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Primary mitochondrial disease describes a diverse group of neuro-metabolic disorders characterised by impaired oxidative phosphorylation. Diagnosis is challenging; >350 genes, both nuclear and mitochondrial DNA (mtDNA) encoded, are known to cause mitochondrial disease, leading to all possible inheritance patterns and further complicated by heteroplasmy of the multicopy mitochondrial genome. Technological advances, particularly next-generation sequencing, have driven a shift in diagnostic practice from ‘biopsy first’ to genome-wide analyses of blood and/or urine DNA. This has led to the need for a reference framework for laboratories involved in mitochondrial genetic testing to facilitate a consistent high-quality service. In the United Kingdom, consensus guidelines have been prepared by a working group of Clinical Scientists from the NHS Highly Specialised Service followed by national laboratory consultation. These guidelines summarise current recommended technologies and methodologies for the analysis of mtDNA and nuclear-encoded genes in patients with suspected mitochondrial disease. Genetic testing strategies for diagnosis, family testing and reproductive options including prenatal diagnosis are outlined. Importantly, recommendations for the minimum levels of mtDNA testing for the most common referral reasons are included, as well as guidance on appropriate referrals and information on the minimal appropriate gene content of panels when analysing nuclear mitochondrial genes. Finally, variant interpretation and recommendations for reporting of results are discussed, focussing particularly on the challenges of interpreting and reporting mtDNA variants.

We describe a patient with chronic progressive external ophthalmoplegia (CPEO) due to a rare mitochondrial genetic variant. Muscle biopsy revealed numerous cytochrome coxidase (COX)-deficient fibres, prompting sequencing of the entire mitochondrial genome in muscle which revealed a rare m.12334G>A variant in the mitochondrial (mt-) tRNALeu(CUN)(MT-TL2) gene. Analysis of several tissues showed this to be a de novomutational event. Single fibre studies confirmed the segregation of high m.12334G>A heteroplasmy levels with the COX histochemical defect, confirming pathogenicity of the m.12334G>A MT-TL2variant. This case illustrates the importance of pursuing molecular genetic analysis in clinically-affected tissues when mitochondrial disease is suspected.

Background/AimThe rare mitochondrial DNA (mtDNA) variant m.8340G>A has been previously reported in the literature in a single, sporadic case of mitochondrial myopathy. In this report, we aim to investigate the case of a 39-year-old male patient with sensorineural deafness who presented to the eye clinic with nyctalopia, retinal pigmentary changes and bilateral cortical cataracts.MethodsThe patient was examined clinically and investigated with autofluorescence, full-field electroretinography, electro-oculogram and dark adaptometry. Sequencing of the mitochondrial genome in blood and muscle tissue was followed by histochemical and biochemical analyses together with single fibre studies of a muscle biopsy to confirm a mitochondrial aetiology.ResultsElectrophysiology, colour testing and dark adaptometry showed significant photoreceptor dysfunction with macular involvement. Sequencing the complete mitochondrial genome revealed a rare mitochondrial tRNALys(MTTK) gene variant—m.8340G>A—which was heteroplasmic in blood (11%) and skeletal muscle (65%) and cosegregated with cytochrome coxidase-deficient fibres in single-fibre studies.ConclusionWe confirm the pathogenicity of the rare mitochondrial m.8340G>A variant the basis of single-fibre segregation studies and its association with an expanded clinical phenotype. Our case expands the phenotypic spectrum of diseases associated with mitochondrial tRNA point mutations, highlighting the importance of considering a mitochondrial diagnosis in similar cases presenting to the eye clinic and the importance of further genetic testing if standard mutational analysis does not yield a result.

