Identification of new variants in MTRNR1 and MTRNR2 genes using whole mitochondrial genome sequencing in a Taiwanese family with MERRF (myoclonic epilepsy with ragged-red fibers) syndrome.

Mitochondrial encephalomyopathy is a multi-system disorder mostly caused by inborn errors of the oxidative phosphorylation (OXPHOS) system and usually manifested as complex neurological disorder and muscle weakness. Myoclonic epilepsy with ragged-red fibers (MERRF) syndrome is one of the major subtypes of mitochondrial disease associated with the m.8344A>G mutation in mitochondrial tRNALys gene. In addition to the symptoms in central nervous and muscle systems, a portion of the patients may develop hearing loss, which has been linked to the genetic mutations of mitochondrial DNA (mtDNA) especially in the mitochondrial ribosome RNA (rRNA) gene. Despite a great number of studies focusing on the consequences of mtDNA mutations, the mechanism of pathogenesis of these overt diseases has remained unclear, and there is no specific and effective treatment for MERRF syndromes. In this study, we developed a high-quality mtDNA sequencing method by next generation sequencing technology to search for the additional pathogenic variations of mtDNA from skin fibroblasts of four members in a Taiwanese family with MERRF syndrome. Through uncovering the signatures of all mtDNA variants in the MERRF family, we identified novel mtDNA variants in the genes encoding mitochondrial 12S and 16S rRNAs. The finding from this study will give us further insight into the molecular mechanisms driving the phenotypic variability and timing of onset of the MERRF syndrome.

Mitochondrial impairment and synaptic dysfunction are associated with neurological defects in iPSCs-derived cortical neurons of MERRF patients.

BACKGROUND: Myoclonic epilepsy with ragged-red fibers (MERRF) syndrome is a rare inherited mitochondrial disease mainly caused by the m.8344A > G mutation in mitochondrial tRNALys gene, and usually manifested as complex neurological disorders and muscle weakness. Currently, the pathogenic mechanism of this disease has not yet been resolved, and there is no effective therapy for MERRF syndrome. In this study, MERRF patients-derived iPSCs were used to model patient-specific neurons for investigation of the pathogenic mechanism of neurological disorders in mitochondrial disease. METHODS: MERRF patient-derived iPSCs were differentiated into excitatory glutamatergic neurons to unravel the effects of the m.8344A > G mutation on mitochondrial bioenergetic function, neural-lineage differentiation and neuronal function. By the well-established differentiation protocol and electrophysiological activity assay platform, we examined the pathophysiological behaviors in cortical neurons of MERRF patients. RESULTS: We have successfully established the iPSCs-derived neural progenitor cells and cortical-like neurons of patients with MERRF syndrome that retained the heteroplasmy of the m.8344A > G mutation from the patients' skin fibroblasts and exhibited the phenotype of the mitochondrial disease. MERRF neural cells harboring the m.8344A > G mutation exhibited impaired mitochondrial bioenergetic function, elevated ROS levels and imbalanced expression of antioxidant enzymes. Our findings indicate that neural immaturity and synaptic protein loss led to the impairment of neuronal activity and plasticity in MERRF neurons harboring the m.8344A > G mutation. By electrophysiological recordings, we monitored the in vivo neuronal behaviors of MERRF neurons and found that neurons harboring a high level of the m.8344A > G mutation exhibited impairment of the spontaneous and evoked potential-stimulated neuronal activities. CONCLUSIONS: We demonstrated for the first time the link of mitochondrial impairment and synaptic dysfunction to neurological defects through impeding synaptic plasticity in excitatory neurons derived from iPSCs of MERRF patients harboring the m.8344A > G mutation. This study has provided new insight into the pathogenic mechanism of the tRNALys gene mutation of mtDNA, which is useful for the development of a patient-specific iPSCs platform for disease modeling and screening of new drugs to treat patients with MERRF syndrome.

Restoration of mitochondrial function through activation of hypomodified tRNAs with pathogenic mutations associated with mitochondrial diseases.

