Our results are in line with deep-sequencing analysis of liver mtDNA that questions the general effect of aging on mtDNA mutagenesis. As we have focused on seven 4-base sequences, we cannot rule out that mutations outside these sites contribute to the age-associated mitochondrial dysfunction. Our data Trihexyphenidyl HCl nevertheless indicate that some sites are more prone to Oxysophocarpine mutagenesis with age, and may correlate with those that undergo progressive clonal expansion to preclude mitochondrial dysfunction. Our method can be used to identify the potential of a position to undergo clonal expansion. Carriers of the common deletion mutation may have up to 60% of mutated mtDNA molecules without manifesting into disease. It would be interesting to address mtRNA integrity in carriers of the common deletion, to correlate this with the onset of disease, known as the Kearns�CSayre syndrome. In this study, we focused on brain mtDNA mutagenesis. Brain and heart were previously shown to accumulate mitochondrial mutations to a similar extent with age. We previously detected equal mutation frequencies in the same sites in liver and brain, but higher in lung. As a rough estimate, the average brain mtDNA mutation frequency in all seven sites is 0.7 610-4 pernt, which is in coherence with results obtained by the deepsequencing analysis of liver mtDNA. Recent ultra-sensitive sequencing of human brain mitochondria has shown that age indeed increase mutagenesis. The apparent contradiction to our data could be that the relative age of the mice in our cohort was relatively much lower than the human cohort in the cited reference. In contrast to mouse brain, the frequency of mtDNA mutations in liver did not change with age in any sites and suggests that age influences mtDNA mutagenesis in a tissue/cell type-specific manner. In particular, this is demonstrated by the reduction in mtDNA mutation level in colorectal cancer. Interesting age-independent relationships between the mutation frequencies within individuals were discovered. We found a positive correlation between Nd1 and Nd3, which might be expected as both sites showed age-associated mutagenesis. However, a stronger correlation was observed between mutation frequencies in 12S and Nd1, as well as in 12S and Nd6, which was independent of age. In comparison, none of the other sitesseemed to be interconnected. Perhaps a subset of mtDNA genes/sequences is particularly important in subregions of the brains, and that the quality of these regions is selected for. An analogy to this is observed during the purifying deselection of nonsynonymous mutations in the protein-coding mtDNA genes during transmission, suggesting that mtDNA mutagenesis is under functional control. An analogue indication of a targeted mutagenesis was suggested in very young human brain samples as well. The presence of nuclear mtDNA fragments could theoretically introduce errors as we analyze total DNA and pseudogenes with altered nucleotide sequence potentially could generate positive signals in our restriction cleavage inhibitionbased assay. However, since the primers used in the qPCR display similar efficiency and the CT values are comparable for the seven sites, it means that the mtDNA copy number is identical independent of the site to quantify. Again, this demonstrates that numts do not contribute significantly to the readout in any of the seven sites.