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616 The American Journal of Human Genetics Volume 80 April 2007 www.ajhg.org
ARTICLE
Disruption of ROBO2 Is Associated with Urinary Tract Anomalies
and Confers Risk of Vesicoureteral Reflux
Weining Lu, 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 Andrews, Vasi Sundaresan, Weimin Bi, Jiong Yan, Jacques C. Giltay, Cisca Wijmenga,
Tom P. V. M. de Jong, Sally A. Feather, Adrian S. Woolf, Yi Rao, James R. Lupski,
Michael R. Eccles, Bradley J. Quade, James F. Gusella, Cynthia C. Morton,
†
and Richard L. Maas
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 path-
way in the pathogenesis of a subset of human VUR.
From the Genetics Division (W.L.; Y.F.; Q.X.; R.L.M.) and Departments of Pathology (S.K.; B.J.Q.; C.C.M.) and Obstetrics, Gynecology andReproductive
Biology (H.F.; C.C.M.), Brigham and Women’s Hospital and Harvard Medical School, Center for Human Genetic Research, Massachusetts GeneralHospital
and Harvard Medical School (F.Q.-R.; H.-G.K.; J.F.G.), and Renal Section, Boston University Medical Center (W.L.; Q.-g.L.), Boston; Departments ofMedical
Genetics and Pediatric Urology, University Medical Center Utrecht, Utrecht, The Netherlands(A.M.v.E.; J.C.G.; C.W.; T.P.V.M.d.J.); NorthwesternUniversity
Institute of Neuroscience, Chicago (X.F.; Y.R.); Genetics Department, Hoˆpital Necker-Enfants Malades, Paris (D.S.); Medical Research Council Centre for
Developmental Neurobiology, King’s College of London (W.A.; V.S.), and Nephro-Urology Unit, Institute of Child Health, University College London
(A.S.W.), London; Department of Molecular and Human Genetics, Baylor College of Medicine, Houston (W.B.; J.Y.; J.R.L.); Department of Pediatric
Nephrology, St. James’ University Hospital, Leeds, United Kingdom (S.A.F.); and Pathology Department, University of Otago, Dunedin, New Zealand
(M.R.E)
Received November 9, 2006; accepted for publication January 15, 2007; electronically published February 14, 2007.
Address for correspondence and reprints: Dr. Richard Maas, Genetics Division, NRB 458, Brigham and Women’s Hospital, Harvard Medical School, 77
Avenue Louis Pasteur, Boston, MA 02115. E-mail: maas@genetics.med.harvard.edu
* Present affiliation: Department of Pathology, The David Geffen School of Medicine at University of California–Los Angeles, Los Angeles.
†
All editorial responsibility for this article was handled by an associate editor of the Journal.
Am. J. Hum. Genet. 2007;80:616–632. 䉷2007 by The American Society of Human Genetics. All rights reserved. 0002-9297/2007/8004-0005$15.00
DOI: 10.1086/512735
Congenital anomalies of the kidney and urinary tract
(CAKUT) make up a family of diseases with a diverse an-
atomical spectrum, including kidney anomalies (e.g., re-
nal dysplasia, duplex kidney, and hydronephrosis) and
ureter anomalies (e.g., vesicoureteral reflux [VUR], me-
gaureter, and ureterovesical junction [UVJ] obstruction).
1,2
In particular, VUR (MIM %193000), a polygenic genetic
disorder with an incidence of ∼1 in 100 infants,
3,4
is one
of the most common clinical manifestations of CAKUT.
VUR is characterized by the reflux of urine from the blad-
der into the ureters and sometimes into the kidneys and
is a risk factor for urinary tract infection (UTI).
5
In com-
bination with intrarenal reflux, the resulting inflamma-
tory reaction may result in renal injury or scarring, also
called “reflux nephropathy.”
6
Extensive renal scarring im-
pairs renal function and may predispose patients to hy-
pertension, proteinuria, and renal insufficiency. Refluxne-
phropathy accounts for as much as 15% of end-stage renal
disease in children and young adults.
7
Primary VUR results
from a developmental defect of the UVJ
8
and is known to
occur in multiple members of families. In siblings and
offspring of affected patients, the prevalence is as high as
50%.
9,10
Despite its high incidence in the pediatric pop-
ulation, 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.
11
On the basis of the mouse
Robo1 mutant phenotype, ROBO1 is a candidate gene for
pulmonary hypoplasia and adenocarcinoma,
12,13
as well as
for dyslexia (DYX5).
14
Mutations in ROBO3 result in hor-
izontal-gaze palsy with progressive scoliosis,
15
whereas ze-
brafish Robo4 is implicated in angiogenesis.
16
Robo2 loss-
of-function mutations in zebrafish and mice result in
retinal and commissural pathfinding defects, respective-
ly.
17,18
Interestingly, Robo2 and Slit2 mouse mutants reveal
an additional key role for SLIT-ROBO signaling in regu-
lating the metanephric expression of glial cell derived neu-
rotrophic factor (Gdnf), which in turn induces ureteric
bud outgrowth from the nephric duct and restricts it to a
single site.
19
However, a role for ROBO2 in human disease
has not been identified elsewhere.
www.ajhg.org The American Journal of Human Genetics Volume 80 April 2007 617
Material and Methods
FISH and Array Comparative Genomic Hybridization
Analyses
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 chro-
mosomes prepared from a t(Y;3)(p11;p12)dn lymphoblastoid cell
line established from patient DGAP107. Array comparative ge-
nomic hybridization (CGH) experiments were performed bySpec-
tral Genomics (SpectralChip 2006 array) and Agilent Technolo-
gies (Human Genome CGH Microarray Kit 44A).
Southern and RT-PCR Analyses
Southern and RT-PCR analyses were performed according to rou-
tine 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 fu-
sion transcripts were ROBO2-F2 (same for all transcripts) and
PCDH11Y-R1, PCDH11Y-R2, and PCDH11Y-R3; those used to am-
plify 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 (ap-
pendix C). All primer sequences are listed in appendix G.
Quantitative Real-Time PCR Analyses
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 b-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 con-
tained 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 trip-
licate RT-PCR amplifications, with each 50-ml reaction containing
200 ng total RNA, 5 mM MgSO
4
, 500 nM forward and reverse
primers, and 200 nM fluorogenic probes. Controls included either
no reverse transcriptase or the substitution of H
2
O 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 compar-
ative threshold cycle methods.
Fusion Proteins
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.
20
Neuronal Migration Assay
The in vitro neuronal migration assay that uses postnatal anterior
subventricular zone (SVZa) cells was described elsewhere.
21
In
brief, P1-6 Sprague-Dawley rat brains devoid of meninges were
placed in 10% fetal calf serum in Dulbecco’s modified Eagle me-
dium (DMEM) and were embedded. Coronal sections of 300 mm
were prepared by vibratome, and tissues within the SVZa borders
were dissected to make SVZa explants 200–300 mm in diameter.
Explants were embedded, together with human embryonic kid-
ney (HEK) cell aggregates in collagen and matrigel (3:2: 1 ratio
of collagen: matrigel:medium), and were cultured in DMEM with
5% CO
2
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 con-
trols, 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% CO
2
for 1–2 h, to form
aggregates. Aggregated cells were washed in DMEM and were
squared with a needle.
Western Blot
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 con-
trol. 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) an-
tibody that detects YFP (Clontech), and Myc-Slit2 and HA-RoboN
were blotted with monoclonal antibodies against Myc and HA,
respectively.
Mutation Analysis
ROBO2 mutation screening employed PCR amplification of each
of the 26 human ROBO2 exons and intron-exon boundaries, fol-
lowed by purification and bidirectional DNA sequencing. Se-
quences 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 con-
firmed by resequencing. National Center for Biotechnology In-
formation RefSeq ROBO2 cDNA sequence (GenInfo identifier [GI]
109254774) and protein sequence (Entrez Protein accession num-
ber NP_002933) (GI 61888896) were used to calculate the nucle-
otide and amino acid positions.
618 The American Journal of Human Genetics Volume 80 April 2007 www.ajhg.org
Figure 1. ROBO2 disrupted in DGAP107. Partial karyogram (A) and idiogram (B) for 46,X,t(Y;3)(p11;p12)dn is shown. VCUG of DGAP107
shows anterior-posterior (C) and lateral (D) views of bilateral grade IV VUR and megaureter at the right UVJ (arrows). bl pBladder;
pe prenal pelvis; ur pureter. E, FISH analysis showing BAC RP11-54A6 (green), which hybridizes to normal chromosome 3, der(3),
and der(Y) and crosses the 3p12 breakpoint. F, Intron-exon structure of ROBO2, with select exons numbered and the relevant BAC
contig. The location of the 3p12 translocation breakpoint is indicated by a red dotted vertical line.
