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

Journal name:
Nature Genetics
Year published:
Published online

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.

At a glance


  1. Mutations in the TMEM216 gene in affected individuals linked to the CORS2 and MKS2 loci.
    Figure 1: Mutations in the TMEM216 gene in affected individuals linked to the CORS2 and MKS2 loci.

    (a) Chromosomal location of the CORS2 and MKS2 loci. (b) TMEM216 genomic organization, depicting start and stop codon, and location of identified base changes. (c) The longest splice isoform encodes a 148-residue tetraspan membrane protein. Mutations in affected individuals cluster toward the middle, with one prevalent Arg73 change occurring repeatedly. Missense, nonsense and splice mutations were identified. (d) Protein sequence alignment showing evolutionary conservation of altered amino acids. (e) Mutations in affected individuals lead to unstable protein products. Shown is a protein blot of whole lysates of cells transfected with a cDNA encoding wild-type TMEM216 or one of the missense mutations found in affected individuals, compared with a control mutant with a benign polymorphism (V71L). Each mutation resulted in the production of 40–50% of wild-type protein levels; α-tubulin was used as a loading control.

  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 [alpha]-tubulin.
    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.

    White dashed line indicates the tubule lumen. Boxes show insets at higher power. Scale bar 5 μm.

  3. TMEM216 mutation or knockdown results in impaired ciliogenesis and centrosome docking.
    Figure 3: TMEM216 mutation or knockdown results in impaired ciliogenesis and centrosome docking.

    (a) Two different TMEM216-mutated fibroblast lines from affected individuals show defective ciliogenesis and impaired centrosome docking (marked by γ-tubulin). Left scale bars, 20 μm; right scale bars, 1 μm. (b) TMEM216 antisera react with a 19-kDa band in control cells, which is reduced in TMEM216 R85X fibroblasts (some residual staining is apparent, probably owing to read-through from geneticin treatment), as well as in siRNA1-treated IMCD3 cells. Fibro., fibroblasts; Nontransf., nontransfected; Scr., scrambled. (c) Transfected IMCD3 cells showing effect of TMEM216 siRNA treatment, with reduced ciliogenesis and centrosome docking (note lack of cilia and lack of apically located centrosomes after knockdown). Top panels, x-y projection; bottom panels, x-z projection; scale bar, 10 μm. (d) Percentage of ciliated cells (defined as having cilia >1 μm long) is reduced after TMEM216 siRNA treatment. Percentage of cells with apical basal bodies (defined as most superior 1-μm sections compared to nuclear position) is similarly reduced. *P < 0.01, **P < 0.001, χ2 test. Bars: s.e.m. (e) Method of quantification at 72 h. Scale bars: white, most apical 1.0 μm; gray, basal 1.5 μm.

  4. TMEM216 complexes with Meckelin, and their loss results in Rho hyperactivation and actin cytoskeleton remodeling.
    Figure 4: TMEM216 complexes with Meckelin, and their loss results in Rho hyperactivation and actin cytoskeleton remodeling.

    (a) TMEM216-GFP (~37 kDa; arrow) is immunoprecipitated (IP) with Meckelin antisera (α) against either the N or C termini from whole-cell extract (input WCE), but not in control immunoprecipitations with an irrelevant antibody (Irr. Ab) or the preimmune antiserum (Preimm.). Arrowhead marks IgG heavy chain. (b) Immunoprecipitation of TMEM216-GFP by anti-GFP pulls down a 60-kDa C terminus–containing isoform of endogenous Meckelin (arrow), but control immunoprecipitations with no antibody (No MAb) or an irrelevant antibody (Irr. MAb) do not. Arrowhead marks IgG heavy chain. (c) MKS2 fibroblast (Fibro.) WCE has increased levels of activated RhoA-GTP compared to normal control. (d) siRNA knockdown of TMEM216 and TMEM67 in IMCD3 cells led to increased RhoA activation compared with scrambled control (Scr.). Total RhoA and β-actin are loading controls. Positive control (+) is loading with nonhydrolyzable GTP-γS; negative control (−) is loading with GDP. (e) RhoA (red) localizes to the basal bodies (γ-tubulin, green) in IMCD3 cells after 24-h treatment with scrambled siRNA, but it mislocalizes to regions adjacent to the basal bodies (arrows; and inset, magnification ×5) and at basolateral surfaces (arrowheads) after TMEM216 knockdown. Mislocalization of γ-tubulin is also apparent (bottom inset). Scale bar, 10 μm. (f) Subcellular phenotypes of fibroblasts cultured from undiseased controls and two fetuses affected by MKS with mutations in TMEM216 (R85X homozygous) and TMEM67 (R217X M261T), as indicated. Actin stress fibers in both mutated cells (arrowheads) were detected by phalloidin staining. Scale bar, 10 μm.

