Life-threatening lactic acidosis occurring in adults with mitochondrial disorders.

Although relatively common in children, severe acute lactic acidosis is rare in adults with mitochondrial myopathies. We report here three cases, aged 27, 32 and 32 years, who developed life-threatening metabolic crisis with severe lactic acidosis, requiring hospitalisation in intensive care unit. Plasma lactates were elevated 10 to 15 fold normal values, necessitating extra-renal dialysis. By contrast CK levels were moderately increased (3 to 5N). No triggering factor was identified, but retrospectively all patients reported long-lasting mild muscle fatigability and weakness before their acute metabolic crisis. All of them recovered after prolonged intensive care but resting lactate levels remained elevated. Muscle biopsy showed ragged-red and COX-negative fibers in two patients and mild lipidosis in the third one. Heteroplasmic pathogenic point mutations were detected in MT-TL1 (m.3280G>A;m.3258C>T) and MT-TK (m.8363A>G). Life-threatening lactic acidosis may thus be a major inaugural clinical manifestation in adults with mitochondrial myopathies. Prolonged intensive care may lead to a dramatic and sustained improvement and is mandatory in such cases.

Myopathy with MTCYB mutation mimicking Multiple Acyl-CoA Dehydrogenase Deficiency.

We describe two patients with mitochondrial DNA mutations in the gene encoding cytochrome b (m.15579A>G, p.Tyr278Cys and m.15045G>A p.Arg100Gln), which presented as a pure myopathic form (exercise intolerance), with an onset in childhood. Diagnosis was delayed, because acylcarnitine profile showed an increase in medium and long-chain acylcarnitines, suggestive of multiple acyl-CoA dehydrogenase deficiency, riboflavin transporter deficiency or FAD metabolism disorder. Implication of cytochrome b in fatty acid oxidation, and physiopathology of the mutations are discussed.

Prediction of long-term prognosis by heteroplasmy levels of the m.3243A>G mutation in patients with the mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes syndrome.

BACKGROUND AND PURPOSE: Our aim was to determine the prognostic value of urine and blood heteroplasmy in patients with the m.3243A>G mutation. METHODS: Adults with the m.3243A>G mutation referred to our institution between January 2000 and May 2014 were retrospectively included. The relationship between their baseline clinical characteristics, their mutation load in urine and blood, and major adverse events (MAEs) during follow-up, defined as medical complications requiring a hospitalization or complicated by death, was studied. RESULTS: Of the 43 patients (age 45.6 ± 13.3 years) included in the study, 36 patients were symptomatic, including nine with evidence of focal brain involvement, and seven were asymptomatic. Over a 5.5 ± 4.0 year mean follow-up duration, 14 patients (33%) developed MAEs. Patients with MAEs had a higher mutation load than others in urine (60.1% ± 13.8% vs. 40.6% ± 26.2%, P = 0.01) and in blood (26.9% ± 18.4% vs. 16.0% ± 12.1%, P = 0.03). Optimal cutoff values for the prediction of MAEs were 45% for urine and 35% for blood. In multivariate analysis, mutation load in urine ≥45% [odds ratio 25.3; 95% confidence interval (CI) 1.1-567.8; P = 0.04], left ventricular hypertrophy (odds ratio 16.7; 95% CI 1.3- 222.5; P = 0.03) and seizures (odds ratio 48.3; 95% CI 2.5-933; P = 0.01) were associated with MAEs. CONCLUSIONS: Patients with the m.3243A>G mutation are at high risk of MAEs, which can be independently predicted by mutation load in urine ≥45%, a personal history of seizures, and left ventricular hypertrophy.

A mutant mitochondrial respiratory chain assembly protein causes complex III deficiency in patients with tubulopathy, encephalopathy and liver failure.

Complex III (CIII; ubiquinol cytochrome c reductase of the mitochondrial respiratory chain) catalyzes electron transfer from succinate and nicotinamide adenine dinucleotide-linked dehydrogenases to cytochrome c. CIII is made up of 11 subunits, of which all but one (cytochrome b) are encoded by nuclear DNA. CIII deficiencies are rare and manifest heterogeneous clinical presentations. Although pathogenic mutations in the gene encoding mitochondrial cytochrome b have been described, mutations in the nuclear-DNA-encoded subunits have not been reported. Involvement of various genes has been indicated in assembly of yeast CIII (refs. 8-11). So far only one such gene, BCS1L, has been identified in human. BCS1L represents, therefore, an obvious candidate gene in CIII deficiency. Here, we report BCS1L mutations in six patients, from four unrelated families and presenting neonatal proximal tubulopathy, hepatic involvement and encephalopathy. Complementation study in yeast confirmed the deleterious effect of these mutations. Mutation of BCS1L would seem to be a frequent cause of CIII deficiency, as one-third of our patients have BCS1L mutations.

