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Mutation-dependent recessive inheritance of NPHS2-associated steroid-resistant nephrotic syndrome

Nature Genetics volume 46, pages 299–304 (2014)Cite this article

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Abstract

Monogenic disorders result from defects in a single gene. According to Mendel's laws, these disorders are inherited in either a recessive or dominant fashion. Autosomal-recessive disorders require a disease-causing variant on both alleles, and according to our current understanding, their pathogenicities are not influenced by each other. Here we present an autosomal-recessive disorder, nephrotic syndrome type 2 (MIM 600995), in which the pathogenicity of an NPHS2 allele encoding p.Arg229Gln depends on the trans-associated mutation. We show that, contrary to expectations, this allele leads to a disease phenotype only when it is associated specifically with certain 3′ NPHS2 mutations because of an altered heterodimerization and mislocalization of the encoded p.Arg229Gln podocin. The disease-associated 3′ mutations exert a dominant-negative effect on p.Arg229Gln podocin but behave as recessive alleles when associated with wild-type podocin. Therefore, the transmission rates for couples carrying the disease-associated mutations and p.Arg229Gln may be substantially different from those expected in autosomal-recessive disorders.

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Figure 1: Membrane targeting of wild-type podocin and p.Arg229Gln podocin as a function of the associated mutation in podocytes stably coexpressing podocin mutants.
Figure 2: Membrane targeting of endogenous podocin in urinary podocytes of cases with SRNS carrying p.Arg229Gln.
Figure 3: Structure of the wild-type podocin homodimer and the effect of p.Arg229Gln.
Figure 4: Superimposed average structures of the non-pathogenic and pathogenic dimers.

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Acknowledgements

We thank S. Lyonnet, L. Abel, S. Harvey and M. Muorah for careful reading and discussion of the manuscript and M. Saleem (Bristol Royal Hospital for Children, University of Bristol) for providing the podocyte cell line. We acknowledge E. Jávorszky, M. Bernáth, M. Krámer and Zs. Nagy for providing assistance, N. Goudin of the Necker Institute Imaging Facility for providing expert knowledge on confocal microscopy, G. Froment, D. Nègre and C. Costa for production of lentivectors (Structure Fédérative de Recherche BioSciences Gerland–Lyon Sud–UMS3444/US8) and the National Information Infrastructure Development Institute (NIIFI) supercomputing center (Hungary). We thank the patients and their family members for participation. Financial support for this work was provided by grants from the Fondation pour la Recherche Médicale (project DMP 2010-11-20-386 to C. Antignac), the Agence Nationale de la Recherche (GenPod project ANR-12-BSV1-0033.01 and 'Investments for the Future' program ANR-10-IAHU-01, both to C. Antignac), the European Community's 7th Framework program grant 2012-305608 (Eurenomics) to C. Antignac, Fondation Association pour la Recherche sur le Cancer (ARC) TÁMOP-4.2.1/B-09/1/KMR-2010-0001 to K.T., OTKA 84087/2010 to T.T., Hungarian Scientific Research Fund (OTKA NK101072 to A.P., K109718 to K.T. and PD101095 to D.K.M.) and a Bolyai János research fellowship of the Hungarian Academy of Sciences to K.T.

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Authors and Affiliations

  1. First Department of Pediatrics, Semmelweis University, Budapest, Hungary

    Kálmán Tory, Andrea Kerti & Tivadar Tulassay

  2. Institut National de la Santé et de la Recherche Médicale (INSERM) Unité Mixte de Recherche (UMR) 1163, Laboratory of Hereditary Kidney Diseases, Paris, France

    Kálmán Tory, Stéphanie Woerner, Fabien Nevo, Olivier Gribouval, Christelle Arrondel, Evelyne Huynh Cong, Géraldine Mollet & Corinne Antignac

  3. Paris Descartes University–Sorbonne Paris Cité, Imagine Institute, Paris, France

    Kálmán Tory, Stéphanie Woerner, Fabien Nevo, Olivier Gribouval, Christelle Arrondel, Evelyne Huynh Cong, Géraldine Mollet & Corinne Antignac

  4. Hungarian Academy of Sciences–Eötvös Loránd University (MTA-ELTE) Protein Modelling Research Group, Budapest, Hungary

    Dóra K Menyhárd, Pál Stráner & András Perczel

  5. Hungarian Academy of Sciences–Semmelweis University (MTA-SE) Pediatrics and Nephrology Research Group, Budapest, Hungary

