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.

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.

Introduction

Polygenic scores (PGS) use the results of genome-association studies (GWAS) to calculate the aggregate effect of trait-associated variants carried by the individual. To date, PGS have primarily been used to model the individual risk of disease onset using summary statistics from susceptibility GWAS, typically with modest clinical relevance. For example, we can identify 1.5% of the population with a threefold increased risk of IBD onset (Khera et al.).
Much less is known about the impact of this onset PGS burden on the presentation and progression of patients with diagnosed IBD. Previous studies have failed to identify an effect of onset PGS on early onset vs late onset presentation (Serra et al.) and CD severity (Lee et al.).

Aims & Methods

We hypothesised that an increased genetic susceptibility burden for IBD has a direct impact on the severity of an individual's IBD disease course.
We genotyped 8179 patients with IBD (3688 CD; 4491 UC), linking them with registry data that captures medical interactions throughout patients' lifetimes. Patients who required 2+ IBD-related hospitalizations, 2+ IBD-related major surgeries, 1 IBD-related hospitalization and 1 IBD-related major surgery or use of systemic corticosteroids (≥5000 mg) in the first 3 years of diagnosis were defined as severe. We conducted a GWAS comparing severe patients (N=1203 UC; N=1165 CD) to those who had a less severe disease course (N=3288 UC; N=2523 CD).
We calculated individual PGS for UC and CD among individuals using the PSG scores from Middha et al. (based on De Lange et al. summary statistics) and association tests controlling for age of onset, year of onset, sex and diagnostic cohorts.

Results

We identified a significant difference in PGS between individuals with less severe and severe disease course for both CD (beta=0.23, p=2.17×10^-9) and UC (beta=0.3, p<1×10^-16).
Next, we split the cohort into quintiles by PGS and generated a stratified Kaplan-Meier curve for time to hospitalisation and surgery. Remarkably, for both outcomes and in both CD and UC the curves were ranked according to their PGS quintiles with lower ones consistently having a lower risk of adverse outcomes compared to the higher quintiles, suggesting an additive effect of genetic susceptibility on IBD severity.
In a time to event proportional hazards analysis, compared to the 20% of patients with lowest PGS, the top 20% had a much quicker time to hospitalization (CD: HR=1.79 (1.55-2.07), p=1.82×10^-15; UC: 1.68 (1.46-1.94), p=7.92×10^-13) and surgery (CD: 2.76 (2.19-3.47), p=4.69×10^-18; UC: 2.03 (1.54-2.68), p=5.57×10^-7).

Conclusion

To our knowledge, this work is the first systematic evidence of a link between IBD disease susceptibility genetics and the severity of the phenotype – patients with a higher genetic burden suffer from a more severe IBD presentation and quicker progression, suggesting that the etiology of the disease susceptibility and severity is not orthogonal.
PGS will not become the singular tool for predicting patient severity, however these are useful to add to the prognostic toolkit. Genotyping required to calculate the PGS can be done for less than €100 and can be combined with known clinically actionable pharmacogenomic variant tests and, in the future, additional targeted PGS of severity, drug response and comorbidity risk.

References

Khera, Amit V., et al. "Genome-wide polygenic scores for common diseases identify individuals with risk equivalent to monogenic mutations." Nature Genetics 50.9 (2018): 1219-1224.
Serra, Eva Gonçalves, et al. "Somatic mosaicism and common genetic variation contribute to the risk of very-early-onset inflammatory bowel disease." Nature Communications 11.1 (2020): 995.
Lee, James C., et al. "Genome-wide association study identifies distinct genetic contributions to prognosis and susceptibility in Crohn's disease." Nature Genetics 49.2 (2017): 262-268.
Middha, Pooja, et al. "Polygenic risk score for ulcerative colitis predicts immune checkpoint inhibitor-mediated colitis." Nature Communications 15.1 (2024): 2568.
De Lange, Katrina M., et al. "Genome-wide association study implicates immune activation of multiple integrin genes in inflammatory bowel disease." Nature Genetics 49.2 (2017): 256-261.