Approximately one third of children with steroid-resistant nephrotic syndrome (SRNS) carry pathogenic variants in one of the many associated genes. The WT1 gene coding for the WT1 transcription factor is among the most frequently affected genes. Cases from the Czech national SRNS database were sequenced for exons 8 and 9 of the WT1 gene. Eight distinct exonic WT1 variants in nine children were found. Three children presented with isolated SRNS, while the other six manifested with additional features. To analyze the impact of WT1 genetic variants, wild type and mutant WT1 proteins were prepared and the DNA-binding affinity of these proteins to the target EGR1 sequence was measured by microscale thermophoresis. Three WT1 mutants showed significantly decreased DNA-binding affinity (p.Arg439Pro, p.His450Arg and p.Arg463Ter), another three mutants showed significantly increased binding affinity (p.Gln447Pro, p.Asp469Asn and p.His474Arg), and the two remaining mutants (p.Cys433Tyr and p.Arg467Trp) showed no change of DNA-binding affinity. The protein products of WT1 pathogenic variants had variable DNA-binding affinity, and no clear correlation with the clinical symptoms of the patients. Further research is needed to clarify the mechanisms of action of the distinct WT1 mutants; this could potentially lead to individualized treatment of a so far unfavourable disease.

Department of Biotechnology University of Chemistry and Technology Prague Czech Republic

Department of Pediatrics 2nd Faculty of Medicine Charles University and Motol University Hospital Prague Czech Republic

Vera Vavrova Lab VIAL Department of Pediatrics 2nd Faculty of Medicine Charles University and Motol University Hospital 5 Uvalu 84 150 06 Prague Czech Republic

Zobrazit více v PubMed

Torban E, Braun F, Wanner N, Takano T, Goodyer PR, Lennon R, et al. From podocyte biology to novel cures for glomerular disease. Kidney Int. 2019;96(4):850–861. doi: 10.1016/j.kint.2019.05.015. PubMed DOI

Dossier C, Lapidus N, Bayer F, Sellier-Leclerc A-L, Boyer O, de Pontual L, et al. Epidemiology of idiopathic nephrotic syndrome in children: Endemic or epidemic? Pediat. Nephrol. 2016;31(12):2299–2308. doi: 10.1007/s00467-016-3509-z. PubMed DOI

McKinney PA, Feltbower RG, Brocklebank JT, Fitzpatrick MM. Time trends and ethnic patterns of childhood nephrotic syndrome in Yorkshire, UK. Pediat. Nephrol. 2001;16(12):1040–1044. doi: 10.1007/s004670100021. PubMed DOI

Tullus K, Webb H, Bagga A. Management of steroid-resistant nephrotic syndrome in children and adolescents. Lancet Child Adolescent Health. 2018;2(12):880–890. doi: 10.1016/S2352-4642(18)30283-9. PubMed DOI

Bierzynska A, McCarthy HJ, Soderquest K, Sen ES, Colby E, Ding WY, et al. Genomic and clinical profiling of a national nephrotic syndrome cohort advocates a precision medicine approach to disease management. Kidney Int. 2017;91(4):937–947. doi: 10.1016/j.kint.2016.10.013. PubMed DOI

Bezdíčka M, Štolbová Š, Seeman T, Cinek O, Malina M, Šimánková N, et al. Genetic diagnosis of steroid-resistant nephrotic syndrome in a longitudinal collection of Czech and Slovak patients: a high proportion of causative variants in NUP93. Pediat. Nephrol. 2018;33(8):1347–1363. doi: 10.1007/s00467-018-3950-2. PubMed DOI

Dong L, Pietsch S, Tan Z, Perner B, Sierig R, Kruspe D, et al. Integration of Cistromic and Transcriptomic Analyses Identifies Nphs2, Mafb, and Magi2 as Wilms' Tumor 1 Target Genes in Podocyte Differentiation and Maintenance. J. Am. Soc. Nephrol. 2015;26(9):2118–2128. doi: 10.1681/ASN.2014080819. PubMed DOI PMC

Hartwig S, Ho J, Pandey P, Macisaac K, Taglienti M, Xiang M, et al. Genomic characterization of Wilms' tumor suppressor 1 targets in nephron progenitor cells during kidney development. Development. 2010;137(7):1189–1203. doi: 10.1242/dev.045732. PubMed DOI PMC

