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Mutations in VIPAR cause an arthrogryposis, renal dysfunction and cholestasis syndrome phenotype with defects in epithelial polarization

Nature Genetics volume 42, pages 303–312 (2010)Cite this article

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A Corrigendum to this article was published on 01 March 2011

This article has been updated

Abstract

Arthrogryposis, renal dysfunction and cholestasis syndrome (ARC) is a multisystem disorder associated with abnormalities in polarized liver and kidney cells. Mutations in VPS33B account for most cases of ARC. We identified mutations in VIPAR (also called C14ORF133) in individuals with ARC without VPS33B defects. We show that VIPAR forms a functional complex with VPS33B that interacts with RAB11A. Knockdown of vipar in zebrafish resulted in biliary excretion and E-cadherin defects similar to those in individuals with ARC. Vipar- and Vps33b-deficient mouse inner medullary collecting duct (mIMDC-3) cells expressed membrane proteins abnormally and had structural and functional tight junction defects. Abnormal Ceacam5 expression was due to mis-sorting toward lysosomal degradation, but reduced E-cadherin levels were associated with transcriptional downregulation. The VPS33B-VIPAR complex thus has diverse functions in the pathways regulating apical-basolateral polarity in the liver and kidney.

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Figure 1: VPS33B interacts with VIPAR.
Figure 2: Intrahepatic defects in individuals with ARC and in Vipar-deficient zebrafish larvae.
Figure 3: Abnormal membrane polarization of mIMCD-3 cells in Vps33b and Vipar deficiencies.
Figure 4: Both Vipar and Vps33b are required for apical junction complex formation.
Figure 5: Abnormalities in cell morphology and growth in Vipar and Vps33b deficiencies.
Figure 6: The VPS33B-VIPAR complex interacts with RAB11A.
Figure 7: Investigation of the intracellular trafficking defects in Vps33b- and Vipar-deficient mIMCD-3 cells.

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Change history

  • 24 February 2011

    In the version of this article initially published, the first and second paragraphs of the Results incorrectly stated that the C14ORF133 gene product was a previously unidentified protein and that no antibody against the C14ORF133 gene product (VIPAR) was available. In fact, an earlier study (ref. 20 in the original manuscript) reported functional analyses of the C14ORF133 gene product (also called SPE-39), described the generation of a polyclonal antibody against human SPE-39 and reported an interaction between SPE-39 and VPS33B, similar to the interaction shown in Figure 1b. These errors have been corrected in the HTML and PDF versions of the article.

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Acknowledgements

A.R.C. holds the Children Liver Disease Foundation PhD Studentship and P.G. is a GlaxoSmithKline Clinician Scientist. A.Z. and F.M. are supported by Framework 6 IP EUTRACC, LSGH CT 2006037445. H.C. is supported by grants from the European Molecular Biology Organization (ASTF 121:2007) and European Science Foundation (Exchange Grant 2008). J.Z.R. is supported by the Biotechnology and Biosciences Research Council (BB/H002308/1). Thanks to F. Lock for her help with the E-cadherin luciferase assay, G. Reynolds for help with E-cadherin immunostaining and R. Knittel for her help with freeze fracture experiments. The authors also wish to thank all the families and clinicians who contributed to this research and thank the ARC syndrome association; Children Living with Inherited Metabolic Diseases (CLIMB); Birmingham Children's Hospital Research Foundation (BCHRF); WellChild; the Wellcome Trust; and the Intramural Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, US National Institutes of Health, for their generous financial support.

Author information

Author notes

  1. Anna Straatman-Iwanowska, Andreas Zaucker and Yoshiyuki Wakabayashi: These authors contributed equally to this work.

Authors and Affiliations

  1. Medical and Molecular Genetics, School of Clinical and Experimental Medicine, University of Birmingham, Birmingham, UK

    Andrew R Cullinane, Anna Straatman-Iwanowska, Andreas Zaucker, Christopher K Bruce, Guanmei Luo, Fatimah Rahman, Hakan Cangul, Ferenc Müller, Eamonn R Maher & Paul Gissen

  2. Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, US National Institutes of Health, Bethesda, Maryland, USA

    Yoshiyuki Wakabayashi & Irwin M Arias

  3. Department of Pediatric Gastroenterology, Hepatology and Nutrition, Hacettepe University, Ankara, Turkey

    Figen Gürakan

  4. Department of Pediatrics, Clinical Genetics Unit, Hacettepe University, Ankara, Turkey

    Eda Utine

  5. Department of Pediatric Gastroenterology, Hepatology and Nutrition, Uludag University School of Medicine, Bursa, Turkey

