Bilateral Renal Agenesis/Hypoplasia/Dysplasia (BRAHD):
Postmortem Analysis of 45 Cases with Breakpoint
Mapping of Two De Novo Translocations
Louise Harewood1.¤, Monica Liu2., Jean Keeling3, Alan Howatson4, Margo Whiteford5, Peter Branney1,
Margaret Evans3, Judy Fantes1, David R. FitzPatrick1,6*
1MRC Human Genetics Unit, Institute of Genetic and Molecular Medicine, Edinburgh, United Kingdom, 2Medical School, University of Edinburgh, Edinburgh, United
Kingdom, 3Department of Paediatric Pathology, New Royal Infirmary, Edinburgh, United Kingdom, 4Department of Paediatric Pathology, Royal Hospital for Sick Children,
Glasgow, United Kingdom, 5Department of Clinical Genetics, Royal Hospital for Sick Children, Glasgow, United Kingdom, 6South-East Scotland Regional Genetics
Services, Western General Hospital, Edinburgh, United Kingdom
Background: Bilateral renal agenesis/hypoplasia/dysplasia (BRAHD) is a relatively common, lethal malformation in humans.
Established clinical risk factors include maternal insulin dependent diabetes mellitus and male sex of the fetus. In the
majority of cases, no specific etiology can be established, although teratogenic, syndromal and single gene causes can be
assigned to some cases.
Methodology/Principal Findings: 45 unrelated fetuses, stillbirths or infants with lethal BRAHD were ascertained through a
single regional paediatric pathology service (male:female 34:11 or 3.1:1). The previously reported phenotypic overlaps with
VACTERL, caudal dysgenesis, hemifacial microsomia and Mu ¨llerian defects were confirmed. A new finding is that 16/45
(35.6%; m:f 13:3 or 4.3:1) BRAHD cases had one or more extrarenal malformations indicative of a disoder of laterality
determination including; incomplete lobulation of right lung (seven cases), malrotation of the gut (seven cases) and
persistence of the left superior vena cava (five cases). One such case with multiple laterality defects and sirelomelia was
found to have a de novo apparently balanced reciprocal translocation 46,XY,t(2;6)(p22.3;q12). Translocation breakpoint
mapping was performed by interphase fluorescent in-situ hybridization (FISH) using nuclei extracted from archival tissue
sections in both this case and an isolated bilateral renal agenesis case associated with a de novo 46,XY,t(1;2)(q41;p25.3). Both
t(2;6) breakpoints mapped to gene-free regions with no strong evidence of cis-regulatory potential. Ten genes localized
within 500 kb of the t(1;2) breakpoints. Wholemount in-situ expression analyses of the mouse orthologs of these genes in
embryonic mouse kidneys showed strong expression of Esrrg, encoding a nuclear steroid hormone receptor.
Immunohistochemical analysis showed that Esrrg was restricted to proximal ductal tissue within the embryonic kidney.
Conclusions/Significance: The previously unreported association of BRAHD with laterality defects suggests that renal
agenesis may share a common etiology with heterotaxy in some cases. Translocation breakpoint mapping identified ESRRG
as a plausible candidate gene for BRAHD.
Citation: Harewood L, Liu M, Keeling J, Howatson A, Whiteford M, et al. (2010) Bilateral Renal Agenesis/Hypoplasia/Dysplasia (BRAHD): Postmortem Analysis of 45
Cases with Breakpoint Mapping of Two De Novo Translocations. PLoS ONE 5(8): e12375. doi:10.1371/journal.pone.0012375
Editor: Pieter H. Reitsma, Leiden University Medical Center, Netherlands
Received May 6, 2010; Accepted July 20, 2010; Published August 25, 2010
Copyright: ? 2010 Harewood et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This project was funded by an intramural program grant in the name of David FitzPatrick and by a grant from the National Institutes of Health (NIH)
P50 DE16215. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
. These authors contributed equally to this work.
¤ Current address: Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland
Bilateral renal agenesis/hypoplasia/dysplasia (BRAHD) is
collective term used here to describe a group of lethal renal
malformations. The EUROCAT registry (a European network of
population-based registries for the epidemiologic surveillance of
congenital anomalies) reports an incidence of 1.3 per 10,000 live
births, fetal deaths/still births and terminations of pregnancy for
fetal anomaly (http://www.eurocat.ulster.ac.uk/pdf/EUROCAT-
Final-EC-Report.pdf). BRAHD shows a male preponderance with
a male:female ratio of 2.5:1. Potter syndrome describes the
constellation of extrarenal clinical features commonly associated
with BRAHD; wide-set eyes, ’squashed’ nose, receding chin, large,
low-set ears deficient in cartilage, deformity of the feet and hands
and hypoplasia of the lungs [1,2]. Potter syndrome is considered to
be the paradigm for a birth defect ‘‘sequence’’ in which a primary
malformation results in a series of secondary birth defects. In the
case of BRAHD, the primary malformation is an intrinsic error of
renal development with the other features of Potter syndrome
resulting from intrauterine deformation due to oligohydramnios. It
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should be noted, however, that Potter syndrome may also be the
result of obstruction of urinary excretion caused by a primary
abnormality of the lower urinary tract .
