Mitochondrial DNA depletion in Leigh syndrome

Leigh syndrome is a heterogenous neurologic disease characterized by seizures, developmental delay, muscle weakness, respiratory abnormalities, optic abnormalities, including atrophy and ophthalmoplegia, and progressive cranial nerve degeneration with early onset in infants and children. Diagnosis can be confirmed by characteristic pathologic findings of necrosis in the basal ganglia, thalamus, and brainstem. Severe dysfunction of mitochondrial energy metabolism is generally present and involved in the etiology of this degenerative central nervous system disease. At the molecular level, a number of point mutations have been located in mitochondrial DNA genes, including ATPase6 and tRNA(Lys) genes, and in nuclear genes encoding subunits of oxidative enzymes, such as pyruvate dehydrogenase. Biochemically these mutations are responsible for enzymatic defects in either respiratory complexes (I, IV, or V) or pyruvate dehydrogenase. We describe here the first case of Leigh syndrome with marked depletion of mitochondrial DNA levels in skeletal muscle and abnormal activities in skeletal muscle of mitochondrial respiratory complexes I, III, IV, and V.     

Mitochondrial DNA depletion in Alpers syndrome

Mitochondrial dysfunction of the energy generating system was suggested in two infants with progressive infantile poliodystrophy characterised by hypotonia, refractory epilepsy, visual impairment, psychomotor retardation, profound brain atrophy, hepatopathy, and increased levels of lactate in blood and cerebrospinal fluid. Histochemical and electron microscopic analyses of liver biopsies revealed cytochrome c oxidase deficiency, microvesicular steatosis, and enormous multiplication of mitochondria of various sizes. In the first patient, the quantitative Southern blot analyses in tissues obtained at autopsy demonstrated reduced content of mtDNA in the liver, brain, and fibroblasts (11 %, 15 %, and 25 % of the mean values in controls) while a normal content of mtDNA was found in muscle and heart. In the second patient, a reduced content of mtDNA was found in the muscle, liver, and brain (15 %, 10 %, and 30 %, respectively, of the mean values in controls). Biochemical studies in the first patient revealed decreased activities of all respiratory chain complexes except complex II in isolated liver mitochondria and decreased amounts of respiratory chain complexes I, III, IV and ATP synthase in liver and frontal cortex, but not in muscle, heart, and fibroblasts. In conclusions, mtDNA depletion associated with Alpers syndrome may be tissue specific.     

Mitochondrial DNA depletion in sporadic inclusion body myositis

Sporadic inclusion body myositis (sIBM) is a late onset disorder of unkown aetiology. Mitochondrial changes such as cytochrome oxidase deficient fibres are a well recognised feature and mitochondrial DNA (mtDNA) deletions have also been reported, but not consistently. Since mtDNA deletions are not present in all cases, we investigated whether other types of mtDNA abnormality were responsible for the mitochondrial changes. We studied 9 patients with sIBM. To control for fibre loss or replacement with inflammatory cells, we compared sIBM patients with necrotising myopathy (n?=?4) as well as with healthy controls. Qualitative anlysis for mtDNA deletions and quantitative measurement of mtDNA copy number showed that muscle from patients with sIBM contained on average 67% less mtDNA than healthy controls (P?=?0.001). The level of mtDNA was also significantly depleted in sIBM when compared to necrotising myopathy. No significant difference in copy number was seen in patients with necrotising myopathy compared to controls. Deletions of mtDNA were present in 4 patients with sIBM, but not all. Our findings suggest that mtDNA depletion is a more consistent finding in sIBM, and one that may be implicated in the pathogenesis of the disease.     

Mitochondrial DNA depletion and dGK gene mutations

Mitochondrial DNA depletion syndrome is a clinically heterogeneous group of disorders characterized by a reduction in mitochondrial DNA copy number. The recent discovery of mutations in the deoxyguanosine kinase (dGK) gene in patients with the hepatocerebral form of mitochondrial DNA depletion syndrome prompted us to screen 21 patients to determine the frequency of dGK mutations, further characterize the clinical spectrum, and correlate genotypes with phenotypes. We detected mutations in three patients (14%). One patient had a homozygous GATT duplication (nucleotides 763-766), and another had a homozygous GT deletion (nucleotides 609-610); both mutations lead to truncated proteins. The third patient was a compound heterozygote for two missense mutations (R142K and E227K) that affect critical residues of the protein. These mutations were associated with variable phenotypes, and their low frequencies suggests that dGK is not the only gene responsible for mitochondrial DNA depletion in liver. The patient with the missense mutations had isolated liver failure and responded well to liver transplantation, which may be a therapeutic option in selected cases.     

