Abstract
Congenital anomalies of the kidney and urinary tract (CAKUT) account for 40–50 % of chronic kidney disease that manifests in the first two decades of life. Thus far, 31 monogenic causes of isolated CAKUT have been described, explaining ~12 % of cases. To identify additional CAKUT-causing genes, we performed whole-exome sequencing followed by a genetic burden analysis in 26 genetically unsolved families with CAKUT. We identified two heterozygous mutations in SRGAP1 in 2 unrelated families. SRGAP1 is a small GTPase-activating protein in the SLIT2–ROBO2 signaling pathway, which is essential for development of the metanephric kidney. We then examined the pathway-derived candidate gene SLIT2 for mutations in cohort of 749 individuals with CAKUT and we identified 3 unrelated individuals with heterozygous mutations. The clinical phenotypes of individuals with mutations in SLIT2 or SRGAP1 were cystic dysplastic kidneys, unilateral renal agenesis, and duplicated collecting system. We show that SRGAP1 is expressed in early mouse nephrogenic mesenchyme and that it is coexpressed with ROBO2 in SIX2-positive nephron progenitor cells of the cap mesenchyme in developing rat kidney. We demonstrate that the newly identified mutations in SRGAP1 lead to an augmented inhibition of RAC1 in cultured human embryonic kidney cells and that the SLIT2 mutations compromise the ability of the SLIT2 ligand to inhibit cell migration. Thus, we report on two novel candidate genes for causing monogenic isolated CAKUT in humans.
Similar content being viewed by others
References
Bertoli-Avella AM, Conte ML, Punzo F, de Graaf BM, Lama G, La Manna A, Polito C, Grassia C, Nobili B, Rambaldi PF, Oostra BA, Perrotta S (2008) ROBO2 gene variants are associated with familial vesicoureteral reflux. J Am Soc Nephrol 19:825–831. doi:10.1681/ASN.2007060692
Boyden LM, Choi M, Choate KA, Nelson-Williams CJ, Farhi A, Toka HR, Tikhonova IR, Bjornson R, Mane SM, Colussi G, Lebel M, Gordon RD, Semmekrot BA, Poujol A, Valimaki MJ, De Ferrari ME, Sanjad SA, Gutkin M, Karet FE, Tucci JR, Stockigt JR, Keppler-Noreuil KM, Porter CC, Anand SK, Whiteford ML, Davis ID, Dewar SB, Bettinelli A, Fadrowski JJ, Belsha CW, Hunley TE, Nelson RD, Trachtman H, Cole TR, Pinsk M, Bockenhauer D, Shenoy M, Vaidyanathan P, Foreman JW, Rasoulpour M, Thameem F, Al-Shahrouri HZ, Radhakrishnan J, Gharavi AG, Goilav B, Lifton RP (2012) Mutations in kelch-like 3 and cullin 3 cause hypertension and electrolyte abnormalities. Nature 482:98–102. doi:10.1038/nature10814
Brakeman P (2008) Vesicoureteral reflux, reflux nephropathy, and end-stage renal disease. Adv Urol. doi:10.1155/2008/508949
Brose K, Bland KS, Wang KH, Arnott D, Henzel W, Goodman CS, Tessier-Lavigne M, Kidd T (1999) Slit proteins bind Robo receptors and have an evolutionarily conserved role in repulsive axon guidance. Cell 96:795–806