Kidney development in vertebrates begins with the formation of
the primary nephric duct - symmetric bilateral cords of epithelial
cells derived from the intermediate mesoderm . In humans, the
nephric duct appears at 22 gestational days (GD). Between 28 and
256 GD, there is transient formation of a functional embryonic
kidney, the mesonephros, which is structurally similar to the
glomerular and collecting systems found in fish . In males the
portion of the nephric duct that drains the mesonephros will form
the epididymis, seminiferous vesicle and vas deferens in post-
embryonic life. The fallopian tubes, uterus and upper vagina - the
Mu ¨llerian structures - develop from paramesonephic tissue in
female embryos . Finally, the definitive kidney or metanephros
is formed when the ureteric bud, an outgrowth of the distal
nephric duct, undergoes extensive branching and induces the
surrounding mesoderm to form glomeruli and nephrons [7,8].
Genetic factors are clearly an important component in the
etiology of BRAHD. There is significant familial aggregation of
cases with an empiric sibling recurrence risk for BRAHD of
approximately 5% [9–12]. Parents or siblings of infants with
BRAHD have an increased incidence of less severe kidney
malformations such as renal agenesis and/or renal dysplasia that
may be bilateral or unilateral, suggesting an autosomal dominant
condition with incomplete penetrance and variable expressivity
[13–15]. Complete non-penetrance has also been reported .
148 different syndrome entities associated with renal agenesis are
listed in the Winter-Barraitser Dysmorphology Database (London
Medical Databases, London). The most commonly reported
BRAHD-associated single gene disorders are Fraser syndrome
(OMIM 219000) [16,17] and Branchio-Oto-Renal syndrome
(BOR, OMIM 113650) .
Recently, mutations in RET have been identified in 7/19
(36.8%) of cases in a cohort of BRAHD . Eight different
mutations were identified in these seven BRAHD cases.
Surprisingly both activating and inactivating mutations in RET
were identified but no convincing mutations were found in the
genes encoding a co-receptor of RET, GFRA, or their ligand,
GDNF. These genes became strong candidates for BRAHD
following the identification of renal agenesis as a prominent
feature in mouse embryos with targeted inactivation of the loci
[20–22]. A missense mutation in the paired domain of PAX2 has
been identified in a single, large autosomal dominant family with
renal dysplasia as an isolated malformation showing highly
variable expression . This is interesting because heterozygous,
loss-of-function mutations in the gene encoding PAX2 have been
associated most commonly associated with renal-coloboma
syndrome (OMIM 120330).
In addition to genetic factors, there is a strong association
between BRAHD and maternal insulin-dependent diabetes
mellitus (IDDM) . This appears be the result of a direct
teratogenic effect of hyperglycaemia on specific tissues within the
cases of BRAHD with particular reference to the associated non-
renal malformations. We identify a strong and previously
unreported association with heterotaxy. We also report interphase
fluorescent in-situ hybridization (FISH) mapping of two de novo
apparently balanced chromosomal rearrangements associated with
BRAHD and determine the embryonic kidney expression of genes
positioned close to the breakpoints. Esrrg showed the strongest
expression during renal development in the mouse and may
represent a candidate gene for future functional and genetic studies.
Cases were ascertained by searches of the fetal and perinatal
post-mortem (PM) reports generated by a single regional pediatric
pathology service over an eleven-year period between 1993–2003.
This work was carried out in a manner consistent with ethical
principles for medical research involving human subjects outlined
in the Declaration of Helsinki and with the approval of the
Lothian Regional Ethics Committee (REF:2000/6/53). All
families had given written consent for research use of tissue and
clinical information at the time of post-mortem (PM) examination.
The PM records were stored in a custom-designed 4D database
(4D, San Jose, USA) using a structured vocabulary for coding
clinical features. All reports with evidence of bilateral renal
malformations or a diagnosis of Potter syndrome were reviewed in
detail. The term ‘agenesis’ was used when no kidney tissue could
be identified on autopsy, and the term ‘dysplasia’ was used when
abnormal kidney structure was described on autopsy or histology
e.g. cystic dysplasia. ‘Renal hypoplasia’ was used to describe a
significantly small kidney without cystic dysplasia. Information on
maternal age and parity, prenatal/birth history and associated
malformations was recorded. Reliable family history information
was generally unobtainable from the PM reports and most parents
had not been referred to clinical genetics services. The criteria for
inclusion in this study were lethal bilateral renal agnesis OR renal
hypoplasia OR renal dysplasia. Infants with unilateral renal
malformation with a normal contralateral kidney were excluded.