  5. TMEM216 disruption results in Dvl1 phosphorylation and planar cell polarity-like phenotypes in zebrafish.
    Figure 5: TMEM216 disruption results in Dvl1 phosphorylation and planar cell polarity–like phenotypes in zebrafish.

    (a) Cells undergoing siRNA knockdown of TMEM216 (left) and TMEM216 R85X fibroblasts from affected individuals (right) show an increase in the upper (phosphorylated) isoform (P-Dvl1) compared to scrambled controls (Scr.). Treatment with Rho inhibitor exoenzyme-C3-transferase (2 μg/ml) alone increased Dvl1 phosphorylation, but increases in P-Dvl1 caused by TMEM216 loss were reversed by Rho inhibition (right). (b) Coimmunoprecipitation of both Dvl1 and RhoA with TMEM216 in TMEM216-GFP–transfected cells. Arrowhead marks IgG heavy chain. (c) Dose-dependent rescue of centrosome docking phenotype in TMEM216 siRNA–treated cells after 0.5 μg/ml (+), 1.0 μg/ml (++) or 2.0 μg/ml (+++) Rho inhibitor treatment. *P < 0.01, **P < 0.001, χ2 test. (d) Injection of translation-blocking morpholino antisense oligonucleotide (MO) to tmem216 compared with scrambled MO caused a ciliary defect phenotype in injected zebrafish embryos (>50 each condition). Injection of human TMEM216 RNA caused no phenotype in wild-type embryos but allowed partial, dose-dependent rescue of the MO phenotype. (e) Lateral (top) and dorsal (bottom) views of zebrafish embryos injected with tmem216 or tmem67 MO at eight-somite stage, showing ciliopathy features. (f) Photos show representative 11-somite-stage embryos hybridized with krox20, pax2 and myoD riboprobes. Convergence to the midline is measured by the width at the fifth rhombomere (horizontal arrow) and extension along the anterior-posterior axis by notochord length (vertical arrow). Graphs quantify the tmem216 morphant phenotype (n = 12–15 embryos per injection). Left graph, dose-dependent rescue by TMEM216 mRNA. Right graph, length and width measurements of tmem216 and tmem67 morphants. Suppression of the tmem216 or tmem67 morphant gastrulation defect causes significant differences in width and length compared to controls (*P < 0.005). Pheno., phenotype; Embry., embryonic; Rhomb., rhombomere. Error bars show s.e.m.


