Ultra-deep duplex sequencing reveals unique features of somatic evolution in the normal tissues of a family with Li-Fraumeni syndrome

Li-Fraumeni Syndrome (LFS) is caused by germline pathogenic variants in <i>TP53</i> which predispose carriers to early onset cancer across multiple tissues. While genomically profiling those cancers has revealed factors contributing to their formation, little is understood about how LFS impacts clonal evolution in healthy tissues preceding cancer. Here, we use ultra-deep duplex sequencing (mean ~15,000× depth) to investigate somatic mutation and selection in a family carrying the germline <i>TP53</i> p.R181H pathogenic variant and a cohort of non-carrier controls. In blood samples, the germline variant was associated with more mutations in a panel designed to capture genomewide mutagenesis, and with reduced positive selection on somatic <i>TP53</i> mutations, despite confounding by chemotherapy treatment in one individual. <i>DNMT3A</i> and <i>TET2</i> mutations were positively selected and <i>GATA2</i> mutations were negatively selected across the cohort, independent of the p.R181H status. Extensive multi-tissue sampling of 22 non-cancerous and 6 cancerous samples was also performed at autopsy in one individual with LFS who succumbed to esophageal cancer. Cross-tissue analysis revealed excess mutations in sun-exposed skin, esophagus and chronically-inflamed stomach tissue, and highly parallel emergence of mutations in the p.R248 hotspot of <i>TP53</i> across most (18/28) tissue samples. Most somatic <i>TP53</i> mutations in LFS that could be assessed for phase arose on the chromosomal copy lacking the p.R181H variant. Our study reveals how the germline p.R181H variant reshapes baseline somatic mutation and selection in normal tissues and highlights the importance of understanding early somatic evolution in LFS prior to cancer development and treatment.