Cytogenetic reports were sought for each of the cases identified
with BRAHD in the searches detailed above using the comput-
erized archive of the clinical cytogenetic laboratory.
Interphase FISH Mapping
Interphase FISH mapping on nuclei isolated from clinically
archived formalin-fixed, paraffin-embedded (FFPE) tissue sections
was performed as previously reported . Briefly, 10 mm sections
were dissociated in pepsin (4 mg/ml in 10 mM HCl at 37uC) for
four hours, filtered using a 40 mm cell strainer (Falcon) and rinsed
through with PBS, before being fixed in 3:1 methanol:acetic acid
fix. BACs, PACs and fosmids were obtained from BACPAC
Resource Center (BPRC) at Children’s Hospital Oakland
Research Institute, or the Wellcome Trust Sanger Institute,
Cambridge. Clones were grown and DNA extracted according to
BPRC protocol. A full list of the clones used to map both
breakpoints is available in Table S2. FISH probes were produced
and FISH performed, according to standard methods, on fixed cell
suspensions  or on FFPE isolated nuclei suspensions (Hare-
wood, 2010). Chromosome paints were a kind gift from Dr Jeff
Interphase FISH mapping of reciprocal translocations requires
the examination of multiple (at least five fully interpretable) nuclei
for each probe to determine consistent co-localization or failure of
co-localization of co-hybridized pairs of BAC probes, or one BAC
probe and a chromosome paint (labeled with different fluoro-
chromes). In reciprocal translocations the presence of a normal
allele in each nucleus provides a useful control. This approach has
been successfully used to map disease-related reciprocal translo-
cations in both peripheral blood leukocyte nuclei  and FFPE
Expression analysis in mouse embryonic kidneys
Wholemount in-situ hybridization (WISH) was performed on
kidneys dissected from 14.5dpc wild-type mouse embryos. PCR of
mouse genomic DNA was used to generate riboprobe DNA
Bilateral Renal Agenesis
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templates for the 1p and 2p breakpoint genes and digoxigenin
(DIG, Roche)-labeled antisense riboprobes generated by in vitro
transcription using T7 RNA polymerase. Embryonic kidneys were
washed in PBST (PBS + 0.1% Tween 20) and permeabilised with
Proteinase K (10 mg/ml) for 20 minutes, then washed twice in
0.1 M triethanolamine, with the addition of acetic anhydride to
the second wash. The kidneys were re-fixed in 4% PFA/0.2%
gluteraldehyde for 20 minutes and washed extensively in PBST.
The kidneys were then placed in 2 ml tubes and prehybridized in
hybridization buffer for 2 hours in a 60uC shaking water bath
before being hybridized for 2 nights at 60uC in fresh hybridization
buffer containing the DIG-labeled probe. Probe solution was
removed and the kidneys washed twice in fresh hybridization
buffer, at 60uC, for ten minutes. Samples were then washed three
times in 2x SSC + 0.1% Tween 20 for 20 minutes per wash and
three times in 0.2x SSC + 0.1% Tween 20 for 30 minutes per wash
(all at 60uC) before being washed twice in maleic acid buffer
(MAB) for 15 minutes per wash at room temperature. MAB was
replaced with a MAB + 2% BMB (Boehringer-Mannheim
blocking reagent) + 20% heat-treated lamb serum solution and
kidneys were left for two hours at room temperature with gentle
agitation. After two hours, a 1/2000 dilution of anti-DIG antibody
coupled to alkaline phosphatase (Roche) in the same solution was
added and left overnight at 4uC, then washed three times in MAB
(5 min per wash) and three times in MAB for one hour per wash.
Color detection was performed using 2 ml of BM purple
precipitating solution (Roche).
Immunohistochemical localization of ESRRG was performed
using a rabbit polyclonal antisera (AbCam, cat ab12988) on
sections of paraffin embedded wild type CD1 mouse 14.5 dpc
embryos at a dilution of 1 in 500. 4–6 mm sections were de-waxed,
rehydrated and microwaved in boiling 10 mM citrate buffer twice
for 30 seconds and left to cool in the buffer for 20 minutes. Heat-
inactivated sheep serum as a 10% solution in PBS was applied for
at least one hour, at room temperarure, to reduce non-specific
binding. The slides were incubated in the same solution, with the
addition of the primary antibody, overnight in a humidified
chamber at 4uC, washed in PBS and PBST (five minutes each) and
the secondary antibody (biotinylated anti-rabbit IgG, 1 in 1000)
applied for one hour at room temperature before being washed as
before. Detection was performed using the Vector Lab ABC kit
with NBT/BCIP and sections were counterstained with eosin.
