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Title: Ultra-Deep Sequencing of Mouse Mitochondrial DNA: Mutational Patterns and Their Origins}
Article:
Abstract:
Somatic mutations of mtDNA are implicated in the aging process, but there is no universally accepted method for their accurate quantification. We have used ultra-deep sequencing to study genome-wide mtDNA mutation load in the liver of normally- and prematurely-aging mice. Mice that are homozygous for an allele expressing a proof-reading–deficient mtDNA polymerase( mtDNA mutator mice) have 10-times-higher point mutation loads than their wildtype siblings. In addition, the mtDNA mutator mice have increased levels of a truncated linear mtDNA molecule, resulting in decreased sequence coverage in the deleted region. In contrast, circular mtDNA molecules with large deletions occur at extremely low frequencies in mtDNA mutator mice and can therefore not drive the premature aging phenotype. Sequence analysis shows that the main proportion of the mutation load in heterozygous mtDNA mutator mice and their wildtype siblings is inherited from their heterozygous mothers consistent with germline transmission. We found no increase in levels of point mutations or deletions in wildtype C57Bl/6N mice with increasing age, thus questioning the causative role of these changes in aging. In addition, there was no increased frequency of transversion mutations with time in any of the studied genotypes, arguing against oxidative damage as a major cause of mtDNA mutations. Our results from studies of mice thus indicate that most somatic mtDNA mutations occur as replication errors during development and do not result from damage accumulation in adult life.
Introduction:
A decline of mitochondrial function has been observed in a variety of aging mammalian tissues and is implicated as a driving force behind the aging process[1]–[2]. A somatic mammalian cell carries thousands of copies of the mitochondrial DNA( mtDNA) chromosome, which encodes essential subunits of the respiratory chain protein complexes as well as rRNAs and tRNAs needed for mitochondrial translation. Expression of mtDNA is required for maintenance of oxidative phosphorylation and accumulation of somatic mtDNA mutations has been suggested as a cause of the observed decrease in respiratory chain function during aging[3]–[4]. A variety of low levels of point mutations and rearrangements of mtDNA are found in aging mammals. Rare mutational events tend to undergo clonal expansion as exemplified by human aging where clonal expansion of somatic mtDNA mutations cause a mosaic respiratory chain deficiency in tissues such as brain, heart, skeletal muscle and large intestine[4]. Point mutations as well as insertions and deletions( indels) of mtDNA are observed in tissues of aging humans[5]–[9], primates[10] and rodents[11], but the relative contribution of each of these different types of mutations to the aging process is unknown. The mtDNA mutator mice( genotype PolgAmut/PolgAmut) express a proof-reading-deficient mtDNA polymerase( PolgAD257A) and have provided experimental support that accumulation of mtDNA mutations can lead to a premature aging syndrome[12]–[14]. These mice contain high levels of point mutations in their mtDNA and high levels of several species of large linear deleted mtDNA molecules. Although the linear deleted molecules are abundant( ∼25–30% of total mtDNA in liver) the corresponding reduction in levels of full-length mtDNA molecules is on its own not sufficient to cause an impairment of respiratory chain function[13]. A detailed molecular characterization of the mtDNA mutator mice has shown that the high levels of point mutations are the likely explanation for the respiratory chain deficiency and the premature aging syndrome[15]. One report claims that the presence of a third type of mtDNA mutation, i. e. circular mtDNA molecules with large deletions, may be of critical importance in driving the premature aging phenotype of mtDNA mutator mice[16]–[17]. However, this finding has been refuted by several other reports showing that the levels of such deleted molecules are extremely low[15],[18]–[21]. In addition, the biochemical phenotype in mtDNA mutator mice can be fully explained by the finding that high levels of point mutations in mtDNA leads to the synthesis of respiratory chain subunits with abundant amino acid replacements, which, in turn, cause instability of the respiratory chain complexes[15]. Oxidative damage has for more than 50 years been proposed as a central mechanism in aging, but the supporting evidence is mainly correlative. Interestingly, the mtDNA mutator mice have no or minor increase in levels of reactive oxygen species( ROS) production and oxidative damage despite a severe decline in oxidative phosphorylation capacity. This finding refutes the popular notion of a vicious cycle whereby somatic mtDNA mutations lead to increased ROS production, which, in turn, creates additional mtDNA mutations that further increase ROS production[12],[14]. Genome-wide estimates of intra-individual mtDNA variability are needed to determine the importance of the various types of mtDNA mutations in aging. Recent studies using next-generation sequencing of the human mitochondrial genome have identified a small number of sites, in which normal individuals carry both the wildtype copy and a high-frequency mutant allele[22]–[23]. In their efforts to eliminate false positive calls, these studies limited their analyses to high-frequency mutations and excluded rare variants. We hypothesised that a DNA sequencing technology that minimize the levels of false positive calls caused by technical errors could be applied to assess the intra-individual mtDNA variability caused by low frequency mutations. We investigated this possibility by using the ABI SOLiD technology to sequence mtDNA of normal aging mice and prematurely aging mtDNA mutator mice. To control for technical errors we sequenced a complete mtDNA genome inserted in a lambda clone.