Pelletier J, Bruening W, Kashtan CE, Mauer SM, Manivel JC, Striegel JE, et al. Germline mutations in the Wilms' tumor suppressor gene are associated with abnormal urogenital development in Denys-Drash syndrome. Cell. 1991;67(2):437–447. doi: 10.1016/0092-8674(91)90194-4. PubMed DOI

Stoll R, Lee BM, Debler EW, Laity JH, Wilson IA, Dyson HJ, et al. Structure of the Wilms tumor suppressor protein zinc finger domain bound to DNA. J. Mol. Biol. 2007;372(5):1227–1245. doi: 10.1016/j.jmb.2007.07.017. PubMed DOI

Rauscher FJ, 3rd, Morris JF, Tournay OE, Cook DM, Curran T. Binding of the Wilms' tumor locus zinc finger protein to the EGR-1 consensus sequence. Science. 1990;250(4985):1259–1262. doi: 10.1126/science.2244209. PubMed DOI

Kann M, Ettou S, Jung YL, Lenz MO, Taglienti ME, Park PJ, et al. Genome-wide analysis of Wilms' tumor 1-controlled gene expression in podocytes reveals key regulatory mechanisms. J. Am. Soc. Nephrol. 2015;26(9):2097–2104. doi: 10.1681/ASN.2014090940. PubMed DOI PMC

Mucha B, Ozaltin F, Hinkes BG, Hasselbacher K, Ruf RG, Schultheiss M, et al. Mutations in the Wilms' tumor 1 gene cause isolated steroid resistant nephrotic syndrome and occur in exons 8 and 9. Pediat. Res. 2006;59(2):325–331. doi: 10.1203/01.pdr.0000196717.94518.f0. PubMed DOI

Lipska BS, Ranchin B, Iatropoulos P, Gellermann J, Melk A, Ozaltin F, et al. Genotype-phenotype associations in WT1 glomerulopathy. Kidney Int. 2014;85(5):1169–1178. doi: 10.1038/ki.2013.519. PubMed DOI

Roca N, Muñoz M, Cruz A, Vilalta R, Lara E, Ariceta G. Long-term outcome in a case series of Denys-Drash syndrome. Clin Kidney. 2019;J12(6):836–839. doi: 10.1093/ckj/sfz022. PubMed DOI PMC

Swiatecka-Urban A, Mokrzycki MH, Kaskel F, Da Silva F, Denamur E. Novel WT1 mutation (C388Y) in a female child with Denys-Drash syndrome. Pediat. Nephrol. 2001;16(8):627–630. doi: 10.1007/s004670100626. PubMed DOI

Hossain A, Saunders GF. The human sex-determining gene SRY is a direct target of WT1. J. Biol. Chem. 2001;276(20):16817–16823. doi: 10.1074/jbc.M009056200. PubMed DOI

Larney C, Bailey TL, Koopman P. Switching on sex: Transcriptional regulation of the testis-determining gene Sry. Development. 2014;141(11):2195–2205. doi: 10.1242/dev.107052. PubMed DOI PMC

Kim J, Prawitt D, Bardeesy N, Torban E, Vicaner C, Goodyer P, et al. The Wilms' tumor suppressor gene (wt1) product regulates Dax-1 gene expression during gonadal differentiation. Mol. Cell Biol. 1999;19(3):2289–2299. doi: 10.1128/mcb.19.3.2289. PubMed DOI PMC

Wilhelm D, Englert C. The Wilms tumor suppressor WT1 regulates early gonad development by activation of Sf1. Genes Dev. 2002;16(14):1839–1851. doi: 10.1101/gad.220102. PubMed DOI PMC

Kreidberg JA, Sariola H, Loring JM, Maeda M, Pelletier J, Housman D, et al. WT-1 is required for early kidney development. Cell. 1993;74(4):679–691. doi: 10.1016/0092-8674(93)90515-r. PubMed DOI

Gadd S, Huff V, Huang CC, Ruteshouser EC, Dome JS, Grundy PE, et al. Clinically relevant subsets identified by gene expression patterns support a revised ontogenic model of Wilms tumor: a Children's Oncology Group Study. Neoplasia. 2012;14(8):742–756. doi: 10.1593/neo.12714. PubMed DOI PMC

