grahambull · Web view(2008) A cryptic duplication 22q13.31 to qter leads to a distinct phenotype...
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22q11.2 duplications in a UK cohort with bladder exstrophy-epispadias complex
Glenda M. Beaman1,2, Adrian S. Woolf3,4 Raimondo M. Cervellione4, David Keene4, Imran
Mushtaq5, Jill E. Urquhart2 , Helen M. Stuart1,2, William G. Newman1,2,6
1Evolution and Genomic Sciences, School of Biological Sciences, Faculty of Biology,
Medicine and Health, University of Manchester, Manchester, UK.
2Manchester Centre for Genomic Medicine, Manchester University NHS Foundation Trust,
Manchester, UK.
3Division of Cell Matrix Biology & Regenerative Medicine, School of Biological Sciences,
Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK.
4Royal Manchester Children’s Hospital, Manchester University NHS Foundation Trust,
Manchester, UK.
5Department of Paediatric Urology, Great Ormond Street Hospital for Children NHS
Foundation Trust, London, UK.
6Peking University Health Sciences Center, Beijing, PR China
Correspondence:
Professor WG Newman,
Manchester Centre for Genomic Medicine,
St Mary’s Hospital,
Manchester M13 9WL, UK.
E-mail: [email protected]
ABSTRACT
The bladder exstrophy-epispadias complex (BEEC) comprises of a spectrum of anterior
midline defects, all affecting the lower urinary tract, the external genitalia and the bony
pelvis. In extreme cases, the gastrointestinal tract is also affected. The pathogenesis of
BEEC is unclear but chromosomal aberrations have been reported. In particular,
duplications of 22q11.2 have been identified in eight unrelated individuals with BEEC. The
current study aimed to identify chromosomal copy number variants in BEEC. Analyses was
performed using the Affymetrix Genome-wide SNP6.0 assay in 92 unrelated patients cared
for by two UK pediatric urology centres. Three individuals had a 22q11.2 duplication, a
significantly higher number than that found in a control group of 12,500 individuals with
developmental delay who had undergone microarray testing (p<0.0001). Sequencing of
CRKL, implicated in renal tract malformations in DiGeorge syndome critical region at 22q11,
in 89 individuals with BEEC lacking 22q11 duplications revealed no pathogenic variants. To
date, 22q11.2 duplication is the genetic variant most commonly associated with BEEC. This
is consistent with the hypothesis that altered expression of a single, yet to be defined, gene
therein is critical to the pathogenesis of this potentially devastating congenital disorder.
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INTRODUCTION
The mammalian urinary bladder serves as a storage organ for urine made by the kidney,
intermittently voiding its contents through the urethra. In humans, the bladder rudiment is
evident at the sixth week of gestation, when the cloaca has become divided into the
urogenital sinus and hindgut. Over the next two months, the bladder wall differentiates into
detrusor smooth muscle, itself invaded by autonomic nerves, with the bladder lumen lined
by a water-tight urothelium (Jenkins et al 2007; Stuart et al 2013). The human bladder is
subject to a range of malformations including: prune belly and megacystis-microcolon-
intestinal hypoperistalsis syndromes, where the detrusor is acontractile; urofacial syndrome,
where there is a functional bladder outflow obstruction; and vesicoureteric reflux, where
urine moves retrogradely from bladder into the upper tract (Woolf et al 2014). Importantly,
such diseases can manifest as monogenic diseases with the implicated genes coding for
molecules affecting neural (Weber et al 2011; Newman and Woolf 2018), smooth muscle
(Thorson et al 2014) and epithelial (Jenkins et al 2005) biology.