  1. Lancaster, M.A. & Gleeson, J.G. The primary cilium as a cellular signaling center: lessons from disease. Curr. Opin. Genet. Dev. 19, 220229 (2009).
  2. Keeler, L.C. et al. Linkage analysis in families with Joubert syndrome plus oculo-renal involvement identifies the CORS2 locus on chromosome 11p12-q13.3. Am. J. Hum. Genet. 73, 656662 (2003).
  3. Valente, E.M. et al. Description, nomenclature, and mapping of a novel cerebello-renal syndrome with the molar tooth malformation. Am. J. Hum. Genet. 73, 663670 (2003).
  4. Valente, E.M. et al. Distinguishing the four genetic causes of Joubert syndrome-related disorders. Ann. Neurol. 57, 513519 (2005).
  5. Baala, L. et al. The Meckel-Gruber syndrome gene, MKS3, is mutated in Joubert syndrome. Am. J. Hum. Genet. 80, 186194 (2007).
  6. Baala, L. et al. Pleiotropic effects of CEP290 (NPHP6) mutations extend to Meckel syndrome. Am. J. Hum. Genet. 81, 170179 (2007).
  7. Delous, M. et al. The ciliary gene RPGRIP1L is mutated in cerebello-oculo-renal syndrome (Joubert syndrome type B) and Meckel syndrome. Nat. Genet. 39, 875881 (2007).
  8. Mougou-Zerelli, S. et al. CC2D2A mutations in Meckel and Joubert syndromes indicate a genotype-phenotype correlation. Hum. Mutat. 30, 15741582 (2009).
  9. Roume, J. et al. A gene for Meckel syndrome maps to chromosome 11q13. Am. J. Hum. Genet. 63, 10951101 (1998).
  10. Gherman, A., Davis, E.E. & Katsanis, N. The ciliary proteome database: an integrated community resource for the genetic and functional dissection of cilia. Nat. Genet. 38, 961962 (2006).
  11. Inglis, P.N., Boroevich, K.A. & Leroux, M.R. Piecing together a ciliome. Trends Genet. 22, 491500 (2006).
  12. Hubner, K., Windoffer, R., Hutter, H. & Leube, R.E. Tetraspan vesicle membrane proteins: synthesis, subcellular localization, and functional properties. Int. Rev. Cytol. 214, 103159 (2002).
  13. Junge, H.J. et al. TSPAN12 regulates retinal vascular development by promoting Norrin- but not Wnt-induced FZD4/β-catenin signaling. Cell 139, 299311 (2009).
  14. Caplan, M.J., Kamsteeg, E.J. & Duffield, A. Tetraspan proteins: regulators of renal structure and function. Curr. Opin. Nephrol. Hypertens. 16, 353358 (2007).
  15. Smith, U.M. et al. The transmembrane protein meckelin (MKS3) is mutated in Meckel-Gruber syndrome and the wpk rat. Nat. Genet. 38, 191196 (2006).
  16. Edvardson, S. et al. Joubert syndrome (JBTS2) in Ashkenazi Jews is associated with a TMEM216 mutation. Am. J. Hum. Genet. 86, 9397 (2010).
  17. Váradi, V., Szabo, L. & Papp, Z. Syndrome of polydactyly, cleft lip/palate or lingual lump, and psychomotor retardation in endogamic gypsies. J. Med. Genet. 17, 119122 (1980).
  18. Dawe, H.R. et al. The Meckel-Gruber Syndrome proteins MKS1 and meckelin interact and are required for primary cilium formation. Hum. Mol. Genet. 16, 173186 (2007).
  19. Wallingford, J.B. et al. Dishevelled controls cell polarity during Xenopus gastrulation. Nature 405, 8185 (2000).
  20. Park, T.J., Haigo, S.L. & Wallingford, J.B. Ciliogenesis defects in embryos lacking inturned or fuzzy function are associated with failure of planar cell polarity and Hedgehog signaling. Nat. Genet. 38, 303311 (2006).
  21. Veeman, M.T., Axelrod, J.D. & Moon, R.T. A second canon. Functions and mechanisms of beta-catenin-independent Wnt signaling. Dev. Cell 5, 367377 (2003).
  22. Winter, C.G. et al. Drosophila Rho-associated kinase (Drok) links Frizzled-mediated planar cell polarity signaling to the actin cytoskeleton. Cell 105, 8191 (2001).
  23. Dawe, H.R. et al. Nesprin-2 interacts with meckelin and mediates ciliogenesis via remodelling of the actin cytoskeleton. J. Cell Sci. 122, 27162726 (2009).
  24. Pan, J., You, Y., Huang, T. & Brody, S.L. RhoA-mediated apical actin enrichment is required for ciliogenesis and promoted by Foxj1. J. Cell Sci. 120, 18681876 (2007).
  25. Park, T.J., Mitchell, B.J., Abitua, P.B., Kintner, C. & Wallingford, J.B. Dishevelled controls apical docking and planar polarization of basal bodies in ciliated epithelial cells. Nat. Genet. 40, 871879 (2008).
  26. Lang, P. et al. Protein kinase A phosphorylation of RhoA mediates the morphological and functional effects of cyclic AMP in cytotoxic lymphocytes. EMBO J. 15, 510519 (1996).
  27. Dutcher, S.K. Elucidation of basal body and centriole functions in Chlamydomonas reinhardtii . Traffic 4, 443451 (2003).
  28. Corbit, K.C. et al. Kif3a constrains β-catenin-dependent Wnt signalling through dual ciliary and non-ciliary mechanisms. Nat. Cell Biol. 10, 7076 (2008).
  29. Leitch, C.C. et al. Hypomorphic mutations in syndromic encephalocele genes are associated with Bardet-Biedl syndrome. Nat. Genet. 40, 443448 (2008).
  30. Badano, J.L. et al. Dissection of epistasis in oligogenic Bardet-Biedl syndrome. Nature 439, 326330 (2006).
  31. Wittwer, C.T. High-resolution DNA melting analysis: advancements and limitations. Hum. Mutat. 30, 857859 (2009).
  32. Budde, B.S. et al. tRNA splicing endonuclease mutations cause pontocerebellar hypoplasia. Nat. Genet. 40, 11131118 (2008).
  33. Wolff, A. et al. Distribution of glutamylated α and β-tubulin in mouse tissues using a specific monoclonal antibody, GT335. Eur. J. Cell Biol. 59, 425432 (1992).
  34. Lancaster, M.A. et al. Impaired Wnt-β-catenin signaling disrupts adult renal homeostasis and leads to cystic kidney ciliopathy. Nat. Med. 15, 10461054 (2009).
  35. Johnson, C.A., Padget, K., Austin, C.A. & Turner, B.M. Deacetylase activity associates with topoisomerase II and is necessary for etoposide-induced apoptosis. J. Biol. Chem. 276, 45394542 (2001).
  36. Trueba, S.S. et al. PAX8, TITF1, and FOXE1 gene expression patterns during human development: new insights into human thyroid development and thyroid dysgenesis-associated malformations. J. Clin. Endocrinol. Metab. 90, 455462 (2005).

