Project Summary/Abstract If we understand the somatic mosaicism of healthy tissues and how this changes through pathogenesis, we will be better equipped to screen for, monitor, and treat disease. Somatic mutations play a central role in disease pathogenesis, from developmental syndromes to cancer, and there is growing consensus that somatic mosaicism in healthy tissue influences fitness and disease predisposition. New methodological breakthroughs enable the accurate detection of somatic mutations, mutation rates, and mutational signatures. Establishing a biological baseline of mosaic profiles in healthy and diseased tissue can facilitate the prediction of genetically-driven disease states and the optimization of disease management. By studying somatic mosaicism in healthy and diseased colon, I will gain insight into colorectal pathogenesis and establish a research framework to apply to other tissues. The colon is ideal for mosaic research, given its monoclonal crypts, elevated mutation rate, and well-studied architecture. There are also significant associations between somatic mosaicism and colorectal disease: somatic mutations are causative in colorectal cancer (CRC) and are proposed to contribute to inflammatory bowel disease (IBD). These diseases have well-established regional presentations, with distinct pathologies along the length of the colon, such as “right” and “left” CRC and differently-clustered subtypes of IBD. However, the genetic etiologies underlying these pathologies are unknown, given previous limitations in mosaic research. This proposal will examine the spatiotemporal dynamics of colonic mosaicism in the context of healthy tissue and pathogenesis. Evaluating mosaicism across the length of the colon will provide insight into whether region-specific mutagenesis, either endogenous or exogenous, explains variation in regional pathogenesis. A preliminary study by Lee-Six et al. reported variation in mutational signature proportions across the colon. However, this analysis was limited by its low sequencing coverage (~15x) and examination of a small number of colonic subregions in a small cohort. By leveraging access to cadaver tissue and colonoscopy samples in tandem with biological and computational advances, I will quantify mosaicism with greater resolution and capacity for hypothesis testing. This proposal will determine individual and cohort-based mutational rates by anatomic subsite of the colon and extract mutational signatures that indicate mutagenic processes, such as cellular proliferation or microbiome variation. I hypothesize that midgut- and hindgut-derived colonic structures will harbor distinct mutational signatures from unique mutagenesis and regionally distinct mutation rates. These mutational landscapes will likely overlap regionally with the spatially-linked findings of CRC and IBD, suggesting a role in pathogenesis. Completion of this proposal will then serve as an ideal framework to examine regional variation a...