Human urinary tract malformations also feature in the bladder exstrophy-epispadias
complex (BEEC) (MIM 600057). The term BEEC describes a spectrum of midline anomalies
of the anterior abdominal wall, bony pelvis, urinary tract, genitalia, hindgut and spine
(Purves and Gearhart, 2010). The mildest form is epispadias, where the urethra opens on
the anterior surface of the penis rather than on its top. The next most severe form is classic
bladder exstrophy (CBE) where the ventral part of the bladder is open, with urothelium
exposed on the body surface. The most severe form is cloacal exstrophy (CE) where there
is an imperforate anus and the gasto-intestinal tract is abnormal.: This variant is also called
the omphalocele, exstrophy, imperforate anus and spinal defects (OEIS) complex. A
European survey of pediatric urology centres revealed that they were caring for 238 babies
that had been born with BEEC over a single year: 71 cases had epispadias, of which 92%
were male; 146 had CBE, of which 66% were male; and 21 had CE, of which 81% were
male (Cervellione et al, 2015). A congenital anomaly survey of 824,368 fetuses, stillbirths
and live-born babies in Northern England calculated the prevalence for BEEC as 5.2 per
100,000 (Jayachrandran et al, 2011). Among 26.3 million international births, CBE had an
overall prevalence of 2.07 per 100,000, with a positive correlation with maternal age (Siffel
et al, 2011). A large international study calculated the prevalence of CE as 0.76 per 100,000
live births, with no relation to maternal age (Feldkamp et al, 2011). Positive associations
have been noted for CE with “use of any fertility medication or assisted reproductive
technology” (Keppler-Noreuil et al, 2017). Only 10% of BEEC cases are detected by routine
antenatal ultrasonography (Jayachrandran et al, 2011).
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Most individuals affected by BEEC have no associated congenital anomalies, and have no
positive family history for BEEC. A twin study reported significantly higher concordance rates
for BEEC in monozygotic (62%) compared with dizygotic (11%) twins (Reutter et al, 2007),
consistent with a strong genetic effect. Wilkins et al, 2012, reported that an insertion /
deletion polymorphism in the P63 promoter, which controls expression of an isoform
ΔNp63, was a significant risk factor for BEEC. A genome-wide association study in CBE
identified a variant at 5q11.1-q11.2 close to ISL1 (ISL LIM homeobox 1) (Zhang et al
2017). This association together with functional studies in mouse embryos and zebrafish
larvae suggest ISL1 as a major susceptibility gene for CBE and as a regulator of urinary
tract development.
Further, a number of different chromosomal aberrations have been reported in individuals
with BEEC. These include: a de novo unbalanced translocation between the long arms of
chromosomes 9 and Y resulting in a 9q34.1-qter deletion (Thauvin-Robinet et al, 2004); a
balanced translocation, 46,XY,t(8;9)(p11.2;q13), with the 9q13 breakpoint disrupting
CNTNAP3 (Boyadjiev et al, 2005); and the deletion del(3)(q12.2q13.2) (Kosaki et al, 2005).
Microduplications involving 22q11.2 have been reported in a number of individuals with
BEEC, including two out of 69 cases versus one of 171 controls (Lundin et al, 2010); out of
66 BEEC cases, one with a de novo microduplication of 22q11.21 and one where the
duplication transmitted from the unaffected mother (Draaken et al, 2010); and 22q11.21
duplications in four of 244 BEEC cases and one of 665 controls. Considering these eight
cases with 22q11.21 duplications (summarized in Table 1 of this paper), it was calculated
that this conferred an odds ratio of 31.86 (Draaken et al, 2014).
The aim of this present study was to identify potentially pathogenic copy number variants
(CNVs) in individuals with BEEC in a previously unstudied UK cohort. We report two
unrelated cases with a 22q11.21 duplication out of 92 patients. In addition, we report a distal
22q11.23 duplication. These findings provide further support that 22q11.2 duplication
predisposes to BEEC.
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MATERIALS AND METHODS
PatientsNinety two individuals with BEEC of white British descent were studied. They were recruited
from a single centre. All 92 had isolated BEEC of which 56 had CBE (37 male, 19 female),
18 had CE (7 male, 11 female), and 18 had epispadias (13 male, 5 female). Following
ethical approval (HRA 68248) written informed consent was taken from all participants or
their parent(s) if they were too young to provide consent. Blood or saliva was collected from
index cases, and from their parents were possible, and genomic DNA was isolated using
standard automated processes. A total of 12,500 unrelated individuals with developmental
delay undergoing microarray analysis from our in house database were used as a
comparator.
