Genome-wide association study and mouse expression data identify a highly conserved 32 kb intergenic region between WNT3 and WNT9b as possible susceptibility locus for isolated classic exstrophy of the bladder

Abstract

Bladder exstrophy-epispadias complex (BEEC), the severe end of the urorectal malformation spectrum, has a profound impact on continence as well as sexual and renal functions. It is widely accepted that for the majority of cases the genetic basis appears to be multifactorial. Here, we report the first study which utilizes genome-wide association methods to analyze a cohort comprising patients presenting the most common BEEC form, classic bladder exstrophy (CBE), to identify common variation associated with risk for isolated CBE. We employed discovery and follow-up samples comprising 218 cases/865 controls and 78 trios in total, all of European descent. Our discovery sample identified a marker near SALL1, showing genome-wide significant association with CBE. However, analyses performed on follow-up samples did not add further support to these findings. We were also able to identify an association with CBE across our study samples (discovery: P = 8.88 × 10(-5); follow-up: P = 0.0025; combined: 1.09 × 10(-6)) in a highly conserved 32 kb intergenic region containing regulatory elements between WNT3 and WNT9B. Subsequent analyses in mice revealed expression for both genes in the genital region during stages relevant to the development of CBE in humans. Unfortunately, we were not able to replicate the suggestive signal for WNT3 and WNT9B in a sample that was enriched for non-CBE BEEC cases (P = 0.51). Our suggestive findings support the hypothesis that larger samples are warranted to identify association of common variation with CBE.

Figure 1.

Manhattan plot for meta-analysis. Results based on fixed effect meta-analysis of GWAS1 and GWAS2 signals. Dashed red line shows threshold for genome-wide significance (P< 5 × 10⁻⁸) and dashed gray line shows cut off for the selection of SNPs for follow-up genotyping (P < 1 × 10⁻⁴).

Figure 2.

Regional association plots for regions on chromosome 6 (EEF1E1/SLC35B3) and chromosome 16 (CYLD/SALL1). The P-values from all imputed SNPs in the regions that passed post-imputation QC procedures are plotted against positions from the February 2009 human reference sequence, annotated by Ref Seq genes. The most associated marker (chromosome 6: rs73374907, P_meta = 6.13 × 10⁻⁸; chromosome 16: rs4785484, P_meta = 2.65 × 10⁻⁸) from the meta-analysis is indicated by a purple dot which is centered in a genomic window of ∼1 Mb. The strength of LD (in r²) between the top SNP and its adjacent markers is demonstrated by the red (high) to dark blue (low) color bar (top right corner). The recombination rate (second y-axis) is plotted in light blue, according to 1000 genomes project data. Plots were generated using Locuszoom (http://csg.sph.umich.edu/locuszoom/) (53).

Figure 3.

Analysis of Wnt3 and Wnt9b expression in mid-gestational mouse embryos by in situ hybridization. (A) Expression of Wnt3 was widely detected from E9.5 to E15.5, with stronger expression observed in the skin epithelia, limb buds and neural tissues. Wnt3 is also present in the genital tubercle at these stages. Expression at E14.5 and E15.5 is not shown. gt, genital tubercle; tl, tail. (B) Expression was detectable between E9.5 and E13.5 in the epithelia of the branchial arches, frontonasal process, limb buds and genital tubercle (including the cloacal membrane between E9.5 and E10.5). Strong expression was also found in the nephric ducts. The expression pattern of E13.5 (not shown) was identical to that at E12.5. ba, branchial arch; cm, cloacal membrane; flb, forelimb bud; fnp, frontonasal process; gt, genital tubercle; hlb, hindlimb bud; nd, nephric duct; nt, neural tube; tl, tail.