Download references

Author information

  1. These authors contributed equally to the work.

    • Enza Maria Valente,
    • Clare V Logan,
    • Soumaya Mougou-Zerelli &
    • Jeong Ho Lee
  2. These authors jointly directed the project.

    • Colin A Johnson,
    • Tania Attié-Bitach &
    • Joseph G Gleeson


  1. Mendel Laboratory, Istituto di Ricovero e Cura a Carattere Scientifico “Casa Sollievo della Sofferenza,” San Giovanni Rotondo, Italy.

    • Enza Maria Valente,
    • Francesco Brancati,
    • Miriam Iannicelli,
    • Lorena Travaglini,
    • Sveva Romani,
    • Barbara Illi &
    • Annalisa Mazzotta
  2. Department of Medical and Surgical Paediatric Sciences, University of Messina, Messina, Italy.

    • Enza Maria Valente,
    • Carmelo D Salpietro &
    • Carmelo Fede
  3. Section of Ophthalmology and Neurosciences, Wellcome Trust Brenner Building, Leeds Institute of Molecular Medicine, St. James's University Hospital, Leeds, UK.

    • Clare V Logan,
    • Matthew Adams,
    • Katarzyna Szymanska,
    • Chris Inglehearn &
    • Colin A Johnson
  4. Département de Génétique, INSERM U781, Hôpital Necker-Enfants Malades, Université Paris Descartes, Paris, France.

