Disruption of ROBO2 Is Associated with Urinary Tract Anomalies and Confers Risk of Vesicoureteral Reflux
2007; Elsevier BV; Volume: 80; Issue: 4 Linguagem: Inglês
10.1086/512735
ISSN1537-6605
AutoresLu W, Albertien M. van Eerde, Xueping Fan, Fabiola Quintero‐Rivera, Shashikant Kulkarni, Heather Ferguson, Hyung‐Goo Kim, Yanli Fan, Qiongchao Xi, Qing-gang Li, Damien Sanlaville, William D. Andrews, Vasi Sundaresan, Weimin Bi, Jiong Yan, Jacques C. Giltay, Cisca Wijmenga, Tom P.V.M. de Jong, Sally Feather, Adrian S. Woolf, Yi Rao, James R. Lupski, Michael R. Eccles, Bradley J. Quade, James F. Gusella, Cynthia C. Morton, Richard L. Maas,
Tópico(s)Cancer-related molecular mechanisms research
ResumoCongenital anomalies of the kidney and urinary tract (CAKUT) include vesicoureteral reflux (VUR). VUR is a complex, genetically heterogeneous developmental disorder characterized by the retrograde flow of urine from the bladder into the ureter and is associated with reflux nephropathy, the cause of 15% of end-stage renal disease in children and young adults. We investigated a man with a de novo translocation, 46,X,t(Y;3)(p11;p12)dn, who exhibits multiple congenital abnormalities, including severe bilateral VUR with ureterovesical junction defects. This translocation disrupts ROBO2, which encodes a transmembrane receptor for SLIT ligand, and produces dominant-negative ROBO2 proteins that abrogate SLIT-ROBO signaling in vitro. In addition, we identified two novel ROBO2 intracellular missense variants that segregate with CAKUT and VUR in two unrelated families. Adult heterozygous and mosaic mutant mice with reduced Robo2 gene dosage also exhibit striking CAKUT-VUR phenotypes. Collectively, these results implicate the SLIT-ROBO signaling pathway in the pathogenesis of a subset of human VUR. Congenital anomalies of the kidney and urinary tract (CAKUT) include vesicoureteral reflux (VUR). VUR is a complex, genetically heterogeneous developmental disorder characterized by the retrograde flow of urine from the bladder into the ureter and is associated with reflux nephropathy, the cause of 15% of end-stage renal disease in children and young adults. We investigated a man with a de novo translocation, 46,X,t(Y;3)(p11;p12)dn, who exhibits multiple congenital abnormalities, including severe bilateral VUR with ureterovesical junction defects. This translocation disrupts ROBO2, which encodes a transmembrane receptor for SLIT ligand, and produces dominant-negative ROBO2 proteins that abrogate SLIT-ROBO signaling in vitro. In addition, we identified two novel ROBO2 intracellular missense variants that segregate with CAKUT and VUR in two unrelated families. Adult heterozygous and mosaic mutant mice with reduced Robo2 gene dosage also exhibit striking CAKUT-VUR phenotypes. Collectively, these results implicate the SLIT-ROBO signaling pathway in the pathogenesis of a subset of human VUR. Congenital anomalies of the kidney and urinary tract (CAKUT) make up a family of diseases with a diverse anatomical spectrum, including kidney anomalies (e.g., renal dysplasia, duplex kidney, and hydronephrosis) and ureter anomalies (e.g., vesicoureteral reflux [VUR], megaureter, and ureterovesical junction [UVJ] obstruction).1Pope 4th, JC Brock 3rd, JW Adams MC Stephens FD Ichikawa I How they begin and how they end: classic and new theories for the development and deterioration of congenital anomalies of the kidney and urinary tract, CAKUT.J Am Soc Nephrol. 1999; 10: 2018-2028PubMed Google Scholar, 2Nakanishi K Yoshikawa N Genetic disorders of human congenital anomalies of the kidney and urinary tract (CAKUT).Pediatr Int. 2003; 45: 610-616Crossref PubMed Scopus (44) Google Scholar In particular, VUR (MIM %193000), a polygenic genetic disorder with an incidence of ∼1 in 100 infants,3Feather SA Malcolm S Woolf AS Wright V Blaydon D Reid CJ Flinter FA Proesmans W Devriendt K Carter J et al.Primary, nonsyndromic vesicoureteric reflux and its nephropathy is genetically heterogeneous, with a locus on chromosome 1.Am J Hum Genet. 