TTC7A mutations disrupt intestinal epithelial apicobasal polarity
Amélie E. Bigorgne, … , Hans Clevers, Geneviève de Saint Basile
Amélie E. Bigorgne, … , Hans Clevers, Geneviève de Saint Basile
Published December 2, 2013
Citation Information: J Clin Invest. 2014;124(1):328-337. https://doi.org/10.1172/JCI71471.
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Research Article Gastroenterology

TTC7A mutations disrupt intestinal epithelial apicobasal polarity

Abstract

Multiple intestinal atresia (MIA) is a rare cause of bowel obstruction that is sometimes associated with a combined immunodeficiency (CID), leading to increased susceptibility to infections. The factors underlying this rare disease are poorly understood. We characterized the immunological and intestinal features of 6 unrelated MIA-CID patients. All patients displayed a profound, generalized lymphocytopenia, with few lymphocytes present in the lymph nodes. The thymus was hypoplastic and exhibited an abnormal distribution of epithelial cells. Patients also had profound disruption of the epithelial barrier along the entire gastrointestinal tract. Using linkage analysis and whole-exome sequencing, we identified 10 mutations in tetratricopeptide repeat domain–7A (TTC7A), all of which potentially abrogate TTC7A expression. Intestinal organoid cultures from patient biopsies displayed an inversion of apicobasal polarity of the epithelial cells that was normalized by pharmacological inhibition of Rho kinase. Our data indicate that TTC7A deficiency results in increased Rho kinase activity, which disrupts polarity, growth, and differentiation of intestinal epithelial cells, and which impairs immune cell homeostasis, thereby promoting MIA-CID development.

Authors

Amélie E. Bigorgne, Henner F. Farin, Roxane Lemoine, Nizar Mahlaoui, Nathalie Lambert, Marine Gil, Ansgar Schulz, Pierre Philippet, Patrick Schlesser, Tore G. Abrahamsen, Knut Oymar, E. Graham Davies, Christian Lycke Ellingsen, Emmanuelle Leteurtre, Brigitte Moreau-Massart, Dominique Berrebi, Christine Bole-Feysot, Patrick Nischke, Nicole Brousse, Alain Fischer, Hans Clevers, Geneviève de Saint Basile

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Figure 5

Rescue of normal phenotype of MIA-CID patient–derived organoids and fibroblasts by ROCK inhibition or WT-TTC7A expression.

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Rescue of normal phenotype of MIA-CID patient–derived organoids and fibr...
(A–E) Immunochemical staining of α6 integrin (green) and F-actin (pink) of intestinal organoids. Control- (A and C) and C3-derived ileum organoids (B, D, and E) were cultured for 5 days with or without Y-27632. Nuclei were stained with DAPI. The inset in E shows abnormal F-actin deposition. Scale bars: 50 μm. (F) Quantification of apicobasal polarity after 5 days of growth with or without Y-27632. Normal polarity, epithelial monolayer with α6 integrin outside and F-actin on the luminal side; aberrant polarity, multilayered epithelium with disturbed α6 integrin and/or F-actin deposition; inverted polarity, F-actin facing outward, α6 integrin clustered inside. 50 organoids total were counted per condition. (G) Western blot analysis of ROCK and downstream effectors P-ERM and P-MLC in C3- and control-derived fibroblasts with or without Y-27632 treatment. Lanes were run on the same gel but were noncontiguous. Data are representative of 2 independent experiments. (H) Rescue of the phenotype of MIA-CID patient fibroblasts by WT-TTC7A expression. E3- or control-derived fibroblasts were transiently transfected with WT-TTC7A or empty vector prior to P-ERM and P-MLC expression analysis by Western blot 24 hours later. Actin was used as a loading control. Data are representative of 3 independent experiments. (I) Recovery of normal adhesion and proliferation of MIA-CID patient fibroblasts by WT-TTC7A expression. Measurement of adhesion followed by proliferation by XCELLigence technology of 2 patient- and 2 control-derived fibroblasts transfected with WT-TTC7A or empty vector. Data are representative of 2 different experiments.