    Tanju B Özkan

  6. Department of Pediatrics, University Hospital of Rostock, Rostock, Germany

    Jonas Denecke

  7. Department of Pediatrics, University Hospital Rebro, Zagreb, Croatia

    Jurica Vukovic

  8. II Pediatric Unit, Gaslini Institute, Genoa, Italy

    Maja Di Rocco

  9. Metabolic Unit, Meyer Children's Hospital, Rambam Medical Center, Technion Faculty of Medicine, Haifa, Israel

    Hanna Mandel

  10. Department of Medical Genetics, Uludag University School of Medicine, Bursa, Turkey

    Hakan Cangul

  11. Division of Gastroenterology, Department of Pediatrics, Hepatology and Nutrition, The Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA

    Randolph P Matthews

  12. Birmingham Platelet Group, School of Clinical and Experimental Medicine, University of Birmingham, Birmingham, UK

    Steve G Thomas

  13. School of Biosciences, University of Birmingham, Birmingham, UK

    Joshua Z Rappoport

  14. Institute of Pathology, University of Tübingen, Tübingen, Germany

    Hartwig Wolburg

  15. Institute of Liver Studies, King′s College Hospital, London, UK

    A S Knisely

  16. The Liver Unit, Birmingham Children's Hospital, Birmingham, UK

    Deirdre A Kelly

  17. West Midlands Regional Genetics Service, Birmingham Women's Hospital, Birmingham, UK

    Eamonn R Maher

  18. The Inherited Metabolic Diseases Unit, Birmingham Children's Hospital, Birmingham, UK

    Paul Gissen

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  1. Andrew R Cullinane
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  2. Anna Straatman-Iwanowska
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Contributions

A.R.C. designed, conducted and interpreted experiments and wrote the manuscript. A.S.-I., A.Z. and Y.W. designed, conducted and interpreted experiments. C.K.B., G.L., F.R. and H.C. performed experiments. F.G., E.U., T.B.Ö., J.D., J.V., M.D.R., H.M. contributed subjects' DNA. R.P.M., S.G.T., J.Z.R., I.M.A., H.W., A.S.K., D.A.K., F.M. and E.R.M. designed and supervised experiments. P.G. conceived and directed the project, obtained funding and wrote the manuscript. All authors edited the manuscript.

Corresponding author

Correspondence to Paul Gissen.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Table (PDF 1815 kb)

Supplementary Movie 1

YFP-VP33B FLIP confocal live-cell microscopy. YFP-VPS33B was transfected into mIMCD-3 cells and a Fluorescence Loss In Photobleaching (FLIP) experiment was carried out in live cells. Bleaching was done using a 514 nm laser set to 100% every 5 seconds in an area of one cell, with an image captured every 2.5 seconds for a total of 5 minutes. Removal of the free cytoplasmic YFP-VPS33B pool (by photobleaching) reveals occasional cytoplasmic clusters, which are not as pronounced as when VPS33B is co-overexpressed with polarin. (WMV 1307 kb)

Supplementary Movie 2

VSVG-YFP (basolaterally targeted protein), Gal-T-GFP-, and A-VSVG-CFP infected cells were incubated overnight at 40°C. Live cell imaging was performed using a Zeiss 510 inverted confocal microscope with an ×20 objective lens. Pinhole was set fully open. Images were taken every minute. Experiment started 10min after the temperature shift to 32°C (time 0). Relative fluorescence intensities associated with the Golgi (RO1), entire cell (RO2) and plasma membrane (RO3) for one representative cell after shift to 32°C are plotted at 1min intervals. VSVG-YFP infected cells were incubated overnight at 40°C. The experiment was started 10 min after the temperature shift to 32°C (time 0). At 0 min VSVG-YFP intracellular localization of fluorescence was noted. After 140min, VSVG-YFP targeted to the plasma membrane although some intracellular fluorescence remained. Relative fluorescence intensity is calculated using the following equation: (Raw F.I. of - background F.I.) ×100/maximum F.I. (MOV 4936 kb)

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Cullinane, A., Straatman-Iwanowska, A., Zaucker, A. et al. Mutations in VIPAR cause an arthrogryposis, renal dysfunction and cholestasis syndrome phenotype with defects in epithelial polarization. Nat Genet 42, 303–312 (2010). https://doi.org/10.1038/ng.538

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