IMPORTANCE: Extrapyramidal movement disorders associated with mitochondrial disease are difficult to treat and can lead to considerable disability. Moreover, potential new treatment trials on the horizon highlight the importance of genotype-phenotype associations and deep phenotyping of the movement disorders related to mitochondrial disease. OBJECTIVE: To describe the phenotype, genetic etiology, and investigation of extrapyramidal movement disorders in a large and well-defined mitochondrial disease cohort. DESIGN, SETTING, AND PARTICIPANTS: An observational cohort study at a single national referral center. Among 678 patients (87% adults) followed up at the Newcastle mitochondrial disease specialized referral center between January 1, 2000, and January 31, 2015, 42 patients (12 pediatric, 30 adult) with genetic or biochemical evidence of mitochondrial disease and with 1 or more predefined extrapyramidal movement disorders (parkinsonism, dystonia, tremor, chorea, and restless legs syndrome) were included. MAIN OUTCOMES AND MEASURES: We investigated the prevalence and genetic causes of dystonia and parkinsonism as well as radiological findings in the context of movement disorders in mitochondrial disease. All patients were interviewed and examined. All available medical notes and clinical, radiological, and genetic investigations were reviewed. RESULTS: Forty-two patients (mean [SD] age, 37 [25] years; 38% female) with mitochondrial disease (12 pediatric [age range, 4-14 years], 30 adult [age range, 20-81 years]) with extrapyramidal movement disorders were identified. Dystonia manifested in 11 pediatric patients (92%), often in the context of Leigh syndrome; parkinsonism predominated in 13 adult patients (43%), among whom 5 (38%) harbored either dominant (n = 1) or recessive (n = 4) mutations in POLG. Eleven adult patients (37%) manifested with either generalized or multifocal dystonia related to mutations in mitochondrial DNA, among which the most common were the m.11778G>A mutation and mutations in MT-ATP6 (3 of 11 patients [27%] each). Bilateral basal ganglia lesions were the most common finding in brain magnetic resonance imaging, usually associated with generalized dystonia or Leigh syndrome. CONCLUSIONS AND RELEVANCE: Dystonia, often associated with Leigh syndrome, was the most common extrapyramidal movement disorder among pediatric patients with mitochondrial disease. Parkinsonism was the most prevalent extrapyramidal movement disorder in adults and was commonly associated with POLG mutations; dystonia was predominantly associated with mitochondrial DNA mutations. These findings may help direct genetic screening in a busy neurology outpatient setting.

IMPORTANCE: Progressive external ophthalmoplegia (PEO) is a common feature in adults with mitochondrial (mt) DNA maintenance disorders associated with somatic mtDNA deletions in muscle, yet the causal genetic defect in many patients remains undetermined. OBSERVATIONS: Whole-exome sequencing identified a novel, heterozygous p.(Gly671Trp) mutation in the AFG3L2 gene encoding an mt protease—previously associated with dominant spinocerebellar ataxia type 28 disease—in a patient with indolent ataxia and PEO. Targeted analysis of a larger, genetically undetermined cohort of patients with PEO with suspected mtDNA maintenance abnormalities identified a second unrelated patient with a similar phenotype and a novel, heterozygous p.(Tyr689His) AFG3L2 mutation. Analysis of patient fibroblasts revealed mt fragmentation and decreased AFG3L2 transcript expression. Western blotting of patient fibroblast and muscle showed decreased AFG3L2 protein levels. CONCLUSIONS AND RELEVANCE: Our observations suggest that AFG3L2 mutations are another important cause, albeit rare, of a late-onset ataxic PEO phenotype due to a disturbance of mtDNA maintenance.

Purpose:Mitochondrial disorders are a common cause of inherited metabolic disease and can be due to mutations affecting mitochondrial DNA or nuclear DNA. The current diagnostic approach involves the targeted resequencing of mitochondrial DNA and candidate nuclear genes, usually proceeds step by step, and is time consuming and costly. Recent evidence suggests that variations in mitochondrial DNA sequence can be obtained from whole-exome sequence data, raising the possibility of a comprehensive single diagnostic test to detect pathogenic point mutations.Methods:We compared the mitochondrial DNA sequence derived from off-target exome reads with conventional mitochondrial DNA Sanger sequencing in 46 subjects.Results:Mitochondrial DNA sequences can be reliably obtained using three different whole-exome sequence capture kits. Coverage correlates with the relative amount of mitochondrial DNA in the original genomic DNA sample, heteroplasmy levels can be determined using variant and total read depths, and—providing there is a minimum read depth of 20-fold—rare sequencing errors occur at a rate similar to that observed with conventional Sanger sequencing.Conclusion:This offers the prospect of using whole-exome sequence in a diagnostic setting to screen not only all protein coding nuclear genes but also all mitochondrial DNA genes for pathogenic mutations. Off-target mitochondrial DNA reads can also be used to assess quality control and maternal ancestry, inform on ethnic origin, and allow genetic disease association studies not previously anticipated with existing whole-exome data sets.Genet Med advance online publication 05 June 2014

