Abstract 1317: Simultaneous identification of candidate melanoma risk variants using massively parallel reporter assay

Abstract

Abstract Recent melanoma Genome-Wide Association Study (GWAS) has identified 20 common susceptibility loci via meta-analysis of ~15,000 cutaneous malignant melanoma cases. While GWAS is extremely powerful in identifying genomic regions contributing to melanoma risk, teasing out functional variants and unraveling molecular mechanisms by which the risk is conferred presents a tremendous challenge due to limited resolution of genetic structure and lack of high-throughput assay system. To simultaneously identify functional risk variants from multiple GWAS loci, we employed recently developed Massively-Parallel Reporter Assays (MPRA). Based on the hypothesis that many GWAS functional variants regulate gene expression through transcriptional mechanism, we systematically examined allele-specific transcriptional activities of genetically indistinguishable candidate variants. Out of ~2,800 melanoma-associated variants (r2>0.4, EUR) from 16 melanoma loci for which transcriptional mechanism could be applied, 835 variants were prioritized based on their relevance as melanocyte-/melanoma-specific cis-element represented by ENCODE annotation as open chromatin or putative promoter/enhancer histone marks in melanocytes and melanoma cell lines. Pooled oligoes of 145bp encompassing each variant with either melanoma risk or protective allele coupled with 10 different unique sequence tags were cloned into luciferase vectors and transfected to melanoma cell lines. Resulting expressed tag counts were subsequently determined using massively parallel sequencing. We were able to identify ~200 potentially functional variants displaying allelic transcriptional activity by combining transfections in a melanoma cell line and HEK293FT cell line. Top functional SNPs from melanoma GWAS loci were also shown as significant eQTL SNPs in human melanocytes including MX2 on chromosome 21. Overlaying MPRA functional SNPs with melanocyte eQTL SNPs further enabled isolation of a functional risk variant among several highly linked MX2 eQTL SNPs. The results from this analysis will greatly accelerate the identification of functional melanoma risk variants and further shed light on molecular mechanisms of genetic susceptibility of melanoma in population. Citation Format: Jiyeon Choi, Tongwu Zhang, Michael Kovacs, Mai Xu, Nghi Lam, Leandro Colli, Kevin Brown. Simultaneous identification of candidate melanoma risk variants using massively parallel reporter assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1317. doi:10.1158/1538-7445.AM2017-1317