Preparation of Robo2
flox
and Robo2
del5
Alleles
Robo2
flox
mice were produced using homologous recombination
in 129 embryotic stem cells and blastocyst injection. After germ-
line transmission, mice were backcrossed to C57BL/6 and were
analyzed thereafter in a mixed C57BL/6-129/Sv background. The
Robo2
flox
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 Robo2
flox
alleles. After
SpeI digestion, the Robo2
flox
amplicon remains uncut, whereas
the wild-type amplicon yields 750-bp and 350-bp products.
Robo2
flox/⫹
mice were bred with Tg
EIIa-Cre
(stock number 003724
[Jackson Laboratory]) to produce the Robo2
del5
allele. The Robo2
del5
allele was amplified by primers Robo2koF and Robo2R, which pro-
duce a 1,100-bp fragment. The wild-type allele was amplified by
primers Robo2wtF and Robo2R, which yield a 1,390-bp fragment.
F2 Robo2
del5/del5
↔Robo2
del5/flox
mosaic mice were prepared as de-
scribed in appendix F and were analyzed for the presence of uri-
nary tract phenotypes. To examine the ureter and kidney defects,
Hoxb7-GFP transgenic mice (gift from Dr. Frank Costantini, Co-
lumbia University) were bred with Robo2 mutants. GFP fluores-
cence was monitored and photographed using a Nikon SMZ-1500
epifluorescence stereomicroscope.
Human and Animal Studies
All human studies were performed under informed consent pro-
tocols approved by the Partners HealthCare System Human Re-
search Committee (Boston), the Human Research Ethics Com-
mittee (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.
Results
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–1Dand 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 mecha-
nism. By metaphase FISH, we identified a BAC clone
(RP11-54A6) that crosses the 3p12 breakpoint, which dis-
rupts intron 2 of ROBO2, which is composed of 26 exons
and spans ∼606 kb of genomic DNA (fig. 1Eand 1F).
We cloned and sequenced the breakpoints on the der(3)
and der(Y) chromosomes (appendix B). In addition to
www.ajhg.org The American Journal of Human Genetics Volume 80 April 2007 619
Figure 2. The t(Y;3) translocation in DGAP107, which generates novel ROBO2 fusion transcripts. A, ROBO2 and PCDH11Y intron-exon
structure surrounding the der(Y) breakpoint. The forward primer F2 in ROBO2 exon 2 (black bar) was used in RT-PCR with three reverse
primers—R2, R1, and R3—in PCDH11Y exons 3, 4, and 5, respectively (blue bars). Dotted lines indicate the observed splicing patterns
of the two fusion transcripts. The red splicing pattern generates Fu-153, which encodes 153 aa, and the blue pattern generates Fu-
129, which encodes 129 aa. B, RT-PCR fusion transcript amplification. Lane 1, F2/R3 primers amplify Fu-153 (641 bp) and Fu-129
transcripts; only the shorter Fu-153 amplicon is shown. Lane 2, F2/R1 primers amplify transcripts for both Fu-129 (456 bp) and Fu-
153 (122 bp). Lane 3, F2/R2 primers amplify only Fu-129 transcripts (347 bp). Lane 4, F2/qR primers amplify only transcripts from the
wild-type nontranslocated ROBO2 allele (606 bp). qR primer is located in exon 7 of ROBO2. C, Real-time RT-PCR quantitation of ROBO2
fusion transcripts Fu-129 and Fu-153 (detected by TaqMan probes shown in panel D) and of ROBO2 nontranslocated allele transcripts
(detected by TaqMan probe across ROBO2 exons 2 and 3) in DGAP107 lymphoblast RNA. D, Exon structure of Fu-153 and Fu-129. Horizontal
bars indicate TaqMan probes used to quantify fusion transcripts. Black boxes indicate ROBO2 exons; blue boxes, PCDH11Y exons; full-
height boxes, coding exons; and half-height boxes, noncoding exons.
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 phe-
notype in DGAP107 is unlikely, however, since PCDH11Y
expression has been detected only in placenta, brain, ret-
ina, and testis
22
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 microde-
letion region pathogenetic in Smith-Magenis syndrome
(SMS [MIM #182290]), a mental retardation syndrome
associated with behavioral and sleep disturbances and
craniofacial and skeletal anomalies.
23
Thus, a role for
del(17)(p11.2) in the sleep, behavioral, and cognitive def-
icits of DGAP107 seems likely. The del(17)(p11.2) micro-
deletion 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 tran-
scripts driven by the ROBO2 promoter (fig. 2B). Each tran-
script contains the first two exons of ROBO2 spliced out
of frame to PCDH11Y downstream exons, resulting in pre-
mature stop codons shortly after ROBO2 exon 2. When
assayed by real-time RT-PCR, these fusion transcripts, de-
noted Fu-129 and Fu-153, are expressed at somewhat re-
duced levels compared with the wild-type ROBO2 tran-
scripts derived from the nontranslocated allele (fig. 2C);
the wild-type transcripts contained no detectable muta-
tions. 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 do-
mains required for SLIT-ROBO signal transduction (fig.
2D).
When expressed without transmembrane and cyto-
plasmic domains, the soluble extracellular Ig domains
of ROBO, denoted as RoboN, are able to bind SLIT ligand.
24
Moreover, the first ROBO Ig domain is necessary and po-
tentially sufficient for SLIT binding.
24
RoboN isoforms
can thus inhibit SLIT-ROBO signaling by competing with
wild-type ROBO for SLIT binding.
25
We therefore hypothe-
sized that the truncated proteins encoded by the ROBO2-
PCDH11Y fusions might act in a dominant-negative man-
ner to block endogenous SLIT-ROBO signaling. To test this
hypothesis, we performed an in vitro neuronal migration
620 The American Journal of Human Genetics Volume 80 April 2007 www.ajhg.org
Figure 3. ROBO2 fusion proteins inhibiting SLIT chemorepulsion. A, YFP-tagged ROBO2 fusion proteins (Fu129-YFP [40 kDa] and
Fu153-YFP [42 kDa]) detected by an anti-YFP antibody, expressed in HEK cell lysates, and secreted into the medium. In the presence
of aggregated cells transfected with Slit2 plus empty vector (B) or Slit2 plus Sema3A (Semaphorin 3A, with no effect on Slit2 repulsive
activity) (C), cells migrate out of SVZa explants and away from the Slit2-expressing cell aggregate (asterisk). In the presence of aggregated
cells transfected with Slit2 plus RoboN (the Robo extracellular domain, which inhibits Slit repulsive activity), cells migrate out of SVZa
explants symmetrically in all directions (D) including toward (arrow) the Slit2 and RoboN-expressing cell aggregate (asterisk). Fu-129
and Fu-153 also effectively block Slit2 repulsive activity (Eand F), allowing symmetrical neuronal migration out of SVZa explants and
toward (arrows) Slit2 and Fu-129 or Slit2– and Fu-153–expressing cell aggregates (asterisks).
assay
21
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 aggre-
gates alone have no effect on SVZa explants, resulting in
a radially symmetric pattern of neuronal outgrowth.
25
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 func-
tion, as a control), the Slit2-expressing cell aggregates
acted upon the SVZa explants to repel SVZa neuronal out-
growth (fig. 3Band 3C). In contrast, in aggregates coex-
pressing 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 neu-
ronal 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 pro-
teins could further compromise ROBO2 function in
DGAP107, which retains only hemizygous ROBO2 ex-
pression from the nontranslocated allele.
Family studies indicate that primary VUR frequently
segregates with autosomal dominant inheritance and in-
complete penetrance.
9,26
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 po-
tential autosomal dominant inheritance. One sequence
change—c.2436TrC, 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 iden-
tified 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 af-
fected 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 TrC
change at position 3477 in coding exon 19 (c.3477TrC)
that would cause a nonconservative missense I945T sub-
stitution in the ROBO2 intracellular domain (ICD) (fig. 4D
and 4E).