2000; 66: 1420-1425Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 4Sargent MA What is the normal prevalence of vesicoureteral reflux?.Pediatr Radiol. 2000; 30: 587-593Crossref PubMed Scopus (190) Google Scholar is one of the most common clinical manifestations of CAKUT. VUR is characterized by the reflux of urine from the bladder into the ureters and sometimes into the kidneys and is a risk factor for urinary tract infection (UTI).5Bailey RR The relationship of vesico-ureteric reflux to urinary tract infection and chronic pyelonephritis-reflux nephropathy.Clin Nephrol. 1973; 1: 132-141PubMed Google Scholar In combination with intrarenal reflux, the resulting inflammatory reaction may result in renal injury or scarring, also called “reflux nephropathy.”6Dillon MJ Goonasekera CD Reflux nephropathy.J Am Soc Nephrol. 1998; 9: 2377-2383PubMed Google Scholar Extensive renal scarring impairs renal function and may predispose patients to hypertension, proteinuria, and renal insufficiency. Reflux nephropathy accounts for as much as 15% of end-stage renal disease in children and young adults.7Kincaid-Smith PS Bastos MG Becker GJ Reflux nephropathy in the adult.Contrib Nephrol. 1984; 39: 94-101PubMed Google Scholar Primary VUR results from a developmental defect of the UVJ8Mackie GG Awang H Stephens FD The ureteric orifice: the embryologic key to radiologic status of duplex kidneys.J Pediatr Surg. 1975; 10: 473-481Abstract Full Text PDF PubMed Scopus (47) Google Scholar and is known to occur in multiple members of families. In siblings and offspring of affected patients, the prevalence is as high as 50%.9Noe HN Wyatt RJ Peeden Jr, JN Rivas ML The transmission of vesicoureteral reflux from parent to child.J Urol. 1992; 148: 1869-1871PubMed Google Scholar, 10Van den Abbeele AD Treves ST Lebowitz RL Bauer S Davis RT Retik A Colodny A Vesicoureteral reflux in asymptomatic siblings of patients with known reflux: radionuclide cystography.Pediatrics. 1987; 79: 147-153PubMed Google Scholar Despite its high incidence in the pediatric population, the genetic basis of VUR remains to be elucidated. Human ROBO1–4 encode homologs of Drosophila Roundabout (Robo), a transmembrane receptor that binds SLIT ligand and transduces a signal to prevent axons from recrossing the CNS midline.11Kidd T Brose K Mitchell KJ Fetter RD Tessier-Lavigne M Goodman CS Tear G Roundabout controls axon crossing of the CNS midline and defines a novel subfamily of evolutionarily conserved guidance receptors.Cell. 1998; 92: 205-215Abstract Full Text Full Text PDF PubMed Scopus (699) Google Scholar On the basis of the mouse Robo1 mutant phenotype, ROBO1 is a candidate gene for pulmonary hypoplasia and adenocarcinoma,12Xian J Aitchison A Bobrow L Corbett G Pannell R Rabbitts T Rabbitts P Targeted disruption of the 3p12 gene, Dutt1/Robo1, predisposes mice to lung adenocarcinomas and lymphomas with methylation of the gene promoter.Cancer Res. 2004; 64: 6432-6437Crossref PubMed Scopus (55) Google Scholar, 13Xian J Clark KJ Fordham R Pannell R Rabbitts TH Rabbitts PH Inadequate lung development and bronchial hyperplasia in mice with a targeted deletion in the Dutt1/Robo1 gene.Proc Natl Acad Sci USA. 2001; 98: 15062-15066Crossref PubMed Scopus (129) Google Scholar as well as for dyslexia (DYX5).14Hannula-Jouppi K Kaminen-Ahola N Taipale M Eklund R Nopola-Hemmi J Kaariainen H Kere J The axon guidance receptor gene ROBO1 is a candidate gene for developmental dyslexia.PLoS Genet. 