A genome-wide association study with tissue transcriptomics identifies genetic drivers for classic bladder exstrophy

Classic bladder exstrophy represents the most severe end of all human congenital anomalies of the kidney and urinary tract and is associated with bladder cancer susceptibility. Previous genetic studies identified one locus to be involved in classic bladder exstrophy, but were limited to a restrict number of cohort. Here we show the largest classic bladder exstrophy genome-wide association analysis to date where we identify eight genome-wide significant loci, seven of which are novel. In these regions reside ten coding and four non-coding genes. Among the coding genes is EFNA1, strongly expressed in mouse embryonic genital tubercle, urethra, and primitive bladder. Re-sequence of EFNA1 in the investigated classic bladder exstrophy cohort of our study displays an enrichment of rare protein altering variants. We show that all coding genes are expressed and/or significantly regulated in both mouse and human embryonic developmental bladder stages. Furthermore, nine of the coding genes residing in the regions of genome-wide significance are differentially expressed in bladder cancers. Our data suggest genetic drivers for classic bladder exstrophy, as well as a possible role for these drivers to relevant bladder cancer susceptibility

ISL1 is a major susceptibility gene for classic bladder exstrophy and a regulator of urinary tract development.

Previously genome-wide association methods in patients with classic bladder exstrophy (CBE) found association with ISL1, a master control gene expressed in pericloacal mesenchyme. This study sought to further explore the genetics in a larger set of patients following-up on the most promising genomic regions previously reported. Genotypes of 12 markers obtained from 268 CBE patients of Australian, British, German Italian, Spanish and Swedish origin and 1,354 ethnically matched controls and from 92 CBE case-parent trios from North America were analysed. Only marker rs6874700 at the ISL1 locus showed association (p = 2.22 × 10-08). A meta-analysis of rs6874700 of our previous and present study showed a p value of 9.2 × 10-19. Developmental biology models were used to clarify the location of ISL1 activity in the forming urinary tract. Genetic lineage analysis of Isl1-expressing cells by the lineage tracer mouse model showed Isl1-expressing cells in the urinary tract of mouse embryos at E10.5 and distributed in the bladder at E15.5. Expression of isl1 in zebrafish larvae staged 48 hpf was detected in a small region of the developing pronephros. Our study supports ISL1 as a major susceptibility gene for CBE and as a regulator of urinary tract development

Genome-wide Association Study and Meta-Analysis Identify ISL1 as Genome-wide Significant Susceptibility Gene for Bladder Exstrophy

Abstract The bladder exstrophy-epispadias complex (BEEC) represents the severe end of the uro-rectal malformation spectrum, and is thought to result from aberrant embryonic morphogenesis of the cloacal membrane and the urorectal septum. The most common form of BEEC is isolated classic bladder exstrophy (CBE). To identify susceptibility loci for CBE, we performed a genome-wide association study (GWAS) of 110 CBE patients and 1,177 controls of European origin. Here, an association was found with a region of approximately 220kb on chromosome 5q11.1. This region harbors the ISL1 (ISL LIM homeobox 1) gene. Multiple markers in this region showed evidence for association with CBE, including 84 markers with genome-wide significance. We then performed a meta-analysis using data from a previous GWAS by our group of 98 CBE patients and 526 controls of European origin. This meta-analysis also implicated the 5q11.1 locus in CBE risk. A total of 138 markers at this locus reached genome-wide significance in the meta-analysis, and the most significant marker (rs9291768) achieved a P value of 2.13 × 10−12. No other locus in the meta-analysis achieved genome-wide significance. We then performed murine expression analyses to follow up this finding. Here, Isl1 expression was detected in the genital region within the critical time frame for human CBE development. Genital regions with Isl1 expression included the peri-cloacal mesenchyme and the urorectal septum. The present study identified the first genome-wide significant locus for CBE at chromosomal region 5q11.1, and provides strong evidence for the hypothesis that ISL1 is the responsible candidate gene in this region. Author Summary The etiology of classic exstrophy of the bladder (CBE) remains unclear. The present genome-wide association study and meta-analysis identified an association between CBE and a region on chromosome 5q11.1. This region contains the gene encoding insulin gene enhancer protein, ISL-1. In this region, 138 single nucleotide polymorphisms (SNPs) reached genome-wide significance, with the SNP rs9291768 showing the lowest P value (p = 2.13 x 10−12). Our findings, as supported by expression analyses in murine models, suggest that ISL1 is a susceptibility gene for CBE

