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Nat Genet. Author manuscript; available in PMC Jan 1, 2011.
Published in final edited form as:
Published online May 30, 2010. doi:  10.1038/ng.594
PMCID: PMC2894012
Mutations in TMEM216 perturb ciliogenesis and cause Joubert, Meckel and related syndromes
Enza Maria Valente,1,2* Clare V. Logan,3* Soumaya Mougou-Zerelli,4,5* Jeong Ho Lee,6* Jennifer L. Silhavy,6 Francesco Brancati,1,7 Miriam Iannicelli,1 Lorena Travaglini,1 Sveva Romani,1 Barbara Illi,1 Matthew Adams,3 Katarzyna Szymanska,3 Annalisa Mazzotta,1 Ji Eun Lee,6 Jerlyn C. Tolentino,6 Dominika Swistun,6 Carmelo D. Salpietro,2 Carmelo Fede,2 Stacey Gabriel,8 Carsten Russ,8 Kristian Cibulskis,8 Carrie Sougnez,8 Friedhelm Hildebrandt,9 Edgar A. Otto,9 Susanne Held,9 Bill H. Diplas,10 Erica Davis,10 Mario Mikula,11 Charles M. Strom,11 Bruria Ben-Ze’ev,12 Dorit Lev,13 Tally Lerman Sagie,13 Marina Michelson,13 Yuval Yaron,14 Amanda Krause,15 Eugen Boltshauser,16 Nadia Elkhartoufi,17 Joelle Roume,18 Stavit Shalev,19 Arnold Munnich,4,17 Sophie Saunier,20 Chris Inglehearn,3 Ali Saad,5 Adila Alkindy,21# Sophie Thomas,4 Michel Vekemans,4,17 Bruno Dallapiccola,22 Nicholas Katsanis,10 Colin A. Johnson,3+ Tania Attié-Bitach,4,17+ and Joseph G. Gleeson6+
1Mendel Laboratory, Istituto di Ricovero e Cura a Carattere Scientifico “Casa Sollievo della Sofferenza”, 71013 San Giovanni Rotondo, Italy
2Department of Medical and Surgical Paediatric Sciences, University of Messina, Messina, Italy
3Section of Ophthalmology and Neurosciences, Wellcome Trust Brenner Building, Leeds Institute of Molecular Medicine, St James’s University Hospital, Leeds, LS9 7TF, U.K.
4Département de Génétique, INSERM U781, Hôpital Necker-Enfants Malades, Université Paris Descartes, Paris, France
5Service de Cytogénétique, Génétique moléculaire et Biologie de la Reproduction, Hôpital Farhat Hached, Sousse, Tunisia
6Neurogenetics Laboratory, Institute for Genomic Medicine, Department of Neurosciences and Pediatrics, Howard Hughes Medical Institute, University of California, San Diego, 92093, USA
7Department of Biomedical Sciences and Aging Research Center, Ce.S.I., G. d’Annunzio University Foundation, Chieti, Italy
8Genome Sequencing and Analysis Program, Broad Institute, Cambridge, MA 02141, USA
9Department of Pediatrics, Howard Hughes Medical Institute, University of Michigan, Ann Arbor, MI 48109-5646, USA
10Center for Human Disease Modeling, Duke University, Durham NC 27710, USA
11Quest Diagnostics Inc. Nichols Institute, San Juan Capistrono, CA 92690, USA
12Safra Pediatric Hospital, Pediatric Neurology Unit, Sheba Medical Center, Ramat-Gan, Israel
13Institute of Medical Genetics, Metabolic Neurogenetics Service, Wolfson Medical Center, Holon 58100 Israel
14Prenatal Diagnosis Unit, Genetic Institute, Tel Aviv Sourasky Medical Center, Tel Aviv 64239, Israel
15Division of Human Genetics, National Health Laboratory Service, School of Pathology, University of the Witwatersrand, Johannesburg 2000, South Africa
16Department of Paediatric Neurology, University Children’s Hospital of Zurich, CH-8032, Zurich, Switzerland
17Département de Génétique, Hôpital Necker-Enfants Malades, Assistance Publique Hôpitaux de Paris (APHP), France
18Génétique Médicale, CHI Poissy, Saint Germain en Laye, France
19The Genetics Institute, Ha’Emek Medical Center, Afula, Israel
20INSERM U983, Hôpital Necker-Enfants Malades, Université Paris Descartes, Hôpital Necker-Enfants Malades, APHP, Paris, France
21University Hospital of Wales, Heath Park, Cardiff, CF14 4XW, U.K.