2005; 1: e50Crossref PubMed Scopus (253) Google Scholar Mutations in ROBO3 result in horizontal-gaze palsy with progressive scoliosis,15Jen JC Chan WM Bosley TM Wan J Carr JR Rub U Shattuck D Salamon G Kudo LC Ou J et al.Mutations in a human ROBO gene disrupt hindbrain axon pathway crossing and morphogenesis.Science. 2004; 304: 1509-1513Crossref PubMed Scopus (277) Google Scholar whereas zebrafish Robo4 is implicated in angiogenesis.16Bedell VM Yeo SY Park KW Chung J Seth P Shivalingappa V Zhao J Obara T Sukhatme VP Drummond IA et al.Roundabout4 is essential for angiogenesis in vivo.Proc Natl Acad Sci U S A. 2005; 102: 6373-6378Crossref PubMed Scopus (172) Google ScholarRobo2 loss-of-function mutations in zebrafish and mice result in retinal and commissural pathfinding defects, respectively.17Fricke C Lee JS Geiger-Rudolph S Bonhoeffer F Chien CB Astray, a zebrafish roundabout homolog required for retinal axon guidance.Science. 2001; 292: 507-510Crossref PubMed Scopus (163) Google Scholar, 18Long H Sabatier C Ma L Plump A Yuan W Ornitz DM Tamada A Murakami F Goodman CS Tessier-Lavigne M Conserved roles for Slit and Robo proteins in midline commissural axon guidance.Neuron. 2004; 42: 213-223Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar Interestingly, Robo2 and Slit2 mouse mutants reveal an additional key role for SLIT-ROBO signaling in regulating the metanephric expression of glial cell derived neurotrophic factor (Gdnf), which in turn induces ureteric bud outgrowth from the nephric duct and restricts it to a single site.19Grieshammer U Le M Plump AS Wang F Tessier-Lavigne M Martin GR SLIT2-mediated ROBO2 signaling restricts kidney induction to a single site.Dev Cell. 2004; 6: 709-717Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar However, a role for ROBO2 in human disease has not been identified elsewhere. Metaphase FISH was performed according to standard methods. RP11 BAC clones were obtained from BACPAC Resources, were labeled as FISH probes, and were hybridized to metaphase chromosomes prepared from a t(Y;3)(p11;p12)dn lymphoblastoid cell line established from patient DGAP107. Array comparative genomic hybridization (CGH) experiments were performed by Spectral Genomics (SpectralChip 2006 array) and Agilent Technologies (Human Genome CGH Microarray Kit 44A). Southern and RT-PCR analyses were performed according to routine protocols. RT-PCR primers used to amplify the 2.8-kb ROBO2 cDNA (GenBank accession number NM_002942) were ROBO2-F1 and ROBO2-R1; those used to amplify ROBO2-PCDH11Y fusion transcripts were ROBO2-F2 (same for all transcripts) and PCDH11Y-R1, PCDH11Y-R2, and PCDH11Y-R3; those used to amplify wild-type ROBO2 transcripts were ROBO2-F2 and ROBO2-qR; and those used to amplify PCDH11Y cDNA (GenBank accession number NM_032971) were PCDH11Y-rtF1 and PCDH11Y-rtR1 (appendix C). All primer sequences are listed in appendix G. PCR primers and TaqMan fluorogenic probes for analysis of the ROBO2 nontranslocated allele and the Fu-129 and Fu-153 fusion transcripts were designed using Primer Express software (Applied Biosystems). TaqMan primers and probes for Gapdh and β-actin were used for normalization. Probe melting temperatures were ∼7°C–10°C higher than those for the matching primer pair. High-pressure liquid chromatography–purified fluorogenic probes contained covalently attached 5′-FAM reporter and 3′-BHQ1 quencher dyes. Sequences of TaqMan primer and probe sets are listed in appendix G. RT-PCR reactions were performed using an iCycler IQ Real-Time Detection System (Bio-Rad). SuperScript One-Step RT-PCR with Platinum Taq kits (Invitrogen) were used for triplicate RT-PCR amplifications, with each 50−μl reaction containing 200 ng total RNA, 5 mM MgSO4, 500 nM forward and reverse primers, and 200 nM fluorogenic probes. Controls included either no reverse transcriptase or the substitution of H2O for RNA for each primer and probe set. The one-step RT-PCR protocol