Evidence for negative selection against somatic mutations induced in normal fibroblasts by N-ethyl-N-nitrosourea

Mutations accumulate with age in most human tissues. While some undergo clonal expansion and contribute to disease, the mutational burden tolerated by a normal cell without functional decline remains unknown. Here, we repeatedly treat proliferating human primary fibroblasts with the point mutagen N-ethyl-N-nitrosourea, and analyze mutation burden by single-cell whole-genome sequencing. Mutation burden increases linearly to ~56,000 single-nucleotide variants per cell, with only a modest reduction in growth rate. We detect negative selection against potentially deleterious coding and noncoding variants, including mutations affecting pathways important for cell growth and maintenance. These findings suggest that selective depletion of harmful variants helps proliferating cells maintain function despite an extreme mutation burden. Because most adult tissues are largely nondividing and cannot remove damaging mutations through a growth disadvantage, somatic mutations that accumulate during aging may have pronounced functional consequences in vivo.