TTC7A mutations disrupt intestinal epithelial apicobasal polarity
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
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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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 4

MIA-CID patient–derived intestinal organoids show defects in proliferation, differentiation, and epithelial polarity associated with abnormally active ROCK pathway.

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MIA-CID patient–derived intestinal organoids show defects in proliferati...
(A) Morphologic images of human ileum–derived organoids. C3-derived cultures formed condensed cell aggregates instead of the budding processes (arrows) and central lumen (asterisk) seen in controls, depending on the presence of Y-27632 (Y). (B) Differentiation and proliferation. Dual immunofluorescence of CK20 (red; showing disrupted apicobasal polarity) and Ki67 (green; showing proliferating epithelial cells). Mutations in TTC7A were associated with strong increased differentiation and reduced proliferation. Addition of Y-27632 restored proliferation and suppressed differentiation in patient cultures, but could not rescue abnormal nuclear positioning (blue; DAPI). (C) Growth profile for control- and patient-derived organoids in the presence or absence of Y-27632. Mean organoid number after passaging of 3 independent wells each. Error bars denote SEM. **P < 0.01, ***P < 0.0001, t test. (D) Western blot analysis of patient-derived organoids showed increased ROCK activity. Membranes were probed with anti-ROCK, anti-P-ERM, anti-Rho, and anti-P-MLC. Actin was used as a loading control. (E) Analysis of cell polarization by immunostaining of α6 integrin (green) and F-actin (red) in the apical brush border and of the tight junction marker ZO-1 (green). Control- and patient-derived organoids were cultured in the absence of Y-27632. Nuclei were stained with DAPI (blue). Scale bars: 100 μm.