Background 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.
Objective 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.
Methods A trio-based whole exome sequencing (WES) strategy was used to identify candidate variants in the TOGARAM1 gene. In silico, in vitro and in vivo (Caenorhabditis elegans) studies were carried out to explore the impact of mutations on protein structure and function, and relevant biological processes.
Results TOGARAM1 encodes a member of the Crescerin1 family of proteins regulating microtubule dynamics. Its orthologue in C. elegans, che-12, 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 TOGARAM1 were generated by CRISPR/Cas9 technology. Although chemotaxis ability on a NaCl gradient was not affected, che-12 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.
Conclusions Our data are in favour of a causative role of TOGARAM1 variants in the pathogenesis of this novel disorder, connecting this gene with primary ciliopathy.
- clinical genetics
- molecular genetics
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Contributors VM conceived and carried out the main bulk of experiments with C. elegans, with help from LP, who also contributed with genome editing to generate the point mutant worm lines. EA performed and interpreted the genetic analyses. KS carried out the in vitro studies with the mutant protein and performed structural modelling. DZ, EG and GG reported the clinical history of the family. MC performed the statistical analyses. LS helped to interpret the results. BD and AN assisted EA with genetic studies and critically revised the manuscript. SM proposed the experiment in discussions with VM and ET, provided critical feedback, and helped to shape the research and to analyse the manuscript. ET conceived and planned the experiments, supervised the findings of this work and wrote the manuscript with input from all authors. All authors discussed the results and contributed to the final manuscript.
Funding This research was supported by grants from Istituto di Ricerca Pediatrica (IRP) (to ET and LS) and the Italian Ministry of Health (GR-2016-02362779) (to ET), Vite Coraggiose (to AN, EA and BD), the Italian Ministry of Health (Ricerca Corrente 2019 to AN, EA) and “UNC Mosley Fund” (to KS).
Competing interests None declared.
Patient consent for publication Not required.
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement Data are available on request.
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