Pervasive Induction of Regulatory Mutation Microclones in Sun-exposed Skin

Abstract Carcinogen-induced mutations are thought near-random, with rare cancer-driver mutations underlying clonal expansion. Using high-fidelity Duplex Sequencing to reach a mutation frequency sensitivity of 4×10 -9 per nt, we report that sun exposure creates pervasive mutations at sites with ∼100-fold UV-sensitivity in RNA-processing gene promoters – cyclobutane pyrimidine dimer (CPD) hyperhotspots – and these mutations have a mini-driver clonal expansion phenotype. Numerically, human skin harbored 10-fold more genuine mutations than previously reported, with neonatal skin containing 90,000 per cell; UV signature mutations increased 8,000-fold in sun-exposed skin, averaging 3×10 -5 per nt. Clonal expansion by neutral drift or passenger formation was nil. Tumor suppressor gene hotspots reached variant allele frequency 0.1-10% via 30-3,000 fold clonal expansion, in occasional biopsies. CPD hyperhotspots reached those frequencies in every biopsy, with modest clonal expansion. In vitro, tumor hotspot mutations arose occasionally over weeks of chronic low-dose exposure, whereas CPD hyperhotspot mutations arose in days at 1000-fold higher frequencies, growing exponentially. UV targeted mini-drivers in every skin cell.