Ultra-deep sequencing of mouse mitochondrial DNA: mutational patterns and their origins

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.

Figure 1. Sequence coverage for the mouse mtDNA samples.

Mitochondrial genome position (x-axis) versus sequence coverage divided by maximum coverage for each sample. The coverage was calculated using a 2 kb sliding window average. The red lines correspond to mtDNA mutator samples. All other mouse samples are in black. The blue line represents the sequence coverage of the λ^mtDNA control. The approximate locations of the origins of light-strand (O_L) and heavy-strand (O_H) replication are indicated by dotted lines with arrows.

Figure 2. Corrected mutational frequencies viewed in the UCSC genome browser.

The scale ranges from low mutation frequencies (light colors) to high mutation frequencies (black). (Abbreviations: wt^B6 = C57Bl/6N animal, wt^mut  =  wildtype sibling of the mtDNA mutator mouse, Hz =  heterozygous sibling of the mtDNA mutator mouse, Mut  =  mtDNA mutator mouse, 30w & 40w  = 30 and 40 weeks of age, respectively).

Figure 3. Number of mutations with a frequency of at least 0.5% in each sample.

(Abbreviations: wt^B6  =  C57Bl/6N animal, wt^mut  =  wildtype sibling of the mtDNA mutator mouse, Hz  =  heterozygous sibling of the mtDNA mutator mouse, Mut  =  mtDNA mutator mouse, 30w & 40w  = 30 weeks of age and 40 weeks of age, respectively).

Figure 4. Mutation frequencies for the two mtDNA mutator mice (Mut 30w and Mut 40w) and the C57Bl/6N 84-week-old mouse (wt^B6) in a region including the control region.

The locations of the control region and the conserved sequence blocks (CSB) 1–3 are indicated by horizontal black lines.

Figure 5. Number of shared high-frequency mtDNA mutation in the C57Bl/6N and mtDNA mutator sibling sets.

A) Number of high frequency mutations (per site frequency >0.5%) in the 30-week-old (30w; blue) and 40-week-old (40w; red) mutator siblings and C57Bl/6N mice (orange). The columns refer to the total number of such positions (“Total in litter”), positions found in only one of the three mice in each group (“in 1 mouse only”), in two of the three mice in each group (“shared by 2 mice”) and finally those shared between all three mice within each of the groups (“shared by 3 mice”). Venn Diagrams showing the distribution of high frequency mutations that are unique mutations and those shared between, B) C57Bl/6N samples, C) the 30-week-old mtDNA mutator sibling set and D) the 40-week-old mtDNA mutator sibling set. (Abbreviations: wt^B6  =  C57Bl/6N animal, wt^mut  =  wildtype sibling of the mtDNA mutator mouse, Hz  =  heterozygous sibling of the mtDNA mutator mouse, Mut  =  mtDNA mutator mouse, 30w and 40w  = 30 weeks of age and 40 weeks of age, respectively).