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1.
Eur J Clin Invest ; 51(9): e13574, 2021 Sep.
Article in English | MEDLINE | ID: mdl-33937992

ABSTRACT

BACKGROUND: Freezing human biopsies is common in clinical practice for storage. However, this technique disrupts mitochondrial membranes, hampering further analyses of respiratory function. To contribute to laboratorial diagnosis of mitochondrial diseases, this study sought to develop a respirometry approach using O2k (Oroboros Ins.) to measure the whole electron transport chain (ETC) activity in homogenates of frozen skeletal muscle biopsies. PATIENTS AND METHODS: We enrolled 16 patients submitted to muscle biopsy in the process of routine diagnostic investigation: four with mitochondrial disease and severe mitochondrial dysfunction; seven with exercise intolerance and multiple deletions of mitochondrial DNA, presenting mild to moderate mitochondrial dysfunction; five without mitochondrial disease, as controls. Whole homogenates of muscle fragments were prepared using grinder-type equipment. O2 consumption rates were normalized using citrate synthase activity. RESULTS: Transmission electron microscopy confirmed mitochondrial membrane discontinuation, indicating increased permeability of mitochondrial membranes in homogenates from frozen biopsies. O2 consumption rates in the presence of acetyl-CoA lead to maximum respiratory rates sensitive to rotenone, malonate and antimycin. This protocol of acetyl-CoA-driven respiration (ACoAR), applied in whole homogenates of frozen muscle, was sensitive enough to identify ETC abnormality, even in patients with mild to moderate mitochondrial dysfunction. We demonstrated adequate repeatability of ACoAR and found significant correlation between O2 consumption rates and enzyme activity assays of individual ETC complexes. CONCLUSIONS: We present preliminary data on a simple, low cost and reliable procedure to measure respiratory function in whole homogenates of frozen skeletal muscle biopsies, contributing to diagnosis of mitochondrial diseases in humans.


Subject(s)
Acetyl Coenzyme A/metabolism , Mitochondria, Muscle/metabolism , Mitochondrial Diseases/diagnosis , Muscle, Skeletal/metabolism , Oxygen Consumption , Adolescent , Adult , Biopsy , Cell Respiration , Child , Clinical Laboratory Techniques/methods , Cryopreservation , Electron Transport , Female , Humans , MELAS Syndrome/diagnosis , MELAS Syndrome/metabolism , Male , Membrane Potential, Mitochondrial , Mitochondrial Diseases/metabolism , Mitochondrial Membranes/metabolism , Muscle, Skeletal/pathology , Ophthalmoplegia, Chronic Progressive External/diagnosis , Ophthalmoplegia, Chronic Progressive External/metabolism , Oxidative Phosphorylation , Permeability , Specimen Handling , Young Adult
2.
Genes (Basel) ; 12(1)2020 12 31.
Article in English | MEDLINE | ID: mdl-33396418

ABSTRACT

Mitochondrial encephalomyopathies comprise a group of heterogeneous disorders resulting from impaired oxidative phosphorylation (OxPhos). Among a variety of symptoms progressive external ophthalmoplegia (PEO) seems to be the most common. The aim of this study is to present clinical and genetic characteristics of Polish patients with PEO. Clinical, electrophysiological, neuroradiological, and morphological data of 84 patients were analyzed. Genetic studies of mitochondrial DNA (mtDNA) were performed in all patients. Among nuclear DNA (nDNA) genes POLG was sequenced in 41 patients, TWNK (C10orf2) in 13 patients, and RNASEH1 in 2 patients. Total of 27 patients were included in the chronic progressive external ophthalmoplegia (CPEO) group, 24 in the CPEO+ group. Twenty-six patients had mitochondrial encephalomyopathy (ME), six patients Kearns-Sayre syndrome (KSS), and one patient sensory ataxic neuropathy, dysarthria, ophthalmoparesis (SANDO) syndrome. Genetic analysis of nDNA genes revealed the presence of pathogenic or possibly pathogenic variants in the POLG gene in nine patients, the TWNK gene in five patients and the RNASEH1 gene in two patients. Detailed patients' history and careful assessment of family history are essential in the diagnostic work-up. Genetic studies of both mtDNA and nDNA are necessary for the final diagnosis of progressive external ophthalmoplegia and for genetic counseling.