Treger TD, Chowdhury T, Pritchard-Jones K, Behjati S. The genetic changes of Wilms tumour. Nat. Rev. Nephrol. 2019;15(4):240–251. doi: 10.1038/s41581-019-0112-0. PubMed DOI

Martínez-Estrada OM, Lettice LA, Essafi A, Guadix JA, Slight J, Velecela V, et al. Wt1 is required for cardiovascular progenitor cell formation through transcriptional control of Snail and E-cadherin. Nat. Genet. 2010;42(1):89–93. doi: 10.1038/ng.494. PubMed DOI PMC

Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial-mesenchymal transitions in development and disease. Cell. 2009;139(5):871–890. doi: 10.1016/j.cell.2009.11.007. PubMed DOI

Essafi A, Webb A, Berry RL, Slight J, Burn SF, Spraggon L, et al. A wt1-controlled chromatin switching mechanism underpins tissue-specific wnt4 activation and repression. Dev. Cell. 2011;21(3):559–574. doi: 10.1016/j.devcel.2011.07.014. PubMed DOI PMC

Costantini F, Kopan R. Patterning a complex organ: Branching morphogenesis and nephron segmentation in kidney development. Dev. Cell. 2010;18(5):698–712. doi: 10.1016/j.devcel.2010.04.008. PubMed DOI PMC

Stark K, Vainio S, Vassileva G, McMahon AP. Epithelial transformation of metanephric mesenchyme in the developing kidney regulated by Wnt-4. Nature. 1994;372(6507):679–683. doi: 10.1038/372679a0. PubMed DOI

Gebeshuber CA, Kornauth C, Dong L, Sierig R, Seibler J, Reiss M, et al. Focal segmental glomerulosclerosis is induced by microRNA-193a and its downregulation of WT1. Nat. Med. 2013;19(4):481–487. doi: 10.1038/nm.3142. PubMed DOI

Chau Y-Y, Brownstein D, Mjoseng H, Lee W-C, Buza-Vidas N, Nerlov C, et al. Acute multiple organ failure in adult mice deleted for the developmental regulator Wt1. PLoS Genet. 2011;7(12):e1002404–e1002404. doi: 10.1371/journal.pgen.1002404. PubMed DOI PMC

Hall G, Gbadegesin RA, Lavin P, Wu G, Liu Y, Oh EC, et al. A novel missense mutation of Wilms' Tumor 1 causes autosomal dominant FSGS. J. Am. Soc. Nephrol. 2015;26(4):831–843. doi: 10.1681/asn.2013101053. PubMed DOI PMC

Smith SI, Down M, Boyd AW, Li CL. Expression of the Wilms' tumor suppressor gene, WT1, reduces the tumorigenicity of the leukemic cell line M1 in C.B-17 scid/scid mice. Cancer Res. 2000;60(4):808–814. PubMed

Loeb DM. WT1 influences apoptosis through transcriptional regulation of Bcl-2 family members. Cell Cycle. 2006;5(12):1249–1253. doi: 10.4161/cc.5.12.2807. PubMed DOI

Ullmark T, Montano G, Gullberg U. DNA and RNA binding by the Wilms' tumour gene 1 (WT1) protein +KTS and -KTS isoforms-From initial observations to recent global genomic analyses. Eur. J. Haematol. 2018;100(3):229–240. doi: 10.1111/ejh.13010. PubMed DOI

Koesters R, Linnebacher M, Coy JF, Germann A, Schwitalle Y, Findeisen P, et al. WT1 is a tumor-associated antigen in colon cancer that can be recognized by in vitro stimulated cytotoxic T cells. Int. J. Cancer. 2004;109(3):385–392. doi: 10.1002/ijc.11721. PubMed DOI

He B, Ebarasi L, Zhao Z, Guo J, Ojala JR, Hultenby K, et al. Lmx1b and FoxC combinatorially regulate podocin expression in podocytes. J. Am. Soc. Nephrol. 2014;25(12):2764–2777. doi: 10.1681/ASN.2012080823. PubMed DOI PMC

Reményi A, Schöler HR, Wilmanns M. Combinatorial control of gene expression. Nat. Struct. Mol. Biol. 2004;11(9):812–815. doi: 10.1038/nsmb820. PubMed DOI

