Biallelic mutations in the <i>TOGARAM1</i> gene cause a novel primary ciliopathy.

Abstract

Dysfunction in non-motile cilia is associated with a broad spectrum of developmental disorders characterised by clinical heterogeneity. While over 100 genes have been associated with primary ciliopathies, with wide phenotypic overlap, some patients still lack a molecular diagnosis. To investigate and functionally characterise the molecular cause of a malformation disorder observed in two sibling fetuses characterised by microphthalmia, cleft lip and palate, and brain anomalies. A trio-based whole exome sequencing (WES) strategy was used to identify candidate variants in the <i>TOGARAM1</i> gene. In silico, in vitro and in vivo (<i>Caenorhabditis elegans</i>) studies were carried out to explore the impact of mutations on protein structure and function, and relevant biological processes. <i>TOGARAM1</i> encodes a member of the Crescerin1 family of proteins regulating microtubule dynamics. Its orthologue in <i>C. elegans</i>, <i>che-12</i>, is expressed in a subset of sensory neurons and localises in the dendritic cilium where it is required for chemosensation. Nematode lines harbouring the corresponding missense variant in <i>TOGARAM1</i> were generated by CRISPR/Cas9 technology. Although chemotaxis ability on a NaCl gradient was not affected, <i>che-12</i> point mutants displayed impaired lipophilic dye uptake, with shorter and altered cilia in sensory neurons. Finally, in vitro analysis of microtubule polymerisation in the presence of wild-type or mutant TOG2 domain revealed a faster polymerisation associated with the mutant protein, suggesting aberrant tubulin binding. Our data are in favour of a causative role of <i>TOGARAM1</i> variants in the pathogenesis of this novel disorder, connecting this gene with primary ciliopathy.