Introduction
Inflammatory bowel disease (IBD) - characterized by chronic inflammation of the gastrointestinal tract – is believed to be caused by an interplay of host genetics, environment and gut microbiome. Recent studies have started investigating the role of somatic DNA mutations in immune-mediated disorders (1, 2). Somatic mutations are mutations that are not present in the germline, but are acquired later in life. We hypothesize that somatic mutations in gut tissue in inflammation-related genes could contribute to the pathogenesis of IBD.
Aims & Methods
Therefore, we investigated somatic mutation patterns in 589 intestinal biopsies of 268 IBD patients and 17 non-IBD controls. We performed whole exome sequencing (WES) on DNA isolated from blood and bulk RNA sequencing (RNAseq) on RNA isolated from intestinal biopsies. Somatic mutations were called using the mutect2 software which compares RNAseq data to the reference genome and the WES data. Somatic mutations were subsequently filtered to reduce the chance of sequencing errors (somatic mutation presence >25% of the RNAseq reads, coverage of at least 10 RNAseq reads). We used 4 gene sets of interest, monogenic very-early-onset IBD (VEO-IBD) genes (n=103), monogenic primary immunodeficiency genes (n=475), the most recent IBD GWAS candidate genes (n=318) and all genes in the IBD GWAS loci (n=3504) and tested whether somatic mutations were more often present in these gene sets compared to the rest of the genome. Finally, we used the MOLGENIS Variant Interpretation Pipeline which uses the CAPICE pathogenicity prediction score and the Clinvar variant database to annotate the called and filtered somatic mutations as benign, likely benign, VUS, likely pathogenic, or pathogenic (3).
Results
We identified 400544 somatic mutations (average 745 per biopsy). We observed a significantly higher accumulation of somatic mutations in the gut biopsies of IBD patients compared to non-IBD controls (p=2e-04). This difference was bigger when only likely pathogenic and pathogenic (LP/P) somatic mutations were considered (p=3e-14). We observed a significant enrichment of somatic mutations in the genes of all 4 gene panels in the biopsies of IBD patients compared to controls (VEO-IBD p=0.0006, PID p=0.0001, GWAS candidate p=0.0015, GWAS loci p=0.0003). Biopsies taken from inflamed tissue contained statistically significantly more LP/P somatic mutations in PID, GWAS candidate and GWAS loci genes. 273 somatic mutations identified in IBD biopsies have been previously reported to the Clinvar database, including stop-gain point mutations in HPS1 c.517C>T and HPS4 c.1132C>T, pathogenic missense mutation IL10RA c.301C>T and pathogenic missense mutation in STAT1 c.884C>A. In-depth analysis of a subset of key genes in the IBD pathogenesis revealed multiple LP/P mutations in several patients in both JAK1 (n=35 LP/P) and JAK2 (n=31 LP/P), primarily in the FERM and protein kinase domains. Additionally, we observed 21 LP/P mutations in PLCG2, a gene in which missense mutations can cause immune dysregulation. (4)
Conclusion
Here, in the largest somatic mutation study in IBD patients to date, we show that there is an increased accumulation of likely pathogenic and pathogenic somatic mutations in gut tissue, particularly in genes in which mutations can cause monogenic very-early-onset IBD and/or primary immune deficiencies. Thus, we show that that somatic mutations in the gut could contribute to the pathogenesis of IBD.
References
1) Nanki, Kosaku, Masayuki Fujii, Mariko Shimokawa, Mami Matano, Shingo Nishikori, Shoichi Date, Ai Takano, et al. 2020. “Somatic Inflammatory Gene Mutations in Human Ulcerative Colitis Epithelium.” Nature 577 (7789): 254–59. https://doi.org/10.1038/s41586-019-1844-5.
2)
Olafsson, Sigurgeir, Rebecca E. McIntyre, Tim Coorens, Timothy Butler, Hyunchul Jung, Philip S. Robinson, Henry Lee-Six, et al. 2020. “Somatic Evolution in Non-Neoplastic IBD-Affected Colon.” Cell 182 (3): 672-684.e11. https://doi.org/10.1016/j.cell.2020.06.036.3) Maassen, W. T. K., L. F. Johansson, B. Charbon, D. Hendriksen, S. van den Hoek, M. K. Slofstra, R. Mulder, et al. 2024. “MOLGENIS VIP: An Open-Source and Modular Pipeline for High-Throughput and Integrated DNA Variant Analysis.” medRxiv. https://doi.org/10.1101/2024.04.11.24305656.
4) Lange, Katrina M. de, Loukas Moutsianas, James C. Lee, Christopher A. Lamb, Yang Luo, Nicholas A. Kennedy, Luke Jostins, et al. 2017. “Genome-Wide Association Study Implicates Immune Activation of Multiple Integrin Genes in Inflammatory Bowel Disease.” Nature Genetics 49 (2): 256–61. https://doi.org/10.1038/ng.3760.