Mitochondrial DNA depletion analysis by pseudogene ratioing

The mitochondrial DNA (mtDNA) depletion status of ρ⁰ cell lines is typically assessed by hybridization or polymerase chain reaction (PCR) experiments, in which the failure to hybridize mtDNA or amplify mtDNA using mtDNA-directed primers suggests thorough mitochondrial genome removal. Here, we report the use of an mtDNA pseudogene ratioing technique for the additional confirmation of ρ⁰ status. Total genomic DNA from a U251 human glioma cell line treated with ethidium bromide was amplified using primers designed to anneal either mtDNA or a previously described nuclear DNA-embedded mtDNA pseudogene (mtDNAψ). The resultant PCR product was used to generate plasmid clones. Sixty-two plasmid clones were genotyped, and all arose from mtDNAψ template. These data allowed us to determine with 95% confidence that the resultant mtDNA-depleted cell line contains less than one copy of mtDNA per 10 cells. Unlike previous hybridization or PCR-based analyses of mtDNA depletion, this mtDNAψ ratioing technique does not rely on interpretation of a negative result, and may prove useful as an adjunct for the determination of ρ⁰ status or mtDNA copy number.     

Mitochondrial DNA copy number threshold in mtDNA depletion myopathy

The authors measured the absolute amount of mitochondrial DNA (mtDNA) within single muscle fibers from two patients with thymidine kinase 2 (TK2) deficiency and two healthy controls. TK2 deficient fibers containing more than 0.01 mtDNA/microm3 had residual cytochrome c oxidase (COX) activity. This defines the minimum amount of wild-type mtDNA molecules required to maintain COX activity in skeletal muscle and provides an explanation for the mosaic histochemical pattern seen in patients with mtDNA depletion syndrome.     

Mitochondrial DNA deletions and depletion within paraspinal muscles

G. R. Campbell, A. Reeve, I. Ziabreva, T. M. Polvikoski, R. W. Taylor, R. Reynolds, D. M. Turnbull and D. J. Mahad (2013) Neuropathology and Applied Neurobiology 39, 377–389 Mitochondrial DNA deletions and depletion within paraspinal muscles Aims: Although mitochondrial abnormalities have been reported within paraspinal muscles in patients with axial weakness and neuromuscular disease as well as with ageing, the basis of respiratory deficiency in paraspinal muscles is not known. This study aimed to determine the extent and basis of respiratory deficiency in paraspinal muscles from cases undergoing surgery for degenerative spinal disease and post mortem cases without a history of spinal disease, where age‐related histopathological changes were previously reported. Methods: Cervical and lumbar paraspinal muscles were obtained peri‐operatively from 13 patients and from six post mortem control cases (age range 18–82 years) without a neurological disease. Sequential COX/SDH (mitochondrial respiratory chain complex IV/complex II) histochemistry was performed to identify respiratory‐deficient muscle fibres (lacking complex IV with intact complex II activity). Real‐time polymerase chain reaction, long‐range polymerase chain reaction and sequencing were used to identify and characterize mitochondrial DNA (mtDNA) deletions and determine mtDNA copy number status. Mitochondrial respiratory chain complex subunits were detected by immunohistochemistry. Results: The density of respiratory‐deficient fibres increased with age. On average, 3.96% of fibres in paraspinal muscles were respiratory‐deficient (range 0–10.26). Respiratory deficiency in 36.8% of paraspinal muscle fibres was due to clonally expanded mtDNA deletions. MtDNA depletion accounted for further 13.5% of respiratory deficiency. The profile of immunohistochemically detected subunits of complexes was similar in respiratory‐deficient fibres with and without mtDNA deletions or mtDNA depletion. Conclusions: Paraspinal muscles appeared to be particularly susceptible to age‐related mitochondrial respiratory chain defects. Clonally expanded mtDNA deletions and focal mtDNA depletion may contribute towards the development of age‐related postural abnormalities.     