Chaki M, Airik R, Ghosh AK, Giles RH, Chen R, Slaats GG, Wang H, Hurd TW, Zhou W, Cluckey A, Gee HY, Ramaswami G, Hong CJ, Hamilton BA, Cervenka I, Ganji RS, Bryja V, Arts HH, van Reeuwijk J, Oud MM, Letteboer SJ, Roepman R, Husson H, Ibraghimov-Beskrovnaya O, Yasunaga T, Walz G, Eley L, Sayer JA, Schermer B, Liebau MC, Benzing T, Le Corre S, Drummond I, Janssen S, Allen SJ, Natarajan S, O’Toole JF, Attanasio M, Saunier S, Antignac C, Koenekoop RK, Ren H, Lopez I, Nayir A, Stoetzel C, Dollfus H, Massoudi R, Gleeson JG, Andreoli SP, Doherty DG, Lindstrad A, Golzio C, Katsanis N, Pape L, Abboud EB, Al-Rajhi AA, Lewis RA, Omran H, Lee EY, Wang S, Sekiguchi JM, Saunders R, Johnson CA, Garner E, Vanselow K, Andersen JS, Shlomai J, Nurnberg G, Nurnberg P, Levy S, Smogorzewska A, Otto EA, Hildebrandt F (2012) Exome capture reveals ZNF423 and CEP164 mutations, linking renal ciliopathies to DNA damage response signaling. Cell 150:533–548. doi:10.1016/j.cell.2012.06.028
Costantini F, Shakya R (2006) GDNF/Ret signaling and the development of the kidney. BioEssays 28:117–127. doi:10.1002/bies.20357
Dobson MG, Darlow JM, Hunziker M, Green AJ, Barton DE, Puri P (2013) Heterozygous non-synonymous ROBO2 variants are unlikely to be sufficient to cause familial vesicoureteric reflux. Kidney Int 84:327–337. doi:10.1038/ki.2013.100
Durbec P, Marcos-Gutierrez CV, Kilkenny C, Grigoriou M, Wartiowaara K, Suvanto P, Smith D, Ponder B, Costantini F, Saarma M et al (1996) GDNF signalling through the Ret receptor tyrosine kinase. Nature 381:789–793. doi:10.1038/381789a0
Fan X, Li Q, Pisarek-Horowitz A, Rasouly HM, Wang X, Bonegio RG, Wang H, McLaughlin M, Mangos S, Kalluri R, Holzman LB, Drummond IA, Brown D, Salant DJ, Lu W (2012) Inhibitory effects of Robo2 on nephrin: a crosstalk between positive and negative signals regulating podocyte structure. Cell Rep 2:52–61. doi:10.1016/j.celrep.2012.06.002
Gbadegesin RA, Brophy PD, Adeyemo A, Hall G, Gupta IR, Hains D, Bartkowiak B, Rabinovich CE, Chandrasekharappa S, Homstad A, Westreich K, Wu G, Liu Y, Holanda D, Clarke J, Lavin P, Selim A, Miller S, Wiener JS, Ross SS, Foreman J, Rotimi C, Winn MP (2013) TNXB mutations can cause vesicoureteral reflux. J Am Soc Nephrol 24:1313–1322. doi:10.1681/ASN.2012121148
Grieshammer U, Le M, Plump AS, Wang F, Tessier-Lavigne M, Martin GR (2004) SLIT2-mediated ROBO2 signaling restricts kidney induction to a single site. Dev Cell 6:709–717 (pii: S153458070400108X)
Halbritter J, Diaz K, Chaki M, Porath JD, Tarrier B, Fu C, Innis JL, Allen SJ, Lyons RH, Stefanidis CJ, Omran H, Soliman NA, Otto EA (2012) High-throughput mutation analysis in patients with a nephronophthisis-associated ciliopathy applying multiplexed barcoded array-based PCR amplification and next-generation sequencing. J Med Genet 49:756–767. doi:10.1136/jmedgenet-2012-100973
Halbritter J, Porath JD, Diaz KA, Braun DA, Kohl S, Chaki M, Allen SJ, Soliman NA, Hildebrandt F, Otto EA (2013) Identification of 99 novel mutations in a worldwide cohort of 1,056 patients with a nephronophthisis-related ciliopathy. Hum Genet 132:865–884. doi:10.1007/s00439-013-1297-0
Humbert C, Silbermann F, Morar B, Parisot M, Zarhrate M, Masson C, Tores F, Blanchet P, Perez MJ, Petrov Y, Khau Van Kien P, Roume J, Leroy B, Gribouval O, Kalaydjieva L, Heidet L, Salomon R, Antignac C, Benmerah A, Saunier S, Jeanpierre C (2014) Integrin alpha 8 recessive mutations are responsible for bilateral renal agenesis in humans. Am J Hum Genet 94:288–294. doi:10.1016/j.ajhg.2013.12.017