Methylene blue induced O2 consumption is not dependent on mitochondrial oxidative phosphorylation: Implications for salvage pathways during acute mitochondrial poisoning.

While administration of the cyclic redox agent methylene blue (MB) during intoxication by mitochondrial poisons (cyanide, hydrogen sulfide, rotenone) increases survival, the mechanisms behind these antidotal properties remain poorly understood. The objective of the studies presented in this paper was to characterize the interactions between the redox properties of MB, the intermediate metabolism and the mitochondrial respiration. We first show that intra-venous administration of micromolar levels of methylene blue in sedated and mechanically ventilated rats, increases not only resting oxygen consumption but also CO2 production (by ~ 50%), with no change in their ratio. This hypermetabolic state could be reproduced in a cellular model, where we found that the rate of electron transfer to MB was of the same order of magnitude as that of normal cellular metabolism. Notably, the large increase in cellular oxygen consumption caused by MB was relatively indifferent to the status of the mitochondrial respiratory chain: oxygen consumption persisted even when the respiratory chain was inhibited or absent (using inhibitors and cells deficient in mitochondrial oxidative phosphorylation); yet MB did not impede mitochondrial ATP production in control conditions. We present evidence that after being reduced into leuco-methylene blue (LMB) in presence of reducing molecules that are physiologically found in cells (such as NADH), the re-oxidation of LMB by oxygen can account for the increased oxygen consumption observed in vivo. In conditions of acute mitochondrial dysfunction, these MB redox cycling properties allow the rescue of the glycolysis activity and Krebs cycle through an alternate route of oxidation of NADH (or other potential reduced molecules), which accumulation would have otherwise exerted negative feedback on these metabolic pathways. Our most intriguing finding is that re-oxidization of MB by oxygen ultimately results in an in vivo matching between the increase in the rate of O2 consumed, by MB re-oxidation, and the rate of CO2, produced by the intermediate metabolism, imitating the fundamental coupling between the glycolysis/Krebs cycle and the mitochondrial respiration.

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.

[Multiple acyl-CoA dehydrogenase deficiency (MADD): a curable cause of genetic muscular lipidosis]. / Déficit multiple en acyl-CoA déshydrogénases : une cause traitable de lipidose musculaire d'origine génétique.

INTRODUCTION: Multiple acyl-CoA dehydrogenase deficiency (MADD) is a rare genetic disease involving fatty acid oxidation. It is due to the deficiency of one of the two electron transporters: electron transfer flavoprotein (ETF) or electron transfer flavoprotein ubiquinone oxydoreductase (ETF-QO). Symptoms begin more often in childhood or in young adulthood with a multisystemic disease with encephalopathy or muscular weakness. CASE REPORTS: We report here two adult cases with ETF-QO deficiency, confirmed by mutation analysis (ETFDH gene), revealed by a muscular weakness associated with muscle lipidosis. One of our patients presented an acute encephalopathy with vomiting ten years before the onset of muscular symptoms. The second patient exhibited a slowly progressive pelvic girdle muscle weakness. Diagnosis was established by characteristic abnormalities of acylcarnitine profile by tandem mass spectrometry. For both patients, a dramatic clinical improvement was observed under treatment with riboflavine and L-carnitine. CONCLUSION: Since it is a treatable disorder, this diagnosis must be considered by performing an acylcarnitine profile in all patients presenting with an unexplained muscular weakness.

MPTP intoxication in mice: a useful model of Leigh syndrome to study mitochondrial diseases in childhood.