Basic Clinical Data
45 cases of bilateral renal agenesis and/or hypoplasia and/or
dysgenesis (BRAHD) were identified from postmortem records: 20
cases (44.4%) had bilateral renal agenesis, 15 (33.3%) had
unilateral renal agenesis (URA) with either hypoplasia or dysplasia
affecting the contralateral kidney, and 10 cases (22.2%) had
bilateral renal dysplasia. In the 15 URA cases, the right kidney was
absent in ten cases and the left kidney absent in five cases. Of the
bilateral dysplastic group there were three specific histological
diagnoses; two cases with bilateral medullary dysplasia (one of
these had Meckel Gruber syndrome) and one case with bilateral
renal tubular dysplasia (this case had trisomy 13). The male:female
ratio in the complete BRAHD cohort is 34:11 (3.1:1). In the cases
with either unilateral or bilateral renal agenesis the ratio is 29:6
(4.8:1). In the ten cases with bilateral renal dyplasia there was an
equal sex distribution (5:5). As expected, oligohydramios-associat-
ed deformations were common - characteristic ‘‘Potter facies’’
were reported in 38/45 (84.4%) cases, lung hypoplasia in 23/45
(51.1%) and talipes equinovarus in 25/45 (55.6%).
Associated Non-renal Malformations
31/45 (68.9%) cases had one or more extrarenal malformations
(male:female ratio 2.44:1). There were 175 extrarenal malforma-
tions recorded in these cases consisting of 80 different malforma-
tion types. A full list of the associated malformations is given in
Table S1. 27 different extrarenal malformations were identified in
two or more cases (Table 1). The most common categories of
major malformations were: GI tract atresias (13 cases of anorectal
atresia, four cases of esophageal atresia, two cases of colonic atresia
and one case of multiple small bowel atresia) and axial skeletal
malformations (seven cases of thoracic vertebral malformation,
three cases with an abnormal number of pairs of ribs, two cases of
lumbosacral vertebral malformations, two cases of sacral agenesis
and one case of cervical rachischisis). A definitive autosomal
recessive syndromal diagnosis could be made on clinical grounds
in three cases; Meckel Gruber syndrome (OMIM 249000), Renal-
Hepatic-Pancreatic Dysplasia syndrome (OMIM 208540) and
Fraser syndrome (OMIM 219000) (Table 2).
The cases with extrarenal malformations were further charac-
terized by whether they had any component of three well known
malformation associations (Table 2) considered to overlap with
BRAHD, namely VACTERL association (vertebral malformation,
anal atresia, cardiovascular anomalies, tracheo-esophageal fistula,
Table 1. Extrarenal malformations seen in more than one
Cervical/thoracic vertebral malformation9
Incomplete lobulation right lung7
Malrotation of gut7
Meckel Diverticulum 4
Persistent left superior vena cava5
Ventricular septal defect (VSD)4
Transposition of the great arteries (TGA)3
Abnormal number of pairs of ribs3
Ductal Plate Malformation2
Lumbosacral vertebral malformations2
Malformation of olivary nucleus2
Postaxial polydactyly of upper limbs2
Preaxial polydactyly of lower limbs2
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Table 2. Analysis of Reported Association in Cases with Extrarenal Malformations.
Mat IDDM Cat
Mosaic trisomy 8
Hypoplastic and Dysplastic
Hypoplastic & Cystic Dysplasia
& Cystic Dysplasia
Hypoplastic & Cystic Dysplasia
Meckel Gruber Syndrome
Abbreviations: V – Vertebral malformations, A – Anal atresia, C – Cardiovascular anomalies, TE – Tracheo-esophogeal fistula/Esophogeal atresia, R – Renal anomalies, L – Limb malformations, Mu ¨ – Mu ¨llerian duct anomalies, CD -
Caudal dysgenesis, HFM – Hemifacial microsomia, Lat - Defect in Laterality Determination A - abdominal laterality defect, R - respiratarory tract laterality defect, C - cardiac laterality defecr Mat IDDM – Maternal insulin-dependent
diabetes mellitus, Cat – Category: Chr – Chromosomal abnormality, AR – Autosomal recessive.doi:10.1371/journal.pone.0012375.t002
Bilateral Renal Agenesis
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esophageal atresia, renal anomalies, limb malformations [OMIM
192350]) , Mu ¨llerian duct anomalies (MURCS association
[OMIM 601076], Mayer-Rokitansky-Kuster-Hauser Syndrome
[OMIM 277000]) [31,32] or hemifacial microsomia (Goldenhar
syndrome/Oculo-auriculo-vertebral syndrome [OMIM 164210])
[33,34]. Excluding the renal malformations for which the cases
were selected, 18/31 (58.1%) cases had at least one other feature
consistent with VACTERL association (and hence a VACTERL
score of 2), 15/31 (48.4%) had two or more other features
(VACTERL score of 3), 10/31 (32.2%) had three or more
(VACTERL 4) and 5/31 (16.1%) had four other components of
VACTERL. Three of the nine female BRAHD cases with
extrarenal malformations had Mu ¨llerian duct anomalies and 3/
31 (9.7%) cases had hemifacial microsomia.