Subject(s)
DNA Helicases/genetics , DNA Polymerase gamma/genetics , Kearns-Sayre Syndrome/genetics , Mitochondrial Diseases/genetics , Mitochondrial Encephalomyopathies/genetics , Mitochondrial Proteins/genetics , Ophthalmoplegia, Chronic Progressive External/genetics , Ribonuclease H/genetics , Adolescent , Adult , Aged , Cerebellum/diagnostic imaging , Cerebellum/metabolism , Cerebellum/pathology , Cerebrum/diagnostic imaging , Cerebrum/metabolism , Cerebrum/pathology , Child , DNA Helicases/metabolism , DNA Polymerase gamma/metabolism , DNA, Mitochondrial/genetics , DNA, Mitochondrial/metabolism , Diagnosis, Differential , Female , Gene Expression , Humans , Kearns-Sayre Syndrome/diagnostic imaging , Kearns-Sayre Syndrome/metabolism , Kearns-Sayre Syndrome/pathology , Male , Middle Aged , Mitochondria/metabolism , Mitochondria/pathology , Mitochondrial Diseases/diagnostic imaging , Mitochondrial Diseases/metabolism , Mitochondrial Diseases/pathology , Mitochondrial Encephalomyopathies/diagnostic imaging , Mitochondrial Encephalomyopathies/metabolism , Mitochondrial Encephalomyopathies/pathology , Mitochondrial Proteins/metabolism , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Ophthalmoplegia, Chronic Progressive External/diagnostic imaging , Ophthalmoplegia, Chronic Progressive External/metabolism , Ophthalmoplegia, Chronic Progressive External/pathology , Pedigree , Poland , Polymorphism, Genetic , Ribonuclease H/metabolism , Sequence Deletion
3.
Neuromuscul Disord ; 29(5): 358-367, 2019 05.
Article in English | MEDLINE | ID: mdl-30962064

ABSTRACT

Chronic progressive external ophthalmoplegia (CPEO) is a frequent clinical manifestation of disorders caused by pathogenic mitochondrial DNA mutations. However, for diagnostic purposes skeletal muscle tissue is used, since extraocular muscle tissue is usually not available for work-up. In the present study we aimed to identify causative factors that are responsible for extraocular muscle to be primarily affected in CPEO. We performed comparative histochemical and molecular genetic analyses of extraocular muscle and skeletal muscle single fibers in a case of isolated CPEO caused by the heteroplasmic m.5667G>A mutation in the mitochondrial tRNAAsn gene (MT-TN). Histochemical analyses revealed higher proportion of cytochrome c oxidase deficient fibers in extraocular muscle (41%) compared to skeletal muscle (10%). However, genetic analyses of single fibers revealed no significant difference either in the mutation loads between extraocular muscle and skeletal muscle cytochrome c oxidase deficient single fibers (extraocular muscle 86% ±â€¯4.6%; skeletal muscle 87.8 %±â€¯5.7%, p = 0.246) nor in the mutation threshold (extraocular muscle 74% ±â€¯3%; skeletal muscle 74% ±â€¯4%). We hypothesize that higher proportion of cytochrome c oxidase deficient fibers in extraocular muscle compared to skeletal muscle might be due to facilitated segregation of the m.5667G>A mutation into extraocular muscle, which may explain the preferential ocular manifestation and clinically isolated CPEO.


Subject(s)
Electron Transport Complex IV/metabolism , Muscle Fibers, Skeletal/metabolism , Oculomotor Muscles/metabolism , Ophthalmoplegia, Chronic Progressive External/genetics , Quadriceps Muscle/metabolism , RNA, Transfer, Asn/genetics , Adolescent , Cytochrome-c Oxidase Deficiency , Electron Transport Chain Complex Proteins/metabolism , Humans , Immunohistochemistry , Male , Muscle Fibers, Skeletal/pathology , Muscle, Skeletal , Oculomotor Muscles/pathology , Ophthalmoplegia, Chronic Progressive External/metabolism , Ophthalmoplegia, Chronic Progressive External/pathology , Quadriceps Muscle/pathology
5.
Free Radic Biol Med ; 126: 235-248, 2018 10.
Article in English | MEDLINE | ID: mdl-30138712

