Strategies for fighting mitochondrial diseases.

Mitochondrial diseases are extremely heterogeneous genetic conditions characterized by faulty oxidative phosphorylation (OXPHOS). OXPHOS deficiency can be the result of mutation in mtDNA genes, encoding either proteins (13 subunits of the mitochondrial complexes I, III, IV and V) or the tRNA and rRNA components of the in situ mtDNA translation. The remaining mitochondrial disease genes are in the nucleus, encoding proteins with a huge variety of functions, from structural subunits of the mitochondrial complexes, to factors involved in their formation and regulation, components of the mtDNA replication and expression machinery, biosynthetic enzymes for the biosynthesis or incorporation of prosthetic groups, components of the mitochondrial quality control and proteostasis, enzymes involved in the clearance of toxic compounds, factors involved in the formation of the lipid milieu, etc. These different functions represent potential targets for 'general' therapeutic interventions, as they may be adapted to a number of different mitochondrial conditions. This is in contrast with 'tailored', personalized therapeutic approaches, such as gene therapy, cell therapy and organ replacement, that can be useful only for individual conditions. This review will present the most recent concepts emerged from preclinical work and the attempts to translate them into the clinics. The common notion that mitochondrial disorders have no cure is currently challenged by a massive effort of scientists and clinicians, and we do expect that thanks to this intensive investigation work and tangible results for the development of strategies amenable to the treatment of patients with these tremendously difficult conditions are not so far away.

SURF1 knockout cloned pigs: Early onset of a severe lethal phenotype.

Leigh syndrome (LS) associated with cytochrome c oxidase (COX) deficiency is an early onset, fatal mitochondrial encephalopathy, leading to multiple neurological failure and eventually death, usually in the first decade of life. Mutations in SURF1, a nuclear gene encoding a mitochondrial protein involved in COX assembly, are among the most common causes of LS. LSSURF1 patients display severe, isolated COX deficiency in all tissues, including cultured fibroblasts and skeletal muscle. Recombinant, constitutive SURF1-/- mice show diffuse COX deficiency, but fail to recapitulate the severity of the human clinical phenotype. Pigs are an attractive alternative model for human diseases, because of their size, as well as metabolic, physiological and genetic similarity to humans. Here, we determined the complete sequence of the swine SURF1 gene, disrupted it in pig primary fibroblast cell lines using both TALENs and CRISPR/Cas9 genome editing systems, before finally generating SURF1-/- and SURF1-/+ pigs by Somatic Cell Nuclear Transfer (SCNT). SURF1-/- pigs were characterized by failure to thrive, muscle weakness and highly reduced life span with elevated perinatal mortality, compared to heterozygous SURF1-/+ and wild type littermates. Surprisingly, no obvious COX deficiency was detected in SURF1-/- tissues, although histochemical analysis revealed the presence of COX deficiency in jejunum villi and total mRNA sequencing (RNAseq) showed that several COX subunit-encoding genes were significantly down-regulated in SURF1-/- skeletal muscles. In addition, neuropathological findings, indicated a delay in central nervous system development of newborn SURF1-/- piglets. Our results suggest a broader role of sSURF1 in mitochondrial bioenergetics.

AAV9-based gene therapy partially ameliorates the clinical phenotype of a mouse model of Leigh syndrome.

Leigh syndrome (LS) is the most common infantile mitochondrial encephalopathy. No treatment is currently available for this condition. Mice lacking Ndufs4, encoding NADH: ubiquinone oxidoreductase iron-sulfur protein 4 (NDUFS4) recapitulates the main findings of complex I (cI)-related LS, including severe multisystemic cI deficiency and progressive neurodegeneration. In order to develop a gene therapy approach for LS, we used here an AAV2/9 vector carrying the human NDUFS4 coding sequence (hNDUFS4). We administered AAV2/9-hNDUFS4 by intravenous (IV) and/or intracerebroventricular (ICV) routes to either newborn or young Ndufs4-/- mice. We found that IV administration alone was only able to correct the cI deficiency in peripheral organs, whereas ICV administration partially corrected the deficiency in the brain. However, both treatments failed to improve the clinical phenotype or to prolong the lifespan of Ndufs4-/- mice. In contrast, combined IV and ICV treatments resulted, along with increased cI activity, in the amelioration of the rotarod performance and in a significant prolongation of the lifespan. Our results indicate that extraneurological organs have an important role in LS pathogenesis and provide an insight into current limitations of adeno-associated virus (AAV)-mediated gene therapy in multisystem disorders. These findings warrant future investigations to develop new vectors able to efficiently target multiple organs.