Partial lateralitydefectswere identified in 16/31 (51.6%) cases. The
most common manifestation of heterotaxy was incomplete lobulation
of right lung (seven cases), malrotation of the gut (seven cases), and
persistence of the left superior vena cava (five cases) (Table 2).
Cytogenetic Analysis and Clinical Details of Translocation
Chromosome studies were obtained in 32 of the 45 cases. In
3/32 (9.4%) of the cases the chromosomal analysis was reported as
abnormal. Two numerical chromosome abnormalities were
identified: PM number 96221 with 46,XX,+13 and 3081 with
47,XY,+8/46XY. One structural chromosomal anomaly
was detected - a de novo, apparently balanced reciprocal
translocation, 46,XY,t(2;6)(?p23;?q14), in a pregnancy that was
terminated at 18 weeks of gestation following an ultrasound
diagnosis of anencephaly with multiple other congenital anoma-
lies. The mother had insulin-dependent diabetes mellitus. On
autopsy, this fetus had craniorachischisis extending to the upper
thoracic region (Figure 1). There was marked facial asymmetry
associated with right-sided anophthalmia and a wide ‘‘irregular’’
cleft of the upper lip. The left upper limb was present and had
mild radial aplasia. The right upper limb was absent with a small
digit attached to the upper thorax. There was sirenomelia
associated with anal atresia and malformed genital tubercle.
There was a single umbilical artery. Internal examination revealed
a normal stomach, duodenum, pancreas and spleen. The lungs
had undergone only rudimentary lobulation. The right kidney was
absent and the left kidney had cystic dysplasia. The ureters were
narrow and inserted into the blind ending bowel. There was
transposition of the great arteries and a ventricular septal defect.
In the course of this study we identified another case of BRAHD
associated with a de novo, apparently balanced reciprocal
translocation. Details of the clinical and cytogenetic features of
this case have been reported elsewhere  although no molecular
mapping of the breakpoints had previously been carried out.
Briefly, this was the first pregnancy of a healthy and non-
consanguineous couple. At 29 weeks, oligohydramnios was noted
and an anomaly scan was suggestive of bilateral renal agenesis. At
32 weeks, lung hypoplasia was apparent and the parents opted to
have labor induced. The baby died an hour after birth. On PM
examination the baby was male and had features of Potter
sequence, including a flattened nose, large squashed ears, rocker
bottom feet and marked skin laxity over the trunk and limbs.
Hemorrhagic masses with no recognizable renal structuring were
found in the place of kidneys. Histology revealed undifferentiated
mesenchyme with foci of cartilage. Cytogenetic analysis identified
an apparently balanced reciprocal translocation, 46,XY,t(1;2)
(q32;p25) which had occurred de novo.
Interphase FISH Mapping
Unfortunately, no metaphase chromosome preparations or viable
cell-lines were available from either of the de novo translocation cases.
We therefore proceeded to map both translocations using interphase
nuclei from paraffin embedded tissue by observing the presence or
absence of co-localization of pairs of fluorescently labeled BAC
case showed that the 1q breakpoint lies between BACs RP4-723P6
(chr 1: 216,265,171-216,278,164 GRCh37) and RP11-239I22
(chr 1: 216,441,980-216,535,735 GRCh37) (Figure 2). The USH2A
gene is therefore directly interrupted. The only other gene in the
region encodes the orphan nuclear hormonereceptor,ESRRG. The
2p breakpoint is flanked by BACs RP11-410L9 (chr 2: 2,912,073-
3,034,544 GRCh37) and RP11-568H24 (chr 2: 3,636,396-
3,769,755 GRCh37). Thus, the molecularly-corrected karyotype is
Figure 1. Clinical and radiological features of t(2;6) case. A. shows a left lateral photograph of this fetus with craniorachischisis and severely
abnormal facial structures. B. Lateral radiograph showing the left upper limb with mild radial aplasia and the absence of the right upper limb. The
axial skeleton is severely abnormal with multiple thoracocostal anomalies. C. AP radiograph showing sirenomelia and extensive axial skeletal
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The breakpoints in the t(2;6)(?p23;?q14) were mapped and the
2p22 breakpoint was found to lie between RP11-257N21 (chr 2:
34,357,572-34,549,712 GRCh37) and RP11-153O16 (chr 2:
35,167,661-35,257,709 GRCh37), and the 6q breakpoint between
RP11-286F19 (chr 6: 66,563,349-66,734,396 GRCh37) and
RP11-712I16 (chr 6: 66,749,259-66,912,466 GRCh37) (Figure 3).
