Disorders Of Mitochondrial DNA Maintenance Research Articles

A stagewise response to mitochondrial dysfunction in mitochondrial DNA maintenance disorders

Mitochondrial DNA (mtDNA) deletions which clonally expand in skeletal muscle of patients with mtDNA maintenance disorders, impair mitochondrial oxidative phosphorylation dysfunction. Previously we have shown that these mtDNA deletions arise and accumulate in perinuclear mitochondria causing localised mitochondrial dysfunction before spreading through the muscle fibre. We believe that mito-nuclear signalling is a key contributor in the accumulation and spread of mtDNA deletions, and that knowledge of how muscle fibres respond to mitochondrial dysfunction is key to our understanding of disease mechanisms.To understand the contribution of mito-nuclear signalling to the spread of mitochondrial dysfunction, we use imaging mass cytometry. We characterise the levels of mitochondrial Oxidative Phosphorylation proteins alongside a mitochondrial mass marker, in a cohort of patients with mtDNA maintenance disorders. Our expanded panel included protein markers of key signalling pathways, allowing us to investigate cellular responses to different combinations of oxidative phosphorylation dysfunction and ragged red fibres.We find combined Complex I and IV deficiency to be most common. Interestingly, in fibres deficient for one or more complexes, the remaining complexes are often upregulated beyond the increase of mitochondrial mass typically observed in ragged red fibres. We further find that oxidative phosphorylation deficient fibres exhibit an increase in the abundance of proteins involved in proteostasis, e.g. HSP60 and LONP1, and regulation of mitochondrial metabolism (including oxidative phosphorylation and proteolysis, e.g. PHB1). Our analysis suggests that the cellular response to mitochondrial dysfunction changes depending on the combination of deficient oxidative phosphorylation complexes in each fibre.

Mitochondrial DNA maintenance disorders in 102 patients from different parts of Russia: Mutational spectrum and phenotypes

Currently, pathogenic variants in more than 25 nuclear genes, involved in mtDNA maintenance, are associated with human disorders. mtDNA maintenance disorders manifest with a wide range of phenotypes, from severe infantile-onset forms of myocerebrohepatopathy to late-onset forms of myopathies, chronic progressive external ophthalmoplegia, and parkinsonism.This study represents the results of molecular genetic analysis and phenotypes of 102 probands with mtDNA maintenance disorders. So far, this is the largest Russian cohort for this group of diseases. Mutations were identified in 10 mtDNA maintenance genes: POLG (n = 59), DGUOK (n = 14), TWNK (n = 14), TK2 (n = 8), MPV17 (n = 2), OPA3 (n = 1), FBXL4 (n = 1), RRM2B (n = 1), SUCLG1 (n = 1) and TYMP (n = 1). We review a mutation spectrum for the DGUOK and TWNK genes, that can be specific for the Russian population. In 34 patients we measured the blood mtDNA copy number and showed its significant reduction. Novel variants were found in 41 cases, which significantly expands the mutational landscape of mtDNA maintenance disorders.

P1 Understanding mitochondrial DNA maintenance disorders at the single muscle fibre level

P1 Understanding mitochondrial DNA maintenance disorders at the single muscle fibre level

Understanding mitochondrial DNA maintenance disorders at the single muscle fibre level.

Clonal expansion of mitochondrial DNA (mtDNA) deletions is an important pathological mechanism in adults with mtDNA maintenance disorders, leading to a mosaic mitochondrial respiratory chain deficiency in skeletal muscle. This study had two aims: (i) to determine if different Mendelian mtDNA maintenance disorders showed similar pattern of mtDNA deletions and respiratory chain deficiency and (ii) to investigate the correlation between the mitochondrial genetic defect and corresponding respiratory chain deficiency. We performed a quantitative analysis of respiratory chain deficiency, at a single cell level, in a cohort of patients with mutations in mtDNA maintenance genes. Using the same tissue section, we performed laser microdissection and single cell genetic analysis to investigate the relationship between mtDNA deletion characteristics and the respiratory chain deficiency. The pattern of respiratory chain deficiency is similar with different genetic defects. We demonstrate a clear correlation between the level of mtDNA deletion and extent of respiratory chain deficiency within a single cell. Long-range and single molecule PCR shows the presence of multiple mtDNA deletions in approximately one-third of all muscle fibres. We did not detect evidence of a replicative advantage for smaller mtDNA molecules in the majority of fibres, but further analysis is needed to provide conclusive evidence.

Mutations in the SPG7 gene cause chronic progressive external ophthalmoplegia through disordered mitochondrial DNA maintenance.