Background: Behr's syndrome is a classical phenotypic description of childhood-onset optic atrophy combined with various neurological symptoms, including ophthalmoparesis, nystagmus, spastic paraparesis, ataxia, peripheral neuropathy and learning difficulties. Objective: Here we describe 4 patients with the classical Behr's syndrome phenotype from 3 unrelated families who carry homozygous nonsense mutations in the C12orf65 gene encoding a protein involved in mitochondrial translation. Methods: Whole exome sequencing was performed in genomic DNA and oxygen consumption was measured in patient cell lines. Results: We detected 2 different homozygous C12orf65 nonsense mutations in 4 patients with a homogeneous clinical presentation matching the historical description of Behr's syndrome. The first symptom in all patients was childhood-onset optic atrophy, followed by spastic paraparesis, distal weakness, motor neuropathy and ophthalmoparesis. Conclusions: We think that C12orf65 mutations are more frequent than previously suggested and screening of this gene should be considered not only in patients with mitochondrial respiratory chain deficiencies, but also in inherited peripheral neuropathies, spastic paraplegias and ataxias, especially with pre-existing optic atrophy.

OBJECTIVE To explore myelin components and mitochondrial changes within the central nervous system in patients with well-characterized mitochondrial disorders due to nuclear DNA or mitochondrial DNA (mtDNA) mutations. DESIGN Immunohistochemical analysis, histochemical analysis, mtDNA sequencing, and real-time and long-range polymerase chain reaction were used to determine the pathogenicity of mtDNA deletions. SETTING Department of Clinical Pathology, Columbia University Medical Center, and Newcastle Brain Tissue Resource. PATIENTS Seventeen patients with mitochondrial disorders and 7 controls were studied from August 1, 2009, to August 1, 2010. MAIN OUTCOME MEASURE Regions of myelin-associated glycoprotein (MAG) loss. RESULTS Myelin-associated glycoprotein loss in Kearns-Sayre syndrome was associated with oligodendrocyte loss and nuclear translocation of apoptosis-inducing factor, whereas inflammation, neuronal loss, and axonal injury were minimal. In a Kearns-Sayre syndrome MAG loss region, high levels of mtDNA deletions together with cytochrome- c oxidase–deficient cells and loss of mitochondrial respiratory chain subunits (more prominent in the white than gray matter and glia than axons) confirmed the pathogenicity of mtDNA deletions. CONCLUSION Primary mitochondrial respiratory chain defects affecting the white matter, and unrelated to inflammation, are associated with MAG loss and central nervous system demyelination.

BACKGROUND Mitochondrial diseases are characterized by wide phenotypic and genetic variability, but presentations in adults with akinetic rigidity and hyperkinetic movement disorders are rare. OBJECTIVES To describe clinically a subject with progressive neurodegeneration characterized by psychosis, dementia, and akinesia-rigidity, and to associate this phenotype with a novel mitochondrial transfer RNAPhe (tRNAPhe) (MTTF) mutation. DESIGN, SETTING, AND PATIENT Case description and detailed laboratory investigations of a 57-year-old woman at a university teaching hospital and a specialist mitochondrial diagnostic laboratory. RESULTS Histopathological findings indicated that an underlying mitochondrial abnormality was responsible for the subject's progressive neurological disorder, with mitochondrial genome sequencing revealing a novel m.586G>A MTTF mutation. CONCLUSIONS The clinical phenotypes associated with mitochondrial disorders may include akinesia-rigidity and psychosis. Our findings further broaden the spectrum of neurological disease associated with mitochondrial tRNAPhe mutations.Arch Neurol. 2010;67(11):1399-1402--