ABSTRACT

A major challenge in mitochondrial diseases (MDs) is the identification of biomarkers that could inform of the mechanisms involved in the phenotypic expression of genetic defects. Herein, we have investigated the protein signature of metabolism and of the antioxidant response in muscle biopsies of clinically and genetically diagnosed patients with Progressive External Ophthalmoplegia due to single large-scale (PEO-sD) or multiple (PEO-mD) deletions of mtDNA and Mitochondrial Encephalopathy Lactic Acidosis and Stroke-like episode (MELAS) syndrome, and healthy donors. A high-throughput immunoassay technique that quantitates the expression of relevant proteins of glycolysis, glycogenolysis, pentose phosphate pathway, oxidative phosphorylation, pyruvate and fatty acid oxidation, tricarboxylic acid cycle and the antioxidant response in two large independent and retrospectively collected cohorts of PEO-sD, PEO-mD and MELAS patients revealed that despite the heterogeneity of the genetic alterations, the three MDs showed the same metabolic signatures in both cohorts of patients, which were highly divergent from those of healthy individuals. Linear Discriminant Analysis and Support Vector Machine classifier provided a minimum of four biomarkers to discriminate healthy from pathological samples. Regardless of the induction of a large number of enzymes involved in ameliorating oxidative stress, the down-regulation of mitochondrial superoxide dismutase (SOD2) and catalase expression favored the accumulation of oxidative damage in patients' proteins. Down-regulation of SOD2 and catalase expression in MD patients is not due to relevant changes in the availability of their mRNAs, suggesting that oxidative stress regulates the expression of the two enzymes post-transcriptionally. We suggest that SOD2 and catalase could provide specific targets to improve the detoxification of reactive oxygen species that affects muscle proteins in these patients.


Subject(s)
DNA, Mitochondrial/genetics , MELAS Syndrome/metabolism , Mitochondrial Diseases/metabolism , Ophthalmoplegia, Chronic Progressive External/metabolism , Adolescent , Adult , Aged , Antioxidants/metabolism , Biomarkers/metabolism , Biopsy , Child , Child, Preschool , Gene Expression Regulation , Glycolysis , Healthy Volunteers , Humans , MELAS Syndrome/genetics , MELAS Syndrome/pathology , Middle Aged , Mitochondrial Diseases/genetics , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Ophthalmoplegia, Chronic Progressive External/genetics , Ophthalmoplegia, Chronic Progressive External/pathology , Oxidative Stress , Reactive Oxygen Species , Superoxide Dismutase/genetics , Support Vector Machine , Young Adult
6.
Mol Psychiatry ; 23(10): 2039-2049, 2018 10.
Article in English | MEDLINE | ID: mdl-29892051

ABSTRACT

Although mitochondrial and serotonergic dysfunctions have been implicated in the etiology of bipolar disorder (BD), the relationship between these unrelated pathways has not been elucidated. A family of BD and chronic progressive external ophthalmoplegia (CPEO) caused by a mutation of the mitochondrial adenine nucleotide translocator 1 (ANT1, SLC25A4) implicated that ANT1 mutations confer a risk of BD. Here, we sequenced ANT1 in 324 probands of NIMH bipolar disorder pedigrees and identified two BD patients carrying heterozygous loss-of-function mutations. Behavioral analysis of brain specific Ant1 heterozygous conditional knockout (cKO) mice using lntelliCage showed a selective diminution in delay discounting. Delay discounting is the choice of smaller but immediate reward than larger but delayed reward and an index of impulsivity. Diminution of delay discounting suggests an increase in serotonergic activity. This finding was replicated by a 5-choice serial reaction time test. An anatomical screen showed accumulation of COX (cytochrome c oxidase) negative cells in dorsal raphe. Dorsal raphe neurons in the heterozygous cKO showed hyperexcitability, along with enhanced serotonin turnover in the nucleus accumbens and upregulation of Maob in dorsal raphe. These findings altogether suggest that mitochondrial dysfunction as the genetic risk of BD may cause vulnerability to BD by altering serotonergic neurotransmission.


