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.

Fig 1. Regional association plot for ISL1 across a 1.0 Mb window.

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 P values (y-axis on the left) are from the meta-analysis. The purple diamond indicates the most significant marker.

Fig 2. Genome-wide association scan in classic bladder exstrophy patients.

Association of SNPs is plotted as −log₁₀(p) against chromosomal position. The y-axis shows the negative log₁₀ P 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.

Fig 3. Expression of Isl1 during mouse development.

Whole-mount in situ hybridization (ISH) for Isl1 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. Isl1 was also detected in the craniofacial- and spinal ganglia.