Mitochondrial myopathy of childhood associated with depletion of mitochondrial DNA.

We have studied five children with mitochondrial myopathy manifesting within or soon after the first year of life. Muscle biopsies showed ragged-red fibers and decreased respiratory chain activity. All five patients had a severe decrease (2 to 34% of normal) in the amount of muscle mitochondrial DNA (mtDNA). The depletion of mtDNA correlated with absence of mtDNA-encoded translation products and with loss of cytochrome c oxidase enzyme activity in individual muscle fibers. This mitochondrial myopathy of childhood illustrates one phenotypic expression of a novel pathogenetic mechanism in mitochondrial diseases, the specific depletion of mtDNA in affected tissues.     

Mitochondrial DNA depletion: mutations in thymidine kinase gene with myopathy and SMA

BACKGROUND: Imaging studies have shown disparities in resting metabolism and in functional activation between cognitively normal individuals at high and low risk for AD. A recent study has shown increased parietal activation in high-risk subjects during a paired associates recall task, which the authors postulated might overlap activation typically observed in verbal fluency. OBJECTIVE: To determine whether parietal activation is altered in a letter fluency task in cognitively normal individuals at high risk for AD. METHODS: fMRI was used to compare cortical activation between two groups of cognitively normal women differing in their risk for developing AD. A letter fluency task was used, which activates left frontal and parietal regions. The risk groups differed in family history of AD and APOE allele status but were matched in age, education, and measures of cognitive performance. Average age of the study participants was 53 years. RESULTS: The regional patterns of brain activation were similar between groups and similar to patterns observed by other investigators. However, the high-risk group showed significantly increased activation in the left parietal region despite identical letter fluency performance between risk groups. CONCLUSIONS: Cognitively normal individuals at high risk for AD show increased brain activation in the left parietal region with letter fluency, a region adjacent to that observed by others using a recall task. This convergence of results indicates disruption of functional circuits involving the left parietal lobe in asymptomatic individuals at increased risk for AD.     

Mitochondrial DNA depletion syndrome due to mutations in the RRM2B gene

Mitochondrial DNA depletion syndrome (MDS) is characterized by a reduction in mtDNA copy number and has been associated with mutations in eight nuclear genes, including enzymes involved in mitochondrial nucleotide metabolism (POLG, TK2, DGUOK, SUCLA2, SUCLG1, PEO1) and MPV17. Recently, mutations in the RRM2B gene, encoding the p53-controlled ribonucleotide reductase subunit, have been described in seven infants from four families, who presented with various combinations of hypotonia, tubulopathy, seizures, respiratory distress, diarrhea, and lactic acidosis. All children died before 4 months of age. We sequenced the RRM2B gene in three unrelated cases with unexplained severe mtDNA depletion. The first patient developed intractable diarrhea, profound weakness, respiratory distress, and died at 3 months. The other two unrelated patients had a much milder phenotype and are still alive at ages 27 and 36 months. All three patients had lactic acidosis and severe depletion of mtDNA in muscle. Muscle histochemistry showed RRF and COX deficiency. Sequencing the RRM2B gene revealed three missense mutations and two single nucleotide deletions in exons 6, 8, and 9, confirming that RRM2B mutations are important causes of MDS and that the clinical phenotype is heterogeneous and not invariably fatal in infancy.     

Mitochondrial DNA depletion in single fibers in a patient with novel TK2 mutations

The mitochondrial DNA (mtDNA) depletion syndrome is a genetically heterogeneous group of diseases caused by nuclear gene mutations and secondary reduction in mtDNA copy number. We describe a patient with progressive muscle weakness and increased creatine kinase and lactate levels. Muscle weakness was first noted at age 1.5 years and he died of respiratory failure and bronchopneumonia at age 3.5 years. The muscle biopsy showed dystrophic features with ragged red fibers and numerous cytochrome c oxidase (COX)-negative fibers. qPCR analysis demonstrated depletion of mtDNA and sequence analysis of the mitochondrial thymidine kinase 2 (TK2) gene revealed two novel heterozygous variants, c.332C > T, p.(T111I) and c.156 + 5G > C. Quantitative analysis of mtDNA in single muscle fibers demonstrated that COX-deficient fibers showed more pronounced depletion of mtDNA when compared with fibers with residual COX activity (P < 0.01, n = 25). There was no evidence of manifestations from other organs than skeletal muscle although there was an apparent reduction of mtDNA copy number also in liver. The patient showed a pronounced, albeit transient, improvement in muscle strength after onset of treatment with coenzyme Q10, asparaginase, and increased energy intake, suggesting that nutritional modulation may be a therapeutic option in myopathic mtDNA depletion syndrome.     