22Istituto di Ricovero e Cura a Carattere Scientifico “Bambino Gesù Hospital”, Rome, Italy
Correspondence should be addressed to J.G.G. (jogleeson/at/, E.M.V. (e.valente/at/, C.A.J. (c.johnson/at/ or T.A-B. (tania.attie/at/
*Contributed equally
+These authors jointly directed this work.
#Current address: Clinical Genetics, College of Medicine & Health Sciences, Sultan Qaboos University Hospital, Al-Khod, Muscat, Oman
AUTHOR CONTRIBUTIONS. J.L.S. performed fine mapping in JBTS2, cDNA sequencing, Northern analysis, and identified the TMEM216 gene as mutated. F.B., M.I., L.T, A.M identified the mutation common to Ashkenazi patients and performed mutation analysis; S.G., C.R., K.C., C.S. performer mutation analysis of candidate genes in the JBTS2/MKS2 locus; C.V.L., S.M.-Z., J.H.L., K.S., F.H., E.A.O., S.H. N.E., N.K., performed mutation analysis of TMEM216 in cohorts of patients with ciliopathies; S.M.-Z and S.Sa. performer cilia analysis; S.T. performed cDNA expression and IHC; J.H.L., J.E.L., B.H.D., E.D. performed zebrafish experiments; C.V.L., J.H.L., S.R., B.I., M.A. 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.Mic., Y.Y, A.K., E.B., J.R., S.S, A.S., A.A., B.D., C.A.J. recruited patients and detailed clinical information for the study; M.M., C.M.S. performed control genotyping in Ashkenazi cohorts; A.Mu., C.I., M.V., B.D. helped devise and supervise genetic analysis and contributed to the manuscript; E.M.V., C.A.J., T.A.-B., J.G.G. wrote the manuscript.
Joubert syndrome (JBTS), related disorders (JSRD) and Meckel syndrome (MKS) are ciliopathies. We now report that MKS2 and JBTS2 loci are allelic and due to mutations in TMEM216, encoding an uncharacterized tetraspan transmembrane protein. JBTS2 patients displayed frequent nephronophthisis and polydactytly, and two cases conformed to the Oro-Facio-Digital type VI phenotype, whereas skeletal dysplasia was common in MKS fetuses. A single p.R73L mutation was identified in all patients of Ashkenazi Jewish descent (n=10). TMEM216 localized to the base of primary cilia, and loss of TMEM216 in patient fibroblasts or following siRNA knockdown caused defective ciliogenesis and centrosomal docking, with concomitant hyperactivation of RhoA and Dishevelled. TMEM216 complexed with Meckelin, encoded by a gene also mutated in JSRD and MKS. Abrogation of tmem216 expression in zebrafish led to gastrulation defects that overlap with other ciliary morphants. The data implicate a new family of proteins in the ciliopathies, and further support allelism between ciliopathy disorders.
The neurological features of JSRD include hypotonia, ataxia, psychomotor delay, irregular breathing pattern and oculomotor apraxia and are variably associated with multiorgan involvement, mainly retinal dystrophy, nephronophthisis (NPH) and congenital liver fibrosis. JSRD are genetically heterogeneous, and all known genes encode proteins localized at or near the primary cilium1. We previously mapped the JBTS2 (also known as CORS2) locus to chromosome 11p12-q13.3 in a large Sicilian family and in three consanguineous pedigrees from the Middle East2-3. Aligning the two datasets suggested a minimal candidate interval between D11S1344 and D11S1883 (46.123-63.130 Mb)4 (Fig. 1a).
Figure 1
Figure 1
Mutations in the TMEM216 gene in patients linked to the JBTS2 and MKS2 loci. (a) Chromosomal location of the JBTS2 and MKS2 loci on Chr. 11cent. (b) TMEM216 genomic organization, depicting start and stop codon, and location of identified base changes. (more ...)
Overlapping with JSRD is MKS, characterized by occipital encephalocele and other posterior fossa defects, cystic dysplastic kidneys, hepatic bile duct proliferation and polydactyly, and the two conditions are known to be allelic at four loci5-8. The MKS2 locus was initially mapped in families of North African and Middle Eastern ancestry to a chromosome 11q region telomeric to JBTS29, but our subsequent identification of additional families, as well as SNP re-analysis of the initial family, indicated allelism with JBTS2 between rs1113480 and rs953894 (48.014-62.518 Mb) (Supplementary Fig. 1). Because JSRD and MKS are considered ciliopathies, of the 200 total candidate genes, we first sequenced the exons and splice sites of genes listed in the cilia proteome databases10-11 in one affected subject from each JBTS2/MKS2 family, but no mutations were identified.