In family B5 with CAKUT-VUR, the proband has bilat-
eral 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 GrA sequence
alteration at position 4349 in coding exon 23 (c.4349GrA)
that would cause a nonconservative missense amino acid
www.ajhg.org The American Journal of Human Genetics Volume 80 April 2007 621
Figure 4. ROBO2 missense mutations in familial CAKUT and VUR. A, Family 2559x with CAKUT-VUR and exon 19 (c.3477TrC) mutation.
Arrow indicates the proband. Blackened and gray symbols indicate patients with CAKUT-VUR and family members with urinary tract
symptoms and radiological evidence of CAKUT. Nucleotide changes are shown under each individual. B, 99mTc-dimercaptosuccinic acid
renogram of proband 25592 showing bilateral renal parenchymal defects (arrow). C, VCUG of proband 25592 showing bilateral reflux
(bidirectional arrow). Chromatograms show TrC change (arrow) in exon 19 of family 2559x (D) and amino acid conservation across
species (E). F, Family B5 with CAKUT-VUR and exon 23 (c.4349GrA) mutation. G, VCUG showing bilateral VUR (bidirectional arrow) and
right duplex kidney (arrow) in proband B5. H, IVP detecting right duplex kidney (arrows) in proband B5. I, US showing suspected duplex
(arrow) in upper pole of the right kidney in D1, an asymptomatic aunt of proband B5. Chromatograms show GrA change (arrow)in
exon 23 of family B5 (J) and amino acid conservation across species (K).
substitution, A1236T, in the ROBO2 ICD (fig. 4Jand 4K).
An additional family member, uncle A1, also has this al-
teration but did not exhibit an ultrasonographically de-
tectable renal phenotype; however, nonpenetrance of
VUR is common.
9
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. 4Eand 4K).
To further assess the likelihood that these sequence
changes represent functional missense variants, as op-
posed to rare neutral variants found in the general pop-
ulation, we sequenced ROBO2 exons 12, 19, and 23 in 180
unrelated, clinically unaffected controls of ethnic back-
grounds similar to those of the affected individuals. Two
occurrences of c.2436TrC 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 se-
quence variation, we resequenced the 26 ROBO2 exons
and intron-exon boundaries in an additional 96 controls.
We found only one reoccurrence of c.2436TrC. Of several
nonvalidated putative synonymous and nonsynonymous
ROBO2 coding SNPs listed in Ensembl v39, we detected
only one—c.737CrA, or R32R—in our own sequencing
efforts. Moreover, this apparent change was found to rep-
resent a sequencing artifact. Thus, in sum, these results
suggest that the two sequence changes in the ICD iden-
tified 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 cel-
lular migration,
27
by creating novel threonine phospho-
rylation sites or by influencing the binding of proteins
that interact with the ROBO ICD.
28
For example, the SH3
domain of srGAP1 binds to the ROBO1 ICD CC3 subdo-
main,
27,28
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,
27
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, de-
scribed elsewhere,
19
with a targeted deletion of exon 1
exhibits a multiple ureter phenotype and fails to survive
622 The American Journal of Human Genetics Volume 80 April 2007 www.ajhg.org
Figure 5. Robo2
del5/del5
homozygous, Robo2
del5/⫹
heterozygous, and Robo2
del5/del5
↔Robo2
del5/flox
mosaic newborn mice expressing striking
CAKUT phenotypes. A, Structures of the mouse Robo2
flox
and Robo2
del5
alleles. The Robo2
flox
allele encodes a wild-type, full-length 1,470-
aa Robo2 protein but contains two loxP sites flanking exon 5. The Robo2
del5
allele is generated from Robo2
flox
by Cre, which deletes
Robo2 exon 5 to produce an aberrant transcript expressed only at low levels. Band C, Wild-type female (B) and male (C) newborn
mouse excretory system. The male excretory system in panel C is illuminated by the Hoxb7-GFP transgene. k pkidney; bl pbladder;
ur pureter; ut puterus; te ptestis. Black bidirectional arrows indicate ureter length in panels B and D. Robo2
del5/del5
newborn
homozygotes display multiplex dysplastic kidneys (D) and, at 25#magnification (E), reveal dysplastic cysts (dc) in the calyces and
an internalized nephrogenic zone (arrow). Hoxb7-GFP transgene–positive Robo2
del5/⫹
heterozygous newborns show megaureter dilation
(F)(bidirectional arrow) and early ureter dilatation (G)(arrow). Robo2
del5/del5
↔Robo2
del5/flox
mosaic newborns show hydronephrosis in the
left kidney (H). At 25#magnification (I), they show megaureter (asterisk). Black bidirectional arrows indicate ureter length in panel
H. pe ppelvis.
after birth. To determine whether heterozygosity forRobo2
loss of function could produce an abnormal urinary tract
phenotype and recapitulate human CAKUT-VUR, we pre-
pared a mouse Robo2 floxed allele, Robo2
flox
, containing
loxP sites flanking Robo2 exon 5 (fig. 5A). Robo2
flox/flox
ho-
mozygotes are viable and fertile and lack urinary tract ab-
normalities. We then produced Robo2
del5/⫹
mice that lack
exon 5, by crossing the floxed allele with the Tg
EIIa-Cre
de-
letor strain.
29
The deletion of Robo2 exon 5 causes a read-
ing frameshift. RT-PCR and in situ hybridization experi-
ments showed that Robo2
del5
transcripts were expressed at
markedly reduced levels compared with transcripts from
the wild-type allele. We thus conclude that Robo2
del5
is,
effectively, a null allele. Consistent with results reported
for the Robo2 null allele described elsewhere,
19
Robo2
del5/
del5
homozygotes uniformly died shortly after birth with
multiplex, dysplastic kidneys and short ureters (fig. 5B–
5E). However, whereas no heterozygous phenotype was
described for the existing Robo2 null allele,
19
when a
Hoxb7-GFP reporter transgene that specifically identifies
the ureteric epithelium
30
was introduced into the Robo2
del5/
⫹
background, 4 (15%) of 26 Robo2
del5/⫹
heterozygous new-
borns exhibited a unilateral CAKUT-VUR phenotype (fig.
5Fand fig. 5G). This heterozygous phenotype included
both massive and lesser degrees of megaureter and a wide-
open UVJ, similar to the pathology identified in DGAP107.
To test whether further reductions in Robo2 gene dosage
could increase CAKUT-VUR penetrance, we took advan-
www.ajhg.org The American Journal of Human Genetics Volume 80 April 2007 623
Figure 6. Adult Robo2
del5/del5
↔Robo2
del5/flox
mosaics exhibiting megaureter, hydronephrosis, and UVJ defects. A–D, Ventral views. k p
kidney; ur pureter; bl pbladder; ua purethra. A, Urinary tract in a wild-type mouse aged 87 d. B, Higher magnification of boxed
region in panel A, indicating normal position of the UVJ. C, Right megaureter (arrow)inaRobo2
del5/del5
↔Robo2
del5/flox
mosaic aged 45
d. D, Higher magnification of boxed region in panel C, demonstrating abnormal bilateral UVJ. The obstructed right UVJ connects to a
caudal site in the bladder close to the urethra, causing megaureter. The left UVJ is located laterally in the bladder, a site commonly
associated with human VUR. Eand F, Dorsal views. E, Right megaureter (arrow) and hydronephrosis in a mosaic aged 45 d. Hydronephrosis
replaces the normal renal parenchyma (k), causing an upper pole cyst (cy). F, Left ureter of a male mosaic mouse aged 77 d that remains
connected to the vas deferens (vd) (arrow), resulting in obstruction and severe hydronephrosis. The left kidney has lost all parenchyma
and is replaced by a large cyst. The right kidney, ureter, and vas deferens are normal in appearance. sv pseminal vesicle; te ptestis.
tage of the variable expression of the EIIa-directed Cre
recombinase in the early preimplantation embryo,
31
to
generate mosaic progeny that consisted of admixtures of
Robo2
del5/del5
(null) and Robo2
del5/flox
(haploinsufficient) cells.
This mosaicism originates from the incomplete, stochastic
action of the EIIa-Cre transgene on the Robo2
flox
allele in
the early embryo before implantation.
31
Remarkably, 4
(40%) of 10 Robo2
del5/del5
↔Robo2
del5/flox
mosaic newborns
(resulting from the union of Robo2
del5
;Tg
EIIa-Cre
and Robo2
flox
gametes) exhibited unilateral urinary tract defects, in-
cluding short ureter, megaureter, and hydronephrosis (fig.
5Hand 5I).
