DOP109Investigating the role of driver mutations in Ulcerative Colitis through paired DNA and RNA sequencing of single colonic crypts

Abstract Background Whilst most mutations cells accumulate are neutral, some can alter cellular functions leading to positive selection and clonal expansions. In inflammatory bowel disease (IBD), epithelial injury-repair cycles raise mutation rates and favour clones with growth advantages. Previous studies 1-3 showed that clones with IL-17 signalling pathway mutations, including PIGR, NFKBIZ and ZC3H12A, recurrently expand in IBD despite being uncommon in colorectal cancer. Organoid work suggested that NFKBIZ mutations provide resistance to IL-17A-induced apoptosis and loss of PIGR was hypothesised to worsen inflammation by promoting dysbiosis. Thus, a positive feedback loop could be established, in which mutant clones drive tissue damage while expanding further. We aim to improve our understanding of how clonal expansions and driver genes influence disease biology in IBD. To achieve this, we performed paired DNA-RNA sequencing of individual human colonic crypts to characterise the transcriptional consequences of driver mutations. Methods Multiomics laser-microdissection of single colonic crypts was possible with an improved low-input protocol. Fresh biopsies were collected during colonoscopies from inflamed and uninflamed colon regions. We obtained over 200 new biopsies from 25 patients which, combined with prior data, enabled selection analysis across 1500+ crypts from 50+ patients. We generated 700+ single crypts with paired DNA-RNA data, enabling differential expression analysis (DGE) between mutant and wild-type crypts. Results Our work confirms elevated mutation rates and characteristic mutational signatures associated with azathioprine treatment. Beyond identifying PIGR, NFKBIZ and ZC3H12A as strongly selected genes, consistent with prior work, we show that NFKBIZ is also highly prevalent in individuals of European ancestry. We also expand the catalogue of putative driver genes, particularly highlighting IL13RA1, ELF3 and MED12. DGE analysis suggests mutations in some genes e.g., NFKBIZ, may protect crypts from inflammatory effects, while others remain influenced by inflammation, potentially expanding primarily through increased crypt fission. Crypts with cancer-related mutations display elevated inflammatory signalling and epithelial-to-mesenchymal transition features compared to crypts with IBD-characteristic mutations e.g., ZC3H12A. These findings reveal how distinct driver mutations shape epithelial behaviour in IBD. Conclusion Overall, paired DNA and RNA sequencing of single colonic crypts enabled us to broaden the catalogue of driver genes implicated in IBD and characterise their transcriptomic consequences in human tissue for the first time. Thus, improving our understanding of how mutations shape the epithelial landscape in IBD. 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Conflict of interest: Torra I Benach, Maria: No conflict of interest Marshall, Henry: No conflict of interest Jung, Hyunchul: No conflict of interest Abascal, Federico: No conflict of interest Lawson, Andrew: No conflict of interest Hu, May: No conflict of interest Goodwin, Scott: No conflict of interest Garri, Wendy: No conflict of interest Harris, Bradley: Personal Fees: I have received honoraria for a presentation at a BridgeBio board meeting. Garcia Salinas, Isaac: No conflict of interest Jarman, Georgee: No conflict of interest El Garwany, Omar: No conflict of interest Hooks, Yvette: No conflict of interest Sanghvi, Rashesh: No conflict of interest O’Neill, Laura: No conflict of interest Yogakanthi, Saiumaeswar: No conflict of interest Brezina, Biljana: No conflict of interest Chappel, Lia: No conflict of interest Cotobal Martin, Cristina: No conflict of interest Rahbari, Raheleh: No conflict of interest Raine, Timothy: Grant: Abbvie, Takeda Personal Fees: TR has received research/educational grants and/or speaker/consultation fees from Abbvie, Alfasigma, Arena, Aslan, AstraZeneca, Boehringer-Ingelheim, BMS, Celgene, Domain Therapeutics, Eli Lilly, Ferring, Galapagos, Gilead, GSK, Heptares, LabGenius, Janssen, MonteRosa, Mylan, MSD, Novartis, Numab, Pfizer, Roche, Sandoz, Scientia, Takeda, UCB and XAP therapeutics Anderson, Carl: Personal Fees: Carl Anderson (CAA) has received consultancy or speaker fees from Genomics plc, BridgeBio Ltd and Glaxo Smith Kline.