CLINICAL RESEARCH www.jasn.org

ROBO2 Variants Are Associated with Familial Vesicoureteral Reflux

Aida M. Bertoli-Avella,* Maria Luisa Conte,*† Francesca Punzo,† Bianca M. de Graaf,* Giuliana Lama,† Angela La Manna,† Cesare Polito,† Carolina Grassia,† Bruno Nobili,† Pier Francesco Rambaldi,‡ Ben A. Oostra,* and Silverio Perrotta†

*Department of Clinical Genetics, Erasmus Medical Center, Rotterdam, Netherlands; and †Department of Pediatrics, and ‡Department of Radiological Sciences, Nuclear Medicine, Second University of Naples, Naples, Italy

ABSTRACT The SLIT2 receptor ROBO2 plays a key role in the formation of the ureteric bud, and its inactivation in mice leads to supernumerary ureteric bud development, lack of ureter remodeling, and improper insertion of the ureters into the bladder. Recently, two heterozygous ROBO2 missense mutations were identified in two families with primary vesicoureteral reflux occurring in combination with congenital anomalies of the kidney and urinary tract (VUR/CAKUT). This study investigated a possible causal role of ROBO2 gene variants in 95 unrelated patients with primary VUR (n ϭ 78) or VUR/CAKUT. Eighty-two percent of all patients had a family history of genitourinary anomalies. Twenty-four ROBO2 gene variants were identified by direct sequencing of all 26 exons and the exon-intron boundaries. Of these, four led to amino acid substitutions: Gly328Ser, Asn515Ile, Asp766Gly, and Arg797Gln. When the families were examined, the missense variants co-segregated with VUR (three families) or VUR/CAKUT (one family). These variants were not found in 190 control subjects, and the affected amino acids have been conserved through evolution. In conclusion, a relatively high frequency of ROBO2 variants (5.1%) was found in familial cases; however, functional studies and validation in other cohorts are warranted.

J Am Soc Nephrol 19: 825–831, 2008. doi: 10.1681/ASN.2007060692

Primary vesicoureteral reflux (VUR; OMIM siblings and offspring of patients.5–7 This is compat- 193000) is a common and complex disorder, occur- ible with autosomal dominant inheritance with re- ring with an incidence of approximately one in 100 duced penetrance.8 Early segregation studies have infants.1 The disease is characterized by the retro- pointed to the role of a single major /gene, grade flow of urine from the bladder into the upper with a dominant mutant allele.9 urinary tract and into the kidney. Renal defects as- Nowadays, it is evident that VUR is a genetically het- sociated with VUR are usually known as reflux ne- erogeneous disorder.10,11 We might be studying a disease phropathy (RN). These defects may result from ei- spectrum that varies from “sporadic” patients in whom ther congenital dysplasia, as a result of a ureteral the disease is caused by a combination of frequent genetic bud abnormality occurring during the embryogen- esis, or postinfectious damage.2 RN may lead to hy- Received June 21, 2007. Accepted November 8, 2007. 3 pertension, proteinuria, and renal insufficiency. Published online ahead of print. Publication date available at Moreover, RN accounts for up to 25% of end-stage www.jasn.org. 4 renal failure (ESRF) in children. A.M.B.-A. and M.L.C. contributed equally to this work. VUR may be primary or secondary and may oc- Correspondence: Dr. Aida M. Bertoli-Avella, Department of Clin- cur isolated or in combination with other congeni- ical Genetics, Erasmus MC Rotterdam, P.O. Box 2040, 3000 CA, tal abnormalities of the kidney/urinary tract (VUR/ Rotterdam, Netherlands. Phone: ϩ31-10-4088136; Fax: ϩ31-10- CAKUT). A high familial occurrence has been 4089461; E-mail: [email protected] described with a prevalence of 27 to 50% among Copyright © 2008 by the American Society of Nephrology

