Abstract
Leber congenital amaurosis (LCA) causes blindness or severe visual impairment at or within a few months of birth. Here we show, using homozygosity mapping, that the LCA5 gene on chromosome 6q14, which encodes the previously unknown ciliary protein lebercilin, is associated with this disease. We detected homozygous nonsense and frameshift mutations in LCA5 in five families affected with LCA. In a sixth family, the LCA5 transcript was completely absent. LCA5 is expressed widely throughout development, although the phenotype in affected individuals is limited to the eye. Lebercilin localizes to the connecting cilia of photoreceptors and to the microtubules, centrioles and primary cilia of cultured mammalian cells. Using tandem affinity purification, we identified 24 proteins that link lebercilin to centrosomal and ciliary functions. Members of this interactome represent candidate genes for LCA and other ciliopathies. Our findings emphasize the emerging role of disrupted ciliary processes in the molecular pathogenesis of LCA.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on SpringerLink
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Dharmaraj, S. et al. A novel locus for Leber congenital amaurosis maps to chromosome 6q. Am. J. Hum. Genet. 66, 319–326 (2000).
Mohamed, M.D. et al. Progression of phenotype in Leber's congenital amaurosis with a mutation at the LCA5 locus. Br. J. Ophthalmol. 87, 473–475 (2003).
Ostrowski, L.E. et al. A proteomic analysis of human cilia: identification of novel components. Mol. Cell. Proteomics 1, 451–465 (2002).
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, 961–962 (2006).
Giessl, A. et al. Differential expression and interaction with the visual G-protein transducin of centrin isoforms in mammalian photoreceptor cells. J. Biol. Chem. 279, 51472–51481 (2004).
Guarguaglini, G. et al. The forkhead-associated domain protein Cep170 interacts with Polo-like kinase 1 and serves as a marker for mature centrioles. Mol. Biol. Cell 16, 1095–1107 (2005).
Quintyne, N.J. et al. Dynactin is required for microtubule anchoring at centrosomes. J. Cell Biol. 147, 321–334 (1999).
Burakov, A., Nadezhdina, E., Slepchenko, B. & Rodionov, V. Centrosome positioning in interphase cells. J. Cell Biol. 162, 963–969 (2003).
Rigaut, G. et al. A generic protein purification method for protein complex characterization and proteome exploration. Nat. Biotechnol. 17, 1030–1032 (1999).
Shinmura, K., Tarapore, P., Tokuyama, Y., George, K.R. & Fukasawa, K. Characterization of centrosomal association of nucleophosmin/B23 linked to Crm1 activity. FEBS Lett. 579, 6621–6634 (2005).
Ma, Z. et al. Interaction between ROCK II and nucleophosmin/B23 in the regulation of centrosome duplication. Mol. Cell. Biol. 26, 9016–9034 (2006).
Zhang, H. et al. B23/nucleophosmin serine 4 phosphorylation mediates mitotic functions of polo-like kinase 1. J. Biol. Chem. 279, 35726–35734 (2004).
Shu, X. et al. RPGR ORF15 isoform co-localizes with RPGRIP1 at centrioles and basal bodies and interacts with nucleophosmin. Hum. Mol. Genet. 14, 1183–1197 (2005).
Ginisty, H., Sicard, H., Roger, B. & Bouvet, P. Structure and functions of nucleolin. J. Cell Sci. 112, 761–772 (1999).
Li, D., Dobrowolska, G. & Krebs, E.G. The physical association of casein kinase 2 with nucleolin. J. Biol. Chem. 271, 15662–15668 (1996).
Hollander, B.A., Liang, M.Y. & Besharse, J.C. Linkage of a nucleolin-related protein and casein kinase II with the detergent-stable photoreceptor cytoskeleton. Cell Motil. Cytoskeleton 43, 114–127 (1999).
Lim, A.C., Tiu, S.Y., Li, Q. & Qi, R.Z. Direct regulation of microtubule dynamics by protein kinase CK2. J. Biol. Chem. 279, 4433–4439 (2004).
Faust, M., Gunther, J., Morgenstern, E., Montenarh, M. & Gotz, C. Specific localization of the catalytic subunits of protein kinase CK2 at the centrosomes. Cell. Mol. Life Sci. 59, 2155–2164 (2002).
Schermer, B. et al. Phosphorylation by casein kinase 2 induces PACS-1 binding of nephrocystin and targeting to cilia. EMBO J. 24, 4415–4424 (2005).
Hu, J., Bae, Y.K., Knobel, K.M. & Barr, M.M. Casein kinase II and calcineurin modulate TRPP function and ciliary localization. Mol. Biol. Cell 17, 2200–2211 (2006).
Mhawech, P. 14–3-3 proteins—an update. Cell Res. 15, 228–236 (2005).
Pietromonaco, S.F., Seluja, G.A., Aitken, A. & Elias, L. Association of 14–3-3 proteins with centrosomes. Blood Cells Mol. Dis. 22, 225–237 (1996).
Chen, C.Y., Olayioye, M.A., Lindeman, G.J. & Tang, T.K. CPAP interacts with 14–3-3 in a cell cycle-dependent manner. Biochem. Biophys. Res. Commun. 342, 1203–1210 (2006).
