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 use the recently identified JBTS-associated protein ARMC9 in tandem-affinity purification and yeast two-hybrid screens to identify a novel ciliary module whose dysfunction underlies JBTS. In addition to known JBTS-associated proteins CEP104 and CSPP1, we identify CCDC66 and TOGARAM1 as ARMC9 interaction partners. We show that TOGARAM1 variants cause JBTS and disrupt TOGARAM1 interaction with ARMC9. Using a combination of protein interaction analyses and characterization of patient-derived fibroblasts, CRISPR/Cas9-engineered zebrafish and hTERT-RPE1 cells, we demonstrate that dysfunction of ARMC9 or TOGARAM1 results in short cilia with decreased axonemal acetylation and polyglutamylation, but relatively intact transition zone function. Aberrant cold-and serum-induced ciliary loss in both ARMC9 and TOGARAM1 patient cell lines suggests a role for this new JBTSassociated protein module in ciliary stability.

Background Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder and is one of the most common muscular dystrophies. There are currently few effective therapies to treat the disease, although many small-molecule approaches are being pursued. Certain histone deacetylase inhibitors (HDACi) have been shown to ameliorate DMD phenotypes in mouse and zebrafish animal models. The HDACi givinostat has shown promise for DMD in clinical trials. However, beyond a small group of HDACi, other classes of epigenetic small molecules have not been broadly and systematically studied for their benefits for DMD. Methods We used an established animal model for DMD, the zebrafish dmd mutant strain sapje. A commercially available library of epigenetic small molecules was used to treat embryonic-larval stages of dmd mutant zebrafish. We used a quantitative muscle birefringence assay in order to assess and compare the effects of small-molecule treatments on dmd mutant zebrafish skeletal muscle structure. Results We performed a novel chemical-combination screen of a library of epigenetic compounds using the zebrafish dmd model. We identified candidate pools of epigenetic compounds that improve skeletal muscle structure in dmd mutant zebrafish. We then identified a specific combination of two HDACi compounds, oxamflatin and salermide, that ameliorated dmd mutant zebrafish skeletal muscle degeneration. We validated the effects of oxamflatin and salermide on dmd mutant zebrafish in an independent laboratory. Furthermore, we showed that the combination of oxamflatin and salermide caused increased levels of histone H4 acetylation in zebrafish larvae. Conclusions Our results provide novel, effective methods for performing a combination of small-molecule screen in zebrafish. Our results also add to the growing evidence that epigenetic small molecules may be promising candidates for treating DMD.

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