An autosomal locus predisposing to deletions of mitochondrial DNA.

The molecular mechanisms by which the nuclear genome regulates the biosynthesis of mitochondrial DNA (mtDNA) are only beginning to be unravelled. A naturally occurring in vivo model for a defect in this cross-talk of two physically separate genomes is a human disease, an autosomal dominant progressive external ophthalmoplegia, in which multiple deletions of mtDNA accumulate in the patients' tissues. The assignment of this disease locus to 10q 23.3-24.3 is the first direct evidence for involvement of both nuclear and mitochondrial genomes in a single disorder.

Human mitochondrial DNA deletions associated with mutations in the gene encoding Twinkle, a phage T7 gene 4-like protein localized in mitochondria.

The gene products involved in mammalian mitochondrial DNA (mtDNA) maintenance and organization remain largely unknown. We report here a novel mitochondrial protein, Twinkle, with structural similarity to phage T7 gene 4 primase/helicase and other hexameric ring helicases. Twinkle colocalizes with mtDNA in mitochondrial nucleoids. Screening of the gene encoding Twinkle in individuals with autosomal dominant progressive external ophthalmoplegia (adPEO), associated with multiple mtDNA deletions, identified 11 different coding-region mutations co-segregating with the disorder in 12 adPEO pedigrees of various ethnic origins. The mutations cluster in a region of the protein proposed to be involved in subunit interactions. The function of Twinkle is inferred to be critical for lifetime maintenance of human mtDNA integrity.

Reversible infantile respiratory chain deficiency is a unique, genetically heterogenous mitochondrial disease.

OBJECTIVES: Homoplasmic maternally inherited, m.14674T>C or m. 14674T>G mt-tRNA(Glu) mutations have recently been identified in reversible infantile cytochrome c oxidase deficiency (or 'benign COX deficiency'). This study sought other genetic defects that may give rise to similar presentations. PATIENTS: Eight patients from seven families with clinicopathological features of infantile reversible cytochrome c oxidase deficiency were investigated. METHODS: The study reviewed the diagnostic features and performed molecular genetic analyses of mitochondrial DNA and nuclear encoded candidate genes. RESULTS: Patients presented with subacute onset of profound hypotonia, feeding difficulties and lactic acidosis within the first months of life. Although recovery was remarkable, a mild myopathy persisted into adulthood. Histopathological findings in muscle included increased lipid and/or glycogen content, ragged-red and COX negative fibres. Biochemical studies suggested more generalised abnormalities than pure COX deficiency. Clinical improvement was reflected by normalisation of lactic acidosis and histopathological abnormalities. The m.14674T>C mt-tRNA(Glu) mutation was identified in four families, but none had the m. 14674T>G mutation. Furthermore, in two families pathogenic mutations were also found in the nuclear TRMU gene which has not previously been associated with this phenotype. In one family, the genetic aetiology still remains unknown. CONCLUSIONS: Benign COX deficiency is better described as 'reversible infantile respiratory chain deficiency'. It is genetically heterogeneous, and patients not carrying the m.14674T>C or T>G mt-tRNA(Glu) mutations may have mutations in the TRMU gene. Diagnosing this disorder at the molecular level is a significant advance for paediatric neurologists and intensive care paediatricians, enabling them to select children with an excellent prognosis for continuing respiratory support from those with severe mitochondrial presentation in infancy.

EFNS guidelines for the molecular diagnosis of neurogenetic disorders: motoneuron, peripheral nerve and muscle disorders.

OBJECTIVES: These EFNS guidelines on the molecular diagnosis of motoneuron disorders, neuropathies and myopathies are designed to summarize the possibilities and limitations of molecular genetic techniques and to provide diagnostic criteria for deciding when a molecular diagnostic work-up is indicated. SEARCH STRATEGY: To collect data about planning, conditions and performance of molecular diagnosis of these disorders, a literature search in various electronic databases was carried out and original papers, meta-analyses, review papers and guideline recommendations reviewed. RESULTS: The best level of evidence for genetic testing recommendation (B) can be found for the disorders with specific presentations, including familial amyotrophic lateral sclerosis, spinal and bulbar muscular atrophy, Charcot-Marie-Tooth 1A, myotonic dystrophy and Duchenne muscular dystrophy. For a number of less common disorders, a precise description of the phenotype, including the use of immunologic methods in the case of myopathies, is considered as good clinical practice to guide molecular genetic testing. CONCLUSION: These guidelines are provisional and the future availability of molecular-genetic epidemiological data about the neurogenetic disorders under discussion in this article will allow improved recommendation with an increased level of evidence.