Mutations in mitochondrial (mt-)tRNAs frequently cause mitochondrial dysfunction. Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), and myoclonus epilepsy associated with ragged red fibers (MERRF) are major clinical subgroups of mitochondrial diseases caused by pathogenic point mutations in tRNA genes encoded in mtDNA. We previously reported a severe reduction in the frequency of 5-taurinomethyluridine (τm5U) and its 2-thiouridine derivative (τm5s2U) in the anticodons of mutant mt-tRNAs isolated from the cells of patients with MELAS and MERRF, respectively. The hypomodified tRNAs fail to decode cognate codons efficiently, resulting in defective translation of respiratory chain proteins in mitochondria. To restore the mitochondrial activity of MELAS patient cells, we overexpressed MTO1, a τm5U-modifying enzyme, in patient-derived myoblasts. We used a newly developed primer extension method and showed that MTO1 overexpression almost completely restored the τm5U modification of the MELAS mutant mt-tRNALeu(UUR). An increase in mitochondrial protein synthesis and oxygen consumption rate suggested that the mitochondrial function of MELAS patient cells can be activated by restoring the τm5U of the mutant tRNA. In addition, we confirmed that MTO1 expression restored the τm5s2U of the mutant mt-tRNALys in MERRF patient cells. These findings pave the way for epitranscriptomic therapies for mitochondrial diseases.

Rapamycin rescues mitochondrial dysfunction in cells carrying the m.8344A > G mutation in the mitochondrial tRNALys.

BACKGROUND: Myoclonus, Epilepsy and Ragged-Red-Fibers (MERRF) is a mitochondrial encephalomyopathy due to heteroplasmic mutations in mitochondrial DNA (mtDNA) most frequently affecting the tRNALys gene at position m.8344A > G. Defective tRNALys severely impairs mitochondrial protein synthesis and respiratory chain when a high percentage of mutant heteroplasmy crosses the threshold for full-blown clinical phenotype. Therapy is currently limited to symptomatic management of myoclonic epilepsy, and supportive measures to counteract muscle weakness with co-factors/supplements. METHODS: We tested two therapeutic strategies to rescue mitochondrial function in cybrids and fibroblasts carrying different loads of the m.8344A > G mutation. The first strategy was aimed at inducing mitochondrial biogenesis directly, over-expressing the master regulator PGC-1α, or indirectly, through the treatment with nicotinic acid, a NAD+ precursor. The second was aimed at stimulating the removal of damaged mitochondria through prolonged rapamycin treatment. RESULTS: The first approach slightly increased mitochondrial protein expression and respiration in the wild type and intermediate-mutation load cells, but was ineffective in high-mutation load cell lines. This suggests that induction of mitochondrial biogenesis may not be sufficient to rescue mitochondrial dysfunction in MERRF cells with high-mutation load. The second approach, when administered chronically (4 weeks), induced a slight increase of mitochondrial respiration in fibroblasts with high-mutation load, and a significant improvement in fibroblasts with intermediate-mutation load, rescuing completely the bioenergetics defect. This effect was mediated by increased mitochondrial biogenesis, possibly related to the rapamycin-induced inhibition of the Mechanistic Target of Rapamycin Complex 1 (mTORC1) and the consequent activation of the Transcription Factor EB (TFEB). CONCLUSIONS: Overall, our results point to rapamycin-based therapy as a promising therapeutic option for MERRF.

A Novel MTTK Gene Variant m.8315A>C as a Cause of MERRF Syndrome

In this study, we report on a novel heteroplasmic pathogenic variant in mitochondrial DNA (mtDNA). The studied patient had myoclonus, epilepsy, muscle weakness, and hearing impairment and harbored a heteroplasmic m.8315A>C variant in the MTTK gene with a mutation load ranging from 71% to >96% in tested tissues. In muscle mitochondria, markedly decreased activities of respiratory chain complex I + III and complex IV were observed together with mildly reduced amounts of complex I and complex V (with the detection of V*- and free F1-subcomplexes) and a diminished level of complex IV holoenzyme. This pattern was previously seen in other MTTK pathogenic variants. The novel variant was not present in internal and publicly available control databases. Our report further expands the spectrum of MTTK variants associated with mitochondrial encephalopathies in adults.