J Am Soc Nephrol 19: 825–831, 2008 ISSN : 1046-6673/1904-825 825 CLINICAL RESEARCH www.jasn.org variants with low phenotypic effect (multifactorial or polygenic) to familial VUR, and 17 (18%) were classified as sporadic because no relatively large families carrying more rare gene variants with a strong evidence of renal diseases was found across their relatives. Fifty- phenotypic impact (monogenic disease). seven (60%) patients were detected as having RN; 43 of them were Recently, Lu et al.12 described a de novo human transloca- familial cases. The majority of the cases (n ϭ 78) showed an iso- tion [46,X,t(Y;3)(p11;p12)] in an individual who exhibited lated primary VUR, whereas 17 patients displayed additional re- multiple congenital anomalies including severe bilateral VUR nal/urinary tract abnormalities in combination with VUR (i.e., with ureterovesical junction defects. The translocation dis- duplex collecting system, ureterocele, renal agenesis). rupts ROBO2 (roundabout, axon guidance receptor, homolog Additional family members, when available and recruited 2[Drosophila]), an ideal functional candidate gene for VUR/ in the study, were considered as “affected” on the basis of the CAKUT. The gene was initially known as an axon guidance presence of reflux documented by voiding cystourethrogram receptor and gatekeeper controlling axon midline crossing.13 (VCUG)/direct radionuclide cystography (RNC; see the Con- Recently, it was shown that SLIT2 and its receptor ROBO2 also cise Methods section) and/or the diagnosis of RN, or the de- play a key role in controlling the ureteric bud (UB) formation, tection of ESRF/renal replacement in absence of other known a process of critical importance for the normal kidney devel- causes. Because VUR may spontaneously disappear during opment. Inactivation of either Slit2 or Robo2 in mice leads to childhood and adolescence, the finding of scintigraphic signs supernumerary UB development, lack of ureter remodeling, of RN in relatives of patients with VUR strongly suggests the and improper insertion into the bladder.14 previous occurrence of reflux. Lu et al.12 identified two novel ROBO2 heterozygous missense changes in two unrelated families (British and Dutch ancestry), in ROBO2 Sequence Analysis which the variants co-segregated with the VUR/CAKUT pheno- Table 2 summarizes all sequencing findings in this study and in- type. Whereas homozygous Robo2-null mice exhibit a multiple cludes several previously described variants12 (NCBI-SNP data- ureter phenotype and dysplastic kidneys and fail to survive after base). We found 24 DNA variants in the coding region and exon- birth, heterozygous and mosaic mutant mice show a CAKUT/ intron boundaries of the ROBO2 gene, including 17 novel VUR phenotype with megaureter, wide-open ureteropelvic junc- variants. From them, six were located within the coding region: tion, and hydronephrosis.12 These findings implicated for the first four nucleotide changes led to an amino acid change (Gly328Ser, time the SLIT-ROBO2 signaling pathway in the pathogenesis of a Asn515Ile, Asp766Gly, and Arg797Gln), and two were synony- subset of patients with VUR/CAKUT. mous, or “silent,” changes (Val745Val and Gly1352Gly). All base We evaluated a possible role of ROBO2 mutations in 95 changes were found in heterozygous state. In two of our patients, unrelated Italian patients who had primary VUR or VUR/ we also found a missense variant, Ile598Thr (exon 12, CAKUT; most of them had a positive family history. We found 1793T3C), previously suggested to represent a polymorphism.12 a relatively high frequency of missense variants in this cohort, We then sequenced exons 7, 11, 12, 15, and 16, in which indicating the importance of evaluating this gene especially missense changes were found, in a set of 190 Italian control when dealing with familial cases. subjects (380 alleles). None of the variants was present in the control group. Furthermore, we found a total of 16 intronic variants located in the proximity of exons, 11 of which were RESULTS novel variants (Table 2). We investigated nine of these intronic variants located within 50 bases from the exons to exclude a Clinical Characterization possible effect on gene splicing (see the Concise Methods sec- A summary of the clinical findings in 95 patients included in this tion); however, none of the splicing programs predicted an study is presented in Table 1. Seventy-eight (82%) patients had effect of these variants on gene splicing.

Table 1. Patients’ clinical characteristics Familial Cases Sporadic Cases a(95 ؍ Index Patients (n (%18 ;17 ؍ n) (%82 ;78 ؍ n) Patients with VUR and RN (n ϭ 57; 60%) 43 (75%) 14 (25%) Patients with VUR and no RN (n ϭ 38; 40%) 35 (92%) 3 (8%) Patients with isolated VUR (n ϭ 78; 82%) 66 (85%) 12 (15%) Patients with VUR and additional abnormalities (n ϭ 17; 18%) 12 (71%) 5 (29%) duplex collecting system (n ϭ 9) 6 3 renal agenesis (n ϭ 2) 2 0 ureterocele (n ϭ 2) 11 renal crossed ectopy (n ϭ 1) 1 0 multicystic kidney (n ϭ 1) 0 1 UPJO (n ϭ 1) 10 hypospadias (n ϭ 1) 1 0 aPhenotype of family members is not included.