Mitochondrial (mt‐) tRNA (MTT) gene mutations are an important cause of human morbidity and are associated with a wide range of pathology, from isolated organ‐specific diseases such as myopathy or hearing loss, through to multisystem disorders with encephalopathy, gastrointestinal dysmotility, and life‐threatening cardiomyopathy. Our understanding of how MTTmutations cause disease remains poor and progress has been hampered by the complex interaction of genotype with phenotype that can result in patients who harbor the same mutation exhibiting starkly contrasting phenotypes, whereas other (genetically heterogeneous) patients manifest clinically identical syndromes. A further complexity is the highly polymorphic nature of mitochondrial DNA (mtDNA), which must temper any reflex assumptions of pathogenicity for novel MTTsubstitutions. Nevertheless significant progress is being made and we shall review the methods employed to identify and characterize MTTmutations as pathogenic. Also important is our understanding of the molecular processes involved and we shall discuss the data available on two of the most studied MTTmutations (m.8344A > G and m.3243A > G) as well as other potential pathogenic mechanisms. Knowledge of factors influencing the inheritance of MTTmutations, and therefore the likelihood of disease transmission, is of particular importance to female patients. At present, the factors determining transmission remain elusive, but we shall examine several possible mechanisms and discuss the evidence for each. Finally, a number of different yeast and mouse models are currently used to investigate mitochondrial disease and we will assess the importance of and difficulties associated with each model as well as the future of possible therapies for patients with mitochondrial disease. Copyright © 2010 John Wiley & Sons, Inc.

Maternally inherited mitochondrial DNA (mtDNA) mutations cause symptoms of Leber hereditary optic neuropathy (LHON) in ∼1 in 30,000 individuals. Most of the affected individuals lack respiratory chain defects1and there is no proven prophylactic treatment.

BACKGROUND Pathogenic mutations of the human mitochondrial genome are associated with well-characterized, progressive neurological syndromes, with mutations in the transfer RNA genes being particularly prominent. OBJECTIVE To describe a novel mitochondrial transfer RNAPro gene mutation in a woman with a myoclonic epilepsy with ragged-red fibers–like disease. DESIGN, SETTING, AND PATIENT Case report of a 49-year-old woman presenting with a myoclonic epilepsy with ragged-red fibers–like disease comprising myoclonic jerks, cerebellar ataxia, and proximal muscle weakness. RESULTS Histochemical analysis of a muscle biopsy revealed numerous cytochrome-c oxidase–deficient, ragged-red fibers, while biochemical studies indicated decreased activity of respiratory chain complex I. Molecular investigation of mitochondrial DNA revealed a new heteroplasmic mutation in the TψC stem of the mitochondrial transfer RNAPro gene that segregated with cytochrome-c oxidase deficiency in single muscle fibers. CONCLUSIONS Our case serves to illustrate the ever-evolving phenotypic spectrum of mitochondrial DNA disease and the importance of performing comprehensive mitochondrial genetic studies in the absence of common mitochondrial DNA mutations.Arch Neurol. 2009;66(3):399-402--

Mutations of mitochondrial DNA (mtDNA) are an important cause of genetic disease, yet rarely present in the neonatal period. Here we report the clinical, biochemical, and molecular genetic findings of an infant who died at the age of 1 mo with marked biventricular hypertrophy, aortic coarctation, and severe lactic acidosis due to a previously described but unusual mtDNA mutation, a 7-bp intragenic inversion within the mitochondrial gene encoding ND1 protein of complex I (MTND1). In direct contrast to the previous case, an adult with exercise intolerance who only harbored the mutation in muscle, the MTND1 inversion in our patient was present at high levels in several tissues including the heart, muscle, liver, and cultured skin fibroblasts. There was no evidence of the mutation or respiratory complex I defect in a muscle biopsy from the patient's mother. Transmitochondrial cytoplasmic hybrids (cybrids) containing high mutant loads of the inversion expressed the biochemical defect but apparently normal levels of the assembled complex. Our report highlights the enormous phenotypic diversity that exists among pathogenic mtDNA mutations and reemphasizes the need for appropriate genetic counseling for families affected by mtDNA disease.