was 15 min at 50°C and 5 min at 95°C, followed by 45 cycles, each consisting of 15 s at 95°C and 1 min at 60°C. IQ Supermix reagent for real-time PCR (Bio-Rad) was used for two-step RT-PCR. Relative gene expression was analyzed using standard curve and comparative threshold cycle methods. cDNA sequences for Fu-129 and Fu-153 were amplified by PCR with the use of forward primer 5′-hR2(E1) and reverse primers 3′-LEVA(X1) and 3′-SRSC(X1) (appendix G) cloned into EcoRI and XhoI sites in pcDNA3, under control of the cytomegalovirus promoter. To express yellow fluorescent protein (YFP)–ROBO2-PCDH11Y fusions, the YFP (Venus) coding sequence was cloned into BamHI and EcoRI sites in pCS, whereas ROBO2-PCDH11Y coding sequences were inserted inframe with YFP at EcoRI and XhoI sites. YFP-ROBO2-PCDH11Y fusion proteins were expressed under the control of the simian cytomegalovirus IE94 promoter. Myc-SLIT and hemagglutinin (HA)–RoboN (Robo1-N) constructs have been described elsewhere.20Li HS Chen JH Wu W Fagaly T Zhou L Yuan W Dupuis S Jiang ZH Nash W Gick C et al.Vertebrate slit, a secreted ligand for the transmembrane protein roundabout, is a repellent for olfactory bulb axons.Cell. 1999; 96: 807-818Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar The in vitro neuronal migration assay that uses postnatal anterior subventricular zone (SVZa) cells was described elsewhere.21Ward ME Rao Y Investigations of neuronal migration in the central nervous system.Methods Mol Biol. 2005; 294: 137-156PubMed Google Scholar In brief, P1-6 Sprague-Dawley rat brains devoid of meninges were placed in 10% fetal calf serum in Dulbecco's modified Eagle medium (DMEM) and were embedded. Coronal sections of 300 μm were prepared by vibratome, and tissues within the SVZa borders were dissected to make SVZa explants 200–300 μm in diameter. Explants were embedded, together with human embryonic kidney (HEK) cell aggregates in collagen and matrigel (3:2:1 ratio of collagen:matrigel:medium), and were cultured in DMEM with 5% CO2 at 37°C for 24 h. Cocultured cells were washed in PBS for 10 min and were fixed in 4% paraformaldehyde at 4°C overnight. To make cell aggregates, HEK cells were transiently transfected to express mouse Slit2 or RoboN, DGAP107 Fu-129 or Fu-153, or pcDNA3 or Semaphorin 3A expression vectors as negative controls, with the use of Effectene Transfection Kit (Qiagen). After 24 h, transfected HEK cells were detached and were collected by brief centrifugation, and cell pellets were resuspended in an equal volume of DMEM. Ten microliters of suspended cells were hung from the dish cover at 37°C, in 5% CO2 for 1–2 h, to form aggregates. Aggregated cells were washed in DMEM and were squared with a needle. HEK cells were transfected to express YFP-ROBO2-PCDH11Y-Fu-129, YFP-ROBO2-PCDH11Y-Fu-153, Myc-Slit, and HA-RoboN. Cells transfected with pCS2 vector were used as a negative control. To remove cell debris 48 h after transfection, media were collected and were centrifuged for 20 min at 4°C. Supernatants were diluted with sixfold protein loading buffer and were heated at 95°C for 20 min. Cells were lysed (1× PBS, 0.5% Triton X-100, and 1× protease inhibitor), were diluted with sixfold protein loading buffer, and were heated at 95°C for 20 min. Proteins were resolved by 12% SDS-PAGE, with YFP (Venus) fusion proteins detected by monoclonal anti–green fluorescent protein (GFP) antibody that detects YFP (Clontech), and Myc-Slit2 and HA-RoboN were blotted with monoclonal antibodies against Myc and HA, respectively. ROBO2 mutation screening employed PCR amplification of each of the 26 human ROBO2 exons and intron-exon boundaries, followed by purification and bidirectional DNA sequencing. Sequences of the ROBO2 PCR primer sets are listed in appendix G. Sequence data were analyzed using Lasergene (DNAStar) sequence analysis software. DNA samples with sequence changes were confirmed by resequencing. National Center for Biotechnology Information RefSeq ROBO2 cDNA sequence (GenInfo identifier [GI] 109254774) and protein sequence (Entrez Protein accession number NP_002933) (GI 61888896) were used to calculate the nucleotide and amino acid positions. Robo2flox mice were produced using homologous recombination in 129 embryotic stem cells and blastocyst injection. After germline transmission, mice were backcrossed to C57BL/6 and were analyzed thereafter in a mixed C57BL/6-129/Sv background. The Robo2flox allele was genotyped by PCR amplification, followed by SpeI restriction digestion with the use of PCR primers Ro2-MEBAC15F and Ro2-MEBAC15R (appendix G), which amplify a 1,100-bp fragment for both wild-type and Robo2flox alleles. After SpeI digestion, the Robo2flox amplicon remains uncut, whereas the wild-type amplicon yields 750-bp and 350-bp products. Robo2flox/+ mice were bred with TgEIIa-Cre (stock number 003724 [Jackson Laboratory]) to produce the Robo2del5 allele. The Robo2del5 allele was amplified by primers Robo2koF and Robo2R, which produce a 1,100-bp fragment. The wild-type allele was amplified by primers Robo2wtF and Robo2R, which yield a 1,390-bp fragment. F2 Robo2del5/del5↔Robo2del5/flox mosaic mice were prepared as described in appendix F and were analyzed for the presence of urinary tract phenotypes. To examine the ureter and kidney defects, Hoxb7-GFP transgenic mice (gift from Dr. Frank Costantini, Columbia University) were bred with Robo2 mutants. GFP fluorescence was monitored and photographed using a Nikon SMZ-1500 epifluorescence stereomicroscope. All human studies were performed under informed consent protocols approved by the Partners HealthCare System Human Research Committee (Boston), the Human Research Ethics Committee (Institute of Child Health, University College London), or the University Medical Center (Utrecht). Mouse protocols were approved by the Institutional Animal Care and Use Committee at Harvard Medical School or Boston University Medical Center, with additional approval from King's College, London. The Developmental Genome Anatomy Project (DGAP) is a collaborative effort to use chromosomal rearrangements associated with developmental disorders to identify the underlying genetic etiology. DGAP107 is a man aged 18 years with a 46,X,t(Y;3)(p11;p12)dn translocation, whose phenotype includes bilateral high-grade VUR and right megaureter at the UVJ (fig. 1A–1D and appendix A). He required ureteral reimplantation surgery at age 9 years and was found to have wide-open right and left ureteral orifices due to bilateral absence of intravesical ureteral segments. Normally, these submucosal ureteral segments obliquely traverse the muscular layers of the bladder to prevent retrograde flow of urine by a flap-valve mechanism. By metaphase FISH, we identified a BAC clone (RP11-54A6) that crosses the 3p12 breakpoint, which disrupts intron 2 of ROBO2, which is composed of 26 exons and spans ∼606 kb of genomic DNA (fig. 1E and 1F). We cloned and sequenced the breakpoints on the der(3) and der(Y) chromosomes (appendix B). In addition to disruption of ROBO2 at 3p12, the protocadherin gene PCDH11Y at Yp11 was also disrupted by the translocation. A contribution of PCDH11Y disruption to the VUR phenotype in DGAP107 is unlikely, however, since PCDH11Y expression has been detected only in placenta, brain, retina, and testis22Blanco P Sargent CA Boucher CA Mitchell M Affara NA Conservation of PCDHX in mammals; expression of human X/Y genes predominantly in brain.Mamm Genome. 