The basal ganglia, which are interconnected in the striato-nigral dopaminergic network, are affected in several childhood diseases including Leigh syndrome (LS). LS is the most common mitochondrial disorder affecting children and usually arise from inhibition of the respiratory chain. This vulnerability is attributed to a particular susceptibility to energetic stress, with mitochondrial inhibition as a common pathogenic pathway. In this study we developed a LS model for neuroprotection trials in mice by using the complex I inhibitor MPTP. We first verified that MPTP significantly inhibits the mitochondrial complex I in the brain (p = 0.018). This model also reproduced the biochemical and pathological features of LS: MPTP increased plasmatic lactate levels (p = 0.023) and triggered basal ganglia degeneration, as evaluated through dopamine transporter (DAT) autoradiography, tyrosine hydroxylase (TH) immunohistochemistry, and dopamine dosage. Striatal DAT levels were markedly decreased after MPTP treatment (p = 0.003). TH immunoreactivity was reduced in the striatum and substantia nigra (p = 0.005), and striatal dopamine was significantly reduced (p < 0.01). Taken together, these results confirm that acute MPTP intoxication in young mice provides a reproducible pharmacological paradigm of LS, thus opening new avenues for neuroprotection research.

[Clinical variability and diagnosis steps in childhood mitochondrial disease]. / Variabilité clinique et conduite diagnostique des cytopathies mitochondriales : à propos d'une série de 18 cas pédiatriques.

OBJECTIVES: Mitochondrial respiratory chain deficiencies are known for their high clinical variability. Difficult to diagnose, the prevalence of these diseases is probably underestimated. METHODS: We report 18 children diagnosed with respiratory chain deficiency at the Tours University Hospital over the past 10 years. RESULTS: Three clinical profiles can be distinguished depending on the age at onset of the first symptoms: the neonatal period (4 cases), between 1 month and 2 years of age (10 cases), and after 10 years (4 cases). However, no clinical feature appears specific of any age group. In contrast, respiratory chain analysis on liver biopsy was very informative for all our patients at any age and with any clinical presentation, even with predominant neurological symptoms. CONCLUSIONS: These biochemical analyses support the diagnosis of mitochondrial disorders in view of molecular analysis, which nevertheless frequently remains inconclusive. These investigations should benefit from the new molecular screening technologies based on DNA chips that can identify the genomic mutations responsible for these severe and relatively frequent diseases.

Modulation of mitochondrial morphology by bioenergetics defects in primary human fibroblasts.

Mitochondria are dynamic organelles with continuous fusion and fission, the equilibrium of which results in mitochondrial morphology. Evidence points to there being an intricate relationship between mitochondrial dynamics and oxidative phosphorylation. We investigated the bioenergetics modulation of mitochondrial morphology in five control cultured primary skin fibroblasts and seven with genetic alterations of oxidative phosphorylation. Under basal conditions, control fibroblasts had essentially filamentous mitochondria. Oxidative phosphorylation inhibition with drugs targeting complex I, III, IV or V induced partial but significant mitochondrial fragmentation, whereas dissipation of mitochondrial membrane potential (D Psi m) provoked complete fragmentation, and glycolysis inhibition had no effect. Oxidative phosphorylation defective fibroblasts had essentially normal filamentous mitochondria under basal conditions, although when challenged some of them presented with mild alteration of fission or fusion efficacy. Severely defective cells disclosed complete mitochondrial fragmentation under glycolysis inhibition. In conclusion, mitochondrial morphology is modulated by D Psi m but loosely linked to mitochondrial oxidative phosphorylation. Its alteration by glycolysis inhibition points to a severe oxidative phosphorylation defect.

Myoblast xenotransplantation as a tool to evaluate the appropriateness of nanoparticular versus cellular trackers.

Myoblast transplantation is being considered as a potential strategy to improve muscle function in myopathies; hence, it is important to identify the transplanted cells and to have available efficient reagents to track these cells. We first validated a human to mouse xenotransplantation model warranting the complete and rapid rejection of the cells. We then used this model to assess the appropriateness of a nanoparticle reagent to track the transplanted cells. Human myoblasts were loaded with ferrite nanoparticles and injected into the tibialis muscle of immunocompetent mice. Upon collection and histological analysis of muscle sections at different time points, we observed the total disappearance of the human cells within 6 days while ferrite particles remained detectable and colocalized with mouse infiltrating and neighboring cells at the injection site. These results suggest that the use of exogenous markers such as ferrite nanoparticles may lead to false-positive results and misinterpretation of cell fate.

Antenatal mitochondrial disease caused by mitochondrial ribosomal protein (MRPS22) mutation.

