astheno (60%), asthenoterato (60%), oligoasthenoterato (70%) spermic groups were significantly higher than normal (40%) group. These results suggest that mtDNA mutations cause infertility through an effect on sperm motility. Therefore, identification of mtDNA mutations and large scale deletions in the pathophysiology of human spermatozoa dysfunction is considered to be important to better understanding of the etiology of idiopathic infertility.

Mitochondrial genome variations in advanced stage breast cancer: A case–control study

Entire mitochondrial DNA (mtDNA) sequencing was carried out in 101 primary breast cancer patients and 90 controls of south Indian origin. We identified 69 novel mutations in breast cancer patients and 637 reported polymorphisms in patients and/or controls. PolyPhen-2 analysis predicted 5 out of 14 novel missense mutations as ‘probably damaging variants’. Haplogrouping analysis identified a significant association between haplogroup M5 and breast cancer risk. Microsatellite instability and tumor specific large scale mtDNA deletions were not observed in tumor tissues from the patients. In conclusion, mtDNA mutations and haplogroups may constitute an inheritable risk factor for pathogenesis of breast cancer.

The generation of oxidative stress-induced rearrangements in Saccharomyces cerevisiae mtDNA is dependent on the Nuc1 (EndoG/ExoG) nuclease and is enhanced by inactivation of the MRX complex

Oxidative stress is known to enhance the frequency of two major types of alterations in the mitochondrial genome of Saccharomyces cerevisiae: point mutations and large deletions resulting in the generation of respiration-deficient petite rhō mutants. We investigated the effect of antimycin A, a well-known agent inducing oxidative stress, on the stability of mtDNA. We show that antimycin enhances exclusively the generation of respiration-deficient petite mutants and this is accompanied by a significant increase in the level of reactive oxygen species (ROS) and in a marked drop of cellular ATP. Whole mitochondrial genome sequencing revealed that mtDNAs of antimycin-induced petite mutants are deleted for most of the wild-type sequence and usually contain one of the active origins of mtDNA replication: ori1, ori2 ori3 or ori5. We show that the frequency of antimycin-induced rhō mutants is significantly elevated in mutants deleted either for the RAD50 or XRS2 gene, both encoding the components of the MRX complex, which is known to be involved in the repair of double strand breaks (DSBs) in DNA. Furthermore, enhanced frequency of rhō mutants in cultures of antimycin-treated cells lacking Rad50 was further increased by the simultaneous absence of the Ogg1 glycosylase, an important enzyme functioning in mtBER. We demonstrate also that rad50Δ and xrs2Δ deletion mutants display a considerable reduction in the frequency of allelic mitochondrial recombination, suggesting that it is the deficiency in homologous recombination which is responsible for enhanced rearrangements of mtDNA in antimycin-treated cells of these mutants. Finally, we show that the generation of large-scale mtDNA deletions induced by antimycin is markedly decreased in a nuc1Δ mutant lacking the activity of the Nuc1 nuclease, an ortholog of the mammalian mitochondrial nucleases EndoG and ExoG. This result indicates that the nuclease plays an important role in processing of oxidative stress-induced lesions in the mitochondrial genome.

Prevalence of the primary LHON mutations in Northern Finland associated with bilateral optic atrophy and tobacco-alcohol amblyopia

Leber hereditary optic neuropathy (LHON) is regarded as the most common mitochondrial disease. We have previously reported comprehensive population-based epidemiological data on common mitochondrial DNA (mtDNA) mutations including m.3243A>G, m.8344A>G and large-scale mtDNA deletions in Northern Finland. Our aim was to investigate the prevalence of primary LHON mutations and mutations in the four mtDNA genes considered hot spots for LHON in the same population. The study population consisted of 42 adult patients with an aetiologically undefined bilateral optic atrophy. The major LHON mutations m.3460G>A, m.11778G>A and m.14484T>C were analysed by restriction fragment length polymorphism (RFLP), and MTND1, MTND6 and MTATP6 genes were sequenced. MTND5 gene was analysed by conformation-sensitive gel electrophoresis (CSGE). No major LHON mutations were found in the population of the province of Northern Ostrobothnia giving the prevalence of these mutations 0-1.36:100 000 (95% CI). However, two main mutations were found elsewhere in Northern Finland, homoplasmic m.11778G>A from Kainuu and heteroplasmic m.3460G>A from Central Ostrobothnia. Furthermore, tobacco-alcohol amblyopia was diagnosed in five patients in the study population and one of them had m.11778G>A. The prevalence of the three major LHON mutations is lower in Northern Finland than elsewhere in Finland or in Western Europe. As LHON and tobacco-alcohol amblyopia have a similar phenotype, we recommend analysing the known LHON-associated mutations before setting tobacco-alcohol amblyopia diagnosis.