2000; 11: 906-914Crossref PubMed Scopus (106) Google Scholar and has not been detected in embryonic kidney (appendix C). By array CGH and FISH, we also identified a 3.4-Mb interstitial deletion at 17p11.2 in DGAP107. This region is within the common microdeletion region pathogenetic in Smith-Magenis syndrome (SMS [MIM #182290]), a mental retardation syndrome associated with behavioral and sleep disturbances and craniofacial and skeletal anomalies.23Potocki L Shaw CJ Stankiewicz P Lupski JR Variability in clinical phenotype despite common chromosomal deletion in Smith-Magenis syndrome [del(17)(p11.2p11.2)].Genet Med. 2003; 5: 430-434Crossref PubMed Scopus (87) Google Scholar Thus, a role for del(17)(p11.2) in the sleep, behavioral, and cognitive deficits of DGAP107 seems likely. The del(17)(p11.2) microdeletion could also contribute to the pathogenesis of VUR in DGAP107. However, for reasons described below and in appendix D, we conclude that ROBO2 disruption alone is sufficient to account for the VUR phenotype observed in DGAP107. The t(Y;3) translocation in DGAP107 juxtaposes ROBO2 and PCDH11Y in the same transcriptional orientation. On the der(Y), the promoter and the first two exons of ROBO2 reside upstream of exons 1d–6 of PCDH11Y (fig. 2A). From RT-PCR experiments that used DGAP107 lymphoblast RNA, we identified two ROBO2-PCDH11Y fusion transcripts driven by the ROBO2 promoter (fig. 2B). Each transcript contains the first two exons of ROBO2 spliced out of frame to PCDH11Y downstream exons, resulting in premature stop codons shortly after ROBO2 exon 2. When assayed by real-time RT-PCR, these fusion transcripts, denoted Fu-129 and Fu-153, are expressed at somewhat reduced levels compared with the wild-type ROBO2 transcripts derived from the nontranslocated allele (fig. 2C); the wild-type transcripts contained no detectable mutations. Fu-129 and Fu-153 encode 129- and 153-residue polypeptides, respectively, containing the first ROBO2 extracellular immunoglobulin (Ig) domain, but they are truncated before the transmembrane and cytoplasmic domains required for SLIT-ROBO signal transduction (fig. 2D). When expressed without transmembrane and cytoplasmic domains, the soluble extracellular Ig domains of ROBO, denoted as RoboN, are able to bind SLIT ligand.24Liu Z Patel K Schmidt H Andrews W Pini A Sundaresan V Extracellular Ig domains 1 and 2 of Robo are important for ligand (Slit) binding.Mol Cell Neurosci. 2004; 26: 232-240Crossref PubMed Scopus (84) Google Scholar Moreover, the first ROBO Ig domain is necessary and potentially sufficient for SLIT binding.24Liu Z Patel K Schmidt H Andrews W Pini A Sundaresan V Extracellular Ig domains 1 and 2 of Robo are important for ligand (Slit) binding.Mol Cell Neurosci. 2004; 26: 232-240Crossref PubMed Scopus (84) Google Scholar RoboN isoforms can thus inhibit SLIT-ROBO signaling by competing with wild-type ROBO for SLIT binding.25Wu W Wong K Chen J Jiang Z Dupuis S Wu JY Rao Y Directional guidance of neuronal migration in the olfactory system by the protein Slit.Nature. 1999; 400: 331-336Crossref PubMed Scopus (466) Google Scholar We therefore hypothesized that the truncated proteins encoded by the ROBO2-PCDH11Y fusions might act in a dominant-negative manner to block endogenous SLIT-ROBO signaling. To test this hypothesis, we performed an in vitro neuronal migration assay21Ward ME Rao Y Investigations of neuronal migration in the central nervous system.Methods Mol Biol. 2005; 294: 137-156PubMed Google Scholar in which SVZa explants were cultured in proximity to HEK cell aggregates secreting Slit2 or Slit2 and either RoboN, Semaphorin 3A, Fu-129, or Fu-153 (fig. 3A). We then examined the directionality of neuronal migration away from the SVZa explants (fig. 3B–3F). HEK cell aggregates alone have no effect on SVZa explants, resulting in a radially symmetric pattern of neuronal outgrowth.25Wu W Wong K Chen J Jiang Z Dupuis S Wu JY Rao Y Directional guidance of neuronal migration in the olfactory system by the protein Slit.Nature. 