Three patients born to the same set of consanguineous parents presented with antenatal skin oedema, hypotonia, cardiomyopathy and tubulopathy. The enzymatic activities of multiple mitochondrial respiratory chain complexes were reduced in muscle. Marked reduction of 12s rRNA, the core of the mitochondrial small ribosomal subunit, was found in fibroblasts. Homozygosity mapping led to the identification of a mutation in the MRPS22 gene, which encodes a mitochondrial ribosomal protein. Transfection of the patient cells with wild-type MRPS22 cDNA increased the 12s rRNA content and normalised the enzymatic activities. Quantification of mitochondrial transcripts is advisable in patients with multiple defects of the mitochondrial respiratory chain.

Nuclear DNA origin of mitochondrial complex I deficiency in fatal infantile lactic acidosis evidenced by transnuclear complementation of cultured fibroblasts.

We have studied complex I (NADH-ubiquinone reductase) defects of the mitochondrial respiratory chain in 2 infants who died in the neonatal period from 2 different neurological forms of severe neonatal lactic acidosis. Specific and marked decrease in complex I activity was documented in muscle, liver, and cultured skin fibroblasts. Biochemical characterization and study of the genetic origin of this defect were performed using cultured fibroblasts. Immunodetection of 6 nuclear DNA-encoded (20, 23, 24, 30, 49, and 51 kDa) and 1 mitochondrial DNA-encoded (ND1) complex I subunits in fibroblast mitochondria revealed 2 distinct patterns. In 1 patient, complex I contained reduced amounts of the 24- and 51-kDa subunits and normal amounts of all the other investigated subunits. In the second patient, amounts of all the investigated subunits were severely decreased. The data suggest partial or extensive impairment of complex I assembly in both patients. Cell fusion experiments between 143B206 rho degrees cells, fully depleted of mitochondrial DNA, and fibroblasts from both patients led to phenotypic complementation of the complex I defects in mitochondria of the resulting cybrid cells. These results indicate that the complex I defects in the 2 reported cases are due to nuclear gene mutations.

Impact on oxidative phosphorylation of immortalization with the telomerase gene.

Skin fibroblasts are essential tools for biochemical, genetic and physiopathological investigations of mitochondrial diseases. Their immortalization has been previously performed to overcome the limited number of divisions of these primary cells but it has never been systematically evaluated with respect to efficacy and impact on the oxidative phosphorylation (OXPHOS) characteristics of the cells. We successfully immortalized with the human telomerase gene 15 human fibroblasts populations, 4 derived from controls and 11 from patients with diverse respiratory chain defects. Immortalization induced significant but mild modification of the OXPHOS characteristics of the cells with lower rates of oxygen consumption and ATP synthesis associated with their loose coupling. However, it never significantly altered the type and severity of any genetic OXPHOS defect present prior to immortalization. Furthermore, it did not significantly modify the cells' dependence on glucose and sensitivity to galactose thus showing that immortalized cells could be screened by their nutritional requirement. Immortalized skin fibroblasts with significant OXPHOS defect provide reliable tools for the diagnosis and research of the genetic cause of mitochondrial defects. They also represent precious material to investigate the cellular responses to these defects, even though these should afterwards be verified in unmodified primary cells.

[Diagnostic investigations of mitochondrial diseases with neurological symptoms]. / Approche diagnostique des maladies mitochondriales à présentation neurologique.

Mitochondrial respiratory chain abnormalities are a cause of neuromuscular diseases. They present with very diverse clinical presentations,involving either the central nervous system, the peripheral nervous system, or skeletal muscle, and may be due to mutations either in mitochondrial or nuclear genome. The aim of this review is to familiarise the clinician with these diseases, to evoke main syndromes, and to give guidelines for their diagnostic investigation.

Task force guidelines handbook: EFNS guidelines on diagnosis and management of fatty acid mitochondrial disorders.

Guidelines in the diagnosis and current dietary treatment of long-chain fatty acid (LCFA) defects have been collected according to evidence-based medicine. Since the identification of carnitine and carnitine palmitoyltransferase deficiency more than 25 years ago, nearly every enzymatic step required for beta-oxidation has been associated with an inherited metabolic disorder. These disorders effectively preclude the use of body fat as an energy source. Clinical consequences can range from no symptoms to severe manifestations including cardiomyopathy, hypoglycaemia, peripheral neuropathy and sudden death. A diet high in carbohydrates, diet with medium-chain triglycerides and reduced amount of LCFA has a beneficial effect (class IV evidence) and in appropriate deficiency states carnitine and riboflavin are used (good practice points).