Genome-wide Association Study and Meta-Analysis Identify <i>ISL1</i> as Genome-wide Significant Susceptibility Gene for Bladder Exstrophy

<div><p>The bladder exstrophy-epispadias complex (BEEC) represents the severe end of the uro-rectal malformation spectrum, and is thought to result from aberrant embryonic morphogenesis of the cloacal membrane and the urorectal septum. The most common form of BEEC is isolated classic bladder exstrophy (CBE). To identify susceptibility loci for CBE, we performed a genome-wide association study (GWAS) of 110 CBE patients and 1,177 controls of European origin. Here, an association was found with a region of approximately 220kb on chromosome 5q11.1. This region harbors the <i>ISL1</i> (<i>ISL LIM homeobox 1</i>) gene. Multiple markers in this region showed evidence for association with CBE, including 84 markers with genome-wide significance. We then performed a meta-analysis using data from a previous GWAS by our group of 98 CBE patients and 526 controls of European origin. This meta-analysis also implicated the 5q11.1 locus in CBE risk. A total of 138 markers at this locus reached genome-wide significance in the meta-analysis, and the most significant marker (rs9291768) achieved a P value of 2.13 × 10⁻¹². No other locus in the meta-analysis achieved genome-wide significance. We then performed murine expression analyses to follow up this finding. Here, Isl1 expression was detected in the genital region within the critical time frame for human CBE development. Genital regions with <i>Isl1</i> expression included the peri-cloacal mesenchyme and the urorectal septum. The present study identified the first genome-wide significant locus for CBE at chromosomal region 5q11.1, and provides strong evidence for the hypothesis that <i>ISL1</i> is the responsible candidate gene in this region.</p></div

Most strongly associated SNP in the bladder exstrophy susceptibility locus 5q11.1.

<p>Relative risks (RRs) are given with the risk allele set as baseline. Chr, chromosome; RAF, risk allele frequency.</p><p>Most strongly associated SNP in the bladder exstrophy susceptibility locus 5q11.1.</p

Regional association plot for <i>ISL1</i> across a 1.0 Mb window.

<p>Association with classic bladder exstrophy of individual SNPs in the meta-analysis GWAS is plotted as −log₁₀(p) against chromosomal position. The y-axis on the right shows the recombination rate estimated from the 1000 Genomes (Mar 2012) EUR populations. All <i>P</i> values (y-axis on the left) are from the meta-analysis. The purple diamond indicates the most significant marker.</p

Expression of <i>Isl1</i> during mouse development.

<p>Whole-mount <i>in situ</i> hybridization (ISH) for <i>Isl1</i> in wildtype mouse embryos between E9.5-E12.5 revealed strong expression in the developing genital region, including the cloaca, cloacal membrane, and emerging genital tubercle. ISH on mid-sagittal paraffin sections at later embryonic stages (E12.5-E14.5) revealed expression throughout the genital tubercle, within the peri-cloacal mesenchyme and urorectal septum. <i>Isl1</i> was also detected in the craniofacial- and spinal ganglia.</p

Genome-wide association scan in classic bladder exstrophy patients.

<p>Association of SNPs is plotted as −log₁₀(p) against chromosomal position. The y-axis shows the negative log₁₀<i>P</i> values of the logistic regression for SNPs from the meta-analysis that passed quality control. Chromosomes are shown in alternating colors along the x-axis. The genome-wide significance level is indicated by a red line.</p