Abstract
Genome-wide association studies (GWAS) have identified ~20 melanoma susceptibility loci, most of which are not functionally characterized. Here we report an approach integrating massively-parallel reporter assays (MPRA) with cell-type-specific epigenome and expression quantitative trait loci (eQTL) to identify susceptibility genes/variants from multiple GWAS loci. From 832 high-LD variants, we identify 39 candidate functional variants from 14 loci displaying allelic transcriptional activity, a subset of which corroborates four colocalizing melanocyte cis-eQTL genes. Among these, we further characterize the locus encompassing the HIV-1 restriction gene, MX2 (Chr21q22.3), and validate a functional intronic variant, rs398