Kearns Sayre syndrome: an unusual form of mitochondrial diabetes.

Kearns Sayre syndrome (KSS) is a mitochondrial disorder characterized by the emergence before age 20 of progressive external ophthalmoplegia, pigmentary retinopathy, together with other heterogeneous clinical manifestations, including cardiac conduction defects, muscle abnormalities and endocrinopathies. KSS is associated with large heteroplasmic deletions in mitochondrial DNA. We report the case of a 43-year-old woman, with diabetes mellitus as a first manifestation at age 19. Later, she exhibited bilateral ptosis and external ophthalmoplegia with progressive worsening. DNA analysis identified a large mitochondrial DNA (mtDNA) deletion, which confirmed the diagnosis of KSS. By reporting this case with diabetes mellitus as first manifestation, we aim at emphasizing problems of diagnosis in these subtypes of mitochondrial diabetes.

[Misdiagnosis of mitochondrial myopathies: a study of 12 thymectomized patients]. / Errance diagnostique dans les myopathies mitochondriales: étude de 12 patients thymectomisés.

INTRODUCTION: Myasthenia gravis and mitochondrial myopathies have common symptoms (fatigability, ophthalmoplegia) that could lead to diagnosis confusion. METHODS: We systematically reviewed medical history and ancillary investigations regarding 12 patients (7F/5M, mean age 47+/-14 years) having a mitochondrial myopathy but who were previously misdiagnosed as autoimmune myasthenia gravis and in whom a thymectomy was performed. RESULTS: Ocular palsy, ptosis and bulbar palsy were present in all patients. Limb fatigability was present in 9 cases. Symptoms were fluctuant but without remission. The misdiagnosis of myasthenia was based on the following arguments: 1) decremental EMG response (2 cases); 2) positive injectable anticholinesterase drugs test (3 cases); 3) partial response to oral anticholinesterase medications (2 cases); 4) AChR antibodies titer of 0.6 nM considered as positive (1 case). A multisystemic involvement was present in 5 patients: peripheral neuropathy (2 cases), deafness (2 cases), cardiopathy (3 cases), cerebellar involvement (2 cases) and myoclonia (1 case). The diagnosis of mitochondrial myopathy (at a mean age of 38+/-12 years) has been certified on the results of muscle biopsy showing mitochondrial proliferation (12 cases) and deleted mitochondrial DNA (8 cases). CONCLUSIONS: In a patient presenting with oculomotor symptoms and muscle fatigability, progressive course and multisystemic involvement are major arguments for a mitochondrial myopathy. In the absence of relevant criteria arguing for Myasthenia Gravis (significant variability of muscle weakness, positive titer of anti-AChR or anti-MuSK antibodies, decremental EMG response), a muscle biopsy is required before indication of thymectomy to exclude a mitochondrial disease.

Functional characterization of novel mutations in the human cytochrome b gene.

The great variability of the human mitochondrial DNA (mtDNA) sequence induces many difficulties in the search for its deleterious mutations. We illustrate these pitfalls by the analysis of the cytochrome b gene of 21 patients affected with a mitochondrial disease. Eighteen different sequence variations were found, five of which were new mutations. Extensive analysis of the cytochrome b gene of 146 controls found 20 supplementary mutations, thus further demonstrating the high variability of the cytochrome b sequence. We fully evaluated the functional relevance of 36 of these 38 mutations using indirect criteria such as the nature of the mutation, its frequency in controls, or the phylogenetic conservation of the mutated amino acid. When appropriate, the mtDNA haplotype, the heteroplasmic state of the mutation, its tissue distribution or its familial transmission were also assessed. The molecular consequences of the mutations, which appeared possibly deleterious in that first step of evaluation, were evaluated on the complex III enzymological properties and protein composition using specific antibodies that we have generated against four of its subunits. Two original deleterious mutations were found in the group of seven patients with overt complex III defect. Both mutations (G15150A (W135X) and T15197C (S151P)) were heteroplasmic and restricted to muscle. They had significant consequences on the complex III structure. In contrast, only two homoplasmic missense mutations with dubious clinical relevance were found in the patients without overt complex III defect.