Despite being a canonical presenting feature of mitochondrial disease, the genetic basis of progressive external ophthalmoplegia remains unknown in a large proportion of patients. Here we show that mutations in SPG7 are a novel cause of progressive external ophthalmoplegia associated with multiple mitochondrial DNA deletions. After excluding known causes, whole exome sequencing, targeted Sanger sequencing and multiplex ligation-dependent probe amplification analysis were used to study 68 adult patients with progressive external ophthalmoplegia either with or without multiple mitochondrial DNA deletions in skeletal muscle. Nine patients (eight probands) were found to carry compound heterozygous SPG7 mutations, including three novel mutations: two missense mutations c.2221G>A; p.(Glu741Lys), c.2224G>A; p.(Asp742Asn), a truncating mutation c.861dupT; p.Asn288*, and seven previously reported mutations. We identified a further six patients with single heterozygous mutations in SPG7, including two further novel mutations: c.184-3C>T (predicted to remove a splice site before exon 2) and c.1067C>T; p.(Thr356Met). The clinical phenotype typically developed in mid-adult life with either progressive external ophthalmoplegia/ptosis and spastic ataxia, or a progressive ataxic disorder. Dysphagia and proximal myopathy were common, but urinary symptoms were rare, despite the spasticity. Functional studies included transcript analysis, proteomics, mitochondrial network analysis, single fibre mitochondrial DNA analysis and deep re-sequencing of mitochondrial DNA. SPG7 mutations caused increased mitochondrial biogenesis in patient muscle, and mitochondrial fusion in patient fibroblasts associated with the clonal expansion of mitochondrial DNA mutations. In conclusion, the SPG7 gene should be screened in patients in whom a disorder of mitochondrial DNA maintenance is suspected when spastic ataxia is prominent. The complex neurological phenotype is likely a result of the clonal expansion of secondary mitochondrial DNA mutations modulating the phenotype, driven by compensatory mitochondrial biogenesis.

ATAXIA AND EPILEPSY DUE TO A RARE AUTOSOMAL MITOCHONDRIAL DISEASE

We report a case of a twenty three year old female presenting with status epilepticus due to a rare mutation in a nuclear gene causing mitochondrial dysfunction. She had previously...

Mesencephalic complex I deficiency does not correlate with parkinsonism in mitochondrial DNA maintenance disorders

Genetic evidence from recessively inherited Parkinson's disease has indicated a clear causative role for mitochondrial dysfunction in Parkinson's disease. This role has long been discussed based on findings that toxic inhibition of mitochondrial respiratory complex I caused parkinsonism and that tissues of patients with Parkinson's disease show complex I deficiency. Disorders of mitochondrial DNA maintenance are a common cause of inherited neurodegenerative disorders, and lead to mitochondrial DNA deletions or depletion and respiratory chain defect, including complex I deficiency. However, parkinsonism associates typically with defects of catalytic domain of mitochondrial DNA polymerase gamma. Surprisingly, however, not all mutations affecting DNA polymerase gamma manifest as parkinsonism, but, for example, spacer region mutations lead to spinocerebellar ataxia and/or severe epilepsy. Furthermore, defective Twinkle helicase, a close functional companion of DNA polymerase gamma in mitochondrial DNA replication, results in infantile-onset spinocerebellar ataxia, epilepsy or adult-onset mitochondrial myopathy, but not typically parkinsonism. Here we sought for clues for this specificity in the neurological manifestations of mitochondrial DNA maintenance disorders by studying mesencephalic neuropathology of patients with DNA polymerase gamma or Twinkle defects, with or without parkinsonism. We show here that all patients with mitochondrial DNA maintenance disorders had neuronopathy in substantia nigra, most severe in DNA polymerase gamma-associated parkinsonism. The oculomotor nucleus was also affected, but less severely. In substantia nigra, all patients had a considerable decrease of respiratory chain complex I, but other respiratory chain enzymes were not affected. Complex I deficiency did not correlate with parkinsonism, age, affected gene or inheritance. We conclude that the cell number in substantia nigra correlated well with parkinsonism in DNA polymerase gamma and Twinkle defects. However, complex I defect is a general consequence of mitochondrial DNA maintenance defects, and does not explain manifestation of parkinsonism or degree of mesencephalic cell death in patients with mitochondrial DNA maintenance disorders.