Mitochondrial DNA depletion associated with partial complex II and IV deficiencies and 3-methylglutaconic aciduria

We report a patient with mitochondrial DNA depletion, partial complex II and IV deficiencies, and 3-methylglutaconic aciduria. Complex II deficiency has not been previously observed in mitochondrial DNA depletion syndromes. The observation of 3-methylglutaconic and 3-methylglutaric acidurias may be a useful indicator of a defect in respiratory chain function caused by mitochondrial DNA depletion.     

Mitochondrial DNA polymerase γ deficiency and mtDNA depletion in a child with Alpers' syndrome

Deficiency of mitochondrial DNA polymerase γ activity was found in a patient with mtDNA depletion and Alpers' syndrome. Metabolic evaluation revealed fasting hypoglycemia, dicarboxylic aciduria, and reduced activity of the electron transport chain in skeletal muscle. The patient died in early childhood of fulminant hepatic failure, refractory epilepsy, lactic acidemia, and coma. mtDNA content was 30% of normal in skeletal muscle and 25% in the liver. The activity of mtDNA polymerase γ was undetectable. Ann Neurol 1999;45:54–58     

Mitochondrial DNA depletion syndrome with a mutation in SLC25A4 developing epileptic encephalopathy: A case report

IntroductionAutosomal dominant mitochondrial DNA depletion syndrome (MTDPS-12A) is characterized by severe hypotonia from birth due to a mutation in the adenine nucleotide translocator 1 (ANT1).Case reportA 4-year-old female patient diagnosed with neonatal-onset mitochondrial disease, who had good cognitive function while receiving antiepileptic treatment, presented with sudden-onset status epilepticus with facial and limb myoclonus persisting for more than 30 min. Subsequently, she developed epileptic encephalopathy. Brain MRI showed progressive ventricular enlargement and marked white matter atrophy. She was unable to perform verbal communication or make eye contact and fingertip movements. She lacked any signs of cardiomyopathy. Sanger sequencing demonstrated a heterozygous de novo mutation of c.239G>A (p.Arg80His) in SLC25A4. Her right quadriceps muscle tissue showed lowered complexes I, III, and IV activities and mitochondria DNA depletion (mitochondria/nuclear DNA: 14.6 ± 2.2%) through the quantitative polymerase chain reaction. She was definitively diagnosed with MTDPS-12A.ConclusionStatus epilepticus causes encephalopathy in patients with MTDPS-12A. Reducing the energy requirement on the cardiac muscle and brain may be a treatment strategy for patients with MTDPS-12A. Therefore, seizure management and preventive treatment of status epilepticus are considered to be important for maintaining neurodevelopmental outcomes.     

Mitochondrial DNA analysis in Parkinson's disease

The reduced form of nicotinamide adenine dinucleotide coenzyme Q reductase (complex I) activity has recently been shown to be deficient in the substantia nigra of patients dying with Parkinson's disease. This biochemical defect is identical to that produced by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which also produces parkinsonism in humans. Complex I comprises 25 polypeptides, seven of which are encoded by mitochondrial DNA. Restriction fragment analysis of substantia nigra DNA from six patients with Parkinson's disease did not show any major deletion. In two cases, there were different novel polymorphisms that were not observed in control brain (n = 6) or blood (n = 34) samples.     

Mitochondrial DNA deletion in oculoskeletal myopathy

The case of a patient showing bilateral ophthalmoplegia with proximal limb weakness, severe dysphagia and short stature, without family history, is described. The diagnosis of Kearns-Sayre syndrome was excluded because of the absence of pigmentary retinopathy and of all other common manifestations except short stature. The analysis of mitochondrial DNA of the patient's muscle revealed a deleted form accounting for 65% of the total mitochondrial DNA. The deletion, undetectable in the mitochondrial DNA of peripheral blood leukocytes, was apparently indistinguishable from that already described by others in a far more severe form of classic Kearns-Sayre syndrome.