Molecular investigations of mitochondrial deletions: Evaluating the usefulness of different genetic tests

Deletions in mitochondrial DNA are a common cause of mitochondrial disorders. The molecular diagnosis of mtDNA deletions for years was based on Southern hybridization later replaced by PCR methods such as PCR with primers specific for a particular deletion (mainly the so-called common deletion of 4977bp) and long PCR.In order to evaluate the usefulness of MLPA (Multiplex Ligation-dependent Probe Amplification) in molecular diagnosis of large scale mtDNA deletions we compare four diagnostic methods: Southern hybridization, PCR, long-PCR and MLPA in a group of 16 patients with suspected deletions. Analysis was performed on blood, muscle and in one case hepatic tissue DNA.The MLPA was not able to confirm all the deletions detected by PCR methods, but due to its relative ease of processing, minimal equipment, low costs and the additional possibility to detect frequent point mtDNA mutations in one assay it is worth considering as a screening method. We recommend to always confirm MLPA results by PCR methods.

MERRF and Kearns-Sayre Overlap Syndrome Due to the Mitochondrial DNA M.3291T>C Mutation (IN7-1.008)

Objective: To report a patient with a complex phenotype of myoclonus epilepsy with ragged-red fibers (MERRF) syndrome and Kearns–Sayre syndrome (KSS), who harbored a m.3291T>C mutation in the tRNALeu(UUR) gene. This mutation has never been reported in association with a MERRF/KSS syndrome. Background Individuals who harbor a pathogenic mtDNA mutation can manifest overlapping features of typical mitochondrial syndromes. MERRF is a multisystem disorder characterized by myoclonic seizures, cerebellar ataxia, mitochondrial myopathy, and ragged-red fibers (RRF) on muscle biopsy, commonly caused by the m.8344A>G mutation in the tRNALys gene. KSS is a mitochondrial disorder characterized by onset before 20 years of age of progressive external ophthalmoplegia or pigmentary retinopathy, together with at least one of a triad of cerebellar ataxia, heart block, and elevated cerebrospinal fluid protein. Typically, KSS is due to single, large-scale deletions of mtDNA. Design/Methods: Clinical data were collected. Muscle histochemical and biochemical analysis was performed. Muscle DNA was screened for mtDNA large-scale rearrangements and point mutations. Mutation level was quantified by restriction fragment length polymorphism (RFLP) analysis. Results: A 48-year-old man presented with progressive myoclonus epilepsy, cerebellar ataxia, hearing loss, myopathic weakness, ophthalmoparesis, pigmentary retinopathy, and bifascicular heart block. Muscle biopsy demonstrated multiple RRF. Biochemical analysis of respiratory chain enzymes was normal. Genetic tests were negative for the common MERRF mtDNA mutations, and no deletions in mitochondrial DNA were detected. Direct sequencing mtDNA revealed a T-to-C transition at nucleotide position 3291 in the tRNALeu(UUR) gene. PCR RFLP showed 92% mutant genome in muscle. Conclusions: This is the first report associating a m.3291T>C mutation in the tRNALeu(UUR) gene with an adult-onset MERRF/KSS overlap. Our data reinforce the well-established concept that, in mtDNA-related disorders, the same genetic abnormality can be associated with a wide spectrum of phenotypes, and the observation that tRNALeu(UUR) is a hotspot for mtDNA mutations. Supported by: The National Institutes of Health (NIH HD32062) and by the Marriott Mitochondrial Disorders Clinical Research Fund (MMDCRF). M.H. was supported by NIH grants (R01HD57543, R01HD056103, and RCNS070232) and by the Muscular Dystrophy Association. Disclosure: Dr. Emmanuele has nothing to disclose. Dr. Silvers has nothing to disclose. Dr. Sotiriou has nothing to disclose. Dr. Tanji has nothing to disclose. Dr. DiMauro has nothing to disclose. Dr. Hirano has received personal compensation for activities with Athena Diagnostics as a speaker.Dr. Hirano has received personal compensation in an editorial capacity for Current Neurology and Neuroscience Reports.Dr. Hirano has received research support from Santhera Pharmaceutical and Edison Pharmaceuticals.