Subject(s)
Adenine Nucleotide Translocator 1/genetics , Adenine Nucleotide Translocator 1/metabolism , Bipolar Disorder/genetics , Animals , Bipolar Disorder/metabolism , Delay Discounting/physiology , Dorsal Raphe Nucleus/metabolism , Female , Humans , Impulsive Behavior , Male , Mice , Mice, Knockout , Mitochondria/genetics , Mitochondria/metabolism , Ophthalmoplegia, Chronic Progressive External/metabolism , Reward , Serotonergic Neurons/metabolism , Serotonergic Neurons/physiology
7.
Neuromuscul Disord ; 28(5): 408-413, 2018 05.
Article in English | MEDLINE | ID: mdl-29567350

ABSTRACT

Muscle dysfunction in mitochondrial myopathy is predominantly caused by insufficient generation of energy. We hypothesise that structural changes in muscles could also contribute to their pathophysiology. The aims of this study were to determine fat fractions and strength in selected muscles in patients with chronic progressive external ophthalmoplegia (CPEO), and compare progression of muscle fat fraction with age in individuals with CPEO vs. healthy controls and patients with the m.3243A>G mutation of mitochondrial DNA (mtDNA). Seventeen patients with CPEO and single large-scale deletions of mtDNA, 52 healthy controls, and 12 patients carrying the m.3243A>G mtDNA mutation were included. Muscle fat fractions were measured from cross-sections of paraspinal and leg muscles. Peak muscle strength was assessed from a static dynamometer. There was a direct correlation between age and fat fraction in all muscle groups in CPEO patients and healthy controls (p < 0.05). Analysis of covariance showed a higher progression rate of fat replacement in CPEO patients vs. healthy controls in studied muscle groups (p < 0.05). Patients with the m.3243A>G mutation had slower progression rates of fat replacement. Muscle strength decreased with increasing muscular fat fraction in CPEO patients, no correlation was seen in other groups. This indicates that structural muscle changes contribute to the phenotype of older patients affected by CPEO and large-scale deletions. It should therefore be considered, along with known energy deficiencies, as the cause of exercise intolerance.


Subject(s)
Adipose Tissue/pathology , DNA, Mitochondrial/genetics , Muscle, Skeletal/pathology , Ophthalmoplegia, Chronic Progressive External/genetics , Adipose Tissue/diagnostic imaging , Adipose Tissue/metabolism , Adult , DNA, Mitochondrial/metabolism , Female , Humans , Magnetic Resonance Imaging , Male , Middle Aged , Muscle Strength/physiology , Muscle, Skeletal/diagnostic imaging , Muscle, Skeletal/metabolism , Ophthalmoplegia, Chronic Progressive External/diagnostic imaging , Ophthalmoplegia, Chronic Progressive External/metabolism , Ophthalmoplegia, Chronic Progressive External/pathology , Sequence Deletion
8.
CNS Neurosci Ther ; 23(8): 686-697, 2017 Aug.
Article in English | MEDLINE | ID: mdl-28695670

ABSTRACT

AIMS: Accumulated evidence indicates that cerebral metabolic features, evaluated by proton magnetic resonance spectroscopy (1 H-MRS), are sensitive to early mitochondrion dysfunction associated with mitochondrial encephalomyopathy (ME). The metabolite ratios of lactate (lac)/Cr, N-acetyl aspartate (NAA)/creatine (Cr), total choline (tCho)/Cr, and myoinositol (mI)/Cr are measured in the infarct-like lesions by 1 H-MRS and may reveal metabolic changes associated with ME. However, the application of this molecular imaging technique in the investigation of the pathology of ME subtypes is unknown. METHODS: In this study, cerebral metabolic features of pathologically diagnosed ME cases, that is, 19 mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS); nine chronic progressive external ophthalmoplegia (CPEO); and 23 healthy controls, were investigated using 1 H-MRS. Receiver operating characteristics (ROC) analysis was used to evaluate the diagnostic power of the cerebral metabolites. Histochemical evaluation was carried out on muscle tissues derived from biopsy to assess the abnormal mitochondrial proliferation. The association between cerebral metabolic and mitochondrial cytopathy was examined by correlation analysis. RESULTS: Patients with MELAS or CPEO exhibited a significantly higher Lac/Cr ratio and a lower NAA/Cr ratio compared with controls. The ROC curve of Lac/Cr ratio indicated prominent discrimination between MELAS or CPEO and healthy control subjects, whereas the NAA/Cr ratio may present diagnostic power in the distinction of MELAS from CPEO. Lower NAA/Cr ratio was associated with higher Lac/Cr in MELAS, but not in CPEO. Furthermore, higher ragged-red fibers (RRFs) percentages were associated with elevated Lac/Cr and reduced NAA/Cr ratios, notably in MELAS. This association was not noted in the case of mI/Cr ratio. CONCLUSIONS: Mitochondrial cytopathy (lactic acidosis and RRFs on muscle biopsy) was associated with neuronal viability but not glial proliferation, notably in MELAS. Mitochondrial neuronopathy and neuronal vulnerability are considered significant causes in the pathogenesis of MELAS, particularly with regard to stroke-like episodes.


