GAPVD1 and ANKFY1 Mutations Implicate RAB5 Regulation in Nephrotic Syndrome
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BASIC RESEARCH www.jasn.org GAPVD1 and ANKFY1 Mutations Implicate RAB5 Regulation in Nephrotic Syndrome Tobias Hermle,1,2 Ronen Schneider,1 David Schapiro,1 Daniela A. Braun,1 Amelie T. van der Ven,1 Jillian K. Warejko,1 Ankana Daga,1 Eugen Widmeier,1 Makiko Nakayama,1 Tilman Jobst-Schwan,1 Amar J. Majmundar,1 Shazia Ashraf,1 Jia Rao,1 Laura S. Finn,3 Velibor Tasic,4 Joel D. Hernandez,5 Arvind Bagga,6 Sawsan M. Jalalah,7 Sherif El Desoky,8 Jameela A. Kari,8 Kristen M. Laricchia,9 Monkol Lek,9 Heidi L. Rehm,9 Daniel G. MacArthur,9 Shrikant Mane,10 Richard P. Lifton,10,11 Shirlee Shril,1 and Friedhelm Hildebrandt1 Due to the number of contributing authors, the affiliations are listed at the end of this article. ABSTRACT Background Steroid-resistant nephrotic syndrome (SRNS) is a frequent cause of CKD. The discovery of monogenic causes of SRNS has revealed specific pathogenetic pathways, but these monogenic causes do not explain all cases of SRNS. Methods To identify novel monogenic causes of SRNS, we screened 665 patients by whole-exome se- quencing. We then evaluated the in vitro functional significance of two genes and the mutations therein that we discovered through this sequencing and conducted complementary studies in podocyte-like Drosophila nephrocytes. Results We identified conserved, homozygous missense mutations of GAPVD1 in two families with early- onset NS and a homozygous missense mutation of ANKFY1 in two siblings with SRNS. GAPVD1 and ANKFY1 interact with the endosomal regulator RAB5. Coimmunoprecipitation assays indicated interaction between GAPVD1 and ANKFY1 proteins, which also colocalized when expressed in HEK293T cells. Silencing either protein diminished the podocyte migration rate. Compared with wild-type GAPVD1 and ANKFY1, the mu- tated proteins produced upon ectopic expression of GAPVD1 or ANKFY1 bearing the patient-derived mu- tations exhibited altered binding affinity for active RAB5 and reduced ability to rescue the knockout-induced defect in podocyte migration. Coimmunoprecipitation assays further demonstrated a physical interaction between nephrin and GAPVD1, and immunofluorescence revealed partial colocalization of these proteins in rat glomeruli. The patient-derived GAPVD1 mutations reduced nephrin-GAPVD1 binding affinity. In Dro- sophila,silencingGapvd1 impaired endocytosis and caused mistrafficking of the nephrin ortholog. Conclusions Mutations in GAPVD1 and probably in ANKFY1 are novel monogenic causes of NS. The discovery of these genes implicates RAB5 regulation in the pathogenesis of human NS. J Am Soc Nephrol 29: 2123–2138, 2018. doi: https://doi.org/10.1681/ASN.2017121312 Received December 21, 2017. Accepted May 24, 2018. Steroid-resistant nephrotic syndrome (SRNS) is T.H. and R.S. contributed equally to this work. characterized by edema, nephrotic-range protein- Published online ahead of print. Publication date available at uria, and hyperlipidemia. Mutations in approxi- www.jasn.org. mately 45 different genes have been discovered as Correspondence: Prof. Friedhelm Hildebrandt, Boston Child- 1–38 monogenic causes of SRNS (Supplemental ren’s Hospital, Harvard Medical School, Enders 561, 300 Long- Figure 1A) and our understanding of the patho- wood Avenue, Boston, MA 02115. Email: friedhelm. physiology of SRNS and podocyte biology in gen- [email protected] eral has been formed by the pathways delineated by Copyright © 2018 by the American Society of Nephrology J Am Soc Nephrol 29: 2123–2138, 2018 ISSN : 1046-6673/2908-2123 2123 BASIC RESEARCH www.jasn.org discovery of these genes (Supplemental Figure 1A).1,3 The first Significance Statement gene to be found was nephrin (NPHS1), which encodes a ma- jor constituent of the slit diaphragm.4 Subsequently, muta- A number of single gene mutations have been identified as causes of tions of several proteins associated with the slit diaphragm nephrotic syndrome (NS). This paper describes the discovery of two GAPVD1 complex were identified. Linking the slit diaphragm to the new monogenic genes with mutations associated with NS, , with definite evidence for causality, and ANKFY1, as probably actin cytoskeleton to maintain the complex podocyte mor- causal. The genes are the first endosomal regulators and known phology seems essential because actin-binding and -regulating RAB5 interactors implicated in NS. Both proteins interact and affect proteins form the most numerous group among the mono- podocyte migration rate. GAPVD1 also interacts with the slit di- 1,3 aphragm protein nephrin. The mutations of GAPVD1 observed in genic causes of SRNS. More recently, mutations in CoQ10- biosynthesis genes,14–16 nucleoporins,39–41 and the KEOPS patients affect binding to nephrin and