P0089Patient-derived TTC7A mutations reveal the role of TTC7A in maintaining intestinal barrier integrity and epithelial polarity

Background Mutations in the Tetratricopeptide Repeat Domain 7A (TTC7A) gene cause an autosomal recessive congenital disorder characterized by severe intestinal inflammation, multiple intestinal atresia, and immunodeficiency which belongs to the spectrum of Very Early Onset Inflammatory Bowel Disease (VEO-IBD). TTC7A functions as a multiprotein scaffold essential for cytoskeletal organization, cell polarity and cell survival, in line with structural abnormalities in intestinal tissues from the affected patients. The mechanisms by which TTC7A modulates paracellular permeability remain largely unknown. This study aimed to characterize the architectural alterations in TTC7A-deficient enterocytes that underlie the loss of intestinal epithelial homeostasis. Methods Two patients with TTC7A deficiency, P1 (c.1709A>G; p.H570R) and P2 (c.189C>G; p.D63E and c.412C>T; p.R138X), with a phenotype of multiple intestinal atresia, gut inflammation and agammaglobulinemia were followed in the Paediatric Gastroenterology Department in Lyon, France. TTC7A-deficient Caco-2 cells were generated using CRISPR/Cas9 system. Patient’s mutations were introduced into a GFP-TTC7A plasmid by site-directed mutagenesis. TTC7A-KO Caco-2 cells were complemented with wild-type or mutant TTC7A constructs and cultured in Matrigel (5 days) to allow cyst formation, or on permeable supports (21 days) to obtain differentiated monolayers. Barrier function was assessed by transepithelial electrical resistance (TEER) and Lucifer Yellow permeability (LYP) assays. Immunostaining for actin, E-cadherin and ezrin was performed to evaluate epithelial architecture and polarity. Results Caco-2 TTC7A KO cysts complemented with patient-specific TTC7A mutations exhibited a significant defect in lumen formation, which was rescued by complementation with WT TTC7A, suggesting impaired cell polarization. In control cysts, E-cadherin staining was sharp and localized to the cell membrane whereas it was dull and diffuse in Caco-2 TTC7A KO. In monolayer cultures, TTC7A KO cells displayed significantly reduced TEER and increased LYP, indicating an increased monolayer permeability. E-cadherin staining appeared irregular and poorly defined in Caco-2 TTC7A KO cells, confirming the disruption of adherens junctions. Moreover, ezrin staining was mislocalized in Caco-2 TTC7A KO monolayers compared to controls, further emphasizing the role of TTC7A in epithelial cell polarization. Conclusion TTC7A is crucial for maintaining epithelial polarity and barrier integrity. Its loss leads to defective junctional organization and increased epithelial permeability, which may contribute to intestinal inflammation and epithelial dysfunction observed in TTC7A-deficient patients. Conflict of interest: Mrs. Petit, Céline: No conflict of interest Rozieres, Aurore: None Viret, Christophe: No conflict of interest Peretti, Noel: No conflict of interest Faure, Mathias: No conflict of interest Nancey, Stéphane: board membership and lecturing fees from Abbvie, Takeda, Celltrion Healthcare, Pfizer, Galapagos, Johnson & Jonshon, Lilly, Fresenius, Amgen, Medac, MSD. Duclaux-Loras, Rémi: No conflict of interest