To determine whether Robo2
del5/del5
↔Robo2
del5/flox
mosaic
newborns with urinary tract defects could survive after
prolonged reflux and obstruction, we followed another
cohort of these mice to adulthood. Seven (70%) of 10
Robo2
del5/del5
↔Robo2
del5/flox
adult mosaics, ranging in age
from 45 d to 77 d, manifested defects involving the UVJ
(fig. 6). These UVJ defects were bilateral and especially
notable, in that one UVJ was typically located laterally
and cephalad in the bladder (fig. 6A–6D), a location com-
monly associated with reflux in humans,
32
whereas the
contralateral UVJ was located caudad in the bladder or
even ectopically in the urethra. The caudal UVJ location
was associated with obstruction, resulting in megaureter
and severe hydronephrosis (fig. 6Cand 6E). In some male
Robo2
del5/del5
↔Robo2
del5/flox
mice, the ureter was connected
to the vas deferens, resulting in massive hydronephrosis
(fig. 6F). The Robo2
del5/del5
↔Robo2
del5/flox
mouse model is
thus consistent with the frequent coexistence of reflux and
obstruction in the same patient with VUR.
33
During em-
bryonic development, the nephric duct undergoes apo-
ptosis, transposing the ureter orifice from the nephric duct
to the urogenital sinus epithelium, to form the UVJ.
34
Mu-
tation in mouse Robo2 causes abnormal sites of ureteric
bud outgrowth,
19
which provides a developmental expla-
nation for the ectopic UVJ sites frequently observed in
VUR.
Discussion
Collectively, these results demonstrate that reduced Robo2
gene dosage can contribute to the pathogenesis of CAKUT-
VUR (table 1 and appendix F). In human primary VUR,
624 The American Journal of Human Genetics Volume 80 April 2007 www.ajhg.org
Table 1. Penetrance of CAKUT in Robo2
del5
Mutant Mice
Genotype
Robo2
del5/⫹
Newborn
Robo2
del5/flox
Mosaic
Newborn
Robo2
del5/flox
Mosaic
Adult
Robo2
del5/del5
Newborn
CAKUT penetrance (%) 15 40 70 100
Total number observed 26 10 10 20
linkage studies have produced inconsistent results,
35
un-
derscoring the need for other methods to identify the re-
sponsible genes. This problem is especially acute for VUR,
since the manifestations may vary during life, progres-
sing or resolving spontaneously.
36
Furthermore, because
asymptomatic individuals cannot be classified as “unaf-
fected,” linkage studies may be inconclusive or yield false-
negative results unless confined to affected individuals.
36
Lastly, intrafamilial phenotypic variability and genetic
heterogeneity
3,35
also exist. Since we identified only two
coding-region changes segregating with VUR in 124 fam-
ilies with VUR, alterations in ROBO2 itself are likely to
account for a small subset of VUR. Interestingly, however,
recent studies indicate that ROBO2 resides in an inherently
unstable genomic region, 3p12.3, that is prone to evolu-
tionary chromosomal rearrangements and to loss of het-
erozygosity in human cancers.
37
This raises the yet-un-
tested possibility that ROBO2 may be subject to frequent
rearrangement or microdeletion and duplication at either
the organismal or cellular level, which could be missed by
direct sequencing. In addition, variants in other genes
whose products function in the ROBO2 signal-transduc-
tion pathway may be implicated in the molecular path-
ogenesis of renal dysplasia and VUR, which may coexist
because of interrelated pathophysiology, common under-
lying genetic abnormality, or both mechanisms.
Acknowledgments
We thank Roxana Peters, Robert Eisenman, Diana Donovan,
Annick Turbe-Doan, and Juan Liu, for technical support; Yiping
Shen, Anne Higgins, and Fowzan Alkuraya, for assistance with
array CGH; Chantal Farra, for referral of the DGAP107 subject;
Frank Costantini, for providing Hoxb7-GFP transgenic mice; Wel-
lington Cardoso and Jining Lu, for help with fluorescence ster-
eomicroscopy; and Natalia Leach, Irfan Saadi, Kate Ackerman,
Azra Ligon, David Harris, Gail Bruns, Grigoriy Kryukov, Shamil
Sunyaev, Monica Banerjee, Maria Bitner-Glindzicz, Sue Malcolm,
Dagan Jenkins, Ramon Bonegio, and David Salant, for helpful
suggestions. This work was supported by National Institutes of
Health grants PO1GM061354 (to C.C.M.) and RO1DK063316 (to
R.L.M.); the Hilda Gershon Sugarman Young Investigator grant
from the National Kidney Foundation, a Department of Medicine
Pilot Project grant, and an Evans Medical Foundation grant (to
W.L.); Dutch Kidney Foundation grant C02.2009 (to J.C.G. and
A.M.v.E.); a Health Research Council grant (to M.R.E.); a Kids
Kidney Appeal grant, a Kidney Research UK grant, and Wellcome
Trust grant 066647 (to A.S.W. and S.A.F.); and a Medical Research
Council Career Establishment grant (to W.A. and V.S.).
Appendix A
DGAP107 Phenotype
Figure A1. Facial and limb abnormalities of the DGAP107 proband. Note the low-set, dysplastic ears and subtle membranous syndactyly
and clinodactyly. Blepharophimosis is also present.
www.ajhg.org The American Journal of Human Genetics Volume 80 April 2007 625
Table A1. Clinical Findings in the DGAP107 Subject at Age 14 Years
Characteristic Patient Phenotype
Weight (kg) 40 (10th percentile)
Height (cm) 146 (!5th percentile)
Visual disorders Daltonism, strabismus, hypermetropia
Limb defects Mild syndactyly, clinodactyly, brachymetacarpia
Urinary tract defects VUR grade IV, bilateral UVJ defects, unilateral megaureter
Learning disabilities Global verbal retardation, verbal IQ 69, performance IQ 46
a
Facial features Blepharophimosis, low-set and dysplastic ears
Dental anomalies Malformed lower incisor (“T” shape)
Genital anomalies Complete left testicular agenesis
Neurological defects Seizures, hyperactivity, sleep disorder
Orthopedic abnormality Hyperlordosis
Growth retardation Delayed puberty
Other abnormalities Bilateral inguinal hernia
a
IQ pintelligence quotient.
Appendix B
The DGAP107 3p12 Breakpoint Disrupts ROBO2
Figure B1. ROBO2 disrupted in DGAP107, with the breakpoint lying within intron 2. A, Restriction map surrounding the 3p12 breakpoint.
The base-pair position of BAC RP11-88K11 (AC131005, within intron 2 of ROBO2 [see BAC contig in fig. 1F]) was used to calculate the
distance between restriction enzyme sites. RP11-88K11 overlaps with BAC clone RP11-54A6 used in FISH and also contains thebreakpoint,
which is between boxed BsrDI and SphI sites, on the basis of the aberrant bands detected by Southern blot analysis. B, Southern blot
analysis of DGAP107 (P) and unaffected control (C) genomic DNA, with use of the designated restriction enzymes and the probe A2-
57 shown in panel A. Aberrant bands (white arrows) are present only in DGAP107 DNA digested with SphI and PvuII. C, Breakpoint
cloning showing the sequence of the junction fragment from der(3) with a 1-bp deletion (g [green]) and a 2-bp insertion (tt [pink]).
There is no gain or loss of nucleotides at the der(Y) breakpoint.
626 The American Journal of Human Genetics Volume 80 April 2007 www.ajhg.org
Appendix C
ROBO2 and PCDH11Y Expression in Human Adult and Fetal Tissues
Figure C1. Expression of ROBO2 and PCDH11Y in human tissues. RT-PCR amplified 2.8-kb ROBO2 cDNAs with the use of primers ROBO2-
F1 and ROBO2-R1. The 3-kb ROBO2 cDNA product (upper band of doublet) contains an alternatively spliced exon 24B. RT-PCR amplification
of 404-bp and 620-bp PCDH11Y cDNAs used primers PCDH11Y-F1 and PCDH11Y-R1. The intensity of the fragments indicates the approximate
expression level of ROBO2 and PCDH11Y in these tissues. Notably, there is no expression of PCDH11Y in the fetal kidney. b-actin was
used as a cDNA loading control.
Appendix D
Analysis of the DGAP107 del(17)(p11.2) Microdeletion
The pleiotropic nature of the DGAP107 phenotype sug-
gests that both the t(Y;3)(p11;p12)dn translocation and
the del(17)(p11.2) microdeletion may contribute to the
overall DGAP107 phenotype. However, we parse the con-
tribution of del(17)(p11.2) to the VUR phenotype as fol-
lows. Point mutations in RAI1, which resides in the SMS
critical deletion region, suggest that RAI1 haploinsuffi-
ciency accounts for many features of SMS.