Multi-system neurological disease is common in patients with OPA1 mutations.

Additional neurological features have recently been described in seven families transmitting pathogenic mutations in OPA1, the most common cause of autosomal dominant optic atrophy. However, the frequency of these syndromal 'dominant optic atrophy plus' variants and the extent of neurological involvement have not been established. In this large multi-centre study of 104 patients from 45 independent families, including 60 new cases, we show that extra-ocular neurological complications are common in OPA1 disease, and affect up to 20% of all mutational carriers. Bilateral sensorineural deafness beginning in late childhood and early adulthood was a prominent manifestation, followed by a combination of ataxia, myopathy, peripheral neuropathy and progressive external ophthalmoplegia from the third decade of life onwards. We also identified novel clinical presentations with spastic paraparesis mimicking hereditary spastic paraplegia, and a multiple sclerosis-like illness. In contrast to initial reports, multi-system neurological disease was associated with all mutational subtypes, although there was an increased risk with missense mutations [odds ratio = 3.06, 95% confidence interval = 1.44-6.49; P = 0.0027], and mutations located within the guanosine triphosphate-ase region (odds ratio = 2.29, 95% confidence interval = 1.08-4.82; P = 0.0271). Histochemical and molecular characterization of skeletal muscle biopsies revealed the presence of cytochrome c oxidase-deficient fibres and multiple mitochondrial DNA deletions in the majority of patients harbouring OPA1 mutations, even in those with isolated optic nerve involvement. However, the cytochrome c oxidase-deficient load was over four times higher in the dominant optic atrophy + group compared to the pure optic neuropathy group, implicating a causal role for these secondary mitochondrial DNA defects in disease pathophysiology. Individuals with dominant optic atrophy plus phenotypes also had significantly worse visual outcomes, and careful surveillance is therefore mandatory to optimize the detection and management of neurological disability in a group of patients who already have significant visual impairment.

Collated mutations in mitochondrial DNA (mtDNA) depletion syndrome (excluding the mitochondrial gamma polymerase, POLG1).

These tables list both published and a number of unpublished mutations in genes associated with early onset defects in mitochondrial DNA (mtDNA) maintenance including C10orf2, SUCLG1, SUCLA2, TYMP, RRM2B, MPV17, DGUOK and TK2. The list should not be taken as evidence that any particular mutation is pathogenic. We have included genes known to cause mtDNA depletion, excluding POLG1, because of the existing database (http://tools.niehs.nih.gov/polg/). We have also excluded mutations in C10orf2 associated with dominant adult onset disorders.

EFNS guidelines on the molecular diagnosis of channelopathies, epilepsies, migraine, stroke, and dementias.

OBJECTIVES: These EFNS guidelines on the molecular diagnosis of channelopathies, including epilepsy and migraine, as well as stroke, and dementia are designed to summarize the possibilities and limitations of molecular genetic techniques and to provide diagnostic criteria for deciding when a molecular diagnostic work-up is indicated. SEARCH STRATEGY: To collect data about planning, conditions, and performance of molecular diagnosis of these disorders, a literature search in various electronic databases was carried out and original papers, meta-analyses, review papers, and guideline recommendations were reviewed. RESULTS: The best level of evidence for genetic testing recommendation (B) can be found for a small number of syndromes, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, severe myoclonic epilepsy of infancy, familial recurrent hemorrhages, familial Alzheimer's disease, and frontotemporal lobar degeneration. Good practice points can be formulated for a number of other disorders. CONCLUSION: These guidelines are provisional, and the future availability of molecular genetic epidemiological data about the neurogenetic disorders under discussion in our article will allow improved recommendation with an increased level of evidence.

EFNS guidelines on the molecular diagnosis of ataxias and spastic paraplegias.