Tetraspan transmembrane proteins are characterized by four hydrophobic, putative transmembrane domains (TM1-TM4), forming two extracellular and one intracellular loop, which regulate signaling and trafficking properties of their partner proteins in multiple cellular contexts12. While little is known about their function, they can act with Wnt receptors13, and their ability to form complexes with a wide variety of membrane and cytosolic proteins14 suggests that they may participate in the formation of membrane domains that regulate signaling and sorting processes. Transmembrane proteins also represented attractive candidates, due to similarities to MKS3/TMEM67 encoding Meckelin, which is mutated both in JSRD and MKS5,15. Therefore we additionally sequenced the eight genes encoding transmembrane proteins, eventually identifying homozygous deleterious mutations in TMEM216 in six of the 12 JSRD/MKS families compatible with linkage to the locus (Table 1). Interestingly, residue p.R73 was mutated both in a Sicilian family with JSRD (COR000, p.R73L) and in a Turkish family, in which MKS and JSRD coexisted in the same sibship (COR114/F37, p.R73H). The p.G77A mutation in two Palestinian families (F56, F58) resulted from a substitution (c.230G>C) that affects the first base of exon 5, leading to the use of an alternative splice site in intron 4, the inclusion of an additional 46bp and resultant premature protein termination (p.T78KfsX30) (Supplementary Fig. 2). None of these mutations were identified in over 500 controls from ethnically matched cohorts.
Table 1
Table 1
Clinical and molecular data of TMEM216 mutated families
We next screened an additional 460 JSRD and 132 MKS probands (Supplementary Note) and identified mutations in 12 and two further cases, respectively (Table 1). Twelve of 14 JSRD families shared the same homozygous p.R73L founder mutation, including two families from Sicily and ten families of Ashkenazi Jewish descent. Saturation of the region surrounding the p.R73L mutation with 17 SNP/microsatellite markers indicated that these families shared the same ancestral haplotype, spanning 472 Kb around the mutation (Supplementary Fig. 3), that could be dated back at least 20 generations. The carrier frequency in the Ashkenazi population was determined to be about 1:100, as we identified two heterozygous healthy unrelated carrier individuals among a screened cohort of 212 Ashkenazi individuals, making carrier detection possible at least in this population. A similar carrier frequency of 1:92 has also been determined for the p.R73L mutation in a distinct study on eight Ashkenazi JBTS2 families and 2766 unaffected controls16. Microsatellite analysis also detected shared haplotypes in the two Palestinian (F56, F58) and in the two Tunisian families (F2, F5), homozygous for the same mutations (Supplementary Fig. 1).
Overall, 20 JSRD patients and 11 MKS fetuses carried TMEM216 mutations (Fig. 1b,Table 1). All of the nonsynonymous changes occurred in evolutionarily conserved residues (Fig. 1c-d), and led to unstable protein when transfected into heterologous cells (Fig. 1e,Supplementary Fig. 4). Although truncating mutations were identified in both the middle and end of the protein, p.R73 transversions predominated (Fig. 1c), with the p.R73L clearly a founder mutation. Among JSRD, the phenotype was characterized by frequent occurrence of NPH (9/20) and polydactyly (9/20), while retinal dystrophy and congenital hepatic fibrosis were never observed. In keeping with this, sequence analysis of 96 patients with Bardet-Biedl syndrome identified no homozygous mutations, since retinopathy is a key feature of this condition. In two JSRD patients (MTI161 and MTI467), polydactyly was associated with either tongue tumors or multiple oral frenula, corresponding to the Oro-Facio-Digital type VI (or Varadi-Papp) syndrome16 (OMIM%277170), indicating that TMEM216 mutations are the first known identified cause. In the 11 MKS fetuses with TMEM216 mutations, distinctive clinical features were skeletal dysplasia, including intrauterine growth retardation or bowing of the long bones in six fetuses, cleft palate in four, and anencephaly in two (Table 1,Supplementary Fig. 5).
TMEM216 is a poorly annotated gene, with RefSeq predicting a protein of just 86 aa, suggesting potential alternative splicing. To characterize this mRNA we performed Northern analysis with a commercial human fetal blot, and found a single major mature isoform at about 1.4 Kb (Supplementary Fig. 6a), agreeing with the predicted 1.3 Kb of the longest representative cDNAs. To interrogate splicing we designed primers complementary to the furthest 5′ and 3′ regions of the known cDNA, and sequenced 48 cloned PCR products from a 20 week gestation human fetal brain library. We identified four major splice isoforms, the longest and most prevalent predicting a protein of 148 aa (Supplementary Fig. 6b), which we consider to be the full-length mRNA. There is also extensive alternative splicing, encoding very short proteins (Supplementary Fig. 6b), the functions of which were not evaluated further. Importantly, we did not find any mutations in any of the putative UTRs.