38
Less fre-
quently observed cardiac, renal, and other defects may
reflect hemizygosity for other genes in the SMS common
deletion region.
39
Of note, RAI1 is included in the 3.4-Mb
del(17)(p11.2) in DGAP107, the boundaries of which were
defined by FISH experiments (not shown). It seems likely
that the del(17)(p11.2) microdeletion contributes to some
aspects of the sleep, behavioral, and cognitive deficits in
DGAP107, because similar phenotypes are observed in pa-
tients with SMS who exhibit 17p11.2 deletions or RAI1
haploinsufficiency. Thus, ROBO2 disruption, 17p11.2 mi-
crodeletion, or both could theoretically account for VUR
in DGAP107. Our human and mouse experimental results
indicate that ROBO2 disruption can contribute substan-
tially to the VUR phenotype. Conversely, our analyses al-
so suggest that, whereas the 17p11.2 microdeletion in
DGAP107 may contribute to the pathogenesis of VUR in
DGAP107, it is not likely to play a primary role. There are
three bases for this conclusion.
First, although we note an isolated case report describing
VUR in SMS,
40
reexamination of the frequency of renal
defects in SMS indicates that these are relatively infre-
quent and less frequent than originally believed.
23,41
Second, in the context of this study, we examined and
observed no kidney or collecting system defects in three
age-matched genetically engineered mouse models of
SMS: SMS
df(11)17/⫹
(D2 Mb), SMS
df(11)17⫺l/⫹
(D500 kb), and
Rai1
⫺/⫺
.
42–44
The first two models represent deletion alleles
that eliminate 2 Mb and 500 kb, respectively, from mouse
chromosome 11. These regions are homologous to and
share conservation of synteny with the 17p11.2 region that
is involved in SMS. Although SMS
df(11)17
and SMS
df(11)17⫺l
ho-
mozygotes die before nephrogenesis (9.5 embryonic d),
examination of 20 SMS
df(11)17⫺l/⫹
mice in the context of this
study revealed no UVJ or other urinary tract defects. In
addition, Rai1
⫺/⫺
are not reported to exhibit any evidence
for renal or ureteral defects,
42
and our own analysis of
these mice confirms this finding.
Third, analysis of the urinary tract in five SMS
df(11)17⫺l/
⫹
;Robo2
del5/⫹
transheterozygotes, the genotype of which
nominally approximates the DGAP107 genotype, revealed
no observable phenotype compared with genetic back-
ground and age-matched littermate controls. Thus, where-
as the 17p11.2 microdeletion could be a contributory factor,
it seems unlikely to play a major role in the pathogenesis
of CAKUT and VUR phenotypes in DGAP107.
www.ajhg.org The American Journal of Human Genetics Volume 80 April 2007 627
Appendix E
Clinical Data for Families 2559x and B5
Clinical Data for Family 2559x
2559x is a white British family. The index patient
(25592) has bilateral VUR and bilateral nephropathy. She
presented at age 3 years with a symptomatic, documented
Escherichia coli UTI, at which time ultrasound (US) revealed
two small kidneys that were each 6.0 cm long (normal
mean for age 7.0 cm). An indirect isotope cystogram with
mercaptoacetyltriglycine (MAG3) showed bilateral VUR.
The patient was treated long term with antibiotics. At age
4 years, her plasma creatinine was 107 mmol/liter (normal
mean for age 56 mmol/liter). Her formal EDTA-glomerular
filtration rate was 31 ml/min/1.73 m
2
(normal mean 190
ml/min/1.73 m
2
), and a formal voiding cystourethrogram
(VCUG) confirmed bilateral VUR. A repeat US confirmed
two small kidneys with dimensions of 4.7 cm (left) and
6.0 cm (right) (50th percentile for age 7.2 cm), both with
scarred upper poles. At age 9 years, she had progressive
renal failure with a plasma creatinine of 306 mmol/liter.
By age 12 years, her plasma creatinine had risen to 407
mmol/liter. Her mother (25593) has a history of VUR that
required ureteral reimplantation surgery.
Clinical Data for Family B5
B5 is a white Dutch family. By VCUG and intravenous
pyelogram (IVP), we determined that the proband B5 had
bilateral VUR and a right kidney duplex system. US in-
vestigations further documented the proband’s double
collecting system on the right side and a single system on
the left, with a slightly dilated upper pole system. Renal
US studies were also performed in all other family mem-
bers except grandfather B1, who is deceased. The US find-
ings for family B5 include the following: A1 (uncle) had
normal kidneys, with an 11.0-cm right kidney and a 12.5-
cm left kidney (normal [ⳲSD] 11.5 Ⳳ1.0 cm); C1 (grand-
mother) had a small, 9.6-cm right kidney, an 11.4-cm left
kidney, a slim collecting system on both sides, a normal
corticomedullary ratio, no signs of a duplex system, and
normal flow in the right renal artery; D1 (aunt) had an
11.8-cm right kidney with an upper pole duplex system
(column of Bertini), an 11.0-cm left kidney, a normal cor-
ticomedullary ratio, and no dilatation or urologic com-
plaints; E1 (father) had a normal kidneys, a slim collecting
system on both sides, a small peripelvic cyst on the right
side, and no urologic complaints; F1 (mother) had a left
kidney with fetal lobulation on the right side, no visible
column from the renal parenchyma to the hilus, a duplex
system that can neither be proven nor excluded, a slim
collecting system on both sides, and febrile UTI history;
and G1 (aunt) had no dilatation on either kidney, a cyst
(4.8#3.5 cm) in the upper pole of the left kidney, and
pyelonephritis at age 8 years.
Controls
All controls for genetic studies of families 2559x and B5
were unrelated. Control samples were from 180 Americans
of white European descent (CEPH) and from 96 whites in
the same geographic region as family B5. These latter 96
samples were subjected both to targeted sequencing of
exons 12, 19, and 23 and to complete resequencing of all
26 coding ROBO2 exons.
Appendix F
Analysis of Robo
del5
Mutant Mice
Figure F1. Generation of F2 Robo2
del5/del5
↔Robo2
del5/flox
mosaics
628 The American Journal of Human Genetics Volume 80 April 2007 www.ajhg.org
Table F1. Phenotype-Genotype Correlation in F2 Robo2
del5/del5
↔Robo2
del5/flox
Mosaics
Identification
Number
Age
(d) Sex Phenotype
a
3235 45 F L and R: megaureter, hydronephrosis
3239 45 F R: megaureter, hydronephrosis; L: normal
404 62 M L: megaureter, hydronephrosis with complete loss of renal parenchyma; R: normal
3218 77 M R: megaureter, hydronephrosis with complete loss of renal parenchyma; L: normal
3236 77 F No discernible gross phenotype
403 62 M No discernible gross phenotype
3372 27 M No discernible gross phenotype
3964 20 F L: megaureter, hydronephrosis; R: normal
3963 20 F L: megaureter, hydronephrosis; R: normal
3962 20 F L and R: megaureter, hydronephrosis
N
OTE
.—Genotyping was performed using PCR of mouse tail DNA. All mosaics were positive for Robo2
del5
,Robo2
flox
,
and Tg
EIIa-Cre
.
a
Lpleft; R pright.
Notes on the Breeding Scheme
In the P0 generation, Robo2
flox/⫹
heterozygotes were
crossed with Tg
EIIa-cre/EIIa-cre
homozygotes. The 50% of the
progeny that receive Tg
EIIa-cre
and Robo2
flox
gametes consti-
tute the F1 generation shown (fig. F1). When transmitted
from males, Tg
EIIa-cre
is variably expressed in the F1 embryos
after fertilization and before implantation,
29,31
resulting in
recombination in some embryonic cells but not others.
Thus, the resulting F1 progeny are mosaic and contain
mixtures of Robo2
flox/⫹
(no recombination) and Robo2
del5/⫹
(recombination) cells. These mosaics are genotypically
denoted Robo2
del5/⫹
↔Robo2
flox/⫹
;Tg
EIIa-cre/⫹
. PCR experiments
demonstrated variable ratios of the Robo2
flox
and the
Robo2
del5
alleles, confirming the mosaicism of the F1 mice
(not shown). As expected, 10 F1 mosaic mice examined
exhibit no observable phenotype, because heterozygous
Robo2
del5/⫹
mice exhibit a phenotype at a low percentage
(i.e., 15%), and, even at the most extreme degree of mo-
saicism (100% Robo2
del5/⫹
and 0% Robo2
flox/⫹
), the overall
reduction in Robo2 gene dosage would not surpass that in
Robo2
del5/⫹
heterozygotes.
