Insertion/Deletion Polymorphisms in the ΔNp63 Promoter Are a Risk Factor for Bladder Exstrophy Epispadias Complex

Simon Wilkins,Karen Aitken,Ke Wei Zhang,Tahmina Banu,Renaud Quantin,Atul Thakre,Kaid Johar,Long Li,Michael Ee,Wei Cheng,Istiak Mahfuz,Wendy Fung ,Darius Bägli ,Nancy Teresa D'cruz ,Stephen Kwok‐Wing Tsui ,F Fong ,E Jaureguízar ,Patrick Kwok Shing Ng ,John M Hutson

doi:10.1371/journal.pgen.1003070

Abstract

Bladder exstrophy epispadias complex (BEEC) is a severe congenital anomaly; however, the genetic and molecular mechanisms underlying the formation of BEEC remain unclear. TP63, a member of TP53 tumor suppressor gene family, is expressed in bladder urothelium and skin over the external genitalia during mammalian development. It plays a role in bladder development. We have previously shown that p63−/− mouse embryos developed a bladder exstrophy phenotype identical to human BEEC. We hypothesised that TP63 is involved in human BEEC pathogenesis. RNA was extracted from BEEC foreskin specimens and, as in mice, ΔNp63 was the predominant p63 isoform. ΔNp63 expression in the foreskin and bladder epithelium of BEEC patients was reduced. DNA was sequenced from 163 BEEC patients and 285 ethnicity-matched controls. No exon mutations were detected. Sequencing of the ΔNp63 promoter showed 7 single nucleotide polymorphisms and 4 insertion/deletion (indel) polymorphisms. Indel polymorphisms were associated with an increased risk of BEEC. Significantly the sites of indel polymorphisms differed between Caucasian and non-Caucasian populations. A 12-base-pair deletion was associated with an increased risk with only Caucasian patients (p = 0.0052 Odds Ratio (OR) = 18.33), whereas a 4-base-pair insertion was only associated with non-Caucasian patients (p = 0.0259 OR = 4.583). We found a consistent and statistically significant reduction in transcriptional efficiencies of the promoter sequences containing indel polymorphisms in luciferase assays. These findings suggest that indel polymorphisms of the ΔNp63 promoter lead to a reduction in p63 expression, which could lead to BEEC.

Highlights

Bladder exstrophy epispadias complex (BEEC; MIM600057) is a serious congenital anomaly present in 1 in 36,000 live births [1]
We have demonstrated that p632/2 mouse embryos exhibit ventral midline defects identical to those of human BEEC, including ventral bladder and abdominal wall defects, separation of external genitalia, separation of pubic bones and rectus abdominis muscles, exomphalos, and ventrally translocated anus [9]. p63 is expressed in the bladder urothelium during development, TAp63 at an earlier stage (E9–11) and DNp63 later (E11–19)
In this study we show the expression of the two different TP63 promoters in BEEC patient tissue, and explore sequence of the DNp63 promoter in BEEC patients and controls

Summary

Introduction

Bladder exstrophy epispadias complex (BEEC; MIM600057) is a serious congenital anomaly present in 1 in 36,000 live births [1]. BEEC is manifested as a cluster of ventral midline defects including: 1) ventral bladder and abdominal wall defects, 2) epispadias (split external genitalia), 3) separation of the pubic bones and the rectus abdominis muscles, 4) exomphalos, and 5), ventrally displaced anus [2]. Untreated BEEC patients develop chronic bladder mucosal irritation and have a 700-fold increased chance of developing bladder cancer. The genetic and molecular mechanisms underlying the formation of BEEC remain unclear. Studies have shown an increased incidence of BEEC in children of older mothers [3], in Caucasian populations [4], and a familial genetic component of pathogenesis [5]. Among siblings and offspring of BEEC patients, the risk of BEEC increase dramatically from 1 in

Author Summary

Materials and Methods

Findings

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