To elucidate roles for TMEM216 in human development, we first examined its expression in human embryonic tissues. In situ hybridization analysis in human embryos confirmed expression in the central nervous system, limb bud, kidney and cartilage (Supplementary Fig. 6c-h), which is similar to the broad and relatively low-level expression pattern of other JSRD/MKS genes. We next raised an anti-TMEM216 polyclonal affinity-purified antibody against aa 81-90, demonstrated specificity (Supplementary Fig. 7a-b), and immunostained two different ciliated cell lines (inner medullary collecting duct [IMCD3] and retinal pigment epithelium [hRPE]). We observed localization with the base of the primary cilium or adjacent basal body in the majority of cells, as marked by either acetylated or glutamylated tubulin staining (Fig. 2a-d). TMEM216 antibody also reacted strongly to the base of cilia in organs like kidney containing ciliated cells (Fig. 2c,Supplementary Fig. 7c), but failed to react with these structures in hTERT-immortalized fetal TMEM216 p.R85X homozygous mutant fibroblasts (Supplementary Fig. 7d). Epitope-tagged TMEM216 showed similar localization to the base of cilia and other microtubule structures (i.e. mitotic spindle in cells undergoing late telophase, Supplementary Fig. 8).
Figure 2
Figure 2
Ciliary localization of TMEM216. (a-d) Overlapping localization of endogenous TMEM216 (green) and acetylated α-tubulin or GT335 (glutamylated tubulin) (red) at the base of the primary cilium (arrows) in IMCD3 (a, b), proximal renal tubules (c) (more ...)
In TMEM216 p.R85X mutant fibroblasts, we noticed a failure in ciliogenesis following 48 hr serum starvation (Fig. 3a) compared with controls. Western analysis of whole cell lysates from control fibroblasts identified a band at 19 kD (Fig. 3b), matching the predicted 148 aa full length protein, whereas this band was attenuated or lost in TMEM216 p.R85X fibroblasts or in IMCD3 cells in which Tmem216 was knockded down.
Figure 3
Figure 3
TMEM216 mutation or knockdown results in impaired ciliogenesis and centrosome docking. (a) Two different TMEM216-mutated patient fibroblasts lines show defective ciliogenesis and impaired centrosome docking (marked by γ-tubulin). Scale bar: left (more ...)
To determine the basis of the ciliogenesis defect, we performed transient transfection of monolayers of IMCD3 cells with two separate Tmem216 siRNA duplexes. Tmem216 knockdown prevented ciliogenesis in polarized cells, and blocked correct docking of centrosomes at the apical cell surface (Fig. 3c), as seen previously for Meckelin and MKS117. This ciliogenesis defect was quantified by comparing the percentage of cells with cilia (defined as > 1 μm length) vs. those without cilia (< 1μm length), and by analyzing the percentage of cells with centrosomes located apical to the nucleus. In cells in which Tmem216 was knocked down, we observed a striking defect in both of these measurements compared with two separate control transfections (Fig. 3d-e, chi-squared test, p<0.001, for 350 cells from each condition), suggesting its requirement in centrosome docking.
The similarities in cellular phenotypes of Mks3 and Tmem216 knockdown, and subcellular localizations of Meckelin and TMEM216, prompted us to ask if the two proteins could interact. Firstly, GFP-tagged TMEM216 was immunoprecipitated with antibodies to either N- or C-terminal portions of Meckelin (Fig. 4a) and, secondly, the reciprocal IP experiment used α-GFP antibody to pull down Meckelin (Fig. 4b). Both assays detected a complex between TMEM216 and Meckelin.
Figure 4
Figure 4
TMEM216 complexes with Meckelin, and their loss results in Rho hyperactivation and actin cytoskeleton remodelling. (a) TMEM216-GFP (~37 kDa; arrow) is immunoprecipitated (IP) with anti-Meckelin antisera against either the N- or C-termini from whole cell (more ...)