Hwang DY, Dworschak GC, Kohl S, Saisawat P, Vivante A, Hilger AC, Reutter HM, Soliman NA, Bogdanovic R, Kehinde EO, Tasic V, Hildebrandt F (2014) Mutations in 12 known dominant disease-causing genes clarify many congenital anomalies of the kidney and urinary tract. Kidney Int 85:1429–1433. doi:10.1038/ki.2013.508
Ichikawa I, Kuwayama F, Pope JC, Stephens FD, Miyazaki Y (2002) Paradigm shift from classic anatomic theories to contemporary cell biological views of CAKUT. Kidney Int 61:889–898. doi:10.1046/j.1523-1755.2002.00188.x
Jeanpierre C, Mace G, Parisot M, Moriniere V, Pawtowsky A, Benabou M, Martinovic J, Amiel J, Attie-Bitach T, Delezoide AL, Loget P, Blanchet P, Gaillard D, Gonzales M, Carpentier W, Nitschke P, Tores F, Heidet L, Antignac C, Salomon R (2011) RET and GDNF mutations are rare in fetuses with renal agenesis or other severe kidney development defects. J Med Genet 48:497–504. doi:10.1136/jmg.2010.088526
Kohl S, Hwang DY, Dworschak GC, Hilger AC, Saisawat P, Vivante A, Stajic N, Bogdanovic R, Reutter HM, Kehinde EO, Tasic V, Hildebrandt F (2014) Mild recessive mutations in six fraser syndrome-related genes cause isolated congenital anomalies of the kidney and urinary tract. J Am Soc Nephrol. doi:10.1681/ASN.2013101103
Li X, Chen Y, Liu Y, Gao J, Gao F, Bartlam M, Wu JY, Rao Z (2006) Structural basis of Robo proline-rich motif recognition by the srGAP1 Src homology 3 domain in the Slit-Robo signaling pathway. J Biol Chem 281:28430–28437. doi:10.1074/jbc.M604135200
Lindenmeyer MT, Eichinger F, Sen K, Anders HJ, Edenhofer I, Mattinzoli D, Kretzler M, Rastaldi MP, Cohen CD (2010) Systematic analysis of a novel human renal glomerulus-enriched gene expression dataset. PLoS One 5:e11545. doi:10.1371/journal.pone.0011545
Lu W, van Eerde AM, Fan X, Quintero-Rivera F, Kulkarni S, Ferguson H, Kim HG, Fan Y, Xi Q, Li QG, Sanlaville D, Andrews W, Sundaresan V, Bi W, Yan J, Giltay JC, Wijmenga C, de Jong TP, Feather SA, Woolf AS, Rao Y, Lupski JR, Eccles MR, Quade BJ, Gusella JF, Morton CC, Maas RL (2007) Disruption of ROBO2 is associated with urinary tract anomalies and confers risk of vesicoureteral reflux. Am J Hum Genet 80:616–632. doi:10.1086/512735
MacArthur DG, Manolio TA, Dimmock DP, Rehm HL, Shendure J, Abecasis GR, Adams DR, Altman RB, Antonarakis SE, Ashley EA, Barrett JC, Biesecker LG, Conrad DF, Cooper GM, Cox NJ, Daly MJ, Gerstein MB, Goldstein DB, Hirschhorn JN, Leal SM, Pennacchio LA, Stamatoyannopoulos JA, Sunyaev SR, Valle D, Voight BF, Winckler W, Gunter C (2014) Guidelines for investigating causality of sequence variants in human disease. Nature 508:469–476. doi:10.1038/nature13127
McPherson E, Carey J, Kramer A, Hall JG, Pauli RM, Schimke RN, Tasin MH (1987) Dominantly inherited renal adysplasia. Am J Med Genet 26:863–872. doi:10.1002/ajmg.1320260413
Nguyen Ba-Charvet KT, Brose K, Marillat V, Kidd T, Goodman CS, Tessier-Lavigne M, Sotelo C, Chedotal A (1999) Slit2-Mediated chemorepulsion and collapse of developing forebrain axons. Neuron 22:463–473
