Negative Selection Allows DNA Mismatch Repair-Deficient Mouse FibroblastsIn Vitroto Tolerate High Levels of Somatic Mutations

Abstract

SummarySubstantial numbers of somatic mutations have been found to accumulate with age in different human tissues. Clonal cellular amplification of some of these mutations can cause cancer and other diseases. However, it is as yet unclear if and to what extent an increased burden of random mutations can affect cellular function without clonal amplification. We tested this in cell culture, which avoids the limitation that an increased mutation burdenin vivotypically leads to cancer. We performed single-cell whole-genome sequencing of primary fibroblasts from DNA mismatch repair (MMR) deficientMsh2-/-mice and littermate control animals after long-term passaging. Apart from analyzing somatic mutation burden we analyzed clonality, mutational signatures, and hotspots in the genome, characterizing the complete landscape of somatic mutagenesis in normal and MMR-deficient mouse primary fibroblasts during passaging. While growth rate ofMsh2-/-fibroblasts was not significantly different from the controls, the number ofde novosingle-nucleotide variants (SNVs) increased linearly up until at least 30,000 SNVs per cell, with the frequency of small insertions and deletions (INDELs) plateauing in theMsh2-/-fibroblasts to about 10,000 INDELS per cell. We provide evidence for negative selection and large-scale mutation-driven population changes, including significant clonal expansion of preexisting mutations and widespread cell-strain-specific hotspots. Overall, our results provide evidence that increased somatic mutation burden drives significant cell evolutionary changes in a dynamic cell culture system without significant effects on growth. Since similar selection processes against mutations preventing organ and tissue dysfunction during aging are difficult to envision, these results suggest that increased somatic mutation burden can play a causal role in aging and diseases other than cancer.