826 Journal of the American Society of Nephrology J Am Soc Nephrol 19: 825–831, 2008 www.jasn.org CLINICAL RESEARCH

Table 2. Summary of sequence changes found in ROBO2a Frequency in Control Exon DNA Change and Positionb Type of Mutation AA Change References Subjects 2 94C3A Silent Arg32Arg 0.24d rs6788280 7 982G3A Missense Gly328Serc 0 11 1544A3T Missense Asn515Ilec 0 12 1793T3C Polymorphism Ile598Thr 0.01e Lu et al.12 15 2235A3C Silent Val745Val NT 15 2297A3G Missense Asp766Glyc 0 16 2390G3A Missense Arg797Glnc 0 25 4056C3T Silent Gly1352Gly NT IVS1 IVS1 ϩ 115 C3T Polymorphism? – NT IVS1 IVS1–53 G3A Polymorphism – 0.23e rs9874095 IVS3 IVS3–3 C3T Polymorphism? – NT IVS4 IVS4 ϩ 109 C3T Polymorphism? – NT IVS6 IVS6–24 G3A Polymorphism? – NT IVS9 IVS9 ϩ 33 A3G Polymorphism – 0.04e rs17525412 IVS14 IVS14 ϩ 23 T3C Polymorphism? – NT IVS20 IVS20–14 G3A Polymorphism? – NT IVS20 IVS20 ϩ 53 A3G Polymorphism? – NT IVS22 IVS22 ϩ 13 del A Polymorphism? – NT IVS22 IVS22 ϩ 120 G3A Polymorphism – 0.35e rs1523768 IVS23 IVS23 ϩ 7G3A Polymorphism? – NT IVS24 IVS24 ϩ 28 C3T Polymorphism – 0.14e rs3821735 IVS24 IVS24 ϩ 42 C3T Polymorphism? – NT IVS25 IVS25 ϩ 149 A3G Polymorphism – 0.39e rs1721189 3ЈUTR 3ЈUTR*121 G3A Polymorphism? – NT aNT, not tested; UTR, untranslated region. bNumbering (position 1) starts at base A from the ATG (Ref. Seq. NM_002942.2 and NP_002933.1 for the , NCBI build 36.2). cNovel likely pathogenic mutations. dReported only in Sub-Saharan African population. eFrequency of the alternative allele in Caucasian population reported in NCBI, build 36.2.

Four Novel Likely Pathogenic Candidate Mutations Asn515Ile. Gly328Ser. We found a heterozygous base change, 1544A3 T, located We found a heterozygous nucleotide change (982G3A) in in exon 11, leading to a nonconservative amino acid change exon 7 of the ROBO2 gene, leading to the missense change (uncharged polar, hydrophilic to nonpolar, hydrophobic) Gly328Ser (nonpolar amino acid to uncharged polar) (Figure (Figure 2, C and D). This change was found in one patient 1, C and D). The variant was found in a patient with a right (Family P, IV-10; Figure 2A) and in her affected twin ureteropelvic junction obstruction (UPJO) and left cystic dys- brother (IV-9). They showed primary bilateral moderate plastic kidney (family A, III-14; Figure 1A). A renal radionu- VUR (Figure 2B) and left VUR (grade III) without RN, re- clide scan with mercaptoacetyltriglycine displayed the right spectively. The asymptomatic mother (III-7) carried the UPJO (delayed washout and no response to furosemide) and same missense variant, and her sister (III-8) showed a bilat- the nonfunctioning left kidney (Figure 1B). RNC performed at eral slight pelvic dilation. The maternal grandmother (II-4, the age of 5 yr did not show presence of VUR. This patient deceased) and her sister (II-5) both had ESRF. Unfortu- underwent the removal of the left kidney at age of 5 yr and nately, no DNA was available from any of these patients. surgical correction of right UPJO at age of 6 yr. Her mother was carrying the same missense variant; she was asymptomatic and Asp766Gly. had a normal 99Tcm-dimercaptosuccinic acid (DMSA) scan. In exon 15, we found a heterozygous 2297A3 G missense Individual III-13, the asymptomatic older sister of the proband change coding for the amino acid glycine (nonpolar, hydro- (20 yr of age), was detected as a carrier of the 982G3A change phobic) instead of aspartic acid (charge polar, hydrophilic) as well; therefore, after informed consent, she was investigated at position 766 of ROBO2, corresponding to the third fi- with RNC and was found to have right moderate VUR. Indi- bronectin type 3 domain (Fn3) of the protein (Figure 3, C vidual III-15, the younger sister of the proband (7 yr of age) and D). The proband was diagnosed as having bilateral VUR and asymptomatic, too, did not carry the variant, and RNC did (moderate at left and severe at right kidney) at the age of 15 not show evidence of reflux. The same missense variant was mo after pyelonephritis (Family G, II-4; Figure 3, A and D). also found in a maternal uncle (II-9) and his son (III-19), af- The same change was also found in her mother (I-2), af- fected with right RN and right VUR, respectively. fected with right VUR, and in the asymptomatic sister (II-