Discussion:
Our analysis has revealed a number of novel aspects of the accumulation of mtDNA mutations in wildtype and mtDNA mutator mice. The mutators show highly elevated point mutation frequencies as compared to their wtmut siblings, consistent with previous results[12]–[14],[16],[21],[29]–[30]. The SOLiD sequencing estimates of mutation loads presented here are similar to the mutation load estimates that we and others previously have obtained by Sanger sequencing of cloned PCR fragments[12]–[14]. In a recent study using a different next-generation DNA sequencing technology, He et al.[22] reported eight sites with a mutation frequency >1. 6% in a human mtDNA sample. If we apply their detection threshold to our data, we identify five such sites in the wtB6 mice. Techniques that enable identification of low frequency variants, such as those used in our study, are likely to uncover additional variability and provide a more complete understanding of the mitochondrial mutation load. We hypothesized that our analysis criteria should also uncover gradual increase in the mutation loads with natural aging, consistent with published results obtained by other mutation detection methods( reviewed in[4]). However, we neither detected a difference in the mtDNA mutation load in liver mtDNA from wtB6 mice at different ages( 30–84 weeks), nor did we see a shift in the mutational spectrum consistent with oxidative damage causing mtDNA mutations in mice of the different genotypes. Together, the data we present here suggest that most mtDNA mutations are due to mtDNA replication errors and that oxidative damage of mtDNA does not drive the aging process in liver. Our estimates of the mutation load in the wtB6 and wtmut mice are in good agreement with estimates based on sequencing of cloned PCR fragments[12]–[14], but they are higher than estimates obtained by a restriction enzyme digestion-based assay[30]( summarized in Table S6). Also, we observed no increase in mutation load in wtB6 mice with age, as reported elsewhere[11],[30]. A possible explanation for this difference from results in the literature is that the next-generation sequencing method has an inherent limitation in detecting extremely low levels of mutations. It remains possible that there is a slight increase of mutation load with age in wtB6 mice, as reported in other studies[11], and the true mutation levels may be below the detection threshold of the SOLiD sequencing method. But in any case, the mutation levels seen in the aging wtB6 mice were very modest in comparison to those of age-matched mtDNA mutator mice. Also, we chose to only analyze mtDNA from mouse liver as this tissue made it possible for us to obtain sufficient quantities of pure mtDNA for direct sequencing, without excess DNA amplification. A continuously dividing tissue like liver may show a different pattern of accumulation of mtDNA point mutations with age in comparison with a postmitotic tissue such as brain or heart. The different coding regions showed similar mutation frequencies, while the control region had a much lower mutation frequency. A reduction in the overall mutation load has previously been observed in the mitochondrial control region of mtDNA mutator mice[12]–[13],[29]. The control region contains crucial sequence elements controlling replication and transcription of mtDNA[31]. It is therefore likely that mutations that inhibit mtDNA maintenance or expression could undergo strong selection and be eliminated from the mtDNA pool. Some of the mtDNA point mutations observed in siblings to mtDNA mutator mice are likely to have been inherited via the maternal gamete of their heterozygous mother. The wtmut mice from this cross carry approximately twice the number of high frequency point mutations( >0. 5%) in comparison with wtB6 mice. In addition, an elevated number of small indel mutations are observed in the wtmut mice, but not in wtB6 mice( Table S4). We cannot exclude that some of the shared point mutations represent extreme mutational hotspots, however, a more likely explanation is that these shared mutations are maternally inherited. A similar phenomenon has been observed in humans, where the variability at several positions was shown to be inherited from the common maternal cytoplasm instead of representing repeated de novo mutational events[22]. In mtDNA mutator mice, approximately 30% of the mtDNA molecules are non-replicating, linear mtDNA molecules with large deletions[13],[19]. It could be speculated that these molecules contain an elevated mutation load and that most of the mutation load is sequestered in these molecules. By ultra-deep