Many aspects of actin-dependent polarized cell behavior, including morphogenetic cell movements18 and ciliogenesis19, are mediated by the planar cell polarity (PCP) pathway of non-canonical Wnt signaling20. We therefore first examined RhoA, since the Rho family of small GTPases are key mediators of this pathway20-21. Consistent with previous results following MKS3 loss22, we found that RhoA signaling was hyperactived in both TMEM216 p.R85X fibroblasts or following Tmem216 knockdown (Fig. 4c-d), despite normal total amounts of RhoA in these cells. Centrosome docking at the apical cell surface is prevented by the interruption of actin remodeling23, and is dependent on both RhoA activation and regulation by the core PCP protein Dishevelled (Dvl)24. We confirmed that RhoA is localized to the basal body in confluent IMCD3 cells but, following Tmem216 knockdown for 24 hr, RhoA was mislocalized to peripheral regions of the basal body and to basolateral cell-cell contacts (Fig. 4e), consistent with translocation of ectopically-activated RhoA to the cytosol25. Tmem216 knockdown also showed evidence of a mislocalization of γ-tubulin at the centrosome/basal body for this timepoint, which suggests a defect in γ-tubulin nucleation, one of the earliest steps in ciliogenesis26. The established role of RhoA in modulating the actin cytoskeleton in the PCP pathway then led us to evaluate MKS2 patient fibroblast lines for alterations. We found a co-localization of actin stress fibers and the actin cross-linker filamin-A in the cytoplasm of these mutant cells, which was absent in control (Fig. 4f).
We next looked at Dvl signaling in cells, since cilia negatively regulate Dvl activation27, and Dvl mediates Rho activation at the apical surface of ciliated epithelial cells24. We found that loss of TMEM216 increased phosphorylation of Dvl1 (Fig. 5aleft and right panel), implying that TMEM216 modulates hyper-responsiveness of signaling pathways mediated by Dvl and RhoA. We found that Rho inhibition also increased the Dvl1 phosphorylation in ciliated cells, supporting the existence of feedback mechanism between Rho and Dvl (Fig. 5a, right panel). Unexpectedly, the constitutive Dvl1 phosphorylation associated with TMEM216 loss was blocked by Rho inhibition (Fig. 5a, right panel), suggesting that this loss in ciliated cells can modify the feedback mechanism. Although this possibility warrants further investigation, our data nevertheless suggest a working model in which Dvl1, RhoA and TMEM216 may serve as part of a complex in the pericentrosomal compartment to mediate cellular polarization and centrosomal apical docking. Previous studies have shown that Dvl and Rho contribute to a core framework for regulating the apical docking of centrosomes24, and we also see evidence of a common complex containing TMEM216, Dvl1 and RhoA in TMEM216-transfected cells (Fig. 5b). Since we saw no difference in the localization of Dvl1 following Tmem216 knockdown (Supplementary Fig. 9), these data predict that the hyperactivation of Rho in the absence of TMEM216 might be responsible for the centrosome docking defect at the apical cellular surface. As expected, we found that the impaired centrosome docking caused by Tmem216 knockdown was rescued in a dose-dependent fashion using Rho inhibitor (Fig. 5c).
Figure 5
Figure 5
TMEM216 disruption results in Dvl1 phosphorylation, and planar cell polarity-like phenotypes in zebrafish. (a) siRNA knockdown of Tmem216 (left panel) and TMEM216 p.R85X patient fibroblasts (right panel) cause an increase in the upper (phosphorylated) (more ...)
Meckelin is proposed to regulate centrosomal docking through the RhoA signaling pathway22, and bears similarity to the Frizzled family of transmembrane Wnt receptors15. Direct evidence of a role for Meckelin in PCP signaling stems from zebrafish embryo morphant phenotypes following morpholino knock-down of mks328. These included defects in gastrulation movement (a shortened body axis, broad notochords and misshapen somites), which are typical of defects in non-canonical (PCP) Wnt signaling, and have been observed in numerous ciliary and basal body morphants28-29. We observed identical ciliary phenotypes in tmem216 morphants, which were largely rescued by RNA encoding human TMEM216 (Fig. 5d), and fully rescued by RNA encoding non-targetable zebrafish tmem216 (not shown). We therefore directly compared the tmem216 and mks3 morphant phenotypes in zebrafish, and noted similar defects in both live embryos and in embryros in which pronephric mesoderm, anterior neural structures, adaxial mesodermal cells, and somites were labeled with a krox20, pax2, and myoD riboprobe cocktail (Fig. 5e-f). Quantification demonstrated alteration of convergence to the midline and extension along the AP axis consistent with a PCP defect, although the AP extension defect was more pronounced in the mks3 compared to the tmem216 morphant.
