Classic Bladder Exstrophy: identification of genetic markers and characterization of its associated <em>ISL1</em> gene

Abstract

Classic bladder exstrophy (CBE) is a congenital anomaly with an occurrence of 1 in 30.000 new born. It is characterized by the protrusion of the bladder plate through an open abdominal wall, often accompanied by kidney and upper urinary tract abnormalities. Its long-term complications include different bladder malignancies. <br /> While the phenotype of CBE is well understood, the genetic and molecular causes remain largely unknown. Previous genetic studies have identified one locus associated with CBE, but these studies had limited sample sizes. The aim of this study is to identify new risk loci and gain a better understanding of their contributions to CBE and CBE-associated cancer. Furthermore, the study aims to elucidate the molecular mechanisms through which the associated <em>ISL1</em> gene contributes to CBE. <br /> For that, this study shows here the largest genome-wide association study (GWAS) conducted on CBE to date, identifying eight genome-wide significant loci, seven of which are novel. Within these loci, ten coding and four non-coding genes are found. RNA-seq analysis of mouse and human embryonic bladder tissues at different developmental stages reveal those genes to be expressed and differentially regulated in bladder development. Furthermore, those genes are differentially expressed in various bladder cancers with down-regulated genes in bladder development being up-regulated in bladder cancer and vice versa. These findings suggest genetic drivers for classic bladder exstrophy and their potential role in CBE-associated bladder cancer susceptibility. <br /> The most significant CBE-associated locus is found on chromosome 5, housing the homeobox transcription factor <em>ISL1</em> gene, known to be involved in the development of different tissues. Mouse models have been recently shown its role in urinary tract and genitalia development, and our RNA-seq also indicates high expression of <em>ISL1</em> during early bladder developmental stages, followed by a strong decreased expression in differentiated bladder tissue. Exome sequencing of <em>ISL1</em> in CBE cohort did not find any pathogenic variants in <em>ISL1</em>, suggesting that its contribution to CBE lies in gene regulation rather than genetic variation. <br /> Our GWAS identified a regulatory variant rs2303751 in <em>ISL1</em> that is suggested to be target of EZH2, recently shown to act as a transcription factor. Here, we describe a novel 1.2 kb intragenic promoter (called Fragment 2) residing between 6.2 and 7.4 kb downstream of the <em>ISL1</em> transcription starting site. This promoter is active in the reverse DNA strand of <em>ISL1</em> and in HEK 293 cells harbours a binding side for EZH2 where the rs2303751 marker reseeds. Here we show, that EZH2 enhance Fragment 2 activity and that its silencing reduces <em>ISL1</em> expression. Further <em>in vivo</em> experiments in zebrafish ezh2^-/- ko larvae display tissues specificity of <em>ISL1</em> regulation with reduced expression that locates specifically in the pronephric region of zebrafish larvae. In addition, a shorter and malformed nephric duct is observed in ezh2^-/- ko zebrafish Tg(wt1ß:eGFP) reporter lines. <br /> Since in human Fragment 2 is active in the reverse orientation of <em>ISL1</em>, we showed that in HEK 293 its EZH2 regulation is not altering the expression of <em>ISL1</em>-DT, the immediate divergent transcript from <em>ISL1</em>, but rather disrupts the ratio of the <em>ISL1</em>/<em>ISL1</em>-DT cassette. Our study proposes that EZH2 is a key regulator of <em>ISL1</em> during early urinary tract formation and suggests tissue specific <em>ISL1</em> dysregulation as an underlying mechanism for CBE formation.