Subject(s)
Brain/metabolism , MELAS Syndrome/metabolism , Mitochondria/metabolism , Ophthalmoplegia, Chronic Progressive External/metabolism , Adult , Brain/diagnostic imaging , Cohort Studies , Female , Humans , MELAS Syndrome/diagnostic imaging , MELAS Syndrome/pathology , Male , Mitochondria/pathology , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Ophthalmoplegia, Chronic Progressive External/diagnostic imaging , Ophthalmoplegia, Chronic Progressive External/pathology , Proton Magnetic Resonance Spectroscopy , ROC Curve
9.
Mol Psychiatry ; 21(1): 39-48, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26481320

ABSTRACT

Depression is a common debilitating human disease whose etiology has defied decades of research. A critical bottleneck is the difficulty in modeling depressive episodes in animals. Here, we show that a transgenic mouse with chronic forebrain expression of a dominant negative mutant of Polg1, a mitochondrial DNA (mtDNA) polymerase, exhibits lethargic behavioral changes, which are associated with emotional, vegetative and psychomotor disturbances, and response to antidepression drug treatment. The results suggested a symptomatic similarity between the lethargic behavioral change that was recurrently and spontaneously experienced by the mutant mice and major depressive episode as defined by DSM-5. A comprehensive screen of mutant brain revealed a hotspot for mtDNA deletions and mitochondrial dysfunction in the paraventricular thalamic nucleus (PVT) with similar defects observed in postmortem brains of patients with mitochondrial disease with mood symptoms. Remarkably, the genetic inhibition of PVT synaptic output by Cre-loxP-dependent expression of tetanus toxin triggered de novo depression-like episodes. These findings identify a novel preclinical mouse model and brain area for major depressive episodes with mitochondrial dysfunction as its cellular mechanism.


Subject(s)
DNA-Directed DNA Polymerase/metabolism , Depressive Disorder/physiopathology , Midline Thalamic Nuclei/metabolism , Animals , Comorbidity , Corticosterone/analysis , DNA Polymerase gamma , DNA-Directed DNA Polymerase/genetics , Depressive Disorder/complications , Depressive Disorder/genetics , Depressive Disorder/pathology , Disease Models, Animal , Feces/chemistry , Female , Humans , Immunohistochemistry , Male , Mice, Transgenic , Midline Thalamic Nuclei/pathology , Mitochondria/metabolism , Motor Activity/physiology , Mutation , Neurons/metabolism , Ophthalmoplegia, Chronic Progressive External/complications , Ophthalmoplegia, Chronic Progressive External/metabolism , Ophthalmoplegia, Chronic Progressive External/pathology
10.
J Pediatr Ophthalmol Strabismus ; 51 Online: e29-32, 2014 May 28.
Article in English | MEDLINE | ID: mdl-26900705

ABSTRACT

The authors report the case of a child with horizontal gaze palsy with progressive scoliosis and keratoconus. ROBO3 analysis identified compound heterozygous mutations. Keratoconus surgical approach resulted in visual acuity improvement in both eyes. The previously unreported occurrence of keratoconus with horizontal gaze palsy with progressive scoliosis suggests that ophthalmologic assessment should search for signs of this ectasia in these patients.