J Am Soc Nephrol 19: 825–831, 2008 ROBO2 Genetic Variants and VUR 827 CLINICAL RESEARCH www.jasn.org

Figure 1. (A) Pedigree of Family A. Filled symbols indicate pres- Figure 2. (A) Pedigree of Family P. Filled symbols indicate ence of VUR/CAKUT. Gray symbol indicates an individual reported presence of VUR. Circle with top right corner filled indicates a to have a slight pelvis dilation. The presence (ϩ) or absence (Ϫ)of slight bilateral pelvis dilation (III-8), dotted symbols (II-4, II-5) the missense variant is indicated. (B) 99Tcm-mercaptoacetyltriglycine indicate ESRF. (B) Direct RNC showing a moderate bilateral reflux renogram displaying the right UPJO and the left nonfunctioning in Family P, IV-10. (C) Chromatogram showing the normal and kidney (III-14). (C) Chromatogram showing the normal (WT, wild- mutated (1544A3T) sequences. Codons and corresponding type) and mutated (982G3A) sequences. Codons and correspond- amino acids are also displayed. (D) Protein conservation across ing amino acids are also indicated. (D) Protein conservation across various species. various species (mammals, bird, and fish). IV-11 of Family C (Figure 4A), who received a diagnosis of left 3). Individual II-3 was investigated with RNC at the age of 7 mild VUR at the age of 1 mo after pyelonephritis. DMSA scan yr, but no evidence of reflux was found. showed a left RN (Figure 4B). His father (III-9), carrying the same mutation, was found to have bilateral VUR grade 4 at the Arg797Gln. age of 18 yr. Then, he developed ESRD and received a trans- The missense 2390G3A heterozygous change in exon 16 of plant. His sister (III-8) also showed the same DNA variant. She ROBO2 leads to a nonconservative change of Arg797Gln had a history of microscopic hematuria and urinary tract in- (charge to uncharged polar) in the Fn3 domain of the ROBO2 fections, but she did not consent to RNC or DMSA scan. The protein (Figure 4, C and D). This variant was found in patient same missense variant was also found in the paternal grandfa-

828 Journal of the American Society of Nephrology J Am Soc Nephrol 19: 825–831, 2008 www.jasn.org CLINICAL RESEARCH

Figure 3. (A) Pedigree of Family G. Filled symbols indicate presence of VUR. The presence (ϩ) or absence (Ϫ) of the mis- sense variant is indicated. (B) Direct RNC showing a severe right and moderate left reflux in Family G, II-4. (C) Chromatogram showing the normal and mutated (2297A3G) sequences. Codons and corresponding amino acids are also displayed. (D) Protein conservation across various species. ther (II-4), who was asymptomatic but had a brother (de- ceased) with ESRF. We carried out an “electronic” analysis of these missense Figure 4. (A) Pedigree of Family C. Filled symbols indicate presence of changes to predict the impact of the amino acid allelic variants VUR. Gray symbols indicate other anomalies/symptoms of the genito- on the protein structure and function based on multiple pro- urinary tract (III-6, stenosis of urethra; III-8, recurrent hematuria and tein alignment and three-dimensional structures (PolyPhen urinary tract infections). (B) DMSA renal scintigraphy showing the left RN program). All but Asn515Ile (predicted to be “benign”) were in Family C, IV-11. (C) Chromatogram showing the normal and mutated 3 classified as “possibly damaging” changes. (2390G A) sequences. Codons and corresponding amino acids are also displayed. (D) Protein conservation across various species. Frequency of the Variants We found a relatively high frequency of missense variants in VUR (familial and sporadic). All four DNA variants were het- ROBO2 in our cohort of patients: 4.2% for all patients with erozygous missense changes, and they were found in the group