Thus, the corrected karyotype is 46,XY, t(2;6)(p22.3;q12). Neither
breakpoint disrupts a known gene or lies within 200 kb of one.
Analysis of both regions using ANCORA (http://ancora.genereg.
net/) revealed no evidence of clustering of conserved non-coding
regions, as is commonly seen in gene desert regions showing long-
range cis-regulatory control. A full list of the clones used to map all
breakpoints is available in Table S2.
Expression Analysis of Breakpoint Genes
Wholemount in-situ hybridization (WISH) was performed on
14.5dpc embryonic mouse kidneys using riboprobes designed to
detect expression of the mouse orthologs of the genes at the t(1;2)
breakpoints. In the 1p BP region, no expression of the gene
disrupted by the breakpoint, Ush2a, was detectable but the
neighboring gene, Esrrg, was strongly expressed (Figure 4).
Immunohistochemical staining of sections of 14.5dpc embryonic
mouse kidneys using an antibody against Esrrg showed expression
in the future collecting ducts and kidney capsule. WISH expression
analysis of six of the genes from the 2p breakpoint region showed
intermediate-strong expression of Adi1, intermediate levels of
expression in Tssc1, Ttc15 and Allc, and no detectable expression of
Rnaseh1 or Collec11.
An important and consistent finding in this study and others
[1,11] is that renal agenesis, renal hypoplasia, dysplastic and cystic
dysplastic kidneys can co-occur in individuals with bilateral lethal
disorders of kidney development. This suggests that these
apparently distinct clinical endpoints may result from identical
or related developmental pathological processes. It is also clear
that, although BRADH does occur as an isolated malformation, in
the majority of cases it is part of a more general developmental
disturbance i.e. the causative genetic or environmental teratogens
affect pathways that are critical to both renal and extrarenal
development. The scientific rationale for studying patterns of
associated abnormalities is to provide clues to specific cellular or
morphogenetic processes that may aid the search for causative
factors of BRADH. Previous studies have indeed highlighted
specific extrarenal malformations that appear to be non-randomly
associated with BRAHD, including skeletal anomalies (hemiver-
tebrae, sacral agenesis , sirenomelia , radial aplasia ),
cloacal anomalies (imperforate anus with rectovaginal fistula
[39,40]) and Mu ¨llerian defects (partial atresia of the vagina,
fallopian tubes and uterus in female cases) ). An overlap has
been noted with the well-known malformation associations:
VACTERL (vertebral malformation, anal atresia, cardiovascular
anomalies, tracheo-esophageal fistula, esophageal atresia, renal
anomalies, limb malformations [OMIM 192350]) , MURCS
association (Mu ¨llerian duct aplasia, unilateral renal agenesis, and
cervicothoracic somite anomalies [OMIM 601076]), hemifacial
microsomia ([OMIM 164210]) [33,34] and caudal dysgenesis
spectrum (sacral agenesis, sirenomelia [OMIM 600145]) .
Our case series supports each of these reported associations but
none of these help in elucidating the underlying developmental
pathologies. The most convincing human genetic current evidence
implicating a specific pathway is the finding of RET mutations in
one third of the BRAHD cases in a single cohort . In the same
study, no convincing mutations werefound in genes encoding either
the RET ligand (GDNF) or its co-receptor, GFRA. No associated
abnormalities were reported in the cases with RET mutations,
although mutations in this gene have been associated with another
malformation, Hirschprung disease (OMIM 142623) . GDNF/
RET signaling is obviously critical for aspects of neural crest and
ureteric bud function but, frustratingly, its relationship to other
developmental signaling cascades is not yet clear. Apart from RET
signaling, there is interesting but relatively weak human genetic
evidence implicating Wnt signaling in the control of planar cell
polarity. A heterozygous mutation in WNT4, encoding a signaling
molecule that is thought to repress tissue-specific androgen
production in female embryos, has been reported in a female with
Mu ¨llerian abnormalities associated with unilateral renal agenesis
. This is supported by in vitro work showing that siRNA knock-
down of Wnt4 in cultured embryonic mouse kidneys has a specific
effect of blocking nephron development . Mutations in
VANGL1, a negative regulator of planar cell polarity/Wnt-signaling
,havebeen identified ina case with caudaldysgenesis.This
association is supported by studies of the Isl1-transgenic mouse
model of caudal dysgenesis, which has been shown to have
abnormal embryonic expression of several genes encoding compo-
nents of the Wnt-signaling pathway. How, or if, WNT4 and
VANGL1 interact in vivo is yet to be defined.