Recent work has implicated the tetraspanin TSPAN12 in the regulation of Norrin signaling by the Wnt receptor Frizzled-4 and coreceptor LRP513. We therefore speculate that TMEM216, a novel tetraspan protein, forms a non-canonical Wnt receptor-coreceptor complex with Meckelin. Our data support a role for both proteins in mediating PCP signaling through the RhoA pathway to cause actin cytoskeleton rearrangements, although whether Rho functions upstream or downstream of Dvl1 remains to be determined. In apical regions of the cell, such actin reorganization would be an essential step before the centrosome/basal body could dock correctly and initiate ciliogenesis. The identification of mutations in TMEM216 as a cause of JSRD and MKS therefore further emphasizes the interrelationship between cell polarity, cellular morphogenesis and signal transduction pathways.
Supplementary Material
We thank Marshfield Clinic Research Foundation, Center for Inherited Disease Research (supported by the NIH NHBLI) for genotyping support, Adam Felsenfeld and the Medical Sequencing Initiative at NHGRI, and support for sequencing from the Broad Institute and the Broad Sequencing Platform, Jennifer Meerloo at the UCSD Neurosciences Microscopy Core (Supported by NINDS P30NS047101). We thank Chaim Jalas at Bonei Olam Center for Rare Jewish Genetic Disorders, Brooklyn, NY, Michael R. Eccles at University of Otago, Dunedin, New Zealand, Heather M. Harville at University of Michigan, Gaetano Tortorella, Silvana Briuglia, Roberto Chimenz, Romina Gallizzi and Marilena Briguglio at University of Messina, Italy, and the SOFFOET (French SOciety of FOETalpathology) for patient referrals. We thank Ewan Morrison, Peter Novick and Susan Ferro-Novick for helpful discussions and Dr. Carsten Janke for GT335 antibody. We thank Emmanuel Genin for linkage analysis, Sophie Audolent, Candice Babarit, Flora Legendre, and Helory-Mael Gaudé for technical help, and Olivier Duc for confocal microscopy. S.M-Z. is supported by INSERM-DGRSRT (CS/RN/2008 n°87), and N.K. is the Brumley Professor. 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 (J.-E.L.), BDF Newlife, the Medical Research Council (G0700073) and the Sir Jules Thorn Charitable Trust (09/JTA C.A.J.), l’Agence National pour la Recherche (ANR 07-MRAR-Fetalciliopathies T.A-B.), National Institutes of Health (R01 DK068306 F.H., R01 DK072301 R01 HD04260, R01 DK075972 N.K., NRSA fellowship F32 DK079541 E.D.D., R01 NS052455 and R01 NS04843 J.G.G., and Burroughs Wellcome Fund, and Howard Hughes Medical Institute (F.H. and J.G.G.).
Research subjects
We used standard methods to isolate genomic DNA from peripheral blood of the affected children and family members or from frozen fetal tissue or amniocytes. Chromosome analysis was performed for at least one patient of each family. Informed consent was obtained from all participating families and the studies were approved by the Ethics Boards of Leeds (East), Casa Sollievo della Sofferenza, Hôpital Necker-Enfants Malades, and UCSD.
Genetic mapping
To refine the MKS2/JBTS2 locus, the 10K Affymetrix SNP array was used to perform a genome-wide linkage search in nine consanguineous families with MKS. We performed multipoint linkage analysis using MERLIN software, assuming a fully penetrant recessive model, a disease allele frequency of 0.001, and allowed for heterogeneity between families. Areas of homozygosity on chromosome 11 were confirmed by performing high-resolution haplotype analysis.
Mutation screening
Mutational screening of TMEM216 was performed by direct sequencing of PCR products of the six coding exons and the adjacent intronic junctions in JSRD/MKS families showing potential linkage to the locus and all MKS cases. To test for TMEM216 mutations in the cohort of 460 JSRD patients we applied the high resolution melting (HRM) technique30 using a LightCycler 480 (Roche Applied Science), with the same primers and optimized PCR conditions (Supplementary Table 1). Segregation of the identified mutations was investigated in all available family members. Missense mutations were excluded in the following ethnically matched controls: wildtype at p.R73 for 227 Italian and 109 Turkish; and wildtype at p.G77 and p.L114 for 158 Palestinian and 112 Tunisian individuals. All mutations were also excluded in 200 Central Asian (predominantly Pakistani), 200 European (predominantly British), as well as a cohort of 96 ethnically diverse individuals.
Founder haplotype analysis
The region surrounding TMEM216 was saturated with 14 single nucleotide polymorphisms and three microsatellite markers in ten patients homozygous for the same p.R73L mutation. Estimation of the mutation age was calculated as reported31.