Clarkson PA, Davies HR, Williams DM, Chaudhary R, Hughes IA, Patterson MN. Mutational screening of the Wilms's tumour gene, WT1, in males with genital abnormalities. J. Med. Genet. 1993;30(9):767–772. doi: 10.1136/jmg.30.9.767. PubMed DOI PMC

Bharathavikru R, Dudnakova T, Aitken S, Slight J, Artibani M, Hohenstein P, et al. Transcription factor Wilms' tumor 1 regulates developmental RNAs through 3' UTR interaction. Genes Dev. 2017;31(4):347–352. doi: 10.1101/gad.291500.116. PubMed DOI PMC

Toska E, Roberts SG. Mechanisms of transcriptional regulation by WT1 (Wilms' tumour 1) Biochem. 2014;J461(1):15–32. doi: 10.1042/bj20131587. PubMed DOI PMC

Štolbová Š, Bezdíčka M, Prohászka Z, Csuka D, Hrachovinová I, Burkert J, et al. Molecular basis and outcomes of atypical haemolytic uraemic syndrome in Czech children. Eur. J. Pediatr. 2020;179(11):1739–1750. doi: 10.1007/s00431-020-03666-9. PubMed DOI

Stenson PD, Mort M, Ball EV, Evans K, Hayden M, Heywood S, et al. The human gene mutation database: Towards a comprehensive repository of inherited mutation data for medical research, genetic diagnosis and next-generation sequencing studies. Hum. Genet. 2017;136(6):665–677. doi: 10.1007/s00439-017-1779-6. PubMed DOI PMC

Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: A joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17(5):405–424. doi: 10.1038/gim.2015.30. PubMed DOI PMC

Kleinberger J, Maloney KA, Pollin TI, Jeng LJ. An openly available online tool for implementing the ACMG/AMP standards and guidelines for the interpretation of sequence variants. Genet. Med. 2016;18(11):1165. doi: 10.1038/gim.2016.13. PubMed DOI PMC

Dostálková A, Kaufman F, Křížová I, Vokatá B, Ruml T, Rumlová M. In Vitro Quantification of the Effects of IP6 and Other Small Polyanions on Immature HIV-1 Particle Assembly and Core Stability. J. Virol. 2020;94(20):1. doi: 10.1128/jvi.00991-20. PubMed DOI PMC

Füzik T, Ulbrich P, Ruml T. Efficient Mutagenesis Independent of Ligation (EMILI) J. Microbiol. Methods. 2014;106:67–71. doi: 10.1016/j.mimet.2014.08.003. PubMed DOI

Team RC. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria (2014). URL http://www.R-project.org/.

Fencl F, Malina M, Stará V, Zieg J, Mixová D, Seeman T, et al. Discordant expression of a new WT1 gene mutation in a family with monozygotic twins presenting with congenital nephrotic syndrome. Eur. J. Pediatr. 2012;171(1):121–124. doi: 10.1007/s00431-011-1497-3. PubMed DOI

Nordenskjöld A, Friedman E, Anvret M. WT1 mutations in patients with Denys-Drash syndrome: a novel mutation in exon 8 and paternal allele origin. Hum. Genet. 1994;93(2):115–120. doi: 10.1007/bf00210593. PubMed DOI

Schumacher V, Schneider S, Figge A, Wildhardt G, Harms D, Schmidt D, et al. Correlation of germ-line mutations and two-hit inactivation of the WT1 gene with Wilms tumors of stromal-predominant histology. Proc. Natl. Acad. Sci. US. 1997;A94(8):3972–3977. doi: 10.1073/pnas.94.8.3972. PubMed DOI PMC

Xiong HY, Alipanahi B, Lee LJ, Bretschneider H, Merico D, Yuen RK, et al. RNA splicing: The human splicing code reveals new insights into the genetic determinants of disease. Science. 2015;347(6218):1206. doi: 10.1126/science.1254806. PubMed DOI PMC

Barrera LA, Vedenko A, Kurland JV, Rogers JM, Gisselbrecht SS, Rossin EJ, et al. Survey of variation in human transcription factors reveals prevalent DNA binding changes. Science. 2016;351(6280):1450–1454. doi: 10.1126/science.aad2257. PubMed DOI PMC