Article Text

Download PDFPDF
Original research
Biallelic mutations in the TOGARAM1 gene cause a novel primary ciliopathy
  1. Valeria Morbidoni1,2,
  2. Emanuele Agolini3,
  3. Kevin C Slep4,
  4. Luca Pannone5,6,
  5. Daniela Zuccarello1,
  6. Matteo Cassina1,
  7. Enrico Grosso7,
  8. Giorgia Gai7,
  9. Leonardo Salviati1,2,
  10. Bruno Dallapiccola8,
  11. Antonio Novelli3,
  12. Simone Martinelli5,
  13. Eva Trevisson1,2
  1. 1Clinical Genetics Unit, Department of Women’s and Children’s Health, University of Padova, Padova, Italy
  2. 2Laboratorio di Genetica ed Epidemiologia Clinica, Instituto di Ricerca Pediatrica Citta della Speranza, Padova, Italy
  3. 3Laboratory of Medical Genetics, Ospedale Pediatrico Bambino Gesù, Roma, Italy
  4. 4Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
  5. 5Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità, Roma, Italy
  6. 6Genetics and Rare Diseases Research Division, Ospedale Pediatrico Bambino Gesù, Roma, Italy
  7. 7Medical Genetics Unit, Azienda Ospedaliera Universitaria Città della Salute e della Scienza di Torino, Torino, Italy
  8. 8Scientific Directorate, Ospedale Pediatrico Bambino Gesù, Roma, Lazio, Italy
  1. Correspondence to Dr Eva Trevisson, Clinical Genetics Units, Women's and Children's Health, University of Padua, 35128 Padova, Veneto, Italy; eva.trevisson{at}unipd.it

Abstract

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
  • developmental
  • molecular genetics
View Full Text

Statistics from Altmetric.com

Footnotes

  • 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.

Request Permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.