Mutation of Growth Arrest Specific 8 Reveals a Role in Motile Cilia Function and Human Disease

PLoS Genet. 2016 Jul 29;12(7):e1006220. doi: 10.1371/journal.pgen.1006220.

Wesley R Lewis ¹, Erik B Malarkey ¹, Douglas Tritschler ², Raqual Bower ², Raymond C Pasek ³, Jonathan D Porath ⁴, Susan E Birket ⁵ ⁶, Sophie Saunier ⁷ ⁸, Corinne Antignac ⁹ ¹⁰, Michael R Knowles ¹¹, Margaret W Leigh ¹², Maimoona A Zariwala ¹³, Anil K Challa ¹⁴, Robert A Kesterson ¹⁴, Steven M Rowe ⁵ ⁶, Iain A Drummond ¹⁵, John M Parant ¹⁶, Friedhelm Hildebrandt ⁴, Mary E Porter ², Bradley K Yoder ¹, Nicolas F Berbari ¹⁷

Affiliations

1 Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
2 Genetics, Cell Biology, and Development, University of Minnesota Medical School, Minneapolis, Minnesota, United States of America.
3 Cell and Developmental Biology, Vanderbilt University, Nashville, Tennessee, United States of America.
4 Division of Nephrology, Department of Medicine, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, United States of America.
5 Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
6 Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
7 INSERM, U-983, Necker Hospital, Paris, France.
8 Paris Descartes University, Sorbonne Paris Cité, Imagine Institute, Paris, France.
9 Institut National de la Santé et de la Recherche Médicale (INSERM) Unité Mixte de Recherche (UMR) 1163, Laboratory of Hereditary Kidney Diseases, Paris, France.
10 Assistance Publique-Hôpitaux de Paris, Department of Genetics, Necker Hospital, Paris, France.
11 Department of Medicine, UNC School of Medicine, Marisco Lung Institute, Chapel Hill, North Carolina, United States of America.
12 Department of Pediatrics, UNC School of Medicine, Marisco Lung Institute, Chapel Hill, North Carolina, United States of America.
13 Department of Pathology and Laboratory Medicine, UNC School of Medicine, Marisco Lung Institute, Chapel Hill, North Carolina, United States of America.
14 Department of Genetics, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
15 Nephrology Division, Massachusetts General Hospital, Charlestown, Massachusetts, United States of America.
16 Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
17 Department of Biology, Indiana University-Purdue University Indianapolis, Indianapolis, Indiana, United States of America.

PMID: 27472056 DOI: 10.1371/journal.pgen.1006220

Abstract

Ciliopathies are genetic disorders arising from dysfunction of microtubule-based cellular appendages called cilia. Different cilia types possess distinct stereotypic microtubule doublet arrangements with non-motile or 'primary' cilia having a 9+0 and motile cilia have a 9+2 array of microtubule doublets. Primary cilia are critical sensory and signaling centers needed for normal mammalian development. Defects in their structure/function result in a spectrum of clinical and developmental pathologies including abnormal neural tube and limb patterning. Altered patterning phenotypes in the limb and neural tube are due to perturbations in the Hedgehog (Hh) signaling pathway. Motile cilia are important in fluid movement and defects in motility result in chronic respiratory infections, altered left-right asymmetry, and infertility. These features are the hallmarks of Primary Ciliary Dyskinesia (PCD, OMIM 244400). While mutations in several genes are associated with PCD in patients and animal models, the genetic lesion in many cases is unknown. We assessed the in vivo functions of Growth Arrest Specific 8 (GAS8). GAS8 shares strong sequence similarity with the Chlamydomonas Nexin-Dynein Regulatory Complex (NDRC) protein 4 (DRC4) where it is needed for proper flagella motility. In mammalian cells, the GAS8 protein localizes not only to the microtubule axoneme of motile cilia, but also to the base of non-motile cilia. Gas8 was recently implicated in the Hh signaling pathway as a regulator of Smoothened trafficking into the cilium. Here, we generate the first mouse with a Gas8 mutation and show that it causes severe PCD phenotypes; however, there were no overt Hh pathway phenotypes. In addition, we identified two human patients with missense variants in Gas8. Rescue experiments in Chlamydomonas revealed a subtle defect in swim velocity compared to controls. Further experiments using CRISPR/Cas9 homology driven repair (HDR) to generate one of these human missense variants in mice demonstrated that this allele is likely pathogenic.

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