Progressive External Ophthalmoplegia in Southwestern Finland: A Clinical and Genetic Study

Background: Progressive external ophthalmoplegia (PEO) is a common phenotype of mitochondrial disease. Molecu- lar etiologies include sporadic, large-scale deletions in mitochondrial DNA (mtDNA), multiple mtDNA deletions secondary to autosomal dominant or recessive mutations and mtDNA point mutations. Methods: We studied the prevalence and clinical and genetic characteristics of PEO in a defined population in southwestern Finland. A total of 620 patients were first identified from the patient registry at the Turku University Hospital over an 18-year period. The medical records of these patients were scrutinized, and those with clinical features compatible with PEO were ascertained. Results: We identified 10 patients with possible PEO. The patients were examined clinically, and DNA was analyzed for mtDNA deletions and for the m.3243A>G and m.8344A>G mtDNA point mutations. The ANT1, PEO1, POLG1 and POLG2 genes were sequenced. We confirmed the clinical diagnosis of PEO in 6 patients. Large-scale mtDNA deletions were detected in 3 out of 6 PEO patients and mutations in the POLG1 gene in 1 out of 6. We did not find any mutations in the ANT1, PEO1 or POLG2 genes. Conclusions: Our results suggest that molecular investigation of patients with PEO, either sporadic or familial, should start with an analysis for mtDNA deletions, followed by an analysis of the POLG1 gene.

Mitochondrial autophagy in cells with mtDNA mutations results from synergistic loss of transmembrane potential and mTORC1 inhibition

Autophagy has emerged as a key cellular process for organellar quality control, yet this pathway apparently fails to eliminate mitochondria containing pathogenic mutations in mitochondrial DNA (mtDNA) in patients with a variety of human diseases. In order to explore how mtDNA-mediated mitochondrial dysfunction interacts with endogenous autophagic pathways, we examined autophagic status in a panel of human cytoplasmic hybrid (cybrid) cell lines carrying a variety of pathogenic mtDNA mutations. We found that both genetic- and chemically induced loss of mitochondrial transmembrane potential (Δψ(m)) caused recruitment of the pro-mitophagic factor Parkin to mitochondria. Strikingly, however, the loss of Δψ(m) alone was insufficient to prompt delivery of mitochondria to the autophagosome (mitophagy). We found that mitophagy could be induced following treatment with the mTORC1 inhibitor rapamycin in cybrids carrying either large-scale partial deletions of mtDNA or complete depletion of mtDNA. Further, we found that the level of endogenous Parkin is a crucial determinant of mitophagy. These results suggest a two-hit model, in which the synergistic induction of both (i) mitochondrial recruitment of Parkin following the loss of Δψ(m) and (ii) mTORC1-controlled general macroautophagy is required for mitophagy. It appears that mitophagy can be accomplished by the endogenous autophagic machinery, but requires the full engagement of both of these pathways.