Subject(s)
Keratoconus/genetics , Mutation , Ocular Motility Disorders/genetics , Ophthalmoplegia, Chronic Progressive External/genetics , Receptors, Immunologic/genetics , Scoliosis/genetics , Child , DNA/genetics , Female , Humans , Keratoconus/metabolism , Male , Ocular Motility Disorders/metabolism , Ophthalmoplegia, Chronic Progressive External/metabolism , Pedigree , Protein Isoforms , Receptors, Cell Surface , Receptors, Immunologic/metabolism , Scoliosis/metabolism
11.
Hum Mol Genet ; 22(12): 2411-22, 2013 Jun 15.
Article in English | MEDLINE | ID: mdl-23446635

ABSTRACT

The POLG1 gene encodes the catalytic subunit of mitochondrial DNA (mtDNA) polymerase γ (POLγ). We here describe a sibling pair with adult-onset progressive external ophthalmoplegia, cognitive impairment and mitochondrial myopathy characterized by DNA depletion and multiple mtDNA deletions. The phenotype is due to compound heterozygous POLG1 mutations, T914P and the intron mutation c.3104 + 3A > T. The mutant genes produce POLγ isoforms with heterozygous phenotypes that fail to synthesize longer DNA products in vitro. However, exon skipping in the c.3104 + 3A > T mutant is not complete, and the presence of low levels of wild-type POLγ explains patient survival. To better understand the underlying pathogenic mechanisms, we characterized the effects of POLγ depletion in vitro and found that leading-strand DNA synthesis is relatively undisturbed. In contrast, initiation of lagging-strand DNA synthesis is ineffective at lower POLγ concentrations that uncouples leading strand from lagging-strand DNA synthesis. In vivo, this effect leads to prolonged exposure of the heavy strand in its single-stranded conformation that in turn can cause the mtDNA deletions observed in our patients. Our findings, thus, suggest a molecular mechanism explaining how POLγ mutations can cause mtDNA deletions in vivo.


Subject(s)
DNA Replication , DNA, Mitochondrial/genetics , DNA-Directed DNA Polymerase/metabolism , Ophthalmoplegia, Chronic Progressive External/enzymology , Ophthalmoplegia, Chronic Progressive External/genetics , Adult , Base Sequence , DNA Polymerase gamma , DNA, Mitochondrial/metabolism , DNA-Directed DNA Polymerase/genetics , Exons , Female , Genes, Dominant , Heterozygote , Humans , Introns , Male , Middle Aged , Molecular Sequence Data , Ophthalmoplegia, Chronic Progressive External/metabolism , Pedigree , Point Mutation , Sequence Deletion
12.
J Neurol Sci ; 320(1-2): 131-5, 2012 Sep 15.
Article in English | MEDLINE | ID: mdl-22743145

ABSTRACT

We identified a double mutation in a patient with chronic progressive external ophthalmoplegia, located in the tRNA(Ala) (m.5628T>C) and tRNA(Lys) (m.8348A>G) genes. Both mutations were previously described separately and considered pathogenic, however the same mutations were also reported as polymorphisms or phenotype modulator. We analyzed the proportion of each mutation in isolated muscle fibers by single fiber-polymerase chain reaction to investigate the contribution of each mutation to mitochondrial deficiency. Our findings demonstrated that the mutations were heteroplasmic in skeletal muscle and both mutations were present in all single muscle fibers. The proportions of the m.5628T>C mutation were not significantly different between normal and cytochrome-c-oxidase (COX) deficient fibers. However, a significant higher proportion of the m.8348A>G mutation was observed in COX deficient fibers. Homoplasmic m.8348A>G was only observed in COX negative fibers. In conclusion, we provide a piece of evidence toward the pathogenicity of the m.8348A>G mutation and suggest that m.5628T>C is probably a neutral polymorphism.


Subject(s)
Muscle Fibers, Skeletal/metabolism , Ophthalmoplegia, Chronic Progressive External/genetics , Point Mutation/genetics , RNA, Transfer, Ala/genetics , RNA, Transfer, Lys/genetics , Adult , Electron Transport Complex IV/metabolism , Female , Humans , Mitochondria/genetics , Mitochondria/metabolism , Ophthalmoplegia, Chronic Progressive External/metabolism
14.
J Clin Neurosci ; 18(4): 535-8, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21277779