The F2 generation was generated by intercross of the
subset of F1 germline mosaics that also carried the Tg
E11a-
cre/⫹
transgene. The F2 generation thus also consisted of
mosaic progeny because of the union of Robo2
del5
;Tg
EIIa-cre
and Robo2
flox
gametes. This particular combination of gam-
etes results in a subset of F2 mosaics that contains both
Robo2
del5/del5
and Robo2
del5/flox
cells—that is, a mixture of
heterozygous and homozygous cells. Cells carrying the
Robo2
del5/del5
genotype in these Robo2
del5/del5
↔Robo2
del5/flox
mosaics derive from the action of Cre in cells that com-
mence embryogenesis with the Robo2
del5/flox
genotype;
Robo2
del5/flox
cells result when Cre activity in those cells is
insufficient to cause recombination. As described in the
text and in table F1, 70% of these mosaics exhibit striking
CAKUT phenotypes, whereas 30% exhibit no observable
phenotype. The presence of a CAKUT phenotype presum-
ably correlates with the percentage of Robo2
del5/del5
cells
that make up the urinary tracts of these mosaics.
The mosaic nature of the F2 mice (at least in the germ-
line) was established by further intercross of a subset of
F2 Robo2
del5/del5
↔Robo2
del5/flox
;Tg
EIIa-cre
mosaics to generate an
F3 generation. In this case, a high percentage of Robo2
del5/
del5
cells in the F2 germline result in a preponderance of
gametes carrying the Robo2
del5
mutant allele. The union
of these Robo2
del5
gametes results in an increased propor-
tion of Robo2
del5/del5
mice (90% of progeny) in the F3 gen-
eration that die at birth from CAKUT phenotypes (not
shown). The remaining 10% retain the Robo2
del5/flox
allele
and survive. The non-Mendelian ratio of these resulting
F3 genotypes confirms germline mosaicism in the F2 mice.
Appendix G
PCR Primers
Table G1. PCR Primers and Probes
Primer or Probe Used
a
Type Location Sequence
RT-PCR analyses:
2.8-kb ROBO2 cDNA
b
:
ROBO2-F1 Forward Exon 9 of ROBO2 5
-GAGCAAGGCACACTGCAGATTA-3
ROBO2-R1 Reverse Exon 26 of ROBO2 5
-AGTCATCACTTCCATGAGTCCG-3
404-bp and 620-bp PCDH11Y cDNA
b
:
PCDH11Y-F1 Forward Exon 1 of PCDH11Y 5
-CAGAAACAACCTCAGCGACTCC-3
PCDH11Y-R1 Reverse Exon 4 of PCDH11Y 5
-GAACACCACGCATACTAGCAGG-3
ROBO2-PCDH11Y fusion transcripts
c
:
ROBO2-F2 Forward Exon 2 of ROBO2 5
-GCAGTGAGTCGAAATGCGTC-3
PCDH11Y-R1 Reverse Exon 4 of PCDH11Y 5
-GAACACCACGCATACTAGCAGG-3
PCDH11Y-R2 Reverse Exon 3 of PCDH11Y 5
-CCCACTGTCATTCAGTCCTCAT-3
PCDH11Y-R3 Reverse Exon 5 of PCDH11Y 5
-TGTTTCAATGACATCGAGGCC-3
606-bp ROBO2 nontranslocated allele
c
:
ROBO2-F2 Forward Exon 2 of ROBO2 5
-GCAGTGAGTCGAAATGCGTC-3
ROBO2-qR Reverse Exon 7 of ROBO2 5
-TGGCCGAACCACAAACTGT-3
Quantitative real-time PCR analyses:
ROBO2 nontranslocated allele
d
:
ROBO2-F2 Forward Exon 2 of ROBO2 5
-GCAGTGAGTCGAAATGCGTC-3
ROBO2-qR2 Reverse Exon 3 of ROBO2 5
-GCTCTCCAGCTGCCACTACAA-3
ROBO2-FAM2 TaqMan probe ROBO2 exon 2–exon 3
junction
5
-CTGGAAGTGGCATTGTTACGAGATGACTTCC-3
Fu-129 transcript
d,e
:
ROBO2-F2 Forward Exon 2 of ROBO2 5
-GCAGTGAGTCGAAATGCGTC-3
PCDH11Y-qR1 Reverse Exon 1d of PCDH11Y 5
-TATTCCATCCTCTTCCATCCATTC-3
TaqMan 129 TaqMan probe ROBO2 exon 2 and
PCDH11Y exon 1d
junction
5
-CTGGAAGTGGCATAGGGTGCTTAAAAAGTACAGA-3
Fu-153 transcript
d,e
:
ROBO2-F2 Forward Exon 2 of ROBO2 5
-GCAGTGAGTCGAAATGCGTC-3
PCDH11Y-qR2 Reverse Exon 4 of PCDH11Y 5
-ATGTACGTCCCGGACAACAAA-3
TaqMan 153 TaqMan probe ROBO2 exon 2 and
PCDH11Y exon 4
junction
5
-TGGAAGTGGCATTGTTGTGCGGGT-3
Genotyping of Robo2
flox
and Robo2
del5
mice:
Robo2
flox
allele
f
:
Ro2-MEBAC15F Forward Intron 5 of Robo2 5
-CCAATCATAGTCTCTCCACG-3
Ro2-MEBAC15R Reverse Intron 5 of Robo2 5
-CCTCTGATTCAATGAGATGC-3
1,180-bp fragment from Robo2
del5
allele:
Robo2koF Forward Intron 4 of Robo2 5
-CCACTATGCTGGCTCTGTCTCACAC-3
Robo2R Reverse Intron 5 of Robo2 5
-GGTTTTGGAGGTCTTACTACGTAGC-3
1,390-bp fragment from Robo2
⫹
wild-type allele:
Robo2wtF Forward Intron 4 of Robo2 5
-CAACTTTTCCTTTTCCGGGAGG-3
Robo2R Reverse Intron 5 of Robo2 5
-GGTTTTGGAGGTCTTACTACGTAGC-3
Synthesis of fusion protein constructs:
Fu-129 ROBO2-PCDH11Y fusion cDNA (129-aa fusion protein):
5
-hR2(E1) Forward Exon 1 of ROBO2 with
5
EcoRI linker
5
-ATCGAATTCATGAGTCTGCTGATGTTTACACAAC-
TACTG-3
3
-LEVA(X1) Reverse Intron 1d of PCDH11Y
with 5
XhoI linker
5
-TTACTCGAGCTATGCCACTTCCAGAGACGCATTTCG-3
Fu-153 ROBO2-PCDH11Y fusion cDNA (153-aa fusion protein):
5
-hR2(E1) Forward Exon 1 of ROBO2 with
5
EcoRI linker
5
-ATCGAATTCATGAGTCTGCTGATGTTTACACAAC-
TACTG-3
3
-SRSC(X1) Reverse Intron 4 of PCDH11Y
with 5
XhoI linker
5
-CCACTCGAGCTAGCAGGACCGCGAAAATGTACGTCC-3
a
Primers and probes were used for amplification and quantitation.
b
As shown in figure C1.
c
As shown in figure 2B.
d
As shown in figure 2C.
e
As shown in figure 2D.
f
PCR amplifies a 1,100-bp fragment from both wild-type and Robo2
flox
alleles. After SpeI digestion, Robo2
flox
allele will not cleave (1,100-bp product),
whereas the wild-type allele will be cleaved by SpeI to yield two smaller products of 750 bp and 350 bp.
