Molecular genetic causes of renal ciliopathies - a candidate gene approach including the survivin signaling pathways

• Applicant: Dr. Tilman Jobst-Schwan
• Subject Area: Nephrology ; Human Genetics
• Term: from 2015 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 281319475

• Final Report Year: 2018

Final Report Abstract

Related to my previous work investigating the antiapoptotic protein survivin in the kidney, the aim of my fellowship in the Hildebrandt lab was to identify novel disease genes for cystic kidney disease (NPHP-RC) by combining whole exome sequencing (WES) with a candidate gene approach related to survivin regulation. NPHP-RC is characterized by a dysfunction of the primary cilium. Novel candidate genes would be characterized in vitro and in vivo with focus on zebrafish animal models. However, analysis of 283 WES datasets for mutations in the gene BIRC5, which encodes for survivin, and its interactors did not yield any convincing variants, potentially because of the important function of survivin and its interactors in cell division that might lead to embryonal lethality in humans. Therefore, an early adjustment of the scientific aims of the project was necessary. The proposal included already zebrafish experiments in the context of the project. However, there was no suitable NPHP-RC candidate gene for zebrafish experiments at that time, but several for steroid resistant nephrotic syndrome (SRNS), a chronic kidney disease that is characterized by edema and protein loss via the urine. The phenotype of SRNS in zebrafish has been described as periorbital edema and whole-body edema which was the primary readout for all our SRNS zebrafish projects. I build up and managed the zebrafish program of the lab reaching a peak population of 10.000 adult zebrafish. I established gene knock-out (KO) in zebrafish by CRISPR/Cas9, a novel efficient gene editing technique and generated more than 40 KO zebrafish lines for SRNS (candidate) genes. We identified mutations in the genes LAGE3, OSGEP, TP53RK and TPRKB as disease causing for Galloway-Mowat-Syndrome, a neuro-renal disorder that is characterized by SRNS and microcephaly (small heads). Characterization of KO lines for the genes osgep and tprkb showed microcephaly and early lethality, partially recapitulated the human disease phenotype. Based on these finding I designed an acute CRISPR/Cas9 based approach that allows to rapidly establish a phenotype in zebrafish larvae. To further investigate the pathogenesis of OSGEP mutations, I established a knock-in approach to specifically model the human mutations in zebrafish. KO of the zebrafish gene magi2a caused a distinct SRNS phenotype that corresponds to the phenotype of MAGI2 mutations in humans. Surprisingly, treatment with steroids, the initial standard therapy attempt in nephrotic syndrome, aggravated the disease which stands in contrast to any other SRNS model. The mechanism of this aggravation remains unclear to date. Aside the zebrafish work, I continued analyzing WES datasets and identified mutations in the gene MAP7D3 in two patients with cystic kidney disease. In collaboration with the Pelletier lab (Toronto, Canada), we could show that at least one of the mutations abrogated the interaction with other ciliary proteins. Finally, in a cohort of patients with distal renal tubular acidosis, I identified two novel candidate genes ATP6V1C2 and SLC4A2. We modeled the ATP6V1C2 mutation in the yeast orthologue VMA5 and could show pathogenicity of the mutation. However, Xenopus oocyte expression assays for SLC4A2 did not show any functional impairment of the encoded anion exchanger.

Publications

• Advillin acts upstream of phospholipase C ϵ1 in steroid-resistant nephrotic syndrome. J Clin Invest. 127:4257, 2017
  Rao J, Ashraf S, Tan W, van der Ven AT, Gee HY, Braun DA, Fehér K, George SP, Esmaeilniakooshkghazi A, Choi WI, Jobst-Schwan T, Schneider R, Schmidt JM, Widmeier E, Warejko JK, Hermle T, Schapiro D, Lovric S, Shril S, Daga A, Nayir A, Shenoy M, Tse Y, Bald M, Helmchen U, Mir S, Berdeli A, Kari JA, El Desoky S, Soliman NA, Bagga A, Mane S, Jairajpuri MA, Lifton RP, Khurana S, Martins JC, Hildebrandt F
  
• Mutations in KEOPS-complex genes cause nephrotic syndrome with primary microcephaly. Nat Genet. 49:1529, 2017
  Braun DA, ... Jobst-Schwan T, ... Hildebrandt F
  
• Acute multi-sgRNA knockdown of KEOPS complex genes reproduces the microcephaly phenotype of the stable knockout zebrafish model. PLoS One. 13:e0191503, 2018
  Jobst-Schwan T, Schmidt JM, Schneider R, Hoogstraten CA, Ullmann JFP, Schapiro D, Majmundar AJ, Kolb A, Eddy K, Shril S, Braun DA, Poduri A, Hildebrandt F
  
• GAPVD1 and ANKFY1 Mutations Implicate RAB5 Regulation in Nephrotic Syndrome. J Am Soc Nephrol. 29:2123, 2018
  Hermle T, Schneider R, Schapiro D, Braun DA, van der Ven AT, Warejko JK, Daga A, Widmeier E, Nakayama M, Jobst-Schwan T, Majmundar AJ, Ashraf S, Rao J, Finn LS, Tasic V, Hernandez JD, Bagga A, Jalalah SM, El Desoky S, Kari JA, Laricchia KM, Lek M, Rehm HL, MacArthur DG, Mane S, Lifton RP, Shril S, Hildebrandt F
  
• Mutations in multiple components of the nuclear pore complex cause nephrotic syndrome. J Clin Invest. 128: 4313, 2018
  Braun, DA, ... Jobst-Schwan, T, ... Hildebrandt, F
  
• Mutations in six nephrosis genes delineate a pathogenic pathway amenable to treatment. Nat Commun. 9:1960, 2018
  Ashraf S, ... Jobst-Schwan T, ... Hildebrandt F