ABSTRACT

Chronic progressive external opthalmoplegia (CPEO) is the most common phenotypic syndrome of the mitochondrial myopathies. Muscle biopsy, which provides important morphological clues for the diagnosis of mitochondrial disorders, is normal in approximately 25% of patients with CPEO, thus necessitating molecular genetic analysis for more accurate diagnosis. We aimed to study the utility of various histochemical stains in the diagnosis of CPEO on muscle biopsy and to correlate these results with genetic studies. Between May 2005 and November 2007 all 45 patients diagnosed with CPEO were included in the study (23 males; mean age at presentation, 35 years). Thirty-nine patients had CPEO only and six had CPEO plus; two had a positive family history but the remaining 39 patients had sporadic CPEO. Muscle biopsy samples were stained with hematoxylin and eosin, modified Gomori's trichrome stain, succinic dehydrogenase (SDH), cytochrome C oxidase (COX) and combined COX-SDH. Ragged red fibers were seen in 27 biopsies; seven showed characteristics of neurogenic atrophy only, and 11 were normal. The abnormal fibers were best identified on COX-SDH stain. A complete mitochondrial genome was amplified in muscle and blood samples of all patients. Mutations were found in transfer RNA, ribosomal RNA, ND, CYTB, COX I, II and III genes. Mitochondrial gene mutations were found in ten of the 11 patients with a normal muscle biopsy. The genetic mutations were classified according to their significance. The observed muscle biopsy findings were correlated with genetic mutations noted. Histological studies should be combined with genetic studies for the definitive diagnosis of CPEO syndrome.


Subject(s)
Muscle, Skeletal/pathology , Ophthalmoplegia, Chronic Progressive External/diagnosis , Ophthalmoplegia, Chronic Progressive External/genetics , Adolescent , Adult , Aged , Biopsy , DNA Mutational Analysis , Female , Humans , Immunohistochemistry , Male , Middle Aged , Muscle, Skeletal/metabolism , Mutation , Ophthalmoplegia, Chronic Progressive External/metabolism , Polymerase Chain Reaction , Staining and Labeling , Young Adult
15.
Mitochondrion ; 11(1): 228-33, 2011 Jan.
Article in English | MEDLINE | ID: mdl-20813205

ABSTRACT

We report a heteroplasmic novel mutation m.5636T>C in the mt-tRNA(Ala) in a patient with bilateral ptosis and ophthalmoparesis in whom a muscle biopsy showed cytochrome c oxdidase (COX) negative and ragged red fibers. Using laser capture microdissection we have isolated COX negative fibers and COX positive fibers from the muscle of the patient and determined that the mutation load was clearly increased in COX negative muscle fibers. Additionally, the mutated m.5636T nucleotide is conserved in all the mammal and non-mammal species analyzed and might be structurally relevant as it is located in a position involved in the formation of tertiary structure of canonical mitochondrial tRNAs.


Subject(s)
Genes, Mitochondrial , Mutation , Ophthalmoplegia, Chronic Progressive External/genetics , RNA, Transfer, Ala/genetics , Adult , Biopsy , DNA, Mitochondrial/genetics , Humans , Male , Microdissection/methods , Mitochondria/metabolism , Mitochondria/pathology , Muscle, Skeletal , Ophthalmoplegia, Chronic Progressive External/metabolism , Ophthalmoplegia, Chronic Progressive External/pathology
17.
Muscle Nerve ; 41(1): 92-9, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19705478

ABSTRACT

Autosomal dominant progressive external ophthalmoplegia (adPEO) is a mitochondrial disorder caused by mutations in nuclear genes. Here we report the clinical and genetic features of adPEO in a Chinese family. All patients had gradual onset of ptosis, with or without ophthalmoplegia, around age 30. Thirteen patients had limb weakness around age 40. Eight patients developed dysphagia around age 50. Four patients died of cardiac abnormalities around age 60. Muscle biopsy of the proband indicated mitochondrial myopathy characterized by ragged-red fibers, cytochrome c oxidase-negative fibers, and multiple deletions of mitochondrial DNA. A heterozygous missense mutation of c.1342A>G in the C10orf2 gene resulting in the p.448N>D mutation in the protein was found in the proband and four other affected family members. In summary, we identified an adPEO family with a novel C10orf2 gene mutation that manifested an age-dependent phenotype. It suggests that greater attention must be paid to cardiac abnormalities in the late stages of this disease.