RAB5. Silencing the ortholog of GAPVD1 in the podocyte-like Drosophila nephrocytes results in 42 complex have been discovered as further monogenic causes mistrafficking of fly nephrin. These findings implicate RAB5 regu- of SRNS. This opened new avenues toward a better under- lation as a novel pathogenetic pathway of NS, potentially critical for standing of the complex pathogenesis of SRNS. nephrin trafficking. A role of endocytosis for podocyte biology has previously been proposed: The slit diaphragm protein nephrin is subject to endo- constructs were generated by PCR. Primers are shown in Sup- cytosis utilizing different branches of the endocytosis pathway.43–48 plemental Table 1. ANKFY1 rescue constructs were obtained In mice, phosphoinositide 3-kinases49,50 and effectors of vesicular by introduction of two synonymous mutations within the fission and clathrin uncoating are essential for the glomerular shRNA target sequences. filtration barrier. Knockout of the murine isoforms of dynamin, The following expression vectors were used: pRK5-N-Myc, synaptojanin, or endophilin each resulted in severe protein- pCDNA6.2-N-GFP,pQCXIPmCherry,andpSirenRetroQ. uria.51,52 In humans, no direct endosomal regulator has previ- Clones reflecting the mutations identified in individuals with ously been implicated in nephrotic syndrome. nephrotic syndrome were introduced into the cDNA constructs using Quik change II XL site-directed mutagenesis kit (Agilent Technologies). The following constructs were obtained from METHODS addgene: mCherry-Rab5CAQ79L (#35138), mCherry-Rab5DNS34N (#35139), mCh-Rab5 (#49201), pRK5myc Rac1 wt (#37030), and Study Approval pSpCas9(BB)-2A-GFP (PX458) (#48138). Approval for human subject research was obtained from the The GAPVD1-andANKFY1-specific and control scram- University of Michigan and the Boston Children’sHospital bled siRNAs were purchased from GE Dharmacon. Institutional Review Boards. All participants or their guard- Overexpression experiments were performed in HEK293T ians provided written informed consent. cells (ATCC biologic resource center). Immortalized human podocytes were a gift from Dr. Moin Saleem (University of Study Participants Bristol, Bristol, UK). After informed consent, clinical data and blood samples were HEK cells were maintained in DMEM, supplemented with obtained from individuals with nephrotic syndrome. Clinical 10% FBS, 50 IU/ml penicillin, and 50 mg/ml streptomycin. data were obtained using an established questionnaire (http:// Podocytes were maintained in RPMI 1640 plus GlutaMAX-I www.renalgenes.org). The diagnosis of NS was made by (pe- (Gibco) supplemented with 10% FBS, 50 IU/ml penicillin/ diatric) nephrologists, on the basis of standardized clinical and 50 mg/ml streptomycin, and insulin-transferrin-selenium-X. renal histologic criteria. Renal biopsy samples were evaluated Plasmids and siRNAs were transfected into HEK293T cells at 37° by renal pathologists. C or podocytes grown at the permissive temperature of 33°C using Lipofectamine 2000 or Lipofectamine RNAiMax, respectively (In- Homozygosity Mapping, Whole-Exome Resequencing, vitrogen). Knockdown in human podocyte cell lines employed and Mutation Calling pSirenRetroQ with 2–3 independent shRNAs directed against hu- Homozygosity mapping, whole-exome resequencing, and mu- man GAPVD1 or ANKFY1 for retroviral transduction. Puromycin tation calling were performed as described previously.22 was used to select transduced cells. Knockdown efficiency was con- firmed for all experiments, shRNA targets are shown in Supple- Plasmids, siRNAs, Cell Culture, and Transfection mental Table 1. For rescue experiments, knockdown podocytes Human full-length GAPVD1 cDNA was subcloned after PCR underwent a transient transfection using murine Gapvd1 or hu- from human full-length cDNA (GenBank BC114937; GE man ANKFY1 constructs that are resistant to shRNA. Dharmacon). Mouse Gapvd1 was subcloned after PCR from murine full-length cDNA (RefSeq NM_025709) that was a gift from Dr. Alan Saltiel53 (University of California, San Diego). Immunoblotting, Immunoprecipitation, Pull-Down Human ANKFY1 isoform 1 cDNA (GenBank BC152991.1) Assay, and Immunofluorescence Staining and human NPHS1 cDNA (GenBank: BC156935.1) were ob- Immunoblotting, immunoprecipitation, and immunofluores- tained from the Harvard PlasmID Repository. Truncation cence staining were performed as described previously.39 2124 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 2123–2138, 2018 www.jasn.org BASIC RESEARCH Briefly, HEK293T cells were lysed and precleared using rec- Drosophila Studies Protein A-Sepharose 4B Conjugate (Life Technologies) over- Drosophila melanogaster stable RNAi stocks Gapvd1-RNAi 1 night. Then, equal amounts of protein were incubated with (#108453) and Gapvd1-RNAi 2 (#19649) were obtained from the EZview Red Anti-c-Myc