Negative selection allows human primary fibroblasts to tolerate high somatic mutation loads induced by N-ethyl-N-nitrosourea

Single-cell sequencing has shown that thousands of mutations accumulate with age in most human tissues. While there is ample evidence that some mutations can clonally amplify and lead to disease, the total burden of mutations a cell tolerates without functional decline remains unknown. Here we addressed this question by exposing human primary fibroblasts to multiple, low doses of N-ethyl-N-nitrosourea (ENU) and analyzed somatic mutation burden using single-cell whole genome sequencing. The results indicate that individual cells can sustain ~60,000 single-nucleotide variants (SNVs) with only a slight adverse effect on growth rate. We provide evidence that such high levels of mutations are only tolerated through negative selection against variants in gene coding regions, and in sequences associated with genetic pathways for maintaining basic cellular function and growth. Since most tissues in adults are non-dividing, these results suggest that somatic mutations in the absence of negative selection may have functionally adverse effects. To determine the impact of increased somatic mutation burden on primary human cells in vitro, we repeatedly treated human fetal lung fibroblasts (IMR-90) a total of 9 times with a sublethal dose of 50 µg/mL of N-ethyl-N-nitrosourea (ENU), followed by a recovery period. We then performed RNA-Sequencing of cells collected at the begining of the experiment (1st cycle) or end of the experiment (9th cycle) from the control or ENU-treated groups. Samples collected from 3 independent replicates.