Nguyen-Ba-Charvet KT, Picard-Riera N, Tessier-Lavigne M, Baron-Van Evercooren A, Sotelo C, Chedotal A (2004) Multiple roles for slits in the control of cell migration in the rostral migratory stream. J Neurosci 24:1497–1506. doi:10.1523/JNEUROSCI.4729-03.2004
Pellegrin S, Mellor H (2008) Rho GTPase activation assays. Curr Protoc Cell Biol 38:14.8.1–14.8.19. doi:10.1002/0471143030.cb1408s38
Pepicelli CV, Kispert A, Rowitch DH, McMahon AP (1997) GDNF induces branching and increased cell proliferation in the ureter of the mouse. Dev Biol 192:193–198. doi:10.1006/dbio.1997.8745
Piper M, Georgas K, Yamada T, Little M (2000) Expression of the vertebrate Slit gene family and their putative receptors, the Robo genes, in the developing murine kidney. Mech Dev 94:213–217
Rasouly HM, Lu W (2013) Lower urinary tract development and disease. Wiley Interdiscip Rev Syst Biol Med 5:307–342. doi:10.1002/wsbm.1212
Saisawat P, Kohl S, Hilger AC, Hwang DY, Yung Gee H, Dworschak GC, Tasic V, Pennimpede T, Natarajan S, Sperry E, Matassa DS, Stajic N, Bogdanovic R, de Blaauw I, Marcelis CL, Wijers CH, Bartels E, Schmiedeke E, Schmidt D, Marzheuser S, Grasshoff-Derr S, Holland-Cunz S, Ludwig M, Nothen MM, Draaken M, Brosens E, Heij H, Tibboel D, Herrmann BG, Solomon BD, de Klein A, van Rooij IA, Esposito F, Reutter HM, Hildebrandt F (2014) Whole-exome resequencing reveals recessive mutations in TRAP1 in individuals with CAKUT and VACTERL association. Kidney Int 85:1310–1317. doi:10.1038/ki.2013.417
Sanchez MP, Silos-Santiago I, Frisen J, He B, Lira SA, Barbacid M (1996) Renal agenesis and the absence of enteric neurons in mice lacking GDNF. Nature 382:70–73. doi:10.1038/382070a0
Sanna-Cherchi S, Sampogna RV, Papeta N, Burgess KE, Nees SN, Perry BJ, Choi M, Bodria M, Liu Y, Weng PL, Lozanovski VJ, Verbitsky M, Lugani F, Sterken R, Paragas N, Caridi G, Carrea A, Dagnino M, Materna-Kiryluk A, Santamaria G, Murtas C, Ristoska-Bojkovska N, Izzi C, Kacak N, Bianco B, Giberti S, Gigante M, Piaggio G, Gesualdo L, Kosuljandic Vukic D, Vukojevic K, Saraga-Babic M, Saraga M, Gucev Z, Allegri L, Latos-Bielenska A, Casu D, State M, Scolari F, Ravazzolo R, Kiryluk K, Al-Awqati Q, D’Agati VD, Drummond IA, Tasic V, Lifton RP, Ghiggeri GM, Gharavi AG (2013) Mutations in DSTYK and dominant urinary tract malformations. N Engl J Med 369:621–629. doi:10.1056/NEJMoa1214479
Smith JM, Stablein DM, Munoz R, Hebert D, McDonald RA (2007) Contributions of the Transplant Registry: the 2006 Annual Report of the North American Pediatric Renal Trials and Collaborative Studies (NAPRTCS). Pediatr Transplant 11:366–373. doi:10.1111/j.1399-3046.2007.00704.x
Tang MJ, Worley D, Sanicola M, Dressler GR (1998) The RET-glial cell-derived neurotrophic factor (GDNF) pathway stimulates migration and chemoattraction of epithelial cells. J Cell Biol 142:1337–1345
Vega QC, Worby CA, Lechner MS, Dixon JE, Dressler GR (1996) Glial cell line-derived neurotrophic factor activates the receptor tyrosine kinase RET and promotes kidney morphogenesis. Proc Natl Acad Sci 93:10657–10661
Vivante A, Kohl S, Hwang DY, Dworschak GC, Hildebrandt F (2014) Single-gene causes of congenital anomalies of the kidney and urinary tract (CAKUT) in humans. Pediatr Nephrol 29:695–704. doi:10.1007/s00467-013-2684-4