1999; 400: 331-336Crossref PubMed Scopus (466) Google Scholar When they were cultured with HEK cell aggregates transfected with vectors expressing Slit2 only or Slit2 and Semaphorin 3A (a molecule having no effect on Slit function, as a control), the Slit2-expressing cell aggregates acted upon the SVZa explants to repel SVZa neuronal outgrowth (fig. 3B and 3C). In contrast, in aggregates coexpressing Slit2 and either RoboN, Fu-129, or Fu-153, the latter molecules abrogated the chemorepulsive effect of Slit2 on the SVZa explant and significantly increased the number of neurons able to migrate towards the Slit2 source, resulting in a radially symmetric pattern of neuronal outgrowth (fig. 3B–3F). These results indicate that the ROBO2-PCDH11Y fusion proteins that result from t(Y;3) can act as dominant-negative molecules to block SLIT-mediated chemorepulsive function. The fusion proteins could further compromise ROBO2 function in DGAP107, which retains only hemizygous ROBO2 expression from the nontranslocated allele. Family studies indicate that primary VUR frequently segregates with autosomal dominant inheritance and incomplete penetrance.9Noe HN Wyatt RJ Peeden Jr, JN Rivas ML The transmission of vesicoureteral reflux from parent to child.J Urol. 1992; 148: 1869-1871PubMed Google Scholar, 26Chapman CJ Bailey RR Janus ED Abbott GD Lynn KL Vesicoureteric reflux: segregation analysis.Am J Med Genet. 1985; 20: 577-584Crossref PubMed Scopus (85) Google Scholar To test whether mutations in ROBO2 are associated with CAKUT and VUR in the general population, we sequenced the 26 exons and intron-exon boundaries of ROBO2 in 124 families with VUR with potential autosomal dominant inheritance. One sequence change—c.2436T→C, or I598T—was observed in exon 12 in the ROBO2 extracellular domain but was also identified in three control DNA samples (see below). It therefore most likely represents a sequence polymorphism and was discounted from further study. In contrast, two novel ROBO2 intracellular coding sequence changes were identified that were not found in 276 controls (see below). These produce nonconservative amino acid substitutions in two independent families with CAKUT-VUR (fig. 4 and appendix E). In family 2559x with CAKUT-VUR, the affected daughter, 25592, has bilateral VUR, hypoplastic kidneys, and nephropathy, whereas her mother, 25593, required ureteral reimplantation because of severe VUR (fig. 4A–4C). Both individuals have a heterozygous T→C change at position 3477 in coding exon 19 (c.3477T→C) that would cause a nonconservative missense I945T substitution in the ROBO2 intracellular domain (ICD) (fig. 4D and 4E). In family B5 with CAKUT-VUR, the proband has bilateral VUR and a right duplex collecting system and kidney. Her mother and two aunts have urinary tract symptoms and ultrasonographical evidence of CAKUT, whereas her grandmother has a unilateral small kidney (fig. 4F–4I). All five family members carry a heterozygous G→A sequence alteration at position 4349 in coding exon 23 (c.4349G→A) that would cause a nonconservative missense amino acid substitution, A1236T, in the ROBO2 ICD (fig. 4J and 4K). An additional family member, uncle A1, also has this alteration but did not exhibit an ultrasonographically detectable renal phenotype; however, nonpenetrance of VUR is common.9Noe HN Wyatt RJ Peeden Jr, JN Rivas ML The transmission of vesicoureteral reflux from parent to child.J Urol. 1992; 148: 1869-1871PubMed Google Scholar Both I945 and A1236 are evolutionarily conserved in all mammals and are only slightly divergent in birds and fish, organisms that lack a urinary bladder and UVJ (fig. 4E and 4K). To further assess the likelihood that these sequence changes represent functional missense variants, as opposed to rare neutral variants found