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.

Figure 1. Immunological characteristics of MIA-CID patients.

(A) Circulating blood cell counts. Shown are lymphocyte, monocyte, and neutrophil counts as well as total CD3⁺, CD4⁺, and CD8⁺ T cell counts. (B) Thymus pathology. H&E staining (top) revealed poor corticomedullary demarcation and a paucity of lymphocytes and Hassall’s bodies in A4 versus a control subject. Also shown is immunohistochemical staining of CD3, CD8, and CD4 as well as CK5 and CK8 staining for medullary (m) and cortical (c) thymic epithelial cells, respectively. (C) Mediastinal lymph node. H&E staining and immunohistochemical staining for CD20, CD3, and CD68 in a control subject and in E3 after autopsy. Original magnification, ×50 (B), ×400 (B, enlarged H&E views), ×200 (C, H&E), ×100 (C, immunostaining).

Figure 2. Pedigree and TTC7A mutations in MIA-CID families.

(A) MIA-CID family pedigree. Square, male; circle, female; completely filled symbols, affected individuals; half-filled symbols, heterozygous carriers. Affected individuals who did not undergo genetic testing are indicated in gray. Slashes indicate deceased persons; double horizontal lines indicate consanguinity. A7 has been described previously (8). (B) TTC7A gene mutations and their predicted effect on the TTC7A protein. TPRs are indicated on the protein sequence, in accordance with NCBI human sequence annotation (accession no. Q9ULTO-1).

Figure 3. Immunohistologic studies of the digestive tract of MIA-CID patients.

(A) Pathology studies of the stomach, intestine, and colon. H&E staining of the stomach (antrum) in a control subject (12 years old) and A9 (6 months old). Asterisks denote pseudostratified epithelium; arrow denotes apoptosis in glands. Compared with the proximal small intestine in a control subject (7 days old), that of E3 (2 days old) showed cysts and protrusions of multilayered epithelial cells as well as infiltration with eosinophils. Colon was stained with H&E in a control subject (12 years old) and A9 (6 months old). (B) Immunochemical staining was performed to detect markers for epithelial differentiation as well as AP — as a marker for intestinal epithelium function — in the proximal small intestine in a control subject (1 day old) and patient (2 days old). Staining showed lamina propria infiltration by T cells (CD3) and macrophages (CD68) and Ki67-positive, proliferating epithelial cells as well as expression of CK20, villin, and AP. Original magnification, ×200 (A, stomach and colon, and B), ×50 (A, small intestine), ×400 (A, small intestine, enlarged view).

Figure 4. MIA-CID patient–derived intestinal organoids show defects in proliferation, differentiation, and epithelial polarity associated with abnormally active ROCK pathway.

(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.

Figure 5. Rescue of normal phenotype of MIA-CID patient–derived organoids and fibroblasts by ROCK inhibition or WT-TTC7A expression.

(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.

Figure 6. ROCK targets responsible for cytoskeleton assembly.

Modulation of cytoskeleton assembly is required for the coordination of cell adhesion, polarization and migration. The active form of RhoA (RhoA-GTP) activates ROCK and interacts with the integrin intracellular signaling pathway. RhoA also modulates cytoskeleton reorganization via focal adhesion kinase (FAK) activation and front-rear polarity via phosphorylation of phosphatase and tensin homolog (PTEN). Phosphorylation of ROCK’s downstream effector MLC is required for the assembly of actomyosin complexes. ROCK downregulates the MLC phosphatase (MLCPase), resulting in an increase in phosphorylated MLC, cell contraction, actin organization, stress fiber formation, and focal adhesions, which confer contractility and migration properties to the cell. Phosphorylation of ROCK’s downstream effector ERM has a role in microvilli formation. Phosphorylated LIM domain kinase (LIM-K) phosphorylates and thus deactivates the actin-depolymerizing protein cofilin. Y-27632 blocks Rho-induced actomyosin activation. These data support a role for TTC7A in the downregulation of ROCK activity. Activation and inactivation are shown as arrowheads and blunted lines, respectively. MLCK, myosin light chain kinase; NHE1, Na⁺/H⁺ exchange protein.