Ward ME, Rao Y (2005) Investigations of neuronal migration in the central nervous system. Methods Mol Biol 294:137–156
Wong K, Ren XR, Huang YZ, Xie Y, Liu G, Saito H, Tang H, Wen L, Brady-Kalnay SM, Mei L, Wu JY, Xiong WC, Rao Y (2001) Signal transduction in neuronal migration: roles of GTPase activating proteins and the small GTPase Cdc42 in the Slit-Robo pathway. Cell 107:209–221
Yamazaki D, Itoh T, Miki H, Takenawa T (2013) srGAP1 regulates lamellipodial dynamics and cell migratory behavior by modulating Rac1 activity. Mol Biol Cell 24:3393–3405. doi:10.1091/mbc.E13-04-0178
Acknowledgments
We thank the physicians and the participating families, Anna Pisarek-Horowitz for assistance with early mouse embryonic kidney dissection, and Nine V. A. M. Knoers for mutation analysis of SRGAP1 in additional affected individuals. F.H. is an Investigator of the Howard Hughes Medical Institute, and the Warren E. Grupe Professor of Pediatrics. This research was supported by grants from the National Institutes of Health (R01DK088767 to FH; R01DK078226 to WL), by the March of Dimes Foundation (6-FY11-241 to FH; 1-FY12-426 to WL), by the Excellence Initiative of the German Federal and State Governments (EXC 294 to TBH), by the Excellence Initiative of the German Research Foundation (GSC-4, Spemann Graduate School to CS), by grants from the Dutch Kidney Foundation (KSTP12_010 to AMvE; CP11.18 to KYR), by Fonds NutsOhra (1303-070 to AMvE), and by the European Community’s Seventh Framework Program FP7/2009 (305608, EURenOmics to GvdH and KYR).
Author information
Authors and Affiliations
Corresponding authors
Additional information
D.-Y. Hwang, S. Kohl and X. Fan contributed equally to this work.
Electronic supplementary material
Below is the link to the electronic supplementary material.
439_2015_1570_MOESM1_ESM.pdf
Supplementary material 1 (PDF 4287 kb) Supplementary Figure 1. Srgap1 is expressed in early mouse developing kidney. Supplementary Figure 2. Srgap1 is expressed in mouse metanephric mesenchyme and developing glomeruli at E13.5 and E14.5. Supplementary Figure 3. Srgap1 and Robo2 are coexpressed in metanephric mesenchyme, cap mesenchyme, and renal corpuscles in mice at E.11.5 and E15.5. Slit2 is expressed in ureteric bud and ureteric tip at E11.5 and E15.5. Supplementary Figure 4. SRGAP1 partially colocalizes with ROBO2 in developing podocytes. Supplementary Figure 5. Overexpression of mutant SRGAP1 does not alter CDC42 activity in cultured HEK293T cells. Supplementary Figure 6. Mutations in SLIT2 detected in individuals with CAKUT reduce the chemorepulsive effect of SLIT2
439_2015_1570_MOESM2_ESM.pdf
Supplementary material 2 (PDF 26 kb) Supplementary Figure 7. Similar amounts of SLIT2 proteins present in the conditioned media used for the SVZa assay (Figure 4)
Rights and permissions
About this article
Cite this article
Hwang, DY., Kohl, S., Fan, X. et al. Mutations of the SLIT2–ROBO2 pathway genes SLIT2 and SRGAP1 confer risk for congenital anomalies of the kidney and urinary tract. Hum Genet 134, 905–916 (2015). https://doi.org/10.1007/s00439-015-1570-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00439-015-1570-5