sequencing, we were able to determine that the mutation load in the region covering the linear molecule did not vary from the global mtDNA mutation load in the mutators. A recent publication suggests the PolgAmut polymerase may pause at the control and OriL regions during mtDNA replication, which may explain the generation of the linear molecules with large deletions[19]. Our results are consistent with a hypothesis that altered processivity of the mutator polymerase and not the point mutations per se, are responsible for the creation of the linear molecules. The physiological consequences of the linear molecules, and their contribution to the premature aging in the mtDNA mutator mice, remain unclear and worthy of further investigation. Circular mtDNA molecules with deletions have been suggested to be the driving force of the aging phenotype in the mutator mice, and are reliably detected in human tissues during aging. High levels of these circular mtDNA molecules with deletions lead to mitochondrial dysfunction in human patients[32] or mice engineered to contain these mutations[33]–[34]. However, we found that the circular mtDNA molecules with deletions are exceedingly rare in mtDNA mutator mice, with only 4 breakpoints being detected in the millions of reads of two mutator samples. Very low levels of this type of deleted molecules have also been reported by studies using different PCR based analyses[15],[20]. Recently, an independent next-generation sequencing analysis detected exceedingly low levels of this type of mutation in brain and heart of mtDNA mutator mice[21]. Large deletion of mtDNA are known to impair mitochondrial translation due to lack of one or more tRNA genes[35]. However, mtDNA mutator mice do not display impaired mitochondrial translation in heart or liver[15] and the levels of deleted mtDNA are much lower than the levels observed in respiratory chain deficient mouse strains with single[33] and multiple[34] deletions of mtDNA. Together, these observations provide strong evidence that the circular deleted mtDNA molecules are not the causative factor in the aging phenotype of the mtDNA mutator mice. A recent study made a remarkable observation that similar low levels of circular deletions were accumulating in a mouse model with a tissue-specific disruption of the mitochondrial fusion process[36]. The presence of very low levels of circular mtDNA molecules with deletions in two very different models of mitochondrial dysfunction suggests these rare events are being generated as a secondary consequence of mitochondrial dysfunction. Another possibility is that these molecules are continuously generated at low frequency during normal mtDNA replication and that mitochondrial dysfunction limit their clearance. The large numbers of point mutations in adult mtDNA mutator mice result in production of highly mutated mtDNA-encoded respiratory chain subunits, causing the experimentally observed instability of the respiratory chain complexes[15],[37]. There is likely a threshold for the tolerance of point mutations, where eventually the combined effect of the many amino acid changes in mtDNA mutator mice cause destabilization of respiratory chain complexes and lead to mitochondrial dysfunction. Our results support the assertion that the accumulation of point mutations has an adversary effect on mitochondrial function and cause the premature aging syndrome in the mtDNA mutator mice.
Mitochondria represent the powerhouses of cells and have their own DNA . Mutations in the mitochondrial genome are associated with a range of human diseases and have also been implicated as a driving force behind the aging process . We have used ultra-deep sequencing to study the genome-wide mutation load in the mitochondrial DNA ( mtDNA ) of liver from normal inbred mice and mice that express a proof-reading–deficient mtDNA polymerase ( mtDNA mutator mice ) that cause premature aging . The mtDNA mutator mice show a dramatic increase of point mutations with age and have 10-times-higher point mutation levels than wildtype siblings or normal C57Bl/6N mice . Circular mtDNA molecules with large deletions occur at very low frequencies in mtDNA mutator mice and are therefore unlikely to contribute to the premature aging phenotype . We found no increase in levels of point mutations or deletions in normal mice with increasing age , arguing against the accumulation of mtDNA mutations as contributing to aging . Our results indicate that most somatic mtDNA mutations occur as replication errors during the rapid amplification of mtDNA during embryogenesis and do not result from damage accumulation in adult life .