Abstract 5061: Post-GWAS functional characterization of the 12p11.23 renal cancer susceptibility locus implicates BHLHE41
Authors: Jessop Lea , Bigot Pierre , Machiela Mitchell , Myers Timothy , Sikdar Nilabja , Colli Leandro , Chanock Stephen 
Published: May 2015

Abstract
Introduction. Two of common variants, rs718314 and rs1049380, were associated with renal cell carcinoma (RCC) at the 12p11.23 locus. Interestingly, rs718314 was also identified as being associated with waist-hip ratio. The objective of the present study is to perform a functional analysis of the 12p11.23 region in relation to RCC risk.
Materials and methods. We performed an imputation analysis within 1 Mb of rs718314 in three different study populations from a previously published GWAS of RCC (4197 cases and 8527 controls). The genotyped and high-quality imputed SNPs were tested for association with RCC. We examined the significantly associated variants for sequence overlap with potential regulatory sites by cross referencing them with ENCODE data. Selected putatively functional SNPs were tested for enhancer activity using a dual luciferase assay in 3 different renal cell lines (UO31, SN12-C, 786-O) and electro-mobility shift assays (EMSA) were used to look for binding of transcription factors to selected SNPs. In addition, the RCC-associated variants present in the TCGA database were examined for an effect on nearby gene expression (ITPR2, SSPN, BHLHE41).
Results. After meta-analysis, 499 of the total 13,159 genotyped and imputed SNPs had association p-values with RCC less than 0.05. Within this group, 44 SNPs demonstrated nominally significant association with RCC risk (p&lt;5.10-5). The two initial GWAS SNPs, rs718314 and rs1049380, were strongly associated with RCC (Padj = 3.44.10-6 and Padj = 5.27.10-6). All nominally significant SNPs were in a non-coding region which contains the 3′-UTR of ITPR2. Six of the 44 variants were in regions enriched for H3K4me1 and H3K27ac, chromatin marks found in enhancers. Only one SNP, rs7132434 which is highly correlated with the initial GWAS signal (rs718314, r2=1), showed allele specific regulatory activity in luciferase assays and allele specific differences in protein binding by EMSA. Five SNPs associated with RCC were present in the TCGA database. All of them were associated with BHLHE41 expression in tumor tissues (p&lt;0.05). The most significant association was found with rs12814794 (p=1.10-8) which is in high linkage disequilibrium with rs718314 (r2=0.956) and rs7132434 (r2=0.956). There was no association between RCC associated SNPs and SSPN or ITPR2 tissue expression. BHLHE41 is a gene which has been recently been shown to be involved in the VHL/HIF pathway in breast cancer. It is also known to regulate adipogenic differentiation and could be a link between RCC and obesity, a known risk factor for RCC.
Conclusion. Our results suggest rs7132434 is the functional SNP responsible for the GWAS signal and that this locus could act as an enhancer of BHLHE41. Further investigations will be necessary to confirm the link between rs7132434 and BHLHE41 and to understand the role of BHLHE41 in renal carcinogenesis.
Citation Format: Lea Jessop, Pierre Bigot, Mitchell Machiela, Timothy Myers, Nilabja Sikdar, Leandro Colli, Stephen Chanock. Post-GWAS functional characterization of the 12p11.23 renal cancer susceptibility locus implicates BHLHE41. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5061. doi:10.1158/1538-7445.AM2014-5061