SNP6 Copy-Number Array AnalysisDNA from index cases was analyzed for copy number variations using the Genome-Wide
Human SNP Array 6.0 (Affymetrix, Inc., Santa Clara, CA). Genomic DNA was hybridized to
a SNP6 array according to the manufacturer’s instructions. Arrays were stained using a
Fluidics Station 450 (Affymetrix, Inc.) and scanned using the GeneChip scanner 3000 7G
system (Affymetrix, Inc.). Copy number data were generated using the SNP 6.0 CN/LOH
Algorithm within the Affymetrix Genotyping console v4.2 and analyzed using Chromosome
Analysis suite v2.1. The copy-number detection threshold was set to 0.02, with a minimum
size of deletion / duplication of 100kb. Chromosomal annotation is based on from NCBI
Build 37/hg19.
Droplet digital PCRDroplet digital PCR (ddPCR) was carried out using the QX100 Droplet Digital PCR system
(Bio-Rad Laboratories, Hemel Hempstead, UK). Patient and control DNA samples were
normalized to 20 ng/µl. Three assays were created for each assessed exon, by adding
patient DNA, control DNA or sterilized water to a solution containing: Bio-Rad ddPCRTM
Supermix for Probes (No dUTP; 2x), water, target probe (FAM), reference target probe
(HEX). Oil droplets were created by mixing with Bio-Rad Automated Droplet Generation Oil
for Probes. Forty cycles of PCR were performed using a Verity Thermal Cycler Droplets
were analyzed using a Bio-Rad QX200 Droplet Digital Reader and CNV status was
calculated with QuantaSoft software by comparing the ratio of fluorescent probes for the
reference region and the region of interest.
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Sanger sequencing of CRKLPrimers were designed for individual exons of CRKL (NM_005207)_ with Primer3
(http://frodo.wi.mit.edu/). PCR was performed on genomic DNA. and products purified using
Agencourt AMPure XP (Beckman Coulter Genomics, Krefeld, Germany). DNA sequencing
was performed using the BigDye Terminator Cycle Sequencing Kit (version 3.1; Life
Technologies, Paisley, United Kingdom). Sequencing products were purified using
Agencourt CleanSEQ (Beckman Coulter Genomics), and sequence analysis was performed
using the ABI 3730xl DNA Analyzer (Life Technologies). Primer sequences and
experimental conditions available on request.
RESULTS
Six CNVs were identified comprising two duplications of 22q11.21, one distal duplication of
22q11.23 and three rare CNVs (Table 2). Screening of the microarray data from the 12,500
individuals in the in-house comparator group, predominantly undergoing testing for
developmental delay, revealed 34 duplications at the 22q11.2 locus. The overall incidence
of 22q11.2 duplications in our total BEEC cohort of 92 patients compared with the total
number of 12,500 controls was 3.3% (cohort) versus 0.27% (controls) (odds ratio 12.36;
95% confidence interval, 3.73-40.98; p<0.0001). Here, and in Tables 1 and 2, we provide
details of individual patients and their genetic results.
1. A male with CBE born to non-consanguineous healthy parents had a duplication at
22q11.21, approximately 2.73Mb (18,884837-21,611,337), encompassing 67 genes from
DGCR6 to BCRP2 (Figure 1a). The child also had bladder polyps and bilateral inguinal
herniae. He has normal upper urinary tracts and a normal plasma creatinine of 19 M/L.
Parental testing revealed that the duplication was inherited from the unaffected mother.
2. A male with epispadias born to non-consanguineous parents had a duplication (Figure
1b) at 22q11.21, estimated to be ~2.41Mb (19,059,071-21,465,835), encompassing 62
genes from DGCR2 to BCRP2. This duplication was inherited from his healthy mother.
Following bladder neck reconstruction he developed upper urinary tract dilatation and
moderate kidney failure, the most recent creatinine being 91M/L. Because of progressive
kidney impairment he underwent an ileal conduit to drain the bladder.
3. A male with CBE born to non-consanguineous parents had a duplication at 22q11.23,
estimated to be 1.4Mb (23,664,430-25,064,964) (Figure 1c) encompassing 39 genes from
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CES5AP1 to POM121L10P. He has normal renal tract and his latest creatinine is 18M/L.
The duplication was inherited from a healthy unaffected father.
The 22q11.21 and 22q11.23 duplications were orthogonally confirmed using ddPCR.
In addition, three CNVs of uncertain significance were identified in three unrelated
individuals, as follows.
4. In a female with CE, a 1.4 Mb duplication was detected at 3p26.3 (955,800-2,101,328).
This duplication encompasses CNTN6, coding for contactin 6. This patient’s latest creatinine
was 22 M/L.