J Am Soc Nephrol 19: 825–831, 2008 ROBO2 Genetic Variants and VUR 829 CLINICAL RESEARCH www.jasn.org of patients with familial VUR (three families) and familial sense variants in our cohort of patients: 5.1% of all familial VUR/CAKUT (one family). Thus, in the familial cases, mis- cases. Moreover, we showed that missense changes in ROBO2 sense variants in ROBO2 were found in 5.1% (four of 78) of the are associated not only with VUR/CAKUT phenotype but also patients. with familial VUR. In fact, most of the patients who were found to carry a ROBO2 missense variant had isolated VUR. The enrichment of familial cases in our sample should not DISCUSSION be the cause for the relatively high frequency of missense vari- ants found. In the first study that implicated the ROBO2 path- To investigate whether alterations in the ROBO2 gene play a way in the pathogenesis of a subset of VUR cases, 124 families causative role in our cohort of patients with VUR, we per- were investigated and only two (1.6%) of them were found to formed a systematic sequence analysis of all 26 exons of the have a missense variant.12 Whether these families presented gene. The majority of the patients belong to the familial pri- with isolated VUR or VUR/CAKUT is not clear; however, VUR mary VUR group followed by the familial VUR/CAKUT set. and VUR/CAKUT might be part of the same spectrum of uro- We describe four novel missense changes in the ROBO2 logic tract malformations. gene, all of which were found in patients with a positive family Although in both studies the patients were of white origin, our history. When the families were examined, co-segregation of sample comes specifically from a south European population. the missense variants with the VUR phenotype was observed. Thus, a possibility remains, as for other genetic disorders, that As reported before, asymptomatic individuals without any ev- ROBO2 mutations are more common within certain geographic idence of disease were also found to carry the sequence change. areas. Because all of the missense variants found are different (nu- This observation is consistent with the reduced penetrance de- cleotide positions and amino acid changes) and are not recurrent scribed in families with VUR. The presence of the missense in unrelated patients, it is unlikely that we are facing a founder variants in available affected family members and absence in effect of an old mutational event. Our findings indicate that mu- control individuals, together with the fact that they affect tational screening of ROBO2 might offer physicians a future tool amino acids conserved through evolution, suggests that these for the detection and management of familial VUR. changes may be pathogenic (Figure 1D, 2D, 3D, and 4D); how- In conclusion, our study independently confirms that ge- ever, functional studies on these candidate mutations and rep- netic alterations in ROBO2 are significantly associated with lication in other cohorts are warranted. familial VUR/CAKUT. Furthermore, we showed that ROBO2 ROBO family members act as transmembrane receptors is implicated in the more common isolated VUR phenotype, and consist of an extracellular region with five predicted Ig and especially in patients with a positive family history. three Fn3 domains, a transmembrane domain, and an intra- cellular region with three cytoplasmic motifs.13,15 All four mis- sense changes described here are located within the ROBO2 CONCISE METHODS protein extracellular region: Gly328Ser is located at the begin- ning of the fourth Ig domain, Asn515Ile is between the last Ig Patients and the first Fn3 domain, and Asp766Gly and Arg797Gln are A cohort of 95 patients with VUR, originating from the same geo- within the third Fn3 domain. Members of the ROBO family graphic region of South Italy, were available for the study. All patients (ROBO1 and ROBO2) are able to interact homophilically and were ascertained on the basis of the presence of VUR, isolated or in heterophilically.16 A reduction in homophilic binding has been combination with additional renal/urinary tract abnormalities, doc- observed when any of the Ig or all Fn domains are missing; umented by VCUG in male patients and direct RNC in female pa- therefore, these domains are important for the homophilic tients and family members when available. Five pediatric nephrolo- binding of molecules such as ROBO1 and ROBO2. Moreover, gists and one radiologist assessed the patients. Grading of VUR extracellular Ig domains 1 and 2 of ROBO1 are shown to be detected with VCUG was made according to the International Grad- important for ROBO-SLIT interaction.17 ing System of Vesicoureteral Reflux.18 Reflux observed with RNC was Lu et al.12 also described a nonconservative change in exon graded as mild (reflux to ureter or renal pelvis), moderate (reflux to 12 of ROBO2 leading to a change in amino acid (Ile598Thr). renal pelvis with mild to moderate dilation), or severe (distended This rare variant was found in 1.09% of the control subjects redundant collecting system associated with ureteral dilation). RN (n ϭ 276). We found the same change in two of our patients was diagnosed by DMSA renal scintigraphy and defined as focal or with VUR and none from the control group (n ϭ 190). The multifocal defects of radionuclide uptake and/or as a split renal iso- amino acid isoleucine 598 is located within the first Fn3 do- tope uptake below 43%.19 In all 95 patients, secondary causes of VUR main of the protein and is also conserved in mammals and or presence of syndromic features were excluded. The majority of the birds (chicken). Whether the Ile598Thr change is a rare (inno- patients (n ϭ 78) had at least one affected relative (familial cases), cent) polymorphism or confers a higher risk in some VUR showing a mode of inheritance compatible with an autosomal domi- cases deserves further investigation in other cohorts of patients nant pattern. In 17 patients, no evidence of renal disease could be and control subjects. found across their relatives (sporadic cases). Informed consent from We found an unexpectedly high frequency of ROBO2 mis- the patients and families’ members (parents for their children) and

