P0030 Multiomic Analysis Reveals Three Distinct subtypes within Perianal Crohn’s Disease, Independent of Concomitant Proctitis
Authors: Abdurahiman S , Sabino J , Verstockt S , Ferrante M , Lenfant M , Raes J , D’Hoore A , Vermeire S , Bislenghi G , Verstockt B 
Published: January 2025

Abstract

Background
Perianal fistulisation (pCD) affects ~20% of Crohn’s disease (CD) patients and often requires combined medical and surgical management. Despite significant progress in management of luminal CD, pCD continues to pose a major unmet need, emphasizing the importance of understanding the molecular and environmental drivers and to discover new therapeutic targets.

Methods
Paired biopsies from the fistula tract and rectal orifice of 81 pCD patients underwent 16S rRNA and RNA sequencing, generating multiomic profiles for both tissues. We applied Multiomics Factor Analysis (MOFA), to identify key molecular signatures, followed by hierarchical clustering1. Gene module scores were derived using Seurat to visualize expression patterns on a public single-cell RNA seq (scRNA-seq) dataset.

Results
The MOFA model showed minimal contribution from the microbiota but accounted for over 80% of transcriptomic variance. Unsupervised clustering based on MOFA identified three distinct patient clusters (C1, C2, C3) with unique molecular phenotypes, independent of concomitant or prior proctitis (p=1.0) (Fig 1A). C3 was associated with positive end of the primary axis of variation, showing a keratinization signature in the fistula, without signs of epithelial-mesenchymal transition (EMT) or rectal keratinization (Fig 1B). In contrast, C1 and C2 were linked to the opposite extreme of this axis, characterized by EMT signatures in the fistula and an absence of keratinization. The rectal profiles differed between these two clusters: C1 lacked keratinization in the rectum, while C2 exhibited concurrent keratinization (Fig 1B). The opposing pattern along the primary axis suggests that keratinization and EMT represent distinct, mutually exclusive processes in fistula pathology. Analysis of publicly available scRNA-seq data from psoriasis patients (a keratinization-hallmark disease) revealed significant upregulation of the pCD-specific keratinization gene signature in lesional keratinocytes compared to non-lesional samples, indicating a psoriasis-like keratinization process in pCD2 (Fig 2)2. Finally, microbial profiling showed no significant associations between bacterial genera and any of the identified clusters in either rectum or fistula (adj. p &gt; 0.05)

Conclusion
Our analysis identified three distinct patient clusters in pCD, revealing diverging keratinization and EMT phenotypes in fistula. This stratification was independent of proctitis and there was little to no contribution of the microbiota. Comparing the expression profiles with the ones observed in patients with psoriasis suggests a psoriasis-like keratinization process in a subset of patients. These findings uncover potential novel mechanisms in pCD pathogenesis.

References
1.Argelaguet R, Arnol D, Bredikhin D, et al. MOFA+: a statistical framework for comprehensive integration of multi-modal single-cell data. Genome Biol. 2020;21(1):111. Published 2020 May 11. doi:10.1186/s13059-020-02015-1 Francis L, McCluskey D, Ganier C, et al.
2.Single-cell analysis of psoriasis resolution demonstrates an inflammatory fibroblast state targeted by IL-23 blockade. Nat Commun. 2024;15(1):913. Published 2024 Jan 30. doi:10.1038/s41467-024-44994-w