    • Soumaya Mougou-Zerelli,
    • Arnold Munnich,
    • Sophie Thomas,
    • Michel Vekemans &
    • Tania Attié-Bitach
  5. Service de Cytogénétique, Génétique Moléculaire et Biologie de la Reproduction, Hôpital Farhat Hached, Sousse, Tunisia.

    • Soumaya Mougou-Zerelli &
    • Ali Saad
  6. Neurogenetics Laboratory, Institute for Genomic Medicine, Department of Neurosciences and Pediatrics, Howard Hughes Medical Institute, University of California, San Diego, California, USA.

    • Jeong Ho Lee,
    • Jennifer L Silhavy,
    • Ji Eun Lee,
    • Jerlyn C Tolentino,
    • Dominika Swistun &
    • Joseph G Gleeson
  7. Department of Biomedical Sciences and Aging Research Center, Center for Excellence in Aging, G. d'Annunzio University Foundation, Chieti, Italy.

    • Francesco Brancati
  8. Genome Sequencing and Analysis Program, Broad Institute, Cambridge, Massachusetts, USA.

    • Stacey Gabriel,
    • Carsten Russ,
    • Kristian Cibulskis &
    • Carrie Sougnez
  9. Department of Pediatrics, Howard Hughes Medical Institute, University of Michigan, Ann Arbor, Michigan, USA.

    • Friedhelm Hildebrandt,
    • Edgar A Otto &
    • Susanne Held
  10. Center for Human Disease Modeling, Duke University, Durham, North Carolina, USA.

    • Bill H Diplas,
    • Erica E Davis &
    • Nicholas Katsanis
  11. Quest Diagnostics Nichols Institute, San Juan Capistrano, California, USA.

    • Mario Mikula &
    • Charles M Strom
  12. Safra Pediatric Hospital, Pediatric Neurology Unit, Sheba Medical Center, Ramat-Gan, Israel.

    • Bruria Ben-Zeev
  13. Institute of Medical Genetics, Metabolic Neurogenetics Service, Wolfson Medical Center, Holon, Israel.

    • Dorit Lev,
    • Tally Lerman Sagie &
    • Marina Michelson
  14. Prenatal Diagnosis Unit, Genetic Institute, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel.

    • Yuval Yaron
  15. Division of Human Genetics, National Health Laboratory Service, School of Pathology, University of the Witwatersrand, Johannesburg, South Africa.

    • Amanda Krause
  16. Department of Paediatric Neurology, University Children's Hospital of Zurich, Zurich, Switzerland.

    • Eugen Boltshauser
  17. Département de Génétique, Hôpital Necker-Enfants Malade, Assistance Publique Hôpitaux de Paris (APHP), Paris, France.

    • Nadia Elkhartoufi,
    • Arnold Munnich,
    • Michel Vekemans &
    • Tania Attié-Bitach
  18. Génétique Médicale, CHI Poissy, Saint Germain en Laye, France.

    • Joelle Roume
  19. The Genetics Institute, Ha'Emek Medical Center, Afula, Israel.

    • Stavit Shalev
  20. INSERM U983, Hôpital Necker-Enfants Malade, Université Paris Descartes, Assistance Publique-Hopitaux de Paris, Paris, France.

    • Sophie Saunier
  21. University Hospital of Wales, Heath Park, Cardiff, UK.

    • Adila Alkindy
  22. Istituto di Ricovero e Cura a Carattere Scientifico “Bambino Gesù Hospital,” Rome, Italy.

    • Bruno Dallapiccola
  23. Current address: Clinical Genetics, College of Medicine & Health Sciences, Sultan Qaboos University Hospital, Al-Khod, Muscat, Oman.

    • Adila Alkindy


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.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary Text and Figures (5M)

    Supplementary Figures 1–11, Supplementary Table 1 and Supplementary Note

Additional data