in the general population, we sequenced ROBO2 exons 12, 19, and 23 in 180 unrelated, clinically unaffected controls of ethnic backgrounds similar to those of the affected individuals. Two occurrences of c.2436T→C in exon 12 were detected, but no nucleotide changes were identified in exons 19 or 23. In addition, to determine the full spectrum of ROBO2 sequence variation, we resequenced the 26 ROBO2 exons and intron-exon boundaries in an additional 96 controls. We found only one reoccurrence of c.2436T→C. Of several nonvalidated putative synonymous and nonsynonymous ROBO2 coding SNPs listed in Ensembl v39, we detected only one—c.737C→A, or R32R—in our own sequencing efforts. Moreover, this apparent change was found to represent a sequencing artifact. Thus, in sum, these results suggest that the two sequence changes in the ICD identified in familial VUR are deleterious missense variants that contribute to the CAKUT-VUR phenotype. Both I945T and A1236T could alter the function of the ROBO ICD, which regulates actin polymerization and cellular migration,27Li X Chen Y Liu Y Gao J Gao F Bartlam M Wu JY Rao Z Structural basis of Robo proline-rich motif recognition by the srGAP1 Src homology 3 domain in the Slit-Robo signaling pathway.J Biol Chem. 2006; 281: 28430-28437Crossref PubMed Scopus (26) Google Scholar by creating novel threonine phosphorylation sites or by influencing the binding of proteins that interact with the ROBO ICD.28Wong K Ren XR Huang YZ Xie Y Liu G Saito H Tang H Wen L Brady-Kalnay SM Mei L et al.Signal transduction in neuronal migration: roles of GTPase activating proteins and the small GTPase Cdc42 in the Slit-Robo pathway.Cell. 2001; 107: 209-221Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar For example, the SH3 domain of srGAP1 binds to the ROBO1 ICD CC3 subdomain,27Li X Chen Y Liu Y Gao J Gao F Bartlam M Wu JY Rao Z Structural basis of Robo proline-rich motif recognition by the srGAP1 Src homology 3 domain in the Slit-Robo signaling pathway.J Biol Chem. 2006; 281: 28430-28437Crossref PubMed Scopus (26) Google Scholar, 28Wong K Ren XR Huang YZ Xie Y Liu G Saito H Tang H Wen L Brady-Kalnay SM Mei L et al.Signal transduction in neuronal migration: roles of GTPase activating proteins and the small GTPase Cdc42 in the Slit-Robo pathway.Cell. 2001; 107: 209-221Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar which is partly conserved in ROBO2. The ROBO2 CC3 subdomain (residues 1193–1201) resides close to A1236, and an extended ROBO1 CC3 peptide binds the srGAP1 SH3 domain much more strongly than does the isolated CC3,27Li X Chen Y Liu Y Gao J Gao F Bartlam M Wu JY Rao Z Structural basis of Robo proline-rich motif recognition by the srGAP1 Src homology 3 domain in the Slit-Robo signaling pathway.J Biol Chem. 2006; 281: 28430-28437Crossref PubMed Scopus (26) Google Scholar suggesting that residues outside CC3 also mediate srGAP binding. I945T and A1236T may act as either dominant gain- or loss-of-function mutations that influence protein binding to the ROBO2 ICD. To establish further the involvement of ROBO2 in the pathogenesis of the CAKUT-VUR phenotype postnatally, we next generated and analyzed a conditional Robo2 mouse mutant. A homozygous Robo2-null mouse, described elsewhere,19Grieshammer U Le M Plump AS Wang F Tessier-Lavigne M Martin GR SLIT2-mediated ROBO2 signaling restricts kidney induction to a single site.Dev Cell. 2004; 6: 709-717Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar with a targeted deletion of exon 1 exhibits a multiple ureter phenotype and fails to survive after birth. To determine whether heterozygosity for Robo2 loss of function could produce an abnormal urinary tract phenotype and recapitulate human CAKUT-VUR, we prepared a mouse Robo2 floxed all
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