Multi-omic sequencing reveals distinctive gene expression and epigenetic alterations associated with primary sclerosing cholangitis development in treatment-naïve paediatric ulcerative colitis

Background: Primary sclerosing cholangitis (PSC) is a progressive cholestatic disease with up to 80% of patients also suffering from ulcerative colitis (PSC-UC). The difficulty in the diagnosis along with the increased risk for developing cancer represent a clinical challenge. Furthermore, the precise molecular factors regulating the phenotype of this disease subtype remain unknown. Methods: We applied methyl-capture sequencing and mRNA sequencing to colonic mucosal biopsies from 3 groups of treatment-naïve children at diagnosis from the DOCHAS study - UC (n=10), PSC-UC (n=10) and healthy controls(n=10). Results: Differential gene expression between UC and PSC-UC identified disease-associated genes that were either up- or down-regulated in UC or PSC relative to controls. Specifically, expression of these genes was regulated by master transcriptional regulators (pro-caspases, IL7RA) and transcription factors (AR, p53, JUND, CEBPA). Importantly, gene expression comparison between UC vs PSC-UC revealed 4 up- and 4 down-regulated genes in PSC-UC patients. Notably, RAB31 and TENM3 identified as upregulated in the PSC-UC patients, are also significantly up-regulated in gastrointestinal (GI) cancers. Differential methylation analysis between controls biopsies vs PSC-UC and UC demonstrated >1000 differentially methylated regions (DMRs) with a large proportion of these DMRs enriched in enhancer regions. Conclusion: Our study, for the first time, identifies distinct gene expression and DNA methylation alterations that differentiate UC from PSC-UC at diagnosis in treatment-naïve paediatric patients, some of which are associated with GI cancers. These findings suggest the potential utility of these molecular markers as predictive biomarkers for PSC development in UC or for assessing dysplasia risk in PSC-UC. Further validation in larger patient cohorts is warranted. Overall design: The study analyzed colonic mucosal biopsies from 30 treatment-naïve pediatric patients, grouped into primary sclerosing cholangitis with ulcerative colitis (PSC-UC), ulcerative colitis (UC-only), and healthy controls, with 10 patients in each group. Samples included both disease and control groups, enabling comparative analyses. Experimental conditions included treatment-naïve status and matched clinical parameters such as age and sex, minimizing confounding factors. Variables investigated were gene expression and DNA methylation alterations to uncover molecular differences between PSC-UC and UC, and the regulatory pathways driving these differences. The inclusion of healthy controls provided a baseline reference for identifying disease-specific changes.