Dysfunction of the ciliary ARMC9/TOGARAM1 protein module causes Joubert syndrome
Brooke L. Latour, … , Ronald Roepman, Dan Doherty
Brooke L. Latour, … , Ronald Roepman, Dan Doherty
Published May 26, 2020
Citation Information: J Clin Invest. 2020;130(8):4423-4439. https://doi.org/10.1172/JCI131656.
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Research Article Genetics

Dysfunction of the ciliary ARMC9/TOGARAM1 protein module causes Joubert syndrome

Abstract

Joubert syndrome (JBTS) is a recessive neurodevelopmental ciliopathy characterized by a pathognomonic hindbrain malformation. All known JBTS genes encode proteins involved in the structure or function of primary cilia, ubiquitous antenna-like organelles essential for cellular signal transduction. Here, we used the recently identified JBTS-associated protein armadillo repeat motif–containing 9 (ARMC9) in tandem-affinity purification and yeast 2-hybrid screens to identify a ciliary module whose dysfunction underlies JBTS. In addition to the known JBTS-associated proteins CEP104 and CSPP1, we identified coiled-coil domain containing 66 (CCDC66) and TOG array regulator of axonemal microtubules 1 (TOGARAM1) as ARMC9 interaction partners. We found that TOGARAM1 variants cause JBTS and disrupt TOGARAM1 interaction with ARMC9. Using a combination of protein interaction analyses, characterization of patient-derived fibroblasts, and analysis of CRISPR/Cas9-engineered zebrafish and hTERT-RPE1 cells, we demonstrated that dysfunction of ARMC9 or TOGARAM1 resulted in short cilia with decreased axonemal acetylation and polyglutamylation, but relatively intact transition zone function. Aberrant serum-induced ciliary resorption and cold-induced depolymerization in ARMC9 and TOGARAM1 patient cell lines suggest a role for this new JBTS-associated protein module in ciliary stability.

Authors

Brooke L. Latour, Julie C. Van De Weghe, Tamara D.S. Rusterholz, Stef J.F. Letteboer, Arianna Gomez, Ranad Shaheen, Matthias Gesemann, Arezou Karamzade, Mostafa Asadollahi, Miguel Barroso-Gil, Manali Chitre, Megan E. Grout, Jeroen van Reeuwijk, Sylvia E.C. van Beersum, Caitlin V. Miller, Jennifer C. Dempsey, Heba Morsy, University of Washington Center for Mendelian Genomics, Michael J. Bamshad, Genomics England Research Consortium, Deborah A. Nickerson, Stephan C.F. Neuhauss, Karsten Boldt, Marius Ueffing, Mohammad Keramatipour, John A. Sayer, Fowzan S. Alkuraya, Ruxandra Bachmann-Gagescu, Ronald Roepman, Dan Doherty

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Figure 4

armc9- and togaram1-mutant zebrafish display ciliopathy-associated phenotypes.

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armc9- and togaram1-mutant zebrafish display ciliopathy-associated phen...
(A–C) Larval phenotype demonstrating kidney cysts in armc9–/– (B) and kidney cysts and a curved body shape in togaram1–/– (C) larvae. Black boxes in A–C show ×3.5 magnification of the glomerulus region in the inset. Dashed lines highlight the kidney cysts in B and C. (D–F) Adult scoliosis phenotype in both mutants (E and F) compared with WT (D). (G–I) Immunofluorescence of the pronephric duct in 3-dpf larvae showing fewer cilia. White arrowheads in I point to the short remaining cilia in the togaram1 mutant. (J–L) Immunofluorescence of midbrain ventricles showed shortened cilia in 3-dpf armc9- and togaram1-mutant zebrafish larvae (K and L). (M–O) Immunofluorescence of 3-dpf zebrafish nose pits showed decreased cilia numbers in both mutants (N and O) compared with WT (M). (P–R) Scanning electron microscopy of 5-dpf zebrafish nose pits confirmed the reduced cilia numbers in armc9–/– (Q) and togaram1–/– (R) larvae. The controls were WT, +/+, or +/– siblings of –/–. Scale bars: 500 μm (A–C), 5 mm (D–F) and 10 μm (G–R).