BACKGROUND AND PURPOSE: These EFNS guidelines on the molecular diagnosis of neurogenetic disorders are designed to provide practical help for the general neurologist to make appropriate use of molecular genetics in diagnosing neurogenetic disorders. METHODS: Literature searches were performed before expert members of the task force wrote proposals, which were discussed in detail until final consensus had been reached among all task force members. RESULTS AND CONCLUSION: This paper provides updated guidelines for molecular diagnosis of two particularly complex groups of disorders, the ataxias and spastic paraplegias. Possibilities and limitations of molecular genetic diagnosis of these disorders are evaluated and recommendations are provided.

Disorders from perturbations of nuclear-mitochondrial intergenomic cross-talk.

In the course of evolution, mitochondria lost their independence, and mitochondrial DNA (mtDNA) became the 'slave' of nuclear DNA, depending on numerous nucleus-encoded factors for its integrity, replication and expression. Mutations in any of these factors may alter the cross-talk between the two genomes and cause Mendelian disorders characterized by qualitative (multiple deletions) or quantitative (depletion) alterations of mtDNA, or by defective translation of mtDNA-encoded respiratory chain components.

A direct repeat is a hotspot for large-scale deletion of human mitochondrial DNA.

Kearns-Sayre syndrome (KSS) and progressive external ophthalmoplegia (PEO) are related neuromuscular disorders characterized by ocular myopathy and ophthalmoplegia. Almost all patients with KSS and about half with PEO harbor large deletions in their mitochondrial genomes. The deletions differ in both size and location, except for one, 5 kilobases long, that is found in more than one-third of all patients examined. This common deletion was found to be flanked by a perfect 13-base pair direct repeat in the normal mitochondrial genome. This result suggests that homologous recombination deleting large regions of intervening mitochondrial DNA, which previously had been observed only in lower eukaryotes and plants, operates in mammalian mitochondrial genomes as well, and is at least one cause of the deletions found in these two related mitochondrial myopathies.

Role of adenine nucleotide translocator 1 in mtDNA maintenance.

Autosomal dominant progressive external ophthalmoplegia is a rare human disease that shows a Mendelian inheritance pattern, but is characterized by large-scale mitochondrial DNA (mtDNA) deletions. We have identified two heterozygous missense mutations in the nuclear gene encoding the heart/skeletal muscle isoform of the adenine nucleotide translocator (ANT1) in five families and one sporadic patient. The familial mutation substitutes a proline for a highly conserved alanine at position 114 in the ANT1 protein. The analogous mutation in yeast caused a respiratory defect. These results indicate that ANT has a role in mtDNA maintenance and that a mitochondrial disease can be caused by a dominant mechanism.

De novo and inherited deletions of the 5q13 region in spinal muscular atrophies.

Spinal muscular atrophies (SMAs) represent the second most common fatal autosomal recessive disorder after cystic fibrosis. Childhood spinal muscular atrophies are divided into severe (type I) and mild forms (types II and III). By a combination of genetic and physical mapping, a yeast artificial chromosome contig of the 5q13 region spanning the disease locus was constructed that showed the presence of low copy repeats in this region. Allele segregation was analyzed at the closest genetic loci detected by markers C212 and C272 in 201 SMA families. Inherited and de novo deletions were observed in nine unrelated SMA patients. Moreover, deletions were strongly suggested in at least 18 percent of SMA type I patients by the observation of marked heterozygosity deficiency for the loci studied. These results indicate that deletion events are statistically associated with the severe form of spinal muscular atrophy.

Clinical and molecular features of mitochondrial DNA depletion syndromes.

Mitochondrial DNA depletion syndromes (MDSs) form a group of autosomal recessive disorders characterized by profoundly decreased mitochondrial DNA copy numbers in affected tissues. Three main clinical presentations are known: myopathic, encephalomyopathic and hepatocerebral. The first is associated with mutations in thymidine kinase 2 (TK2) and p53-induced ribonucleotide reductase B subunit (RRM2B); the second with mutations in succinate synthase A (SUCLA2) and B (SUCLG1); the third with mutations in Twinkle (PEO1), pol-gammaA (POLG1), deoxyguanosine kinase (DGUOK) and MPV17 (MPV17). In this work, we review the MDS-associated phenotypes and present our own experience of 32 MDS patients, with the aim of defining the mutation frequency of the known genes, the clinical spectrum of the diseases, and the genotype-phenotype correlations. Five of our patients carried previously unreported mutations in one of the eight MDS genes.

