Tobacco exposure and somatic mutations in normal human bronchial epithelium

Tobacco smoking causes lung cancer(1–3), driven by the 60+ carcinogens in cigarette smoke that directly damage and mutate DNA(4,5). The profound effects of tobacco on the lung cancer genome have been well documented(6–10), but we lack equivalent data for normal bronchial cells. We sequenced whole genomes of 632 colonies derived from single bronchial epithelial cells across 16 subjects. Tobacco smoking was the major influence on mutation burden, adding 1000-10,000+ mutations/cell, massively increasing both within-subject and between-subject variance, and generating several distinct signatures of substitutions and indels. A population of cells in subjects with smoking history had mutation burdens equivalent to that expected for never-smokers: these cells had less damage from tobacco-specific mutational processes, were four-fold more frequent in ex-smokers than current smokers, and had significantly longer telomeres than their more mutated counterparts. Driver mutations increased in frequency with age, affecting 4-14% of cells in middle-aged never-smokers. In current smokers, ≥25% of cells carried driver mutations and 0-6% cells had 2 or even 3 drivers. Thus, tobacco smoking increases mutation burden, cell-to-cell heterogeneity and driver mutations, but quitting promotes replenishment of bronchial epithelium from mitotically quiescent cells that have avoided tobacco mutagenesis.