Mutations of mitochondrial DNA (mtDNA) are an important cause of genetic disease, yet rarely present in the neonatal period. Here we report the clinical, biochemical, and molecular genetic findings of an infant who died at the age of 1 mo with marked biventricular hypertrophy, aortic coarctation, and severe lactic acidosis due to a previously described but unusual mtDNA mutation, a 7-bp intragenic inversion within the mitochondrial gene encoding ND1 protein of complex I (MTND1). In direct contrast to the previous case, an adult with exercise intolerance who only harbored the mutation in muscle, the MTND1inversion in our patient was present at high levels in several tissues including the heart, muscle, liver, and cultured skin fibroblasts. There was no evidence of the mutation or respiratory complex I defect in a muscle biopsy from the patient's mother. Transmitochondrial cytoplasmic hybrids (cybrids) containing high mutant loads of the inversion expressed the biochemical defect but apparently normal levels of the assembled complex. Our report highlights the enormous phenotypic diversity that exists among pathogenic mtDNA mutations and reemphasizes the need for appropriate genetic counseling for families affected by mtDNA disease.

A cornerstone of mitochondrial genetics, strict maternal inheritance, has been challenged recently by the study of a patient with mitochondrial myopathy due to a sporadic 2bp deletion. The mitochondrial DNA (mtDNA) harboring the mutation was paternal in origin, whereas the patient's blood was identical to the maternal genotype. To determine whether this is a common phenomenon, we studied mtDNA sequence variation between muscle and blood from 35 patients with sporadic mitochondrial myopathies, but detected no evidence of paternal mtDNA transmission. Our findings suggest that paternal transmission of mtDNA is rare and should not alter our genetic advice to families.

A cornerstone of mitochondrial genetics, strict maternal inheritance, has been challenged recently by the study of a patient with mitochondrial myopathy due to a sporadic 2bp deletion. The mitochondrial DNA (mtDNA) harboring the mutation was paternal in origin, whereas the patient's blood was identical to the maternal genotype. To determine whether this is a common phenomenon, we studied mtDNA sequence variation between muscle and blood from 35 patients with sporadic mitochondrial myopathies, but detected no evidence of paternal mtDNA transmission. Our findings suggest that paternal transmission of mtDNA is rare and should not alter our genetic advice to families.

Background: Acquired human mitochondrial genome (mtDNA) deletions are symptoms and drivers of focal mitochondrial respiratory deficiency, a pathological hallmark of aging and late-onset mitochondrial disease. Results: To decipher connections between these processes, we create LostArc, an ultrasensitive method for quantifying deletions in circular mtDNA molecules. LostArc reveals 35 million deletions (~ 470,000 unique spans) in skeletal muscle from 22 individuals with and 19 individuals without pathogenic variants in POLG. This nuclear gene encodes the catalytic subunit of replicative mitochondrial DNA polymerase γ. Ablation, the deleted mtDNA fraction, suffices to explain skeletal muscle phenotypes of aging and POLG-derived disease. Unsupervised bioinformatic analyses reveal distinct age- and disease-correlated deletion patterns. Conclusions: These patterns implicate replication by DNA polymerase γ as the deletion driver and suggest little purifying selection against mtDNA deletions by mitophagy in postmitotic muscle fibers. Observed deletion patterns are best modeled as mtDNA deletions initiated by replication fork stalling during strand displacement mtDNA synthesis.

OBJECTIVE To describe an unusual clinical phenotype in an adult harboring 2 compound heterozygous polymerase γ (POLG) mutations. DESIGN Case report. SETTING University-based outpatient neurology clinic and pathology and genetics laboratory. PATIENT A 27-year-old man presenting with isolated distal myopathy of the upper extremities in the absence of sensory disturbances. RESULTS Histochemical analysis of a muscle biopsy specimen showed numerous cytochrome c oxidase–deficient fibers. Molecular analysis revealed marked depletion of muscle mitochondrial DNA in the absence of multiple mitochondrial DNA deletions. Sequence analysis of the POLG gene revealed heterozygous sequence variants in compound c.1156C>T (p.R386C) and c.2794C>T (p.H932Y) segregating with clinical disease in the family. The p.R386C change appears to be a novel mutation. CONCLUSION Our case broadens the phenotypic spectrum of disorders associated with POLG mutations and highlights the complex relationship between genotype and phenotype in POLG-related disease.Arch Neurol. 2010;67(9):1144-1146--