5. A male with CBE was found to carry a 3.3 Mb deletion on chromosome 4p15.2
(27,110,192-30,421,576) encompassing MIR4275 coding for microRNA 4275. This deletion
is not listed in Decipher. This patient’s latest creatinine was 47 M/L.
6. A female with CE and a lumbosacral lipomeningocele was found to have a 0.6 Mb
duplication at 22q13.31 (47,114,520-47,757,314). This duplication included CERK, coding
for ceramide kinase, and TBC1D22A, coding for TBC1 domain family member 22A. The
patient’s most recent creatinine level was 42 M/L.
Loss of function variants in CRKL have been reported in individuals with congenital
anomalies of the kidneys and urinary tract malformations (Lopez-Rivera et al 2017). Notably
CRKL lies in the 22q11 deletion critical region. In the current study, Sanger sequencing
CRKL in the 89 individuals without a 22q11 duplication did not identify any putative
pathogenic variants.
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DISCUSSION
The 22q11.2 locus is susceptible to copy number variation mediated by inter- or intra-
chromosomal non-allelic homologous recombination (NAHR) caused by misalignment of
segmental duplications or low-copy repeats (LCRs) (Edelmann et al, 1999; Shaikh et al,
2007). The most common rearrangements involving eight LCRs (LCR22A – LCR22H)
(Edelmann et al, 1999) are deletions implicated in genomic diseases including
velocardiofacial syndrome (VCFS) (MIM 192430) (Shprintzen et al 1978) and reciprocal
duplication resulting in 22q11.2 duplication syndrome (MIM 608363) (Portnoi, 2009).
Phenotypes of individuals with 22q11.2 microduplications are extremely variable, with
healthy asymptomatic individuals to those with multiple problems, including learning
difficulties, congenital cardiac malformations, velopharyngeal insufficiency, with or without
cleft palate, dysmorphic features, growth and developmental delay, behavioural problems
and hearing loss (Firth, 2009, Adam et al, 2013).
In our cohort of 92 individuals with BEEC, we identified five duplications and a single large
chromosomal deletion. Our identification of three 22q11.2 duplications (~3.3%), is similar to
that of the ~2.5% previously reported (Ludin et al, 2010; Draaken et al, 2010 and 2014) and
contrasts with a background frequency of 0.3% (van Campenhout et al, 2010) and 0.27% in
our own data in individuals with developmental delay. The affected individuals had no other
clinical features that would have indicated a duplication at this locus. Of note, one individual
with a 22q11.21 duplication had an epispadias, a milder form of the BEEC spectrum not
previously associated with this duplication. In both cases reported here, the duplications
were inherited from a healthy mother suggesting that additional factors are required for
phenotypic expression. Of the reports of 22q11.21 duplication associated with BEEC before
the current report, five have arisen de novo, four have been inherited maternally and one
paternally (Table 1). Although the parent of origin of the allele on which the de novo
duplications arose was not characterized, there appears to be a preponderance of female
inherited duplications which contrasts with the findings of another study (van Campenhout et
al, 2010) where in a series of ten cases of 22q11 duplication associated with developmental
delay, four arose de novo, four were paternally inherited and two were maternally inherited.
Although these are small numbers from which to draw conclusions the parent of origin of
such a duplication may predispose to the likelihood of BEEC rather than other 22q11
duplication associated phenotypes (van Campenhout et al, 2010).
A comparison of eight previously reported 22q11.21 duplications in individuals with CBE
revealed a 414kb phenocritical region harboring ten candidate protein coding genes
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phosphatidylinositol 4‐kinase, catalytic, alpha (PI4KA); serpin peptidase inhibitor, clade D
(heparin cofactor) member 1 (SERPIND1); synaptosomal‐associated protein, 29kDa
(SNAP29); v‐crk sarcoma virus CT10 oncogene homolog (avian)‐like (CRKL); apoptosis‐inducing factor, mitochondrion‐associated, 3 (AIFM3); leucine‐zipper‐like transcription
regulator 1 (LZTR1); THAP domain containing 7 (THAP7); purinergic receptor P2X, ligand‐gated ion channel, 6 (P2RX6); solute carrier family 7 (orphan transporter) member 4
(SLC7A4); and breakpoint cluster region pseudogene 2 (BCRP2). (Draaken et al, 2014).
