The Forgotten Frontier: Epithelial-Based Diagnostics for Pediatric Inflammatory Bowel Disease

Pediatric inflammatory bowel disease (IBD) represents a significant clinical challenge, characterized by chronic gastrointestinal inflammation with more severe presentations than adult-onset disease. Affected children frequently experience growth failure, delayed puberty, and substantial psychosocial burden. Notably, monogenic IBD, resulting from mutations in a single gene, predominantly manifests in the pediatric population, demanding specialized diagnostic and therapeutic approaches. Despite the recognized importance of intestinal barrier dysfunction in IBD development, research has predominantly focused on dysregulation of immune cells, leaving intrinsic epithelial defects poorly understood and therapeutically underexploited. This thesis addresses this critical knowledge gap by employing patient-derived three-dimensional intestinal epithelial organoids (IEOs) and epithelial cell lines to investigate how inherent epithelial abnormalities, particularly those arising from monogenic mutations, contribute to pediatric IBD pathogenesis and may serve as personalized therapeutic targets.
We established a comprehensive cohort of IEOs derived from 94 pediatric IBD patients and 46 controls, generating the largest RNA sequencing dataset for pediatric IBD to date. Transcriptional profiling was performed at baseline and following bacterial lysate challenge to simulate immunological stress. Co-expression network analysis revealed that the IBD cohort lacked uniform transcriptional signatures. Instead, distinct gene expression patterns emerged in response to bacterial stimulation and within specific monogenic IBD subtypes, including variants in TTC7A, STXBP2, and LRBA. Notably, different TTC7A variants exhibited diverse transcriptional profiles, whereas STXBP2 and LRBA mutations shared overlapping signatures characterized by disrupted epithelial homeostasis, IL-1 signaling activation, and altered zinc trafficking pathways, suggesting epithelial-intrinsic mechanisms of injury.
Given TTC7A's critical role in epithelial function, we conducted detailed investigations of three pediatric patients harboring heterozygous TTC7A missense variants. Patient-derived IEOs demonstrated remarkable heterogeneity in growth characteristics, morphology, apoptosis rates, forskolin-induced swelling responses, apicobasal polarity, and therapeutic responses to ROCK inhibitor and leflunomide. These observations revealed substantial variability in both clinical outcomes and molecular/cellular phenotypes across different TTC7A variants, emphasizing the necessity for variant-specific therapeutic strategies.
To systematically characterize TTC7A variant effects, we overexpressed 11 individual missense mutations in genome-edited Caco-2 epithelial cell lines lacking endogenous TTC7A. Through integrated RNA sequencing and imaging flow cytometry, we assessed transcriptional changes, protein expression, and endoplasmic reticulum stress responses. Our analyses revealed that specific mutations produced unique phenotypes, some displayed similar effects, while others showed no detectable abnormalities. Based on phenotypic severity, we developed a classification system for TTC7A variants to facilitate clinical interpretation and guide targeted molecular therapy development.
Collectively, this thesis provides crucial insights into epithelial dysfunction in early-onset IBD through comprehensive transcriptomic and functional characterization. By identifying distinct epithelial phenotypes that are associated with specific genotypes, particularly TTC7A variants, we have uncovered novel potential therapeutic targets. These findings establish a foundation for personalized treatment strategies applicable not only to monogenic IBD cases but also to idiopathic IBD patients exhibiting comparable epithelial molecular signatures, potentially benefiting patients across diverse age groups and disease etiologies.