    Tivadar Tulassay

  6. Laboratory of Structural Chemistry and Biology, Eötvös Loránd University, Budapest, Hungary

    András Perczel

  7. Department of Genetics, Assistance Publique–Hôpitaux de Paris, Necker Hospital, Paris, France

    Corinne Antignac

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  1. Kálmán Tory
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Contributions

C. Antignac, T.T., A.P. and K.T. designed the study based on a hypothesis generated by K.T. O.G., K.T. and A.K. performed the genetic studies. S.W., F.N., G.M., K.T., C. Arrondel and E.H.C. performed the functional studies. D.K.M., P.S. and A.P. modeled the podocin dimerization. K.T., D.K.M., S.W., G.M., A.P. and C. Antignac wrote the paper. All the authors agreed to publish the paper.

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Correspondence to Kálmán Tory or Corinne Antignac.

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Integrated supplementary information

Supplementary Figure 1 Pedigree of the two individuals carrying p.[V290M];[R229Q] with no proteinuria in adulthood

Supplementary Figure 2 Membrane-targeting of wild-type podocin and podocin R299Q as a function of the associated mutation in transiently transfected podocytes

(a,b) Wild-type podocin (columns 1–2) and podocin R229Q (columns 3–4) are shown in red, the coexpressed GFP-tagged podocin proteins (ordered according to the position of the mutation) in green. The plasma membrane is labeled with WGA and shown in blue. Both wild-type podocin and podocin R229Q are localized to the plasma membrane when coexpressed with either wild-type podocin, podocin R238S (panel a, rows 1, 3) or podocin V290M (panel b, row 1) (white pixels correspond to the merge of red, green and blue). Similarly, despite retention of podocin R138Q in the ER, both wild-type podocin and podocin R229Q are localized to the plasma membrane (panel a, row 2, magenta pixels correspond to red and blue). In contrast, while wild-type podocin reaches the plasma membrane (magenta pixels) in cells coexpressing podocin A284V, podocin A288T (panel a, rows 4, 5) podocin R291W, podocin A297V or podocin E310K (panel b, rows 2-4) podocin R229Q is retained in cytoplasmic compartments. b, last row, 3rd column: The arrow indicates a cell which does not express podocin E310K but only podocin R299Q, which is therefore well targeted to the plasma membrane. Percentages in green and red indicate the proportion of the WGA-labeled perimembranous area that colocalizes with GFP- or HA-tagged podocin proteins, respectively, within the presented cell. Similar results were found in 6-7 cells per group (c,d). Scale bars = 5 μm. (c,d) Membrane targeting of podocin proteins shown as the percentage of the plasma membrane (WGA) that is positive for GFP- (c) or HA-tagged podocin proteins (d), within 6-7 cells per group. GFP- and HA-tagged podocin proteins are indicated in green and red, respectively. The coexpressed podocin proteins are indicated in subscript. ♯ P ≤ 0.0027 vs. wtwt (wt-GFP coexpressed with wt-HA); * P ≤ 0.0039 vs. R229Qwt (R229Q-HA coexpressed with wt-GFP)

Source data

Supplementary Figure 3 Synaptopodin and podocin staining of urinary podocytes

Both synaptopodin and podocin staining confirm the presence of podocytes in the culture of urine sediments. In contrast to control patients with non-NPHS2 associated glomerulopathies (C1-C3), podocin is not targeted to the plasma membrane in the patient with p.[R229Q];[A284V] (Pt), as also shown in Figure 2. Scale bar = 20 μm.

Supplementary Figure 4 Punnett squares illustrating non-Mendelian transmission rates in theoretical couples with R229Q and the ‘associated mutations’

Individuals with expected early- or late-onset SRNS are in dark and light gray, respectively.

Supplementary Figure 5 Sequence alignment for human podocin and Pyrococcus horikoshii stomatin

There is a 35% identity and 65% homology for the modeled region (podocin residues 161-332): colored boxes show identities. Arrows above the sequence indicate the domain structure. Cyan corresponds to the N-terminal cytosolic fragment, magenta to the intra-membrane fragment and green to the C-terminal cytosolic fragment of podocin.

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Supplementary Table 1 and Supplementary Figures 1–5 (PDF 4784 kb)

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Tory, K., Menyhárd, D., Woerner, S. et al. Mutation-dependent recessive inheritance of NPHS2-associated steroid-resistant nephrotic syndrome. Nat Genet 46, 299–304 (2014). https://doi.org/10.1038/ng.2898

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