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: william.newman@manchester.ac.uk

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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