Table G2. PCR Primer Sets Used to Amplify 26 Human ROBO2 Exons and Intron-Exon
Boundaries in Mutation Analysis
Exon and Primer
Amplicon
Size
(bp)
Location in
ROBO2 Sequence
1: 286
Forward 5
-UTR 5
-TTTGCTCTTCTTGACTTTAATTAGTATCTAGG-3
Reverse Introns 1–2 5
-TATAACCCACATCAAATTCAAAAAGAAAT-3
2: 501
Forward Introns 1–2 5
-CGAAGAGTTTAATTTCCCCATCA-3
Reverse Introns 2–3 5
-GCGTCTATGGGAACACATCAAAA-3
3: 264
Forward Introns 2–3 5
-TTGTACAACAAAAAGCCTAAGTTACTGTC-3
Reverse Introns 3–4 5
-AAAATTCAATCTCTCTGGGCCAT-3
4: 280
Forward Introns 3–4 5
-TAATGACCTTTATTTTCTATTCTGTTCCTTT-3
Reverse Introns 4–5 5
-T TATATGGCCCAGTTTTTAATGTTAGTAATACT-3
5: 291
Forward Introns 4–5 5
-CTTTTTTCATAATGTACTTAAAGCATGCA-3
Reverse Introns 5–6 5
-GTGGCATTGTAGCTGTCCTTTTATT-3
6: 427
Forward Introns 5–6 5
-TTGCACTTTGTGGCTGATTTG-3
Reverse Introns 6–7 5
-TAATTTTATTTCAACTAATGATAGAGAGGACAC-3
7: 289
Forward Introns 6–7 5
-CAACATAGTACCATATTTTCTCCTTGACATA-3
Reverse Introns 7–8 5
-AAGCAAGGCAAGCTTTCAGG-3
8: 336
Forward Introns 7–8 5
-CCCACTGTATTCCTTAATTGTAGTAGCTT-3
Reverse Introns 8–9 5
-TCCACATGGTTAACGTGTATCTAGAAA-3
9: 351
Forward Introns 8–9 5
-TTCAGTGTCAATATATCAAGCCTACTGA-3
Reverse Introns 9–10 5
-CACTATGCAATTTTTCCATAGAGCAG-3
10: 231
Forward Introns 9–10 5
-TGGCTGTCATTGAGTAATTATTCTGC-3
Reverse Introns 10–11 5
-TCCCCCCTTAACTTATTATTTGATATTG-3
11: 343
Forward Introns 10–11 5
-CTGTCTAGGTCAGGTCCTTTAGTAGACTG-3
Reverse Introns 11–12 5
-CAGCAGGATAGTTCAGGTGACATT-3
12: 324
Forward Introns 11–12 5
-AACCTTTGTCATTGATACCCAACTC-3
Reverse Introns 12–13 5
-TCCTCATCAAGCCCCTCGT-3
13: 298
Forward Introns 12–13 5
-AGTTCTAAAGACATGAGGTTGATTTACATAA-3
Reverse Introns 13–14 5
-CACTCTTTGTTCATTTGCATTTTCC-3
14: 409
Forward Intron 13–14 5
-AGGACAGAAATGGGACAAATGAA-3
Reverse Intron 14–15 5
-TTCTAAGGAAGATAACAAATAGGTACTGTAACA-3
15: 294
Forward Introns 14–15 5
-AGTCTCCTGCAACTTGTCTTTATACTCAT-3
Reverse Introns 15–16 5
-TCATTCGTGAGACACTGAGATTTCT-3
16: 321
Forward Introns 15–16 5
-AATATTTGATCAGTTACAGTAGTCTCGTTACC-3
Reverse Introns 16–17 5
-TGCAAAATCATCATCCACCTTG-3
17: 338
Forward Introns 16–17 5
-TCTTCATTTTTGATGCACCATGT-3
Reverse Introns 17–18 5
-TTTCTGTTCCTTCCATTCATTTCAT-3
18: 211
Forward Introns 17–18 5
-CCTCAGCTCTAAACTAAGGGCCA-3
Reverse Introns 18–19 5
-TCTTACTATAGAGTTTCCCCAGTCCTG-3
19: 240
Forward Introns 18–19 5
-AAATCTTCCATTTCTTAACGCTTTATATTG-3
Reverse Introns 19–20 5
-AAAAACACAACTTACCTCCACGG-3
20: 432
(continued)
www.ajhg.org The American Journal of Human Genetics Volume 80 April 2007 631
Table G2. (continued)
Exon and Primer
Amplicon
Size
(bp)
Location in
ROBO2 Sequence
Forward Introns 19–20 5
-GATAGTTTTGGGCTTCCGGTG-3
Reverse Introns 20–21 5
-TGAATCACTAAGTCAAACAACAAATACTAATT-3
21: 306
Forward Introns 20–21 5
-CATAAATACACCTTGCCATCTGATG-3
Reverse Introns 21–22 5
-TGGCAAAAATGAACAACAGAGAG-3
22: 411
Forward Introns 21–22 5
-TGCATGTATGTGTATATGTATTTGTGTCA-3
Reverse Introns 22–23 5
-TGTAGTTCTATCAGAATCTCTTGTGAATTTATT-3
23: 379
Forward Introns 22–23 5
-AAGACAGTATGAGTTACTATAGCATGCATTT-3
Reverse Introns 23–24 5
-GGAAGTAGTTGACTTTTGATGCATTTTA-3
24: 325
Forward Introns 23–24 5
-AGGTAGATTTACAGGTTAGTCATAGTGCA-3
Reverse Introns 24–25 5
-CATGGAGCACGTCTCTTCAGC-3
25: 351
Forward Introns 24–25 5
-TGGTAAAGTAGGCCATTCACAGTG-3
Reverse Introns 25–26 5
-CAAGATTCTTTCTGAATCACGATAGC-3
26: 574
Forward Introns 25–26 5
-TCACAAACTCATCTATCTGAAGACCTTAT-3
Reverse 3
-UTR 5
-AAAATTGCAGTGCAAAATTTAAACA-3
Web Resources
Accession numbers and URLs for data presented herein are as
follows:
BACPAC Resources, http://bacpac.chori.org/
DGAP, http://dgap.harvard.edu/
Ensembl, http://www.ensembl.org/Homo_sapiens/index.html
Entrez Protein, http://www.ncbi.nlm.nih.gov/entrez/query.fcgi
?dbpProtein (for ROBO2 [accession number NP_002933])
GenBank, http://www.ncbi.nlm.nih.gov/Genbank/ (for ROBO2
[accession number NM_002942] and PCDH11Y [accession
number NM_032971])
Online Mendelian Inheritance in Man (OMIM), http://www.ncbi
.nlm.nih.gov/Omim/ (for VUR and SMS)
References
1. Pope JC 4th, Brock JW 3rd, Adams MC, Stephens FD, Ichikawa
I (1999) 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 10:2018–2028
2. Nakanishi K, Yoshikawa N (2003) Genetic disorders of human
congenital anomalies of the kidney and urinary tract
(CAKUT). Pediatr Int 45:610–616
3. Feather SA, Malcolm S, Woolf AS, Wright V, Blaydon D, Reid
CJ, Flinter FA, Proesmans W, Devriendt K, Carter J, et al (2000)
Primary, nonsyndromic vesicoureteric reflux and its nephro-
pathy is genetically heterogeneous, with a locus on chro-
mosome 1. Am J Hum Genet 66:1420–1425
4. Sargent MA (2000) What is the normal prevalence of vesi-
coureteral reflux? Pediatr Radiol 30:587–593
5. Bailey RR (1973) The relationship of vesico-ureteric reflux to
urinary tract infection and chronic pyelonephritis-reflux ne-
phropathy. Clin Nephrol 1:132–141
6. Dillon MJ, Goonasekera CD (1998) Reflux nephropathy. JAm
Soc Nephrol 9:2377–2383
7. Kincaid-Smith PS, Bastos MG, Becker GJ (1984) Reflux ne-
phropathy in the adult. Contrib Nephrol 39:94–101
8. Mackie GG, Awang H, Stephens FD (1975) The ureteric orifice:
the embryologic key to radiologic status of duplex kidneys.
