Mutations in TMEM216 perturb ciliogenesis and cause Joubert, Meckel and related syndromes

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Joubert syndrome (JBTS), related disorders (JSRDs) and Meckel syndrome (MKS) are ciliopathies. We now report that MKS2 and CORS2 (JBTS2) loci are allelic and caused by mutations in TMEM216, which encodes an uncharacterized tetraspan transmembrane protein. Individuals with CORS2 frequently had nephronophthisis and polydactyly, and two affected individuals conformed to the oro-facio-digital type VI phenotype, whereas skeletal dysplasia was common in fetuses affected by MKS. A single G218T mutation (R73L in the protein) was identified in all cases of Ashkenazi Jewish descent (n = 10). TMEM216 localized to the base of primary cilia, and loss of TMEM216 in mutant fibroblasts or after knockdown caused defective ciliogenesis and centrosomal docking, with concomitant hyperactivation of RhoA and Dishevelled. TMEM216 formed a complex with Meckelin, which is encoded by a gene also mutated in JSRDs and MKS. Disruption of tmem216 expression in zebrafish caused gastrulation defects similar to those in other ciliary morphants. These data implicate a new family of proteins in the ciliopathies and further support allelism between ciliopathy disorders.

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Figure 1: Mutations in the TMEM216 gene in affected individuals linked to the CORS2 and MKS2 loci.
Figure 2: Localization of endogenous TMEM216 to the base of primary cilia in IMCD cells (a), proximal renal tubule (b) and hRPE cells (c), costained for GT335 (glutamylated tubulin) or acetylated α-tubulin.
Figure 3: TMEM216 mutation or knockdown results in impaired ciliogenesis and centrosome docking.
Figure 4: TMEM216 complexes with Meckelin, and their loss results in Rho hyperactivation and actin cytoskeleton remodeling.
Figure 5: TMEM216 disruption results in Dvl1 phosphorylation and planar cell polarity–like phenotypes in zebrafish.


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We thank Marshfield Clinic Research Foundation, Center for Inherited Disease Research (supported by the US National Institutes of Health National Heart, Lung, and Blood Institute) for genotyping support and A. Felsenfeld and the Medical Sequencing Initiative at the National Human Genome Research Institute. We acknowledge support for sequencing from the Broad Institute and the Broad Sequencing Platform and J. Meerloo at the University of California, San Diego Neurosciences Microscopy Core (Supported by National Institute of Neurological Disorders and Stroke grant P30NS047101). For patient referrals, we thank C. Jalas at Bonei Olam Center for Rare Jewish Genetic Disorders; M.R. Eccles at the University of Otago; H.M. Harville at University of Michigan; G. Tortorella, S. Briuglia, R. Chimenz, R. Gallizzi and M. Briguglio at University of Messina; E. Bertini and the International JSRD Study Group; and the French Society of Foetal Pathology. We thank E. Morrison, P. Novick and S. Ferro-Novick for helpful discussions; C. Janke (Macromolecular Biochemistry Research Center) for GT335 antibody; P. Robinson (University of Leeds) for siRNA duplexes against Hhari; E. Genin for linkage analysis, S. Audolent, C. Babarit, F. Legendre and H.-M. Gaudé for technical help and O. Duc for confocal microscopy. S.M.-Z. is supported by INSERM-DGRSRT (CS/RN/2008 no. 87). This work was supported by the Italian Ministry of Health (RC2010, Ricerca Finalizzata 2006), Telethon Foundation Italy (GGP08145 to E.M.V.), Pierfranco and Luisa Mariani Foundation (E.M.V.), American Heart Association grant O9POST2250641 (J.E.L.), BDF Newlife, the Medical Research Council (G0700073) and the Sir Jules Thorn Charitable Trust (09/JTA to C.A.J.), l'Agence National pour la Recherche (ANR 07-MRAR-Fetalciliopathies to T.A.-B.), the US National Institutes of Health (R01 DK068306 to F.H.; R01 DK072301, R01 HD04260 and R01 DK075972 to N.K.; National Research Service Award fellowship F32 DK079541 to E.E.D., R01 NS052455 and R01 NS04843 to J.G.G.), Burroughs Wellcome Fund and Howard Hughes Medical Institute (F.H. and J.G.G.).

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J.L.S. performed fine mapping in CORS2, cDNA sequencing and RNA blot analysis and identified the TMEM216 gene as mutated. F.B., M.I., L.T. and A. Mazzotta identified the mutation common to affected Ashkenazi individuals and performed mutation analysis. S.G., C.R., K.C. and C.S. performed mutation analysis of candidate genes in the CORS2/MKS2 locus; C.V.L., S.M.-Z., J.H.L., K.S., F.H., E.A.O., S.H., N.E. and N.K. performed mutation analysis of TMEM216 in cohorts of individuals with ciliopathies; S.M.-Z. and S. Saunier performed cilia analysis. S.T. performed cDNA expression and immunohistochemistry. J.H.L., J.E.L., B.H.D. and E.E.D. performed zebrafish experiments. C.V.L., J.H.L., S.R., B.I., M.A. and C.A.J. did confocal microscopy and biochemical assays. E.M.V., J.C.T., D.S., C.D.S., C.F., B.B.-Z., D.L., T.L.S., M. Michelson, Y.Y., A.K., E.B., J.R., S. Shalev, A.S., A.A., B.D. and C.A.J. recruited patients and gathered detailed clinical information for the study. M. Mikula and C.M.S. performed control genotyping in Ashkenazi cohorts. A. Munnich, C.I., M.V. and B.D. helped devise and supervise genetic analysis and contributed to the manuscript. E.M.V., C.A.J., T.A.-B. and J.G.G. wrote the manuscript.

Correspondence to Enza Maria Valente or Colin A Johnson or Tania Attié-Bitach or Joseph G Gleeson.

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