RNA array data from embryonic mice in GenitoUrinary Development Molecular Anatomy
Project (GUDMAP); http://www.gudmap.org, detected: PI4KA in embryonic bladder and
genital tubercule, SERPIND1 in embryonic ureter, SNAP29 in embryonic bladder, ureter and
kidney, CRKL in embryonic kidney mesenchyme and interstitium, AIFM3 in embryonic
bladder, LZTR1 in embryonic kidney mesenchyme, interstitium and tubules, and embryonic
ureter, THAP7 in embryonic kidney mesenchyme, interstitium and tubules, embryonic ureter,
and embryonic bladder, P2RX6 in embryonic bladder, SLC7A4 in embryonic bladder and
kidney and BCRP2 in embryonic bladder, ureter and kidney. Studies are needed to seek
and map the expression of these genes in RNA and protein levels in the developing urinary
tract and its precursors, including the uro-rectal septum and surrounding tissues. It is worth
noting that an ‘exstrophy’ gene may play its biological role even before the bladder becomes
a morphological unit, and perhaps in surrounding embryonic tissue that are themselves not
destined to form the urinary tract. Further work will be needed to assess expression of
candidate genes in such early and extra-renal tract tissues.
The 22q11.21 duplications in our two cases encompass and are larger than those reported
by Draaken and so do not refine the critical interval. In the phenocritical region CRKL,
THAP7 and LZTR1 are expressed in the mouse embryonic renal tract as assessed by in situ
hybridisation and are potential candidates genes for CBE (Draaken et al, 2014).
Subsequently, loss of function variants in CRKL have been reported in individuals with
congenital anomalies of the kidneys and urinary tract (Lopez-Rivera et al, 2017) and the
functional importance of Crkl has been demonstrated in murine genitouruinary tract
development (Haller et al 2017). We did not identify any sequence variants in CRKL in
individuals with BEEC without a 22q11 duplication.
The 1.4 Mb duplication detected on chromosome 22q11.23 has not previously been
reported in association with BEEC. Many studies have been undertaken to unravel the
developing pathogenesis of the complex phenotypes arising from CNV abnormalities within
the 22q11.2 region (Wentzel et al, 2008). Human studies and mouse models suggest that
sensitivity to gene dosage changes during embryogenesis and the existence of modifying
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factors determine the specific phenotype. Further work will be required to define these
complex temporospatial relationships during normal and altered development.
For the three other larger CNVs that were identified in this cohort there is no prior evidence
of these being associated with disorders of the lower urinary tract. The 3p26.3 duplication is
in a region where microduplication has been associated with neurodevelopmental disorders
(Hu et al, 2015). Deletions at 4p15.2 have been observed in the Database of Genomic
Variants (Redon et al; 2006, Cooper et al, 2011; Coe at el, 2014; Mills et al, 2011) without
an associated phenotype. Larger duplications overlapping the 22q13.31 locus have been
reported associated with neurodevelopmental disorders (Peeters et al, 2008).
In conclusion, we provide further evidence that microduplications at 22q11.2 may manifest
with non-syndromic CBE and increase the likelihood that altered expression of a gene or
genes within, or near to, this locus are key to the pathogenesis of this devastating congenital
disorder.
AcknowledgementsWe thank all the patients and their family members for their cooperation and continued
support in these studies. Thanks to Jeanette Rothwell and Rebekah Brown for help with
participant recruitment. The study was supported by grants from Kidneys for Life, Kids
Kidney Research, the Wolfson Foundation and Newlife (15-16/06).
Conflict of interest statementThe authors declare no conflict of interest.
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Table 1. 22q11.21 duplications previously reported and those found in the current study associated with BEEC.
Table 2. CNVs over 0.5 Mb detected in BEEC cases in the current study.n/a – parents samples not available
Figure 1. SNP microarray results showing predicted duplications (indicated by red arrow) a. in a male proband with CBE: 22q11.21(18,884,837-21,611,337); b. in a male proband with epispadias: 22q11.21(19,059,071-21,465,835); c. in a male proband with CBE: 22q11.23(23,664,430-25,064,964). Tracks were arranged from top to bottom showing copy number states (segments), Log2 ratio, and smooth signal.
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