Mitochondrial DNA copy number and function decrease with age in the short‐lived fish Nothobranchius furzeri

Among vertebrates that can be kept in captivity, the annual fish Nothobranchius furzeri possesses the shortest known lifespan. It also shows typical signs of aging and is therefore an ideal model to assess the role of different physiological and environmental parameters on aging and lifespan determination. Here, we used Nothobranchius furzeri to study whether aging is associated with mitochondrial DNA (mtDNA) alterations and changes in mitochondrial function. We sequenced the complete mitochondrial genome of N.furzeri and found an extended control region. Large-scale mtDNA deletions have been frequently described to accumulate in other organisms with age, but there was no evidence for the presence of detectable age-related mtDNA deletions in N.furzeri. However, mtDNA copy number significantly decreased with age in skeletal muscle, brain, liver, skin and dorsal fin. Consistent with this finding, expression of Pgc-1α that encodes a transcriptional coactivator of mitochondrial biogenesis and expression of Tfam and mtSsbp both encoding mtDNA binding factors was downregulated with age. The investigation of possible changes in mitochondrial function revealed that the content of respiratory chain complexes III and IV was reduced in skeletal muscle with age. In addition, ADP-stimulated and succinate-dependent respiration was decreased in mitochondria of old fish. These findings suggest that despite the short lifespan, aging in N.furzeri is associated with a decline in mtDNA copy number, the downregulation of mtDNA-associated genes and an impairment of mitochondrial function.

Prevalence and severity of voice and swallowing difficulties in mitochondrial disease

Mutations of mitochondrial DNA (mtDNA) cause a broad spectrum of clinical phenotypes. Anecdotal evidence suggests that voice and swallow problems are a common feature of these diseases. To characterize accurately the prevalence and severity of voice and swallow problems in a large cohort of patients with mitochondrial disease. Patients with proven mitochondrial disease were sent validated questionnaires to assess both voice and swallow function. The presence of voice and swallow symptoms was correlated with other clinical features of mitochondrial disease in affected patients. From the original 177 patients contacted, 98 swallowing status questionnaires and 96 Voice Handicap Index questionnaires were returned, response rates of 55% and 54%, respectively. Swallow: 48% of patients reported more difficulties with swallowing than control participants. Patients with single mtDNA deletions were most likely to report problems (65.2%), with patients with an m.8344A>G point mutation least likely (33.3%). All genotypes had a mean severity score in excess of the normal range, the highest mean score being found in the single large-scale mtDNA deletion group (10.12), the lowest in the m.3243A>G group (5.56) Voice; 48% of patients reported some difficulty with voice. Patients with single large-scale deletions showed the highest prevalence (65.2%), patients with the m.3243A>G mutation the lowest (33%). The most severe voice difficulties were reported by patients with an m.8344A>G point mutation. Patients with an m.3243A>G point mutation had the mildest and lowest incidence of voice problems. All genotypes scored outside of the normal range expected on the VHI overall (≥11.5 in control trials). Patients with an m.8344A>G point mutation reported a significantly higher degree of physical voice handicap than m.3243A>G patients (13.13 versus 4.40, p = 0.02). In patients with either single or multiple mtDNA deletions the likely pathophysiological mechanism is of proximal muscle weakness, whereas in patients with the m.8344A>G mutation cerebellar ataxia is the likely cause. Dysphagia and dysarthria have been identified as symptoms in previous research, however the prevalence and pathophysiology of these symptoms have not been explored. This paper indicates that voice and swallow problems are a common, though predominantly mild feature of mitochondrial disease and that there is a core group of pathophysiological symptoms linked to the presence of voice and swallowing problems. This paper recommends early referral to speech and language therapists to identify emerging dysphonia and dysphagia and to provide appropriate intervention.

Large-scale mitochondrial DNA deletions in weak goslings

1. Evidence has accumulated in mammals to support the idea that mitochondrial DNA (mtDNA) deletions and mutations might contribute to ageing and reproductive failure. White Roman geese were monitored to evaluate the effect of large-scale deletions of mtDNA in an avian species. 2. A total of 340 samples from 114 dead embryos, 111 weak goslings, and 115 normal goslings were used in this experiment. The regions of these two large-scale mtDNA deletions, ΔmtDNA6829 and ΔmtDNA6992, were between the COI and ND5 genes. A 3·6% (4 out of 111) positive sample was detected in the weak goslings. In contrast, no large-scale mitochondrial DNA deletions were detected in either the dead embryos (0 out of 114) or the normal goslings (0 out of 115). 3. Large-scale mtDNA deletions may be a factor causing weak goslings.