TBC1D32 variants disrupt retinal ciliogenesis and cause retinitis pigmentosa
Béatrice Bocquet, Caroline Borday, Nejla Erkilic, Daria Mamaeva, Alicia Donval, Christel Masson, Karine Parain, Karolina Kaminska, Mathieu Quinodoz, Irene Perea-Romero, Gema Garcia-Garcia, Carla Jimenez-Medina, Hassan Boukhaddaoui, Arthur Coget, Nicolas Leboucq, Giacomo Calzetti, Stefano Gandolfi, Antonio Percesepe, Valeria Barili, Vera Uliana, Marco Delsante, Francesca Bozzetti, Hendrik P.N. Scholl, Marta Corton, Carmen Ayuso, Jose M. Millan, Carlo Rivolta, Isabelle Meunier, Muriel Perron, Vasiliki Kalatzis
Béatrice Bocquet, Caroline Borday, Nejla Erkilic, Daria Mamaeva, Alicia Donval, Christel Masson, Karine Parain, Karolina Kaminska, Mathieu Quinodoz, Irene Perea-Romero, Gema Garcia-Garcia, Carla Jimenez-Medina, Hassan Boukhaddaoui, Arthur Coget, Nicolas Leboucq, Giacomo Calzetti, Stefano Gandolfi, Antonio Percesepe, Valeria Barili, Vera Uliana, Marco Delsante, Francesca Bozzetti, Hendrik P.N. Scholl, Marta Corton, Carmen Ayuso, Jose M. Millan, Carlo Rivolta, Isabelle Meunier, Muriel Perron, Vasiliki Kalatzis
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Research Article Genetics Ophthalmology

TBC1D32 variants disrupt retinal ciliogenesis and cause retinitis pigmentosa

Abstract

Retinitis pigmentosa (RP) is the most common inherited retinal disease (IRD) and is characterized by photoreceptor degeneration and progressive vision loss. We report 4 patients presenting with RP from 3 unrelated families with variants in TBC1D32, which to date has never been associated with an IRD. To validate TBC1D32 as a putative RP causative gene, we combined Xenopus in vivo approaches and human induced pluripotent stem cell–derived (iPSC-derived) retinal models. Our data showed that TBC1D32 was expressed during retinal development and that it played an important role in retinal pigment epithelium (RPE) differentiation. Furthermore, we identified a role for TBC1D32 in ciliogenesis of the RPE. We demonstrated elongated ciliary defects that resulted in disrupted apical tight junctions, loss of functionality (delayed retinoid cycling and altered secretion balance), and the onset of an epithelial-mesenchymal transition–like phenotype. Last, our results suggested photoreceptor differentiation defects, including connecting cilium anomalies, that resulted in impaired trafficking to the outer segment in cones and rods in TBC1D32 iPSC-derived retinal organoids. Overall, our data highlight a critical role for TBC1D32 in the retina and demonstrate that TBC1D32 mutations lead to RP. We thus identify TBC1D32 as an IRD-causative gene.

Authors

Béatrice Bocquet, Caroline Borday, Nejla Erkilic, Daria Mamaeva, Alicia Donval, Christel Masson, Karine Parain, Karolina Kaminska, Mathieu Quinodoz, Irene Perea-Romero, Gema Garcia-Garcia, Carla Jimenez-Medina, Hassan Boukhaddaoui, Arthur Coget, Nicolas Leboucq, Giacomo Calzetti, Stefano Gandolfi, Antonio Percesepe, Valeria Barili, Vera Uliana, Marco Delsante, Francesca Bozzetti, Hendrik P.N. Scholl, Marta Corton, Carmen Ayuso, Jose M. Millan, Carlo Rivolta, Isabelle Meunier, Muriel Perron, Vasiliki Kalatzis

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Figure 8

EMT-like phenotype in human TBC1D32 iPSC-derived RPE.

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EMT-like phenotype in human TBC1D32 iPSC-derived RPE. IF studies and MIP...
IF studies and MIP confocal imaging of control (A) and TBC1D32 (B) iPSC-derived RPE using antibodies directed against vimentin and MERTK. The boxed area in B indicates areas of upregulated vimentin and disrupted MERTK expression. Scale bars = 20 µm. IF studies and MIP confocal imaging of control (C) and TBC1D32 (D) iPSC-derived RPE using antibodies directed against SMA and ZO1. Scale bars = 20 μm. qPCR analysis of CDH1 (E), CDH3 (F), CTNNB1 (G), SNAI1 (H), and FN1 (I) in control and TBC1D32 RPE. Data are represented as mean ± SEM; n = 3 technical replicates; *P < 0.05; 2-tailed Mann-Whitney test. Representative Western blot analysis of E-cadherin (J), P-cadherin (K), and β-catenin (L) expression and quantification relative to the β-actin loading control; the same membrane was hybridized with 2 different primary antibodies in panels J and L. Data represented as mean ± SEM; n = 3 blots; *P < 0.05; 2-tailed Mann-Whitney test. (M) Representative Western blot analysis and quantification of 2 independent blots of β-catenin distribution in the cytosol versus membrane fractions of control and TBC1D32 iPSC-derived RPE relative to the β-actin loading control and displayed on a logarithmic scale. Data are represented as mean ± SEM.