EFNS guidelines on the molecular diagnosis of mitochondrial disorders.

OBJECTIVES: These European Federation of Neurological Sciences (EFNS) guidelines are designed to provide practical help for the general neurologist to make appropriate use of molecular genetics for diagnosing mitochondrial disorders (MIDs), which gain increasing attention and are more frequently diagnosed due to improved diagnostic tools. BACKGROUND: Since the publication of the first EFNS guidelines on the molecular diagnosis of inherited neurological diseases in 2001, rapid progress has been made in this field, necessitating the creation of an updated version. SEARCH STRATEGY: To collect data about the molecular diagnosis of MIDs search for literature in various electronic databases, such as Cochrane library, MEDLINE, OMIM, GENETEST or Embase, were carried out and original papers, meta-analyses, review papers, and guideline recommendations were reviewed. RESULTS: The guidelines summarise the possibilities and limitations of molecular genetic diagnosis of MIDs and provide practical recommendations and diagnostic criteria in accordance with the EFNS Scientific Committee to guide the molecular diagnostic work-up of MIDs. RECOMMENDATIONS: The proposed guidelines suggest an approach to the molecular diagnosis of MIDs in a manner accessible to general neurologists.

EFNS guidelines on the molecular diagnosis of neurogenetic disorders: general issues, Huntington's disease, Parkinson's disease and dystonias.

BACKGROUND AND PURPOSE: These EFNS guidelines on the molecular diagnosis of neurogenetic disorders are designed to provide practical help for the general neurologist to make appropriate use of molecular genetics in diagnosing neurogenetic disorders. Since the publication of the first two EFNS-guideline papers on the molecular diagnosis of neurological diseases in 2001, rapid progress has been made in this field, necessitating an updated series of guidelines. METHODS: Literature searches were performed before expert members of the task force wrote proposals, which were discussed in detail until final consensus had been reached among all task force members. RESULTS AND CONCLUSION: This paper provides updated guidelines for molecular diagnosis of Huntington's disease, Parkinson's disease and dystonias as well as a general introduction to the topic. Possibilities and limitations of molecular genetic diagnosis of these disorders are evaluated and recommendations are provided.

Further pitfalls in the diagnosis of mtDNA mutations: homoplasmic mt-tRNA mutations.

BACKGROUND: Mitochondrial DNA (mtDNA) mutations are important causes of human genetic disease, with mutations in tRNA genes particularly prevalent. In many patients, mutations are heteroplasmic, affecting a population of mtDNA molecules. Establishing the pathogenicity of homoplasmic mitochondrial tRNA (mt-tRNA) mutations, in which the mutation is present in every mtDNA molecule, is extremely difficult. These mutations must conform to specific pathogenic criteria, documenting unequivocally a functional defect of the mutant mt-tRNA. AIMS: To investigate the pathogenic nature of two homoplasmic mt-tRNA(Thr) deletions, m.15940delT (previously reported as pathogenic) and m.15937delA, by assessing the steady state levels of the mutant mt-tRNA in tissue and cell-line samples from six unrelated families, in which affected individuals were thoroughly investigated for mitochondrial DNA disease on the basis of clinical presentations. Rates of de novo mitochondrial protein synthesis were also examined in control and m.15937delA mutant fibroblasts. RESULTS: Our data strongly suggest that both single nucleotide deletions are neutral polymorphisms; no obvious defects were apparent in either steady state mt-tRNA(Thr) levels or rates of mitochondrial protein synthesis. CONCLUSIONS: These findings have important implications for the investigation of other families with suspected mtDNA disease, in particular the requirement to fulfil strict and established pathogenic criteria in order to avoid misattribution of pathogenicity to mt-tRNA variants.

Identification of new mutations in the ETHE1 gene in a cohort of 14 patients presenting with ethylmalonic encephalopathy.

BACKGROUND: Ethylmalonic encephalopathy (EE) is a rare autosomal recessive metabolic disorder characterised by progressive encephalopathy, recurrent petechiae, acrocyanosis and chronic diarrhoea, with a fatal outcome in early in life. METHODS: 14 patients with EE were investigated for mutations in the ETHE1 gene. RESULTS: Of the 14 patients, 5 were found to carry novel mutations. CONCLUSIONS: This work expands our knowledge of the causative mutations of EE.