It can be seen from the above data that very large gaps remain
in our knowledge of the embryopathology that results in human
BRAHD. The motivation for the present study was to find clinical
and cytogenetic clues that may identify other molecular players in
this disease. Probably the most interesting result was the previously
unrecognized finding that one third of cases in our cohort had one
or more extrarenal malformations, indicative of a disorder in
laterality determination. Disorders of laterality can be divided into
Figure 2. Mapping of the t(1;2) breakpoints using interphase FISH analysis. As no metaphase spreads or viable cells were available from
this case, interphase mapping was performed using dissociated nuclei from archival tissue sections. The mapping of each clone was confirmed on
multiple nuclei to confirm the presence or absence of co-localization. A. Two representative nuclei used to map probes flanking the 1q breakpoint.
On the left is the merged color image with the green and red channels of the same image shown in the middle and the right-hand-side respectively.
The top nucleus shows co-localization of the FISH signals for RP11-22M7 and RP4-723P6 on both chromosomes (white arrows), which together with
other results shown in Table S2, can be used to map the more telomeric probe (RP4-723P6) to the der(1). The lower nucleus demonstrates the utility
of chromosome arm paints in interphase mapping. The green channel image shows three clear 1q chromosomal paint domains within the nucleus.
On the basis of the relative volume of these domains and with knowledge of the cytogenetic location of the translocation breakpoint, it is possible to
assign these domains to the normal chromosome 1 (1(N), the largest domain) and the derivative chromosomes (der(1), middle-sized domain and
der(2), smallest domain). It can be seen that the RP11-239I22 probe maps to the der(2) and is therefore distal to the 1q breakpoint. B. The physical
map of the breakpoint region of 1q with the orange box indicating the region of genomic DNA that could plausibly contain the breakpoint. There are
only two genes in this region with one of these, USH2A, being directly disrupted by the breakpoint. C. Two representative nuclei used in mapping the
2p breakpoint. The overall configuration is identical to A. The upper nucleus shows colocalization of RP11-410L9 with a probe (RP11-352J11) that has
been shown to map telomeric of the 2p breakpoint. The lower nucleus shows failure of colocalization of RP11-568H24 on one allele, showing that this
maps to the der(2). D. The physical map of the breakpoint region (orange box) on 2p. Unfortunately, it was not possible to map this breakpoint
further. Five genes could plausibly be disrupted by this breakpoint; TSSC1, TTC15, ADI1, RNASEH1 and RPS7.
Bilateral Renal Agenesis
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Figure 3. Mapping of the t(2;6) breakpoints using interphase FISH analysis. A. Cohybridization of mini-paints (groups of adjacently
mapping BACs hybridized together) for 2p25.1 and 2p22.1 demonstrating that the normal (2p) and derivative chromosomes (der(2) and der(6)) can
be clearly distinguished on interphase mapping. B and C. Representative images showing mapping of the clones flanking the 2p breakpoint with
RP11-257N21 mapping to the der(6) and RP11-153O16 to the der(2) when co-hybridized with the 2p22.1 and 2p25.1 paint respectively, which are on
opposite sides of the breakpoint. D. Diagrammatic representation of the 2p breakpoint region with the orange boxed region containing the
breakpoint. There are no known protein-coding genes in this region of DNA. E and F. Mapping of the 6q breakpoint showing the centromeric flanking
clone, RP11-286F19, showing colocalization with the centromeric probe (6cen) and the absence of colocalization with the 6q12 paint (which is
telomeric to the breakpoint) on one allele. G. Colocalization of the telomeric flanking clone, RP11-712I16, with the 6q12 paint. H. Diagrammatic
representation of the 6q genomic region with the orange box representing the location of the translocation breakpoint. This region contains no
known protein coding genes.
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complete reversal of normal visceral situs (situs inversus) or partial
reversal - left isomerism, right isomerism or heterotaxy. Situs
inversus is strongly associated with primary ciliary dyskinesias
[47,48] and there is also a well known association between
ciliopathies and renal cystic disease. Heterotaxy is character-
ized by specific malformations of abdominal structures (malrota-
tion of the gut, polysplenia, midline liver etc.), early heart
development (doubling or loss of normally asymmetrical struc-
tures) and the lungs (abnormal lobulation). The strong association
of these malformation categories with BRADH suggests that they
may share a common etiology. This is particularly interesting
given the very striking excess of male cases in this group because
mutations in the gene encoding the zinc finger transcription factor,
ZIC3, have been shown to segregate with cardiac and abdominal
heterotaxy as an X-linked recessive trait . Whilst there are no
previous reports of renal agenesis associated with this gene, it is
interesting to note that one affected male in the original report had
renal hypoplasia. Other genes and pathways have been implicated
in human heterotaxy, for example intragenic mutations have been
identified in CFC  and ACVR2B , which are both
components of a transforming growth factor-beta signaling system.