J Pediatr Surg 10:473–481
9. Noe HN, Wyatt RJ, Peeden JN Jr, Rivas ML (1992) The trans-
mission of vesicoureteral reflux from parent to child. J Urol
148:1869–1871
10. Van den Abbeele AD, Treves ST, Lebowitz RL, Bauer S, Davis
RT, Retik A, Colodny A (1987) Vesicoureteral reflux in asymp-
tomatic siblings of patients with known reflux: radionuclide
cystography. Pediatrics 79:147–153
11. Kidd T, Brose K, Mitchell KJ, Fetter RD, Tessier-Lavigne M,
Goodman CS, Tear G (1998) Roundabout controls axon
crossing of the CNS midline and defines a novel subfamily
of evolutionarily conserved guidance receptors. Cell 92:205–
215
12. Xian J, Aitchison A, Bobrow L, Corbett G, Pannell R, Rabbitts
T, Rabbitts P (2004) Targeted disruption of the 3p12 gene,
Dutt1/Robo1, predisposes mice to lung adenocarcinomas and
lymphomas with methylation of the gene promoter. Cancer
Res 64:6432–6437
13. Xian J, Clark KJ, Fordham R, Pannell R, Rabbitts TH, Rabbitts
PH (2001) Inadequate lung development and bronchial hy-
perplasia in mice with a targeted deletion in the Dutt1/Robo1
gene. Proc Natl Acad Sci USA 98:15062–15066
14. Hannula-Jouppi K, Kaminen-Ahola N, Taipale M, Eklund R,
Nopola-Hemmi J, Kaariainen H, Kere J (2005) The axon guid-
ance receptor gene ROBO1 is a candidate gene for develop-
mental dyslexia. PLoS Genet 1:e50
15. Jen JC, Chan WM, Bosley TM, Wan J, Carr JR, Rub U, Shattuck
D, Salamon G, Kudo LC, Ou J, et al (2004) Mutations in a
human ROBO gene disrupt hindbrain axon pathway crossing
and morphogenesis. Science 304:1509–1513
16. Bedell VM, Yeo SY, Park KW, Chung J, Seth P, Shivalingappa
V, Zhao J, Obara T, Sukhatme VP, Drummond IA, et al (2005)
Roundabout4 is essential for angiogenesis in vivo. Proc Natl
Acad Sci U S A 102:6373–6378
632 The American Journal of Human Genetics Volume 80 April 2007 www.ajhg.org
17. Fricke C, Lee JS, Geiger-Rudolph S, Bonhoeffer F, Chien CB
(2001) Astray, a zebrafish roundabout homolog required for
retinal axon guidance. Science 292:507–510
18. Long H, Sabatier C, Ma L, Plump A, Yuan W, Ornitz DM,
Tamada A, Murakami F, Goodman CS, Tessier-Lavigne M
(2004) Conserved roles for Slit and Robo proteins in midline
commissural axon guidance. Neuron 42:213–223
19. 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
20. Li HS, Chen JH, Wu W, Fagaly T, Zhou L, Yuan W, Dupuis S,
Jiang ZH, Nash W, Gick C, et al (1999) Vertebrate slit, a se-
creted ligand for the transmembrane protein roundabout, is
a repellent for olfactory bulb axons. Cell 96:807–818
21. Ward ME, Rao Y (2005) Investigations of neuronal migration
in the central nervous system. Methods Mol Biol 294:137–
156
22. Blanco P, Sargent CA, Boucher CA, Mitchell M, Affara NA
(2000) Conservation of PCDHX in mammals; expression of
human X/Y genes predominantly in brain. Mamm Genome
11:906–914
23. Potocki L, Shaw CJ, Stankiewicz P, Lupski JR (2003) Variability
in clinical phenotype despite common chromosomal dele-
tion in Smith-Magenis syndrome [del(17)(p11.2p11.2)]. Ge-
net Med 5:430–434
24. Liu Z, Patel K, Schmidt H, Andrews W, Pini A, Sundaresan V
(2004) Extracellular Ig domains 1 and 2 of Robo are important
for ligand (Slit) binding. Mol Cell Neurosci 26:232–240
25. Wu W, Wong K, Chen J, Jiang Z, Dupuis S, Wu JY, Rao Y
(1999) Directional guidance of neuronal migration in the ol-
factory system by the protein Slit. Nature 400:331–336
26. Chapman CJ, Bailey RR, Janus ED, Abbott GD, Lynn KL (1985)
Vesicoureteric reflux: segregation analysis. Am J Med Genet
20:577–584
27. 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 sig-
naling pathway. J Biol Chem 281:28430–28437
28. Wong K, Ren XR, Huang YZ, Xie Y, Liu G, Saito H, Tang H,
Wen L, Brady-Kalnay SM, Mei L, et al (2001) Signal trans-
duction in neuronal migration: roles of GTPase activating
proteins and the small GTPase Cdc42 in the Slit-Robo path-
way. Cell 107:209–221
29. Lakso M, Pichel JG, Gorman JR, Sauer B, Okamoto Y, Lee E,
Alt FW, Westphal H (1996) Efficient in vivo manipulation of
mouse genomic sequences at the zygote stage. Proc Natl Acad
Sci USA 93:5860–5865
30. Srinivas S, Goldberg MR, Watanabe T, D’Agati V, al-Awqati
Q, Costantini F (1999) Expression of green fluorescent protein
in the ureteric bud of transgenic mice: a new tool for the
analysis of ureteric bud morphogenesis. Dev Genet 24:241–
251
31. Holzenberger M, Lenzner C, Leneuve P, Zaoui R, Hamard G,
Vaulont S, Bouc YL (2000) Cre-mediated germlinemosaicism:
a method allowing rapid generation of several alleles of a
target gene. Nucleic Acids Res 28:E92
32. Tanagho EA, Guthrie TH, Lyon RP (1969) The intravesical
ureter in primary reflux. J Urol 101:824–832
33. Stauss J, Connolly LP, Connolly SA, Zurakowski D, Treves ST,
Peters CA (2003) Dynamic renal scintigraphy in children with
vesicoureteral reflux and suspected coexisting ureteropelvic
junction obstruction. J Urol 170:1966–1970
34. Batourina E, Tsai S, Lambert S, Sprenkle P, Viana R, Dutta S,
Hensle T, Wang F, Niederreither K, McMahon AP, et al (2005)
Apoptosis induced by vitamin A signaling is crucial for con-
necting the ureters to the bladder. Nat Genet 37:1082–1089
35. Sanna-Cherchi S, Reese A, Hensle T, Caridi G, Izzi C, Kim YY,
Konka A, Murer L, Scolari F, Ravazzolo R, et al (2005) Familial
vesicoureteral reflux: testing replication of linkage in seven
new multigenerational kindreds. J Am Soc Nephrol 16:1781–
1787
36. Eccles MR, Bailey RR, Abbott GD, Sullivan MJ (1996) Unrav-
elling the genetics of vesicoureteric reflux: a common familial
disorder. Hum Mol Genet 5:1425–1429
37. Yue Y, Grossmann B, Galetzka D, Zechner U, Haaf T (2006)
Isolation and differential expression of two isoforms of the
ROBO2/Robo2 axon guidance receptor gene in humans and
mice. Genomics 88:772–778
38. Slager RE, Newton TL, Vlangos CN, Finucane B, Elsea SH
(2003) Mutations in RAI1 associated with Smith-Magenis syn-
drome. Nat Genet 33:466–468
39. Bi W, Yan J, Stankiewicz P, Park SS, Walz K, Boerkoel CF,
Potocki L, Shaffer LG, Devriendt K, Nowaczyk MJ, et al (2002)
Genes in a refined Smith-Magenis syndrome critical deletion
interval on chromosome 17p11.2 and the syntenic region of
the mouse. Genome Res 12:713–728
40. Chou IC, Tsai FJ, Yu MT, Tsai CH (2002) Smith-Magenis syn-
drome with bilateral vesicoureteral reflux: a case report. J For-
mos Med Assoc 101:726–728
41. Greenberg F, Lewis RA, Potocki L, Glaze D, Parke J, Killian J,
Murphy MA, Williamson D, Brown F, Dutton R, et al (1996)
Multi-disciplinary clinical study of Smith-Magenis syndrome
(deletion 17p11.2). Am J Med Genet 62:247–254
42. Bi W, Ohyama T, Nakamura H, Yan J, Visvanathan J, Justice
MJ, Lupski JR (2005) Inactivation of Rai1 in mice recapitulates
phenotypes observed in chromosome engineered mouse
models for Smith-Magenis syndrome. Hum Mol Genet 14:
983–995
43. Walz K, Caratini-Rivera S, Bi W, Fonseca P, Mansouri DL,
Lynch J, Vogel H, Noebels JL, Bradley A, Lupski JR (2003)
Modeling del(17)(p11.2p11.2) and dup(17)(p11.2p11.2) con-
tiguous gene syndromes by chromosome engineering in
mice: phenotypic consequences of gene dosage imbalance.
Mol Cell Biol 23:3646–3655
44. Yan J, Keener VW, Bi W, Walz K, Bradley A, Justice MJ, Lupski
JR (2004) Reduced penetrance of craniofacial anomalies as a
function of deletion size and genetic background in a chro-
mosome engineered partial mouse model for Smith-Magenis
syndrome. Hum Mol Genet 13:2613–2624