Genetic location is based on Human Genome Browser build 36.3 ( The ciliary proteome was searched using web-based tools10-11. RefSeq and Pfam were respectively accessed at ( and (
Full-length TMEM216 was cloned into the pcDNA3.0 vector, and then shuttled into the mCherry-, EGFP-, and FLAG- containing vectors. Mutations were introduced into TMEM216-pEGFP-N3 by QuickChange mutagenesis (Stratagene). TMEM216 open reading frame was also cloned into pCS2+ vector in order to make RNA for injection into zebrafish embryos.
Cells and antibodies
Mouse inner medullary collecting duct (IMCD3), human hTERT-immortalized retinal pigmentary epithelial (hRPE), and human embryonic kidney (HEK293) cells were grown in Dulbecco’s minimum essential medium (DMEM)/Ham’s F12 supplemented with 10% fetal calf serum at 37°C/5% CO2. Fibroblasts were immortalized with the hTERT system, and maintained in Fibroblast Growth Medium (Genlantis) supplemented with 10% fetal calf serum and 0.2 mg/ml geneticin. Normal, undiseased control fibroblasts were gestationally-age matched to fibroblasts from MKS patients22. Primary antibodies used were: mouse α-EGFP antibody (Covance MMS-118R); mouse anti-GFP and rabbit A.V. peptide (“Living Colors”, Clontech); mouse anti-α-tubulin, mouse anti-γ-tubulin, mouse anti-acetylated-tubulin (Sigma-Aldrich); mouse-anti-glutamylated tubulin (GT335)32, rabbit-anti-γ-tubulin, rabbit-anti-Meckelin, mouse anti-β actin (Abcam); mouse anti-filamin A (AbNova); and mouse anti-Dvl1 (Santa Cruz Biotechnology SC-8025); mouse anti-RhoA (Cytoskeleton). Rabbit-anti-Meckelin C-terminus, raised against amino acids 982-995, has been described previously17. Rabbit-anti-Meckelin N-terminus, raised against amino acids 100-113, has also been described22. Secondary antibodies were Alexa-Fluor 488-Alexa-Fluor 594- and Alexa-Fluor 568-conjugated goat anti-mouse IgG and goat anti-rabbit IgG (Molecular Probes), and HRP-conjugated goat anti-mouse and goat anti-rabbit (Dako). Alexa-Fluor 488 and 633 phalloidin conjugate (Molecular Probes) was used to visualize F-actin.
Biochemical assays
Constructs encoding wildtype or mutant TMEM216 were transfected into 293T cells in a ratio of 20:1 TMEM216:TK-βgal vector. Cells were lysed after 48h and samples analyzed by Western blot with α-EGFP antibody (1:500) and β-gal assay to standardize transfection efficiency33. Normalized loading levels were confirmed by blotting with α-tubulin (1:2000).
Rabbit-anti-TMEM216 antiserum was raised against the peptide sequence NLCQRKMPLS(C), comprising amino acids 81-90, by GenScript (Piscataway, NJ, USA). Antiserum was affinity-purified, and co-immunoprecipitation was performed as described previously34. Whole cell extracts (WCE) were prepared from confluent untransfected HEK293 cells, or IMCD3 cells that had been transiently transfected with 1.0 μg plasmid constructs in 90mm tissue culture dishes, or scaled down as appropriate. WCE supernatants were processed for immunoprecipitation experiments by using 5 μg affinity-purified mouse anti-GFP, or 5 μg MAbs, or 5-10 μg purified IgG fractions from rabbit polyclonal antisera, coupled to protein G- and/or protein A-sepharose beads (GE Healthcare) Immunoprecipitations were performed in reduced salt incubation buffer (20 mM Tris, pH7.5, 25 mM NaCl, 2 mM EDTA, 0.5 mM EGTA, 0.02% [w/v] NaN3, 10% [v/v] glycerol, 10% [v/v] ethanol, 0.1% [v/v] protease inhibitor cocktail). For assessing Dvl1 phosphorylation status, extraction and wash buffers were supplemented with phosphatase inhibitor cocktail (Sigma Aldrich).
In situ hybridization in human embryos
Human embryos were collected from terminated pregnancies using the mefiprestone protocol in agreement with French bioethics laws (94-654 and 04-800). Embryos were fixed in 11% formaldehyde, 60% ethanol and 10% acetic acid, embedded in paraffin and sectioned at 5 μm. Primers (Supplementary Table 1) were selected for RT-PCR amplification on RNA extracted from a whole C12 (4w) embryo to be used as template for generating the riboprobes, as described previously35. Sections were hybridized with a Digoxygenin labeled probe at 70°C overnight, and digoxygenin was detected with an anti-DIG-Fab’ antibody (Roche) at 1:1000.
