Mutations in ARMC9, which Encodes a Basal Body Protein, Cause Joubert Syndrome in Humans and Ciliopathy Phenotypes in Zebrafish

Abstract

Joubert syndrome (JS) is a recessive neurodevelopmental disorder characterized by hypotonia, ataxia, abnormal eye movements, and variable cognitive impairment. It is defined by a distinctive brain malformation known as the "molar tooth sign" on axial MRI. Subsets of affected individuals have malformations such as coloboma, polydactyly, and encephalocele, as well as progressive retinal dystrophy, fibrocystic kidney disease, and liver fibrosis. More than 35 genes have been associated with JS, but in a subset of families the genetic cause remains unknown. All of the gene products localize in and around the primary cilium, making JS a canonical ciliopathy. Ciliopathies are unified by their overlapping clinical features and underlying mechanisms involving ciliary dysfunction. In this work, we identify biallelic rare, predicted-deleterious ARMC9 variants (stop-gain, missense, splice-site, and single-exon deletion) in 11 individuals with JS from 8 families, accounting for approximately 1% of the disorder. The associated phenotypes range from isolated neurological involvement to JS with retinal dystrophy, additional brain abnormalities (e.g., heterotopia, Dandy-Walker malformation), pituitary insufficiency, and/or synpolydactyly. We show that ARMC9 localizes to the basal body of the cilium and is upregulated during ciliogenesis. Typical ciliopathy phenotypes (curved body shape, retinal dystrophy, coloboma, and decreased cilia) in a CRISPR/Cas9-engineered zebrafish mutant model provide additional support for ARMC9 as a ciliopathy-associated gene. Identifying ARMC9 mutations as a cause of JS takes us one step closer to a full genetic understanding of this important disorder and enables future functional work to define the central biological mechanisms underlying JS and other ciliopathies.

ARMC9 and TOGARAM1 define a Joubert syndrome-associated protein module that regulates axonemal post-translational modifications and cilium stability

“…Single larvae were lysed for genotyping which revealed a very high mutation efficiency (94% of sequenced clones from 7 larvae had small insertions-deletions, the majority of which were frameshift mutations). Among the mutant F0 fish, a striking scoliosis phenotype was observed in juveniles (Supplemental Figure 5E), reminiscent of other ciliopathy mutants including armc9 CRISPR-F0 fish (24). Taken together, these results confirm that loss of togaram1 causes ciliopathy phenotypes in zebrafish and supports a role for togaram1 in ciliary function.…”

“…In this study, we identified a new JBTS-associated protein module that can be distinguished physically and functionally from the previously proposed JBTS protein network at the transition zone of primary cilia (41). Several components of this new module localize at the ciliary basal body (24) and at the proximal end of the ciliary axoneme (25,42). Mutations in the genes encoding two directly interacting members of the module, ARMC9 and TOGARAM1, result in defects in cilium length, microtubule post-translational modifications (acetylation and polyglutamylation), and ciliary stability in patient-derived fibroblasts, zebrafish mutants, and genetically edited hTERT-RPE1 cell lines (Summary Figure 9).…”

“…This unique composition is achieved in and assess its role in the pathology of JBTS, we map the interaction domains and evaluate cellular defects in cultured human cells and zebrafish mutants. We find that the proteins of this module, previously shown to associate with microtubules (24,(29)(30)(31), are required for appropriate post-translational modification of ciliary microtubules and cilium stability, processes that are compromised by loss of both ARMC9 and TOGARAM1.…”

“…This is consistent with a dominant negative effect of the TOG3 domain variant, since this effect is observed in control cells expressing endogenous TOGARAM1. Since both long and short cilia have been identified in fibroblasts from patients with different genetic causes of JBTS, no simple correlation between cilium length and JBTS disease mechanism can be made (23,24,57,58). Work in Tetrahymena indicated that TOGARAM1 and ARMC9 orthologs may have opposite effects on B-tubule length (21).…”

Tectonic Proteins Are Important Players in Non-Motile Ciliopathies

“…According to a recent study, about 55%~60% patients with Joubert syndrome are caused by the known gene mutations [62]. To date, there are about 30 genes to be associated with Joubert syndrome or Joubert syndrome related disorders (NPHP1, AHI1, ARMC9, CEP290, RPGRIP1L, TMEM67, CC2D2A, ARL13B, INPP5E, OFD1, TMEM216, CEP41, TMEM237, TCTN2, KIAA0556, KIF7, TCTN1, TMEM138, MKS1, C5ORF42, TMEM231, TCTN3, CSPP1, PDE6D, IFT172, ZNF423, TTC21B, B9D1, B9D2, and C2CD3) by target sequencing [63][64][65], indicating genetic heterogeneity of Joubert syndrome to a large extent.…”