830 Journal of the American Society of Nephrology J Am Soc Nephrol 19: 825–831, 2008 www.jasn.org CLINICAL RESEARCH approval from the Ethic Committee at Second University of Naples ureteric reflux and renal scarring. Kidney Int Suppl 4: S65–S72, 1975 was obtained previously. 3. Thomsen HS: Vesicoureteral reflux and reflux nephropathy. Acta Ra- diol Diagn (Stockh) 26: 3–13, 1985 4. Ardissino G, Dacco V, Testa S, Bonaudo R, Claris-Appiani A, Taioli E, Sequencing Analysis Marra G, Edefonti A, Sereni F: Epidemiology of chronic renal failure in All 26 exons and exon-intron boundaries of the ROBO2 gene were children: Data from the ItalKid project. Pediatrics 111: e382–e387, amplified using newly design intronic primers (Supplementary Table 2003 3). Direct sequencing of both strands was performed using the BigDye 5. Noe HN: The long-term results of prospective sibling reflux screening. terminator chemistry (version 3.1; Applied Biosystems, Foster City, J Urol 148: 1739–1742, 1992 6. Van den Abbeele AD, Treves ST, Lebowitz RL, Bauer S, Davis RT, Retik CA). Products were loaded on an ABI 3100 genetic analyzer and ex- A, Colodny A: Vesicoureteral reflux in asymptomatic siblings of pa- amined with the SeqScape software version 2.5 (Applied Biosystems). tients with known reflux: Radionuclide cystography. Pediatrics 79: A negative control was included in all reactions. 147–153, 1987 The frequency of potential pathogenic variants was assessed by 7. Connolly LP, Treves ST, Connolly SA, Zurakowski D, Share JC, Bar- direct sequencing in a panel of 190 control individuals (380 alleles) Sever Z, Mitchell KD, Bauer SB: Vesicoureteral reflux in children: Incidence and severity in siblings. J Urol 157: 2287–2290, 1997 from the same geographic area of Italy. In the familial cases, these 8. Devriendt K, Groenen P, Van Esch H, van Dijck M, Van de Ven W, novel variants were also tested in all available family members. Fryns JP, Proesmans W: Vesico-ureteral reflux: A genetic condition? The program PolyPhen (Polymorphism Phenotyping, http:// Eur J Pediatr 157: 265–271, 1998 genetics.bwh.harvard.edu/pph/index.html) was used to predict 9. Chapman CJ, Bailey RR, Janus ED, Abbott GD, Lynn KL: Vesi- the possible impact of an amino acid substitution on the structure coureteric reflux: Segregation analysis. Am J Med Genet 20: 577–584, 1985 and function of ROBO2 protein. This prediction is based on 10. Sanna-Cherchi S, Reese A, Hensle T, Caridi G, Izzi C, Kim YY, Konka straightforward empiric rules that are applied to the sequence, A, Murer L, Scolari F, Ravazzolo R, Ghiggeri GM, Gharavi AG: phylogenetic, and structural information characterizing the sub- Familial vesicoureteral reflux: Testing replication of linkage in seven stitution. The nonsynonymous changes are classified as benign, new multigenerational kindreds. J Am Soc Nephrol 16: 1781–1787, possibly or probably damaging, and unknown. Four different 2005 11. Feather SA, Malcolm S, Woolf AS, Wright V, Blaydon D, Reid CJ, computer programs—NetGene2 (Center for Biological