Immunofluorescence and confocal microscopy
IMCD3 or hTERT-immortalized fetal fibroblasts were seeded at 20 × 103 cells/well on glass coverslips in six-well plates and fixed in ice-cold methanol (5 minutes at 4°C) or 2% paraformaldehyde (20 minutes at room-temp) as described previously17,22. Confocal images were obtained using a Nikon Eclipse TE2000-E system, controlled by EZ-C1 3.50 (Nikon) software. Images were processed in Metamorph, and figures were assembled using Adobe Photoshop CS3.
Transfection and siRNA
For transfection with plasmids, cells at 90% confluency were transfected using Lipofectamine 2000 (Invitrogen). Cells were incubated for 24 to 72 hrs prior to lysis or immunostaining. For RNAi knockdown in IMCD3 cells, siRNA duplexes (Supplementary Table 1) were designed against different regions of the mouse Tmem216 (“Stealth Select”, Invitrogen). Mks3 siRNA reagents have been described previously22. The medium or low GC non-targeting negative controls (Invitrogen) were used as scrambled siRNA controls. Irrelevant siRNA duplexes against Hhari were used as a second negative control (a gift from P. Robinson, University of Leeds, UK). Individual duplexes (20 nM) or siRNA pools (total 60 nM) were transfected into IMCD3 cells at 60-80% confluency using Lipofectamine 2000 RNAiMAX (Invitrogen). The efficiency of siRNA transfections, as determined with BLOCK-iT Fluorescent Oligo (Invitrogen), was >60%. Further assays were carried out at 72 hours after transfection.
Gene expression analyses with quantitative real-time PCR
For the relative quantitation of gene expression we used quantitative real-time PCR for the standard curve method. Total RNA (1 μg) was reverse-transcribed using the Superscript III first strand cDNA system (Applied Biosystems). PCR analysis of cDNA was performed using dHPLC-purified primers (Supplementary Table 1) specific for mouse Tmem216 (Invitrogen), following optimization to eliminate primer-dimers and subsequent confirmation by analysis of amplimer dissociation curves following a qPCR run. Each reaction was run in triplicate. Amplimer levels were quantified continuously with the SYBR GreenER qPCR system (Invitrogen) using an ABI 7500 instrument, essentially as described previously15. Hprt RNA was amplified for normalization, quantified in Supplementary Figure 10.
RhoA activation assay
The activated GTP-bound isoform of RhoA was specifically assayed in pull-down assays using a GST fusion protein of the Rho effector rhotekin (Cytoskeleton), using conditions recommended by the manufacturers. Rho activity was inhibited by treating cells with cell permeable exoenzyme-C3-transferase (Cytoskeleton) at 2 μg/ml for 5 hr under standard cell culture conditions. Results shown are representative of three separate experiments. Full scans of all Western blots are shown in Supplementary Figure 11.
Identification of ciliary defect phenotypes in Zebrafish
To knockdown tmem216 in zebrafish, a translational blocking morpholino antisense oligonucleotide (MO) or control (Gene Tools) or control was microinjected (4ng/nl) into one-two cell stage embryos, obtained from natural spawning of wild-type (AB) zebrafish lines. The mRNA encoding full-length human TMEM216 was co-injected where indicated. Endogenous mks3 was suppressed with a splice-blocking MO described previously (3 ng/nl)28. For assessment of gastrulation phenotypes (shortened body axis, wide undulating notochord, thin and elongated somites, and small anterior structures), mid-somitic embryos were scored blind at 8 somites (live; 80-100 embryos/injection), or 10-11 somites (morphometric analyses). Embryos were fixed overnight in 4% PFA, hybridized in situ with DIG-labeled krox20, pax2, and myoD riboprobes according to standard protocols, and flat-mounted for imaging and analysis. At 3 days postfertilization, morphological phenotype of morphants were quantified under bright-field microscopy based upon ciliary defects (hydrocephalus, small brain, heart edema, and curved tail) or embryonic lethal phenotypes.
DATABASE ACCESSION NUMBERS. Full length TMEM216 open reading frame is encoded in EST BI910875. Accession number for human TMEM216 protein sequence is NP_001167462.
COMPETING FINANCIAL INTERESTS. The authors declare that they have no competing financial interests.
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