Mitochondrial myopathy in progressive external ophthalmoplegia (PEO) has been associated with POLG1 mutations. POLG1 encodes the catalytic alpha subunit of polymerase gamma and is the only polymerase known to be involved in mtDNA replication. It has two functionally different domains, one polymerase domain and one exonuclease domain with proofreading activity. In this study we have investigated whether mtDNA point mutations are involved, directly or indirectly, in the pathogenesis of PEO. Muscle biopsy specimens from patients with POLG1 mutations, affecting either the exonuclease or the polymerase domain, were investigated. Single cytochrome c oxidase (COX)-deficient muscle fibers were dissected and screened for clonally expanded mtDNA point mutations using a sensitive denaturing gradient gel electrophoresis analysis, in which three different regions of mtDNA, including five different tRNA genes, were investigated. To screen for randomly distributed mtDNA point mutations in muscle, two regions of mtDNA including deletion breakpoints were investigated by high-fidelity PCR, followed by cloning and sequencing. Long-range PCR revealed multiple mtDNA deletions in all the patients but not the controls. No point mutations were identified in single COX-deficient muscle fibers. Cloning and sequencing of muscle homogenate identified randomly distributed point mutations at very low frequency in patients and controls (<1:50 000). We conclude that mtDNA point mutations do not appear to be directly or indirectly involved in the pathogenesis of mitochondrial disease in patients with different POLG1 mutations.
"dominant or recessive progressive external ophthalmoplegia (PEO) syndromes (Van Goethem et al., 2001; Lamantea et al., 2002), autosomal recessive sensory ataxic neuropathy with dysarthria and ophthalmoplegia (SANDO) (Van Goethem et al., 2003a; Mancuso et al., 2004), adult-or juvenile-onset mixed sensory and cerebellar ataxic syndrome with epileptic seizures and myoclonus (Van Goethem et al., 2004; Winterthun et al., 2005), a myoclonic epilepsy and ragged red fibers-like phenotype (Van Goethem et al., 2003b), Parkinson's disease (Luoma et al., 2004; Davidzon et al., 2006), spinocerebellar ataxia (Hakonen et al., 2005), and a classic Alpers–Huttenlocher syndrome (AHS), an autosomal recessive hepatocerebral syndrome (Naviaux et al., 1999; Naviaux & Nguyen, 2004; Davidzon et al., 2005; Ferrari et al., 2005; Nguyen et al., 2005, 2006; Horvath et al., 2006). POLG1 mutations have been reported to be associated with mtDNA deletions (Van Goethem et al., 2001; Di Fonzo et al., 2003), depletion (Naviaux et al., 1999; Naviaux & Nguyen, 2004; Tesarova et al., 2004; Davidzon et al., 2005; Ferrari et al., 2005; Horvath et al., 2006) or point mutations (Kollberg et al., 2005), which at least partly determine the clinical phenotype. "
[Show abstract][Hide abstract] ABSTRACT: Polymerase gamma (POLG) is the sole enzyme in the replication of mitochondrial DNA (mtDNA). Numerous mutations in the POLG1 gene have been detected recently in patients with various phenotypes including a classic infantile-onset Alpers-Huttenlocher syndrome (AHS). Here we studied the molecular etiology of juvenile-onset AHS manifesting with status epilepticus and liver disease in three teenagers.
We examined 14- and 17-year-old female siblings (patients 1 and 2) and an unrelated 15-year-old girl (patient 3) with juvenile-onset AHS, sequenced POLG1, and the entire mtDNA, examined mtDNA deletions by amplification of the full-length mtDNA with the long PCR method and used real-time PCR to quantify mtDNA in the tissue samples.
The initial manifestations were migraine-like headache and epilepsy, and the terminal manifestations status epilepticus and hepatic failure. A homozygous W748S mutation in POLG1 was detected in the three patients. No deletions or pathogenic point mutations were found in mtDNA, but all three patients had mtDNA depletion.
POLG mutations should be considered in cases of teenagers and young adults with a sudden onset of intractable seizures or status epilepticus, and acute liver failure. The W748S POLG1 mutation seems to lead to tissue-specific, partial mtDNA depletion in patients with juvenile-onset Alpers syndrome. Valproic acid should be avoided in the treatment of epileptic seizures in these patients.
"The mtDNA also encodes for two rRNAs and 22 tRNAs necessary for mitochondrial protein synthesis. The replication of mtDNA requires several nuclearencoded factors such as mtRNA polymerase, mitochondrial transcription factor A (mtTFA), DNA polymerase gamma (POLG), deoxyguanosine kinase (DGUOK), and thymidine kinase 2 (TK2) (Larsson and Clayton, 1995; Larsson and Luft, 1999; Kollberg et al., 2005). mtDNA is more vulnerable to mutation than nuclear DNA and the mutation rate is much higher in mtDNA. "
[Show abstract][Hide abstract] ABSTRACT: Mitochondrial OXPHOS disorders are caused by mutations in mitochondrial or nuclear genes, which directly or indirectly affect mitochondrial oxidative phosphorylation (OXPHOS). Primary mtDNA abnormalities in children are due to rearrangements (deletions or duplications) and point mutations or insertions. Mutations in the nuclear-encoded polypeptide subunits of OXPHOS result in complex I and II deficiency, whereas mutations in the nuclear proteins involved in the assembly of OXPHOS subunits cause defects in complexes I, III, IV, and V. Here, we review recent progress in the identification of mitochondrial and nuclear gene defects and the associated clinical manifestations of these disorders in childhood.
"Interactions between tRNA sequence and gamma polymerase might resemble those between tRNA and tRNA synthetase. Such confounding effects would explain why associations between genotypes (in any of the different genes involved) and diseases remain elusive (as an example, in polymerase gamma; Kollberg et al., 2005). "
[Show abstract][Hide abstract] ABSTRACT: The heavy strand of vertebrate mitochondrial genomes accumulates deaminations proportionally to the time it spends single-stranded during replication. A previous study showed that the strength of genome-wide deamination gradients originating from tRNA gene's locations increases with their capacities to form secondary structures resembling mitochondrial origins of light strand replication (OL), suggesting an alternative function for tRNA sequences. We hypothesize that this function is frequently pathogenic for those tRNA genes that normally do not form OL-like structures, because this could cause excess mutations in genome regions unadapted to tolerate them. In human mitochondrial genomes, pathogenic tRNA variants usually form less OL-like structures than non-pathogenic ones in cases where the normal non-pathogenic tRNA variant can function as OL, as evolutionary analyses reveal. For tRNAs lacking the putative OL-like functioning capacity, pathogenic variants form more OL-like secondary structures, particularly structures that might invoke bi-directional replication (true for 14 among 21 tRNA species, p<0.05, sign test; significantly at p<0.05 (1 tailed test) for 7 tRNA species), but not more unidirectional replication invoking structures. Accounting for the functional cloverleaf-like structure-forming capacities of tRNAs yields similar results. Rare, non-pathogenic tRNA mutants tend to form more OL-like structures than the common, non-pathogenic ones, suggesting weak directional selection also among non-pathogenic variants. The duration spent single stranded by a region of the heavy strand (D(ssH)) during replication, estimated by integrating over all regions that can function as OL in Homo sapiens mitochondrial genomes, increases with distance of that region from the Dloop. This suggests convergence of single-strandedness during replication and transcription, and explains conserved locations of tRNA species in mitochondrial genomes and bacterial operons. These locations minimize deamination costs only in anticodons and not in other tRNA regions, during replication and transcription. Therefore, putative functioning as OLs by tRNA sequences is normal at some locations and pathogenic at others.
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