Sequence Flinter FA, Proesmans W, Devriendt K, Carter J, Warwicker P, Good- Analysis, Denmark), GeneSplicer (Center for Bioinformatics and ship TH, Goodship JA: Primary, nonsyndromic vesicoureteric reflux Computational Biology, University of Maryland), SpliceView (In- and its nephropathy is genetically heterogeneous, with a locus on stitute for Biomedical Technologies, National Research Council, 1. Am J Hum Genet 66: 1420–1425, 2000 Italy), and BDGP Splice Site Prediction (Berkeley Drosophila Ge- 12. 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 nome Project)—were used to predict possible consequences on V, Bi W, Yan J, Giltay JC, Wijmenga C, de Jong TP, Feather SA, Woolf splicing of several novel intronic variants found in the proximity of AS, Rao Y, Lupski JR, Eccles MR, Quade BJ, Gusella JF, Morton CC, the exon-intron boundaries. Maas RL: Disruption of ROBO2 is associated with urinary tract anom- alies and confers risk of vesicoureteral reflux. Am J Hum Genet 80: 616–632, 2007 13. Kidd T, Brose K, Mitchell KJ, Fetter RD, Tessier-Lavigne M, Goodman ACKNOWLEDGMENTS CS, Tear G: Roundabout controls axon crossing of the CNS midline and defines a novel subfamily of evolutionarily conserved guidance This work was in part supported by the Nierstichting Nederland receptors. Cell 92: 205–215, 1998 (Dutch Kidney Foundation, grant C05.2146), the Progetti di Ril- 14. Grieshammer U, Le M, Plump AS, Wang F, Tessier-Lavigne M, Martin GR: SLIT2-mediated ROBO2 signaling restricts kidney induction to a evante Interesse Nazionale, the Fondo per gli Investimenti della single site. Dev Cell 6: 709–717, 2004 Ricerca di Base, and Regione Campania. 15. Sundaresan V, Roberts I, Bateman A, Bankier A, Sheppard M, Hobbs We are indebted to the patients and family members for coopera- C, Xiong J, Minna J, Latif F, Lerman M, Rabbitts P: The DUTT1 gene, tion and interest. We thank Tom de Vries-Lentsch for the artwork. a novel NCAM family member is expressed in developing murine neural tissues and has an unusually broad pattern of expression. Mol Cell Neurosci 11: 29–35, 1998 16. Hivert B, Liu Z, Chuang CY, Doherty P, Sundaresan V: Robo1 and DISCLOSURES Robo2 are homophilic binding molecules that promote axonal growth. None. Mol Cell Neurosci 21: 534–545, 2002 17. Liu Z, Patel K, Schmidt H, Andrews W, Pini A, Sundaresan V: Extracel- lular Ig domains 1 and 2 of Robo are important for ligand (Slit) binding. Mol Cell Neurosci 26: 232–240, 2004 REFERENCES 18. Duckett JW, Bellinger MF: A plea for standardized grading of vesi- coureteral reflux. Eur Urol 8: 74–77, 1982 1. Scott JE, Swallow V, Coulthard MG, Lambert HJ, Lee RE: Screening of 19. Polito C, Rambaldi PF, Signoriello G, Mansi L, La Manna A: Permanent newborn babies for familial ureteric reflux. Lancet 350: 396–400, 1997 renal parenchymal defects after febrile UTI are closely associated with 2. Smellie J, Edwards D, Hunter N, Normand IC, Prescod N: Vesico- vesicoureteric reflux. Pediatr Nephrol 21: 521–526, 2006

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