Myoclonic Epilepsy with Ragged-red Fibers with Intranuclear Inclusions.

We herein report a case of myoclonic epilepsy with ragged-red fibers (MERRF) harboring a novel variant in mitochondrial cysteine transfer RNA (MT-TC). A 68-year-old woman presented with progressive myoclonic epilepsy with optic atrophy and peripheral neuropathy. A skin biopsy revealed p62-positive intranuclear inclusions. No mutations were found in the causative genes for diseases known to be related to intranuclear inclusions; however, a novel variant in MT-TC was found. The association between intranuclear inclusions and this newly identified MERRF-associated variant is unclear; however, the rare complication of intranuclear inclusions in a patient with typical MERRF symptoms should be noted for future studies.

Lipomatosis and optic neuropathy clinches the diagnosis of myoclonic epilepsy with ragged red fibres (MERRF) syndrome.

We present a rare case of myoclonic epilepsy with ragged red fibres with high level of heteroplasmy presenting with optic neuropathy and a rare phenotype of lipomatosis. Cutaneous lipomas are typically thought of as a benign/isolated entity and this case emphasises importance of considering mitochondrial disease in all patients with lipomatosis especially in the presence of other systemic abnormalities.

Perioperative Management of an Adult Patient With Myoclonic Epilepsy With Ragged Red Fibers Syndrome: A Case Report.

Myoclonic epilepsy with ragged red fibers (MERRF) syndrome is a rare mitochondrial disease potentially associated with increased sensitivity to anesthesia and metabolic decompensation. We present the perioperative management in a 59-year-old man with MERRF scheduled for lipomatosis cure under general anesthesia (GA). Following a reduced fasting period, the patient had an uneventful balanced GA with propofol, sevoflurane, and rocuronium. The patient did not present metabolic decompensation nor malignant hyperthermia but prolonged neuromuscular blockade. Propofol and sevoflurane may be used in asymptomatic MERRF adult patients. Such patients present high risk of residual neuromuscular blockade that should be monitored and reversed.

Diagnosing MERRF requires clinical and genetic evidence.

The interesting case about a patients with myoclonic epilepsy with ragged-red fibers (MERRF) syndrome due to the variant m.8344A>G with a heteroplasmy rate of 95% reported by Felczak et al. expands the phenotypic spectrum of MERRF syndrome. The authors reported a pituitary adenoma, calcium deposits in arterial walls, and an intra-cerebral lipoma in the corpus callosum in their patient. Shortcomings of the study are that the diagnostic criteria for MERRF were not accomplished, that the patient should be rather diagnosed as a mitochondrial, multiorgan disorder syndrome (MIMODS), that no pedigree and heteroplasmy rates in first degree relative were provided, that hormone levels were not provided despite obvious endocrinological involvement, and that no serum or cerebrospinal fluid (CSF) lactate levels were reported.

"Myo-neuropathy" is commonly associated with mitochondrial tRNALysine mutation.

The mitochondrial tRNALys (mt-tRNALys) mutation is initially associated with myoclonic epilepsy and ragged-red fibers (MERRF). The clinical, laboratory, morphologic and molecular findings from 22 mt-tRNALys mutation carriers from local database in East China were analyzed retrospectively. We identified 13 symptomatic and 9 asymptomatic individuals with a known pathogenic mitochondrial tRNALys mutation. The most common mutations were m.8344 A>G (81.8%), m.8363G>A (9.1%), m.8356 T>C (4.5%) and m.8356 T>G (4.5%). The degree of mutation heteroplasmy in blood was high both in symptomatic (mean 64.5%, range 41-82%) and asymptomatic individuals (mean 53.1%, range 21-78%). Age at onset ranged from 6 year-old to the age of 66 years (mean 35.8 ± 16.4 years old). The most frequent symptoms were muscle weakness (76.9%), exercise intolerance (76.9%), elevated creatine kinase levels (61.5%), peripheral neuropathy (69.2%) and cerebellar ataxia (61.5%), while myoclonus was only present in 23.1% of symptomatic patients. A diagnosis of mitochondrial myopathy (MM) and neuropathy ataxia and retinitis pigmentosa (NARP/NARP-like) syndrome was made in 77% of symptomatic patients, whereas the classic syndrome of myoclonic epilepsy with ragged-red fibers (MERRF) was rare (23%). In this cohort of patients with mt-tRNALys mutation, more than one third of our patients did not develop signs and symptoms of central nervous system involvement even in later stages of the disease, indicating the necessity to investigate the mt-tRNALys gene in 'pure' mitochondrial 'myo-neuropathy'.

Ganglionopathies Associated with MERRF Syndrome: An Original Report.

Neuropathies in Myoclonic Epilepsy with Ragged Red Fibers (MERRF) syndrome are frequent but ganglionopathies have never been reported. We retrospectively identified 24 patients with MERRF mutations in the neuromuscular center Nord/Est/Ile de France (Pitié-Salpêtrière, Paris, France). Seventeen nerve conduction studies (NCS) were available. Five patients had MERRF syndrome and ganglionopathy, a pure sensory neuropathy. All of them displayed ataxia and mild clinical sensory abnormalities. Ganglionopathies have been reported in mitochondrial diseases but never in MERRF syndrome. We suggest that patients presenting with ganglionopathy, especially if associated with myopathy, lipomatosis or epilepsy, should be screened for MERRF mutations.

Clinical phenotype of mitochondrial diabetes due to rare mitochondrial DNA mutations.

OBJECTIVE: While the most frequent mutation responsible for mitochondrial diabetes is the point mutation m.3243 A>G of mitochondrial DNA (mtDNA), few data are available about the role of rare mtDNA mutations in the pathophysiology of diabetes. The main objective of our study was to describe the phenotypic characteristics of patients suffering from diabetes linked to rare mtDNA mutations. RESEARCH DESIGN AND METHODS: We performed a post-hoc analysis of a prospective multicenter cohort of 743 patients with mitochondrial disorder (previously published by the French Network of Mitochondrial Diseases), associated to a literature review of the PubMed database from 1992 to May 2016. We extracted all reported patients with diabetes and identified rare mtDNA mutations and described their clinical and metabolic phenotypes. RESULTS: The 50 identified patients (10 from the princeps study; 40 from the review of the literature) showed a heterogeneous metabolic phenotype in terms of age, symptoms prior to diagnosis, treatments, and associated clinical and biological signs. However, neurological symptoms were more frequent in case of rare mtDNA mutations compared to the classical m.3243 A>G mutation (P=0.024). In contrast, deafness (65% vs. 95%, P=3.7E-5), macular pattern dystrophy (20% vs. 86%, P=1.6E-10) and nephropathy (8% vs. 28%, P=0.018) were significantly less frequent than in case of the classical m.3243 A>G mutation. CONCLUSION: Although no specific metabolic phenotype could be identified suggesting or eliminating implication of rare mtDNA mutations in diabetes, clinical phenotypes featured more frequent neurological signs.

Tumors masquerading as neurological diseases: A caution for clinicians in planning diagnosis and treatment.

INTRODUCTION: Neurological diseases can be due to direct diseases of the central nervous system (CNS) or peripheral nervous system (PNS) or be a bystander syndrome of systemic diseases. Treatment options depend on the cause. Toxic, metabolic and nutritional, and immune-mediated consequences of clinically occult neoplasms produce a spectrum of neurological diseases, recognition of which has therapeutic and prognostic importance. PATIENTS AND METHODS: Children, as well as adults who presented to the authors in the last 5 years with neurological diseases and later their diseases could be diagnosed or attributed to neoplasms which were occult, were included for the study. OBSERVATION: 28 patients were seen by the authors in the last 5 years with neurological manifestation and hidden tumor. Maximum incidence was in the age of above 60 years followed by the age group of 21-40 years. The commonest neurological presentation was muscle and nerve in adults and seizure in children. DISCUSSION: Short duration, rapid progression, severe weight loss, and poor response to treatment given for nontumor associated neurological syndrome are the red flags which point to the diagnosis. CONCLUSION: Seizures and psychosis formed the commonest features in children, muscle and nerve in adults. Short duration, rapid progression, and resistance to treatment are the markers for possible underlying neoplasm.