Chang, B. et al. In-frame deletion in a novel centrosomal/ciliary protein CEP290/NPHP6 perturbs its interaction with RPGR and results in early-onset retinal degeneration in the rd16 mouse. Hum. Mol. Genet. 15, 1847–1857 (2006).
den Hollander, A.I. et al. Mutations in the CEP290 (NPHP6) gene are a frequent cause of Leber congenital amaurosis. Am. J. Hum. Genet. 79, 556–561 (2006).
Sayer, J.A. et al. The centrosomal protein nephrocystin-6 is mutated in Joubert syndrome and activates transcription factor ATF4. Nat. Genet. 38, 674–681 (2006).
Valente, E.M. et al. Mutations in CEP290, which encodes a centrosomal protein, cause pleiotropic forms of Joubert syndrome. Nat. Genet. 38, 623–625 (2006).
van Wijk, E. et al. The DFNB31 gene product whirlin connects to the Usher protein network in the cochlea and retina by direct association with USH2A and VLGR1. Hum. Mol. Genet. 15, 751–765 (2006).
Roepman, R. et al. Interaction of nephrocystin-4 and RPGRIP1 is disrupted by nephronophthisis or Leber congenital amaurosis-associated mutations. Proc. Natl. Acad. Sci. USA 102, 18520–18525 (2005).
Brandstatter, J.H., Koulen, P., Kuhn, R., van der, P.H. & Wassle, H. Compartmental localization of a metabotropic glutamate receptor (mGluR7): two different active sites at a retinal synapse. J. Neurosci. 16, 4749–4756 (1996).
Acknowledgements
We thank the LCA families for their participation; H. Brunner, C. Johnson, N. Knoers and H. Kremer for discussions; C.J. Gloeckner for the TAPe constructs; T. Goldmann, E. Sehn, J. Hehir-Kwa, I. Janssen, K. Voesenek, A. Schumacher and S. Schöffmann for technical assistance; K. Klima, R. Pigeon and C. Robert for organizing the clinical data from affected individuals; S. Yzer, L.I. van den Born, S. Kohl, B. Wissinger, E. de Baere, B.P. Leroy, W. Bergen, K. Rohrschneider and C.B. Hoyng for sharing patient samples; and the Marshfield Mammalian Genotyping Service for carrying out genotyping in the Pakistani families. This research was supported by grants from The Netherlands Organisation for Scientific Research (916.56.160 to A.I.d.H.); the Foundation Fighting Blindness USA (BR-GE-0606-0349-RAD to A.I.d.H.); the Dutch Kidney Foundation (C04.2112 to R.R.); Landelijke Stichting voor Blinden en Slechtzienden (to A.I.d.H. and F.P.M.C.); Algemene Nederlandse Vereniging ter Voorkoming van Blindheid (to A.I.d.H., R.R. and F.P.M.C); Rotterdamse Vereniging Blindenbelangen (to R.R. and F.P.M.C.); Stichting Blindenhulp (to R.R. and F.P.M.C.); Stichting OOG (to R.R. and F.P.M.C.); the British Retinitis Pigmentosa Society (GR552 to R.R.); the European Union 6th Framework RETNET (MRTNCT-2003-504003 to F.P.M.C. and M.U.), EVI-GENORET (LSHG-CT-2005 512036 to M.U., F.P.M.C. and R.R.) and INTERACTION PROTEOME (LSHG-CT-2003-505520 to M.U.); the Wellcome Trust (061682 and 073477 to C.F.I. and M.D.M., and 068579 to M.E.C.); Yorkshire Eye Research (006 to C.F.I.); the Foundation Fighting Blindness Canada (to R.K.K. and F.P.M.C.); the Fonds de la Recherche en Santé Québec; TD Financial Group (to R.K.K.); Foundation of Retinal Research; The Grousbeck Foundation; The Edel and Krieble Funds; the Ort Family Foundation (to I.H.M., S.D. and R.K.K.); Pro Retina Germany (to M.U. and R.R.); Deutsche Forschungsgemeinschaft (Wo 548-6 to U.W.), FAUN-Stiftung (to U.W.) and Forschung contra Blindheit (to U.W.).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary Fig. 1
Sequences of LCA5 mutations identified in LCA families. (PDF 26 kb)
Supplementary Fig. 2
Evolutionary conservation of lebercilin and C21ORF13 proteins. (PDF 21 kb)
Supplementary Fig. 3
Expression of the LCA5 gene in human tissues and mammalian cell lines, and detection of lebercilin in mouse tissues. (PDF 467 kb)
Supplementary Fig. 4
Expression of the recombinant lebercilin proteins in ARPE-19 and COS-1 cells. (PDF 328 kb)
Supplementary Fig. 5
Expression of the TAPe constructs and immunoprecipitation of lebercilin. (PDF 422 kb)
Supplementary Table 1
Refinement of the LCA5 interval in three Pakistani LCA families. (PDF 49 kb)
Supplementary Table 2
Homozygous chromosomal regions in patients 27240 and 28609. (PDF 46 kb)
Supplementary Table 3
Protein/peptide summaries of LC-MSMS analysis of tandem affinity-purified lebercilin protein complexes. (PDF 489 kb)
Supplementary Table 4
Primer sequences for amplification of the exons and splice junctions of the LCA5 gene. (PDF 48 kb)
Rights and permissions
About this article
Cite this article
den Hollander, A., Koenekoop, R., Mohamed, M. et al. Mutations in LCA5, encoding the ciliary protein lebercilin, cause Leber congenital amaurosis. Nat Genet 39, 889–895 (2007). https://doi.org/10.1038/ng2066
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/ng2066