On reviewing the available cytogenetic results for our BRAHD
cohort we identified one male case with a de novo, apparently
balanced reciprocal translocation 46,XY,t(2;6)(p22.3;q12). This
case had a severe phenotype with multiple malformations
associated with BRAHD including sirenomelia and cardiac,
abdominal and pulmonary evidence of heterotaxy. We mapped
this translocation to determine if either breakpoint disrupted a
gene that could be a plausible candidate for the associated
phenotype. However, both breakpoints mapped to gene-free
regions. There are several possible explanations for this finding:
First, the translocation could be coincidental - the fact that the
mother of this fetus had insulin-dependent diabetes mellitus, a
known risk factor for BRAHD , may make this more likely;
Second, one or both of the breakpoint regions may be critical for
the cis-regulation of one or more neighboring genes, although
Figure 4. Wholemount in-situ hybridization (WISH) of breakpoint genes on embryonic mouse kidneys. A. Diagram of the 1q genomic
region with orange box indicating the breakpoint region. B. WISH of the mouse orthologs of the 1q breakpoint genes with representative kidneys from
14.5dpcmouseembryosshowingundetectable expression ofUsh2a andstrong expression ofEsrrg. C.ImmunohistochemicalanalysisofEsrrg in14.5dpc
mouse kidney sections. Strong staining is seen in the collecting ducts (arrows) with weaker staining in the mesothelial capsule (arrow heads) of the
kidney(kid)andadrenalgland(adr).D.Diagramof2pgenomicregionwithorangeboxindicatingthe breakpoint region.E.WISHofthemouseorthologs
of the 2p breakpoint genes with representative kidneys from 14.5dpc mouse embryos showing undetectable expression of Colec11 and Rnaseh1,
intermediate expression of Tssc1, Ttc15 & Allc and stronger expression of Adi1. No satisfactory probe template could be amplified for Tmsl2 or Rps7.
Bilateral Renal Agenesis
PLoS ONE | www.plosone.org9 August 2010 | Volume 5 | Issue 8 | e12375
evidence against this comes from the lack of clustering of
conserved non-coding regions in these regions or chromatin
evidence of enhancer activity in publicly available datasets; Third,
the causative mutation may be a deletion in cis with one of the
breakpoints, which is a relatively common finding in de novo
translocations . However, we were unable to perform an
array-based comparative genomic hybridization on this case as
there was no high quality genomic DNA available.
We also report the mapping of another de novo translocation in
an unrelated male case of isolated BRAHD. We reported clinical
and cytogenetic details of this case some years earlier . The
1q41 breakpoint was found to disrupt USH2A, a gene that is
mutated in the autosomal recessive disorder Usher syndrome 2A
(OMIM 276901). This gene was not a good candidate for
BRAHD as it was not expressed in the embryonic kidney and
homozygous or compound heterozygous loss-of-function muta-
tions do not cause a renal phenotype in humans or knockout mice
(Dominic Cosgrove, personal communication). However, we
found that the mouse ortholog of the neighboring gene, ESRRG,
was strongly expressed in mouse embryonic kidneys. Immunohis-
tochemical analysis shows that Esrrg localizes to developing
collecting ducts in the 14.5dpc embryonic kidney. ESRRG,
therefore, seemed to be a reasonable candidate gene for BRAHD.
However, targeted inactivation of Esrrg in mouse  revealed
that most homozygous mice die in the first week of life with heart
failure, and that no renal phenotype has been noted in these
animals. At the 2p25.3 breakpoint several genes could be disrupted
or deleted. Expression analyses in embryonic mouse kidneys
showed that only Adi1 was moderately highly expressed. ADI1
encodes a protein with homology to bacterial aci-reductone
dioxygenase, which functions in the methionine recycling
pathway. ADI1 localizes to both the cytoplasm and nucleus and
has been implicated in both mRNA processing and induction of
apoptosis [55,56]. No mouse knockout for Adi1 is currently
available but it is a potential candidate gene for BRAHD.
This clinical and cytogenetic review of a large cohort of
BRAHD cases from a single center has confirmed much of the
previously reported clinical associations. In addition, we have
identified a strong association of BRAHD with heterotaxy, which
implicates a series of signaling pathways and transcription factors
that are involved in establishing laterality. The translocation
mapping in a case of isolated BRAHD has yielded two plausible
candidate genes that merit further investigation. We plan to
continue to examine ESRRG as a candidate gene by performing
functional analyses in embryonic cultured kidneys and assessing
renal development in the ‘‘knock-out’’ mouse. We hope that our
study will stimulate interest in this common lethal human
spectrum of malformations.
Found at: doi:10.1371/journal.pone.0012375.s001 (0.12 MB
Full list of associated malformations in BRAHD cases.
Found at: doi:10.1371/journal.pone.0012375.s002 (0.13 MB
Mapping details of all BAC probes used for Interphase
We would like to express our sincere thanks to the families of the cases
reported here for agreeing to research use of the anonymised data and
Conceived and designed the experiments: DRF. Performed the experi-
ments: LH ML PB JF. Analyzed the data: JWK DRF. Contributed
reagents/materials/analysis tools: JWK AH MW ME. Wrote the paper:
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