Reply: MFN2, a new gene responsible for mitochondrial DNA depletion

Sir, we recently showed in Brain that the MFN2 gene, typically responsible for autosomal dominant axonal Charcot–Marie–Tooth disease, is a novel gene associated with ‘mitochondrial DNA breakage’ syndrome (Rouzier et al. , 2012). We described a novel MFN2 heterozygous missense mutation in Tunisian patients who presented with optic atrophy beginning in early childhood, axonal neuropathy and mitochondrial myopathy in adult life with accumulation of mitochondrial DNA deletions in muscle. Renaldo et al. (2012) confirm that MFN2 is involved in mitochondrial DNA maintenance disorders. Indeed, they show that this gene is responsible for a severe phenotype in a child who presented a developmental delay with hypotonia and failure to thrive at the age of 6 months. Evolution was marked by the gradual appearance of ataxia, areflexia, abnormal movements and axonal sensorimotor neuropathy. At the age of 9 years, weight, height and head circumference were <3 SD. The child presented a bilateral optic atrophy and was deaf. Brain MRI was normal. A mitochondrial myopathy, with cytochrome c-oxidase negative fibres, was diagnosed on muscle biopsy and a multiple respiratory chain deficiency was found both in muscle and fibroblasts. Furthermore, Renaldo et al. (2012) found mitochondrial DNA depletion in the patient’s muscle with a low amount of mitochondrial DNA (28% of controls). The association of optic atrophy and axonal neuropathy led the authors to analyse the MFN2 gene and to identify a novel missense heterozygous mutation (p.D210Y) that affects the same aspartic acid residue modified in the Tunisian family that we described previously (p.D210V). A defect in the maintenance of mitochondrial genome can lead to a reduction of mitochondrial DNA copy number and/or multiple mitochondrial DNA deletions. In mouse models, conditional inactivation of Mfn2 in muscle leads to a mitochondrial myopathy with a severe mitochondrial DNA depletion above …

Mitochondrial disease and epilepsy

Mitochondrial respiratory chain disorders are relatively common inborn errors of energy metabolism, with a combined prevalence of one in 5000. These disorders typically affect tissues with high energy requirements, and cerebral involvement occurs frequently in childhood, often manifesting in seizures. Mitochondrial diseases are genetically heterogeneous; to date, mutations have been reported in all 37 mitochondrially encoded genes and more than 80 nuclear genes. The major genetic causes of mitochondrial epilepsy are mitochondrial DNA mutations (including those typically associated with the mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes [MELAS] and myoclonic epilepsy with ragged red fibres [MERRF] syndromes); mutations in POLG (classically associated with Alpers syndrome but also presenting as the mitochondrial recessive ataxia syndrome [MIRAS], spinocerebellar ataxia with epilepsy [SCAE], and myoclonus, epilepsy, myopathy, sensory ataxia [MEMSA] syndromes in older individuals) and other disorders of mitochondrial DNA maintenance; complex I deficiency; disorders of coenzyme Q(10) biosynthesis; and disorders of mitochondrial translation such as RARS2 mutations. It is not clear why some genetic defects, but not others, are particularly associated with seizures. Epilepsy may be the presenting feature of mitochondrial disease but is often part of a multisystem clinical presentation. Mitochondrial epilepsy may be very difficult to manage, and is often a poor prognostic feature. At present there are no curative treatments for mitochondrial disease. Individuals with mitochondrial epilepsy are frequently prescribed multiple anticonvulsants, and the role of vitamins and other nutritional supplements and the ketogenic diet remain unproven.

Dysfunctional mitochondrial maintenance: what breaks the circle of life?

Despite being recognized as the first Mendelian-inherited mitochondrial disorders (Zeviani et al ., 1990), it has taken over a quarter of a century to realize that disorders of mitochondrial DNA maintenance form a large proportion of the patients seen in adult neurogenetics clinics. Most conditions were initially identified in families presenting with the ‘classical’ mitochondrial phenotype of late-onset chronic progressive external ophthalmoplegia and bilateral ptosis. However, several papers published in Brain in the last 2 years have shown that the field may have been barking up the wrong (phenotypic) tree. The clinical manifestations associated with disorders of mitochondrial DNA maintenance are proving to be much more heterogeneous than was initially understood; and, interestingly, one of the more recently implicated nuclear genetic defects, OPA1 , is a major player in the stability of the mitochondrial network. This principle comes to the fore yet again in this issue of Brain , strongly suggesting that the burden of human disease associated with the accumulation of somatic mitochondrial DNA abnormalities and disturbed mitochondrial dynamics will expand further, whilst providing valuable mechanistic insight of much broader pathophysiological relevance. First defined at the molecular level in 2001 (Van Goethem et al ., 2001), large pedigrees with autosomal dominant chronic progressive external ophthalmoplegia due to mutations in POLG are well established and now feature routinely in neurology and ophthalmology textbooks. POLG codes for the only DNA polymerase present within mitochondria, polymerase γ, and mutations in this nuclear gene lead to point mutations and the accumulation of mitochondrial DNA deletions throughout life (Chan and Copeland, 2009). A combination of clonal expansion and recurrent mutation drive the level of these mutations upwards within individual cells (see Box 1 for a detailed explanation). When the percentage level exceeds a critical threshold, the cells express a biochemical defect that can affect …