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Mutations in the gene encoding the basal body protein RPGRIP1L, a nephrocystin-4 interactor, cause Joubert syndrome

Abstract

Protein-protein interaction analyses have uncovered a ciliary and basal body protein network that, when disrupted, can result in nephronophthisis (NPHP), Leber congenital amaurosis, Senior-Løken syndrome (SLSN) or Joubert syndrome (JBTS)1,2,3,4,5,6. However, details of the molecular mechanisms underlying these disorders remain poorly understood. RPGRIP1-like protein (RPGRIP1L) is a homolog of RPGRIP1 (RPGR-interacting protein 1), a ciliary protein defective in Leber congenital amaurosis7,8. We show that RPGRIP1L interacts with nephrocystin-4 and that mutations in the gene encoding nephrocystin-4 (NPHP4) that are known to cause SLSN disrupt this interaction. RPGRIP1L is ubiquitously expressed, and its protein product localizes to basal bodies. Therefore, we analyzed RPGRIP1L as a candidate gene for JBTS and identified loss-of-function mutations in three families with typical JBTS, including the characteristic mid-hindbrain malformation. This work identifies RPGRIP1L as a gene responsible for JBTS and establishes a central role for cilia and basal bodies in the pathophysiology of this disorder.

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Figure 1: Structure of RPGRIP1L and RPGRIP1 and their mRNA expression in different tissues.
Figure 2: RPGRIP1L interaction with nephrocystin-4.
Figure 3: Colocalization of RPGRIP1L and nephrocystin-4 in the basal bodies of mammalian cells.
Figure 4: Localization of RPGRIP1L in the retina.
Figure 5: Localization of RPGRIP1L in brain and kidney.
Figure 6: Mapping and sequence analysis of families carrying RPGRIP1L mutations.

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Acknowledgements

We thank A.I. den Hollander and H. Kremer for discussions, the University of Washington Center for Ecogenetics and Environmental Health (CEEH) for SNP mapping, C. Hutter for biostatistical advice and C. Beumer, S. van der Velde-Visser, G. Merkx and E. Sehn for expert technical assistance. We thank all the participating families with Joubert syndrome and S. Aysun, P.F. Chance, D. Knutzen and J. Adkins for clinical and technical support. We thank Y. Xiumin (Department of Cell Biology, Max Planck Institute of Biochemistry) for the antibodies to CEP290 and ninein; J. Salisbury (Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine) for the anti-pan-centrin and B.K. Yoder (Department of Cell Biology, University of Alabama at Birmingham Medical Center) for rabbit anti-polaris. This work was supported by grants from the Dutch Kidney Foundation (C04.2112 to N.K. and R.R.), Pro Retina Germany (to R.R.), EVI-GENORET (LSHG-CT-2005 512036 to F.P.M.C. and R.R.), Rotterdamse Vereniging Blindenbelangen (to F.P.M.C. and R.R.), Stichting Blindenhulp (to F.P.M.C. and R.R.), Stichting OOG (to F.P.M.C. and R.R.), Algemene Nederlandse Vereniging ter Voorkoming van Blindheid (to F.P.M.C. and R.R.), Landelijke Stichting voor Blinden en Slechtzienden (to A.K. and F.P.M.C.), FAUN-Stiftung (to U.W.), Forschung contra Blindheit (to T.M. and U.W.), the US National Institutes of Health (grants K23-NS45832 to M.A.P., K24-HD46712 to I.A.G., and K12-RR023265 to D.D., the University of Washington Center on Human Development and Disability (NICHD P30 HD02274 to D.D., M.A.P. and I.A.G.), the University of Washington CEEH, the US National Institute of Environmental Health Sciences (P30ES07033 to F.M.F.) and the March of Dimes Endowment for Healthier Babies at Children's Hospital in Seattle to D.D., M.A.P. and I.A.G.).

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Authors and Affiliations

Authors

Contributions

The study was designed by N.V.A.M.K. and R.R., who share senior authorship of this paper. H.H.A., S.E.C.v.B., S.J.F.L., T.A.P., T.M., A.K. and U.W. analyzed protein expression and localization; T.M. and U.W. performed immuno-electron microscopy; H.H.A. and S.J.F.L. analyzed protein-protein interactions; S.E.C.v.B. analyzed gene expression; M.A.P., H.O., H.Y.K. and N.V.A.M.K. acquired clinical data; D.D., N.T.G and F.M.F. performed mapping of microsatellites and SNPs; S.E.C.v.B., N.T.G. and K.V. performed sequence analysis of families; D.D., H.G.B., F.P.M.C., I.A.G., N.V.A.M.K. and R.R. supervised the work and H.H.A., D.D. and R.R. wrote the manuscript, with assistance from most of the coauthors.

Corresponding author

Correspondence to Ronald Roepman.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Reciprocal GST pulldown analysis of RPGRIP1L and nephrocystin-4. (PDF 319 kb)

Supplementary Fig. 2

Localization of eCFP-nephrocystin-4 and mRFP-RPGRIP1LC2-N-end in COS-1 cells. (PDF 531 kb)

Supplementary Fig. 3

Confirmation of the specificity of the anti-RPGRIP1L antibody SNC040. (PDF 364 kb)

Supplementary Fig. 4

Confirmation of the specificity of staining by the anti-RPGRIP1L antibody SNC040 in the retina, brain and kidney. (PDF 121 kb)

Supplementary Fig. 5

Localization of RPGRIP1L and nephrocystin-4 in the retina. (PDF 167 kb)

Supplementary Fig. 6

Joubert syndrome–associated mutations in RPGRIP1L disrupt the interaction with nephrocystin-4. (PDF 209 kb)

Supplementary Table 1

Affected individuals with RPGRIP1L mutations show features of Joubert syndrome and Meckel-Gruber syndrome. (PDF 95 kb)

Supplementary Table 2

Oligonucleotide primers used for RT-PCR and sequencing of RPGRIP1L and for RT-PCR of RPGRIP1, NPHP4 and GAPDH. (PDF 87 kb)

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Arts, H., Doherty, D., van Beersum, S. et al. Mutations in the gene encoding the basal body protein RPGRIP1L, a nephrocystin-4 interactor, cause Joubert syndrome. Nat Genet 39, 882–888 (2007). https://doi.org/10.1038/ng2069

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