Germline mutation rates and the long-term phenotypic effects of mutation accumulation in wild-type laboratory mice and mutator mice

The germline mutation rate is an important parameter that affects the amount of genetic variation and the rate of evolution. However, neither the rate of germline mutations in laboratory mice nor the biological significance of the mutation rate in mammalian populations is clear. Here we studied genome-wide mutation rates and the long-term effects of mutation accumulation on phenotype in more than 20 generations of wild-type C57BL/6 mice and mutator mice, which have high DNA replication error rates. We estimated the base-substitution mutation rate to be 5.4 × 10⁻⁹ (95% confidence interval = 4.6 × 10⁻⁹–6.5 × 10⁻⁹) per nucleotide per generation in C57BL/6 laboratory mice, about half the rate reported in humans. The mutation rate in mutator mice was 17 times that in wild-type mice. Abnormal phenotypes were 4.1-fold more frequent in the mutator lines than in the wild-type lines. After several generations, the mutator mice reproduced at substantially lower rates than the controls, exhibiting low pregnancy rates, lower survival rates, and smaller litter sizes, and many of the breeding lines died out. These results provide fundamental information about mouse genetics and reveal the impact of germline mutation rates on phenotypes in a mammalian population.Germline mutations are the ultimate source of congenital diseases, individual phenotypic variations, and evolutionary phenotypic changes. The per generation de novo mutation rate affects genetic variability and the speed of evolution (Kimura 1983; Drake et al. 1998). Advancements in high-throughput sequencing have made it possible to determine the mutation rates in various organisms. For example, the mean germline base-substitution mutation rate is calculated at 1.2 × 10⁻⁸ per nucleotide per generation for humans (Conrad et al. 2011; Kong et al. 2012; Campbell and Eichler 2013), 1.2 × 10⁻⁸ for chimpanzees (Venn et al. 2014), 2.8 × 10⁻⁹ for Drosophila melanogaster (Keightley et al. 2014), and 2.7 × 10⁻⁹ for Caenorhabditis elegans (Denver et al. 2009). The per generation mutation rate varies between species (Lynch 2010a) and within them; for example, the rate in humans varies several-fold between individuals, and is influenced by age, sex, and other genetic or environmental factors (Conrad et al. 2011; Kong et al. 2012; Campbell and Eichler 2013).More recently, the importance of understanding the risks and effects of germline mutagens, such as environmental chemicals and radiation, on the future health of animal populations, including humans, has become clear (Yauk et al. 2015). However, only limited information about the long-term effects of mutation-rate differences is currently available. For example, we know that an increased mutation rate will increase the frequency of congenital disease. However, the overall phenotypic effects of accumulated mutations on future populations living under higher mutation-rate conditions are largely unknown. Furthermore, the range of germline mutation rates that will permit the long-term survival of mammalian populations is unclear. Thus, here we established a new experimental model for assessing germline mutation rates and their phenotypic effects on future populations living under higher mutation-rate conditions.For this model, we raised two lines of mice, wild-type C57BL/6 mice (control mice) and homozygous Pold1^exo/exo mice (mutator mice), for more than 20 generations with phenotypic inspection, and established a set of mutation accumulation (MA) lines. Pold1^exo/exo mice lack the 3′-5′ exonuclease activity of DNA polymerase delta (of which a catalytic subunit is encoded by the Pold1 gene) on a C57BL/6 background (Uchimura et al. 2009) and have a high rate of DNA replication errors. DNA polymerase delta, a major enzyme in DNA replication and genome maintenance, contributes to faithful DNA synthesis, mainly lagging-strand synthesis, through its intrinsic 3′-5′ exonuclease proofreading activity (Burgers 2009; Prindle and Loeb 2012). Disrupting its exonuclease activity increases the spontaneous mutation (mainly base-substitution) rates in yeast (Simon et al. 1991) and mouse somatic cells (Albertson et al. 2009) and increases tumor susceptibility in mice (Goldsby et al. 2002) and humans (Palles et al. 2013). In the current study, ∼30% of the mutator mice died from thymic lymphoma at 3–8 mo of age, but the tumor rate did not change over the generations studied. The mice were bred by one-to-one natural mating between siblings, without artificial selection. At the time of this writing, we had studied up to 24 generations of these breeding lines (Fig. 1).Open in a separate windowFigure 1.Pedigrees of control and mutator mice in a long-term breeding study. All breeding lines that were separated by five or more generations from the original line are shown: blue, wild-type; black, surviving mutator lines; and red, extinct mutator lines. Green arrows indicate whole-genome sequencing was performed on mice from the indicated generations. The first appearance of a conspicuous phenotypic anomaly is shown for each breeding line. Gray shading indicates body weight was analyzed for the generations indicated (see Fig. 3, legend).For the current study, we performed whole-genome sequencing of the mouse MA lines and estimated the per generation mutation rate in the control and mutator mice. This is the first application of high-throughput sequencing to determine the spontaneous germline mutation rate of wild-type mice and provides the first direct estimate of a genome-wide germline mutation rate in laboratory mice. We also recorded detailed phenotypic data through several generations and found that the different germline mutation rates between the mutator and control mice had a significant impact on the health of their descendants. Our findings indicate that the combination of genome sequencing and MA analyses in mice is a promising tool for understanding the biological significance of mutation rate and de novo mutations in a population at the whole-genome level.