Tobias Hermle1,2, Ronen Schneider1, David Schapiro1, Daniela A Braun1, Amelie T van der Ven1, Jillian K Warejko1, Ankana Daga1, Eugen Widmeier1, Makiko Nakayama1, Tilman Jobst-Schwan1, Amar J Majmundar1, Shazia Ashraf1, Jia Rao1, Laura S Finn3, Velibor Tasic4, Joel D Hernandez5, Arvind Bagga6, Sawsan M Jalalah7, Sherif El Desoky8, Jameela A Kari8, Kristen M Laricchia9, Monkol Lek9, Heidi L Rehm9, Daniel G MacArthur9, Shrikant Mane10, Richard P Lifton10,11, Shirlee Shril1, Friedhelm Hildebrandt12. 1. Department of Medicine, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts. 2. Renal Division, University Medical Center Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany. 3. Department of Pathology, Seattle Children's Hospital, University of Washington, Seattle, Washington. 4. Department of Pediatric Nephrology, Medical Faculty Skopje, University Children's Hospital, Skopje, Macedonia. 5. Department of Pediatric Nephrology, Providence Sacred Heart Medical Center and Children's Hospital, Spokane, Washington. 6. Division of Nephrology, All India Institute of Medical Sciences, New Delhi, India. 7. Department of Pathology, College of Medicine, and. 8. Pediatric Nephrology Center of Excellence and Pediatric Department, Faculty of Medicine, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia. 9. Broad Center for Mendelian Genetics, Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, Massachusetts. 10. Department of Genetics, Yale University School of Medicine, New Haven, Connecticut; and. 11. Laboratory of Human Genetics and Genomics, The Rockefeller University, New York, New York. 12. Department of Medicine, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts; friedhelm.hildebrandt@childrens.harvard.edu.
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 sequencing. 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 mutated proteins produced upon ectopic expression of GAPVD1 or ANKFY1 bearing the patient-derived mutations 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 Drosophila, silencing Gapvd1 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.
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 sequencing. 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 mutated proteins produced upon ectopic expression of GAPVD1 or ANKFY1 bearing the patient-derived mutations 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 Drosophila, silencing Gapvd1impaired 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 humanNS.
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