Investigating the role of circulating cell-free dna as a mechanistic biomarker in inflammatory bowel disease: development of an integrated precision-medicine enabled platform in Scotland

Abstract

BACKGROUND: Circulating cell-free DNA (cfDNA) represents a class of biological molecules whose role in inflammation remains poorly understood. Inflammatory bowel disease (IBD), from ulcerative colitis to Crohn’s disease, comprises a spectrum of chronic immune-mediated conditions with complex pathogenic mechanisms that manifest primarily as gut-mucosal inflammation. There remains an unmet need that requires a greater understanding of disease mechanisms to find better treatments for patients.

HYPOTHESIS/METHODS: cfDNA is a biomarker in IBD that captures a dimension of disease activity not covered by current clinical biomarkers. Mitochondrial cfDNA may identify a subset of patients whose disease is driven by immune-mediated recognition of mitochondrial cfDNA. cfDNA metagenomics may provide new insights into disease biology.

Two multi-centre translational cohort studies were set up – GI-DAMPs (cross-sectional) and MUSIC (longitudinal) and the execution of which is discussed. Clinical sampling was performed, and subsequent analysis was carried out using Qubit for total quantification, digital polymerase chain reaction (dPCR) for COX3, ND2 and GAPDH genes, fragment analysis with the Agilent BioAnalyzer, and cfDNA sequencing using both Nanopore and Illumina platforms.

RESULTS: Patients with highly active IBD (requiring admission to hospital) had significantly higher total cfDNA (median 0.52 ng/uL, Kruskal-Wallis p<0.001), mitochondrial ND2 (median 359 copies/uL, Kruskal-Wallis p<0.05) and genomic GAPDH levels (median 8.7 copies/uL, Kruskal-Wallis p<0.01) compared to patients with active disease or remission. Digital PCR techniques provide better resolution compared to Qubit. cfDNA fragment analysis shows an increase in the 160bp peak and the release of longer fragments in highly active disease, suggesting increased apoptosis and necrosis, compared to patients in remission or healthy controls. cfDNA sequencing and bioinformatic analysis were feasible. cfDNA metagenomics reveals that patients with active disease have reduced alpha diversity (median Chao1 2612, p=0.07 and median Shannon 0.06, p=0.43) and significantly different beta diversity profiles (permanova R2 0.766, p<0.01) compared to patients in remission or healthy controls.

CONCLUSION: The analysis of cfDNA with modern advances in technology is an unexplored dimension of inflammation biology. cfDNA correlates with IBD activity and further study is required to validate its use as a clinical and mechanistic biomarker. Further scientific work in cfDNA could unlock new insights into both cfDNA and IBD biology, potentially allowing the development of better mechanistic and predictive biomarkers, new therapeutics, and general insights into other inflammatory diseases.