Subject(s)
DNA Helicases/genetics , DNA/genetics , Muscle, Skeletal/metabolism , Mutation, Missense , Ophthalmoplegia, Chronic Progressive External/genetics , Adult , Aged , Biopsy , Blotting, Southern , DNA Helicases/metabolism , Female , Humans , Immunohistochemistry , Male , Middle Aged , Mitochondrial Proteins , Muscle, Skeletal/pathology , Ophthalmoplegia, Chronic Progressive External/metabolism , Ophthalmoplegia, Chronic Progressive External/pathology , Phenotype , Polymerase Chain Reaction
19.
Neuromuscul Disord ; 19(4): 297-9, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19285865

ABSTRACT

We sequenced all mitochondrial tRNA genes in a 61-year-old man with chronic progressive external ophthalmoplegia and mitochondrial myopathy but without mtDNA rearrangements, and identified a heteroplasmic m.3244G>A mutation in the tRNA(Leu(UUR)) gene. This mutation had been previously associated with the MELAS phenotype, but not described in any detail. The mutation load in muscle was 84% and COX-negative fibers harbored greater levels of mutant genomes than COX-positive fibers. The m.3244G>A mutation affects a highly conserved nucleotide in the dihydrouridine loop and has been associated with a wobble modification deficiency of the mutant tRNA.


Subject(s)
DNA, Mitochondrial/genetics , Genetic Predisposition to Disease/genetics , Mitochondrial Diseases/genetics , Mutation/genetics , Ophthalmoplegia, Chronic Progressive External/genetics , RNA, Transfer, Leu/genetics , Base Sequence , Conserved Sequence/genetics , DNA Mutational Analysis , Disease Progression , Humans , Male , Middle Aged , Mitochondrial Diseases/metabolism , Mitochondrial Diseases/physiopathology , Muscle Fibers, Skeletal/metabolism , Muscle Fibers, Skeletal/pathology , Nucleotides/genetics , Oculomotor Muscles/metabolism , Oculomotor Muscles/physiopathology , Ophthalmoplegia, Chronic Progressive External/metabolism , Ophthalmoplegia, Chronic Progressive External/physiopathology , Quadriceps Muscle/metabolism , Quadriceps Muscle/pathology
20.
Hum Mol Genet ; 18(2): 328-40, 2009 Jan 15.
Article in English | MEDLINE | ID: mdl-18971204

ABSTRACT

Mutations in the mitochondrial helicase Twinkle underlie autosomal dominant progressive external ophthalmoplegia (PEO), as well as recessively inherited infantile-onset spinocerebellar ataxia and rare forms of mitochondrial DNA (mtDNA) depletion syndrome. Familial PEO is typically associated with the occurrence of multiple mtDNA deletions, but the mechanism by which Twinkle dysfunction induces deletion formation has been under debate. Here we looked at the effects of Twinkle adPEO mutations in human cell culture and studied the mtDNA replication in the Deletor mouse model, which expresses a dominant PEO mutation in Twinkle and accumulates multiple mtDNA deletions during life. We show that expression of dominant Twinkle mutations results in the accumulation of mtDNA replication intermediates in cell culture. This indicated severe replication pausing or stalling and caused mtDNA depletion. A strongly enhanced accumulation of replication intermediates was evident also in six-week-old Deletor mice compared with wild-type littermates, even though mtDNA deletions accumulate in a late-onset fashion in this model. In addition, our results in cell culture pointed to a problem of transcription that preceded the mtDNA depletion phenotype and might be of relevance in adPEO pathophysiology. Finally, in vitro assays showed functional defects in the various Twinkle mutants and broadly agreed with the cell culture phenotypes such as the level of mtDNA depletion and the level of accumulation of replication intermediates. On the basis of our results we suggest that mtDNA replication pausing or stalling is the common consequence of Twinkle PEO mutations that predisposes to multiple deletion formation.


Subject(s)
DNA Helicases/genetics , DNA Replication , DNA, Mitochondrial/genetics , Mutation , Ophthalmoplegia, Chronic Progressive External/genetics , Animals , Cell Line , DNA Helicases/metabolism , DNA, Mitochondrial/metabolism , Genes, Dominant , Humans , Mice , Mice, Transgenic , Mitochondrial Proteins , Ophthalmoplegia, Chronic Progressive External/metabolism
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