Following the first division of the zygote, somatic mutations begin to accumulate in all human cells. Daughter cells become increasingly mutated leading to mosaic tissues, composed of genetically heterogeneous clonal units. In recent years, large scale sequencing efforts have begun to characterise the process of mutagenesis in normal tissues. These observations have improved our understanding of how somatic cells evolve oncogenic phenotypes and hinted that somatic changes may contribute to the development of age- related phenotypes and degenerative disease.

To accurately characterise the mutational processes in normal tissues, we developed nanorate sequencing (NanoSeq), a duplex sequencing protocol with error rates of less than five errors per billion base pairs, allowing for the accurate identification of mutations in single DNA molecules. Using NanoSeq we describe the mutational processes in the nuclear and mitochondrial genome of three distinct cell types across the brain and cardiac muscle. We show that post-mitotic tissues accumulate somatic mutations at comparable rates, and by similar processes to dividing cells. We explore the relationship of mutation rates to transcription and chromatin state, and reveal patterns of transcription-coupled damage and repair in neuronal genomes. In the analysis of somatic mutations in cardiomyocytes, we identify a novel mutational signature that we believe may be caused by hypoxia-induced oxidative stress. Lastly, we demonstrate that neurons isolated from Alzheimer’s disease brains have a lower mutation burden than healthy neurons.

To further investigate the role of somatic mutations in degenerative disease, we characterised somatic mutagenesis in rheumatoid arthritis and osteoarthritis. Using laser capture microdissection, we isolated 2000 microbiopsies of intimal lining, sublining, and lymphocytes for whole exome sequencing. Using these results we show that the synovium is a mostly polyclonal tissue with the capacity for large clonal expansions. We explore how the histopathology of rheumatoid arthritis and osteoarthritis relates to driver mutations, clonal expansions, and immune infiltration. Using NanoSeq, we characterise the mutational processes in isolated synovial cell types. Finally we show the power of NanoSeq for high- throughput driver discovery and identify 15 genes under positive selection in the synovium.

This thesis offers novel insights into the mutation rate of distinct cell types in healthy and disease states. This work contributes to a growing body of literature characterising somatic evolution and provides a foundation for further functional studies to directly investigate the role of mutant clones in disease states.