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Nature Communications May 2018It has been generally thought that tRNA modifications are stable and static, and their frequencies are rarely regulated. N-threonylcarbamoyladenosine (tA) occurs at...
It has been generally thought that tRNA modifications are stable and static, and their frequencies are rarely regulated. N-threonylcarbamoyladenosine (tA) occurs at position 37 of five mitochondrial (mt-)tRNA species. We show that YRDC and OSGEPL1 are responsible for tA37 formation, utilizing L-threonine, ATP, and CO/bicarbonate as substrates. OSGEPL1-knockout cells exhibit respiratory defects and reduced mitochondrial translation. We find low level of tA37 in mutant mt-tRNA isolated from the MERRF-like patient's cells, indicating that lack of tA37 results in pathological consequences. Kinetic measurements of tA37 formation reveal that the Km value of CO/bicarbonate is extremely high (31 mM), suggesting that CO/bicarbonate is a rate-limiting factor for tA37 formation. Consistent with this, we observe a low frequency of tA37 in mt-tRNAs isolated from human cells cultured without bicarbonate. These findings indicate that tA37 is regulated by sensing intracellular CO/bicarbonate concentration, implying that mitochondrial translation is modulated in a codon-specific manner under physiological conditions.
Topics: Adenosine; Apoptosis Regulatory Proteins; Base Pairing; Bicarbonates; CRISPR-Cas Systems; Carbon Dioxide; Cell Line; Cell Respiration; Fibroblasts; GTP-Binding Proteins; Gene Deletion; HEK293 Cells; HT29 Cells; HeLa Cells; Humans; MERRF Syndrome; Mitochondria; Models, Biological; Myoblasts; Nucleic Acid Conformation; Proteins; RNA Processing, Post-Transcriptional; RNA, Transfer; RNA-Binding Proteins
PubMed: 29760464
DOI: 10.1038/s41467-018-04250-4 -
Molecular Medicine (Cambridge, Mass.) Aug 2022Myoclonus, Epilepsy and Ragged-Red-Fibers (MERRF) is a mitochondrial encephalomyopathy due to heteroplasmic mutations in mitochondrial DNA (mtDNA) most frequently...
BACKGROUND
Myoclonus, Epilepsy and Ragged-Red-Fibers (MERRF) is a mitochondrial encephalomyopathy due to heteroplasmic mutations in mitochondrial DNA (mtDNA) most frequently affecting the tRNA gene at position m.8344A > G. Defective tRNA 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.
Topics: DNA, Mitochondrial; Humans; MERRF Syndrome; Mitochondria; Mitochondrial Proteins; Mutation; RNA, Transfer, Lys; Sirolimus
PubMed: 35922766
DOI: 10.1186/s10020-022-00519-z -
Frontiers in Neurology 2019Lipomas have often been associated with mtDNA mutations and were mainly observed in patients with mutation in mitochondrial tRNAlysine which is also the most frequent...
Lipomas have often been associated with mtDNA mutations and were mainly observed in patients with mutation in mitochondrial tRNAlysine which is also the most frequent mutation associated with MERRF. Up to date, no systematic studies have been developed in order to assess the incidence of lipomas in large cohorts of mitochondrial patients.The aim of this study is to analyze the incidence and characteristics of lipomas among an Italian cohort of patients with mitochondrial diseases. A retrospective, database-based study (Nation-wide Italian Collaborative Network of Mitochondrial Diseases) of patients with lipomas was performed. A total of 22 (1.7%) patients with lipomas have been identified among the 1,300 mitochondrial patients, enrolled in the Italian database. In about 18% multiple systemic lipomatosis (MSL) was the only clinical manifestation; 54% of patients showed a classical MERRF syndrome. Myopathy, alone or in association with other symptoms, was found in 27% of patients. Lactate was elevated in all the 12 patients in which was measured. Muscle biopsy was available in 18/22 patients: in all of them mitochondrial abnormalities were present. Eighty six percent had mutations in mtDNA coding for tRNA lysine. In most of patients, lipomas were localized along the cervical-cranial-thoracic region. In 68% of the patients were distributed symmetrically. Only two patients had lipomas in a single anatomical site (1 in right arm and 1 in gluteus maximum). MSL is often overlooked by clinicians in patients with mitochondrial diseases where the clinical picture could be dominated by a severe multi-systemic involvement. Our data confirmed that MSL is a rare sign of mitochondrial disease with a strong association between multiple lipomas and lysine tRNA mutations. MSL could be considered, even if rare, a red flag for mitochondrial disorders, even in patients with an apparently isolated MSL.
PubMed: 30873109
DOI: 10.3389/fneur.2019.00160 -
Ophthalmic Genetics 2017Mitochondrial disease often manifests with ophthalmologic signs and symptoms. Due to the important role of mitochondria in aerobic metabolism, the eyes are among the...
BACKGROUND
Mitochondrial disease often manifests with ophthalmologic signs and symptoms. Due to the important role of mitochondria in aerobic metabolism, the eyes are among the more preferentially involved organs. The clinical diagnosis of mitochondrial disease can be facilitated by an improved knowledge of the types and magnitude of their various manifestations. The aim of this study was to describe the ophthalmological manifestations of patients with mitochondrial diseases that are currently not well elucidated.
METHODS
From a database of patients with verified primary mitochondrial disease (n = 81) who had visited our institution we identified 74 patients who had ophthalmologic examinations. Demographic, clinical, and ophthalmologic data were collected. Institutional review board approval was obtained.
RESULTS
A total of 74 patients were identified with Leigh disease, MELAS, MERRF, CPEO, Pearson/Kearns-Sayre syndrome, as well as other mtDNA point mutations, deletions, depletions, and mutations. Overall, 26 of the 74 patients (35%) had one or more ophthalmological abnormalities. Retinal pigmentary changes were present in 12/74 (16%) of patients. Partial or total optic atrophy (OA) was noted in 8/74 (10%) of patients. Decreased extra-ocular movement (EOM) was noted in 6/74 (8%) of patients. Other ophthalmological findings included macular atrophy (2/74), macular dystrophy (1/74), and visual field defects (1/74).
CONCLUSIONS
Over one-third of our cohort of patients with mitochondrial disorders had ophthalmological manifestations, some of which affected vision significantly. Eye examinations are critical in patients with mitochondrial disease so that complete assessments of the effects of the underlying mutations are uncovered and the appropriate counseling and care are given.
Topics: Adult; DNA, Mitochondrial; Eye Diseases; Female; Humans; Kearns-Sayre Syndrome; Leigh Disease; MELAS Syndrome; MERRF Syndrome; Male; Mitochondrial Diseases; Ophthalmoplegia, Chronic Progressive External; Point Mutation; Retrospective Studies; Sequence Deletion
PubMed: 27029465
DOI: 10.3109/13816810.2015.1130153 -
Cell Death & Disease Apr 2018Degeneration or loss of inner ear hair cells (HCs) is irreversible and results in sensorineural hearing loss (SHL). Human-induced pluripotent stem cells (hiPSCs) have...
ATOH1/RFX1/RFX3 transcription factors facilitate the differentiation and characterisation of inner ear hair cell-like cells from patient-specific induced pluripotent stem cells harbouring A8344G mutation of mitochondrial DNA.
Degeneration or loss of inner ear hair cells (HCs) is irreversible and results in sensorineural hearing loss (SHL). Human-induced pluripotent stem cells (hiPSCs) have been employed in disease modelling and cell therapy. Here, we propose a transcription factor (TF)-driven approach using ATOH1 and regulatory factor of x-box (RFX) genes to generate HC-like cells from hiPSCs. Our results suggest that ATOH1/RFX1/RFX3 could significantly increase the differentiation capacity of iPSCs into MYO7A-positive cells, upregulate the mRNA expression levels of HC-related genes and promote the differentiation of HCs with more mature stereociliary bundles. To model the molecular and stereociliary structural changes involved in HC dysfunction in SHL, we further used ATOH1/RFX1/RFX3 to differentiate HC-like cells from the iPSCs from patients with myoclonus epilepsy associated with ragged-red fibres (MERRF) syndrome, which is caused by A8344G mutation of mitochondrial DNA (mtDNA), and characterised by myoclonus epilepsy, ataxia and SHL. Compared with isogenic iPSCs, MERRF-iPSCs possessed ~42-44% mtDNA with A8344G mutation and exhibited significantly elevated reactive oxygen species (ROS) production and CAT gene expression. Furthermore, MERRF-iPSC-differentiated HC-like cells exhibited significantly elevated ROS levels and MnSOD and CAT gene expression. These MERRF-HCs that had more single cilia with a shorter length could be observed only by using a non-TF method, but those with fewer stereociliary bundle-like protrusions than isogenic iPSCs-differentiated-HC-like cells could be further observed using ATOH1/RFX1/RFX3 TFs. We further analysed and compared the whole transcriptome of M1-HCs and M1-HCs after treatment with ATOH1 or ATOH1/RFX1/RFX3. We revealed that the HC-related gene transcripts in M1-iPSCs had a significantly higher tendency to be activated by ATOH1/RFX1/RFX3 than M1-iPSCs. The ATOH1/RFX1/RFX3 TF-driven approach for the differentiation of HC-like cells from iPSCs is an efficient and promising strategy for the disease modelling of SHL and can be employed in future therapeutic strategies to treat SHL patients.
Topics: Adolescent; Basic Helix-Loop-Helix Transcription Factors; Catalase; Cell Differentiation; Cilia; DNA, Mitochondrial; Embryoid Bodies; Female; Hair Cells, Auditory, Inner; Humans; Induced Pluripotent Stem Cells; MERRF Syndrome; Myosin VIIa; Point Mutation; Reactive Oxygen Species; Regulatory Factor X Transcription Factors; Regulatory Factor X1; Superoxide Dismutase
PubMed: 29740017
DOI: 10.1038/s41419-018-0488-y -
Mitochondrial DNA. Part A, DNA Mapping,... 2016Vietnamese patients (106) tentatively diagnosed with encephalomyopathy were screened for the presence of 15 common point mutations in mitochondria using PCR-RFLP. The...
Vietnamese patients (106) tentatively diagnosed with encephalomyopathy were screened for the presence of 15 common point mutations in mitochondria using PCR-RFLP. The screened mutations include A3243G, T3271C and T3291C for Mitochondrial Encephalopathy, Lactic Acidosis and Stroke-like episodes (MELAS); A8344G and T8356C for Myoclonus Epilepsy and Rag-Red Fibers (MERRF); G11778A, G3460A and T14484C for Leber's Hereditary Optic Neuropathy (LHON); T8993G/C and T9176G for Leigh syndrome; A1555G for deafness syndrome; G4298A, T10010C, T14728C and T14709C for neuromuscular syndrome. As a result, 6 cases of A3243G (5.7%) and 2 cases of T14727C (3.9%) were found. The 6 cases of A3243G mutation were heteroplasmic at different levels (4.23-80.85%). The T14727C change was discovered for the first time in the MTTE gene encoding for tRNA(Glu) and showed homoplasmy. The T14727C change was probably a mutation because it was further confirmed as vertically inherited from the mother and not the result of isolated polymorphism.
Topics: Asian People; DNA, Mitochondrial; Female; Genome, Mitochondrial; Humans; Leigh Disease; Male; Mitochondrial Encephalomyopathies; Point Mutation; Polymorphism, Genetic
PubMed: 24708131
DOI: 10.3109/19401736.2014.900665 -
Child Neurology Open 2021In the field of mitochondrial medicine, correlation of clinical phenotype with mutation heteroplasmy remains an outstanding question with few, if any, clear thresholds...
In the field of mitochondrial medicine, correlation of clinical phenotype with mutation heteroplasmy remains an outstanding question with few, if any, clear thresholds corresponding to a given phenotype. The m.8344A>G mutation is most commonly associated with myoclonus epilepsy and ragged red fiber syndrome (MERRF) at varying levels of heteroplasmy. However, a handful of cases been previously reported in which individuals homoplasmic or nearly homoplasmic for this mutation in the blood have presented with multiple bulbar palsies, respiratory failure, and progressive neurologic decline almost uniformly following a respiratory illness. MRI brain in all affected individuals revealed symmetric T2 hyperintense lesions of subcortical gray matter structures, consistent with Leigh syndrome. Here, we present 3 cases with clinical, biochemical, and neuro-imaging findings with the additional reporting of spinal lesions. This new phenotype supports a heteroplasmy-dependent phenotype model for this mutation and recognition of this can help clinicians with diagnosis and anticipatory clinical guidance.
PubMed: 33718511
DOI: 10.1177/2329048X21991382 -
Journal of Neurology Mar 2015The objective of the study was to better characterize the clinical phenotype associated with the A8344G "MERRF" mutation of mitochondrial DNA. Fifteen mutated patients...
The objective of the study was to better characterize the clinical phenotype associated with the A8344G "MERRF" mutation of mitochondrial DNA. Fifteen mutated patients were extensively investigated. The frequency of main clinical features was: exercise intolerance and/or muscle weakness 67 %, respiratory involvement 67 %, lactic acidosis 67 %, cardiac abnormalities 53 %, peripheral neuropathy 47 %, myoclonus 40 %, epilepsy 40 %, ataxia 13 %. A restrictive respiratory insufficiency requiring ventilatory support was observed in about half of our patients. One patient developed a severe and rapidly progressive cardiomyopathy requiring cardioverter-defibrillator implantation. Five patients died of overwhelming, intractable lactic acidosis. Serial muscle MRIs identified a consistent pattern of muscle involvement and progression. Cardiac MRI showed non-ischemic late gadolinium enhancement in the left ventricle inferolateral part as early sign of myocardial involvement. Brain spectroscopy demonstrated increased peak of choline and reduction of N-acetylaspartate. Lactate was never detected in brain areas, while it could be documented in ventricles. We confirm that muscle involvement is the most frequent clinical feature associated with A8443G mutation. In contrast with previous reports, however, about half of our patients did not develop signs of CNS involvement even in later stages of the disease. The difference may be related to the infrequent investigation of A8344G mutation in 'pure' mitochondrial myo-cardiomyopathy, representing a bias and a possible cause of syndrome's underestimation. Our study highlights the importance of lactic acidosis and respiratory muscle insufficiency as critical prognostic factors. Muscle and cardiac MRI and brain spectroscopy may be useful tools in diagnosis and follow-up of MERRF.
Topics: Adolescent; Adult; Aged; Aspartic Acid; Brain; Cardiomyopathies; DNA, Mitochondrial; Female; Humans; MERRF Syndrome; Magnetic Resonance Imaging; Male; Middle Aged; Muscle, Skeletal; Mutation; Spectrum Analysis
PubMed: 25559684
DOI: 10.1007/s00415-014-7632-0 -
Stem Cell Research Jul 2018MERRF syndrome is predominantly caused by A8344G mutation in the mitochondrial DNA (mtDNA), affecting MT-TK gene, which impairs the mitochondrial electron transport...
MERRF syndrome is predominantly caused by A8344G mutation in the mitochondrial DNA (mtDNA), affecting MT-TK gene, which impairs the mitochondrial electron transport chain function. Here, we report the generation of two isogenic induced pluripotent stem cell (iPSC) lines, TVGH-iPSC-MRF-M and TVGH-iPSC-MRF-M, from the skin fibroblasts of a female MERRF patient harboring mtDNA A8344G mutation by using retrovirus transduction system. Both cell lines share the same genetic background except containing different proportions of mtDNA with the A8344G mutation. Both cell lines exhibited the pluripotency and capacity to differentiate into three germ layers.
Topics: Adolescent; Animals; DNA, Mitochondrial; Female; Humans; Induced Pluripotent Stem Cells; MERRF Syndrome; Mice; Mutation
PubMed: 29960149
DOI: 10.1016/j.scr.2018.05.011 -
European Journal of Neurology Jun 2016Peripheral neuropathy in mitochondrial diseases (MDs) may vary from a subclinical finding in a multisystem syndrome to a severe, even isolated, manifestation in some...
BACKGROUND AND PURPOSE
Peripheral neuropathy in mitochondrial diseases (MDs) may vary from a subclinical finding in a multisystem syndrome to a severe, even isolated, manifestation in some patients.
METHODS
To investigate the involvement of the peripheral nervous system in MDs extensive electrophysiological studies were performed in 109 patients with morphological, biochemical and genetic diagnosis of MD [12 A3243G progressive external ophthalmoplegia (PEO)/mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), 16 myoclonic epilepsy with ragged-red fibres (MERRF), four mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), 67 PEO with single or multiple deletions of mitochondrial DNA, 10 others].
RESULTS
A neuropathy was found in 49 patients (45%). The incidence was very high in MNGIE (100%), MELAS (92%) and MERRF (69%), whilst 28% of PEO patients had evidence of peripheral involvement. The most frequent abnormality was a sensory axonal neuropathy found in 32/49 patients (65%). A sensory-motor axonal neuropathy was instead detected in 16% of the patients and sensory-motor axonal demyelinating neuropathy in 16%. Finally one Leigh patient had a motor axonal neuropathy. It is interesting to note that the great majority had preserved tendon reflexes and no sensory disturbances.
CONCLUSIONS
In conclusion, peripheral involvement in MD is frequent even if often mild or asymptomatic. The correct identification and characterization of peripheral neuropathy through electrophysiological studies represents another tile in the challenge of MD diagnosis.
Topics: Adolescent; Adult; Aged; Aged, 80 and over; Axons; Child; Female; Humans; Male; Median Nerve; Middle Aged; Mitochondria; Mitochondrial Diseases; Peripheral Nervous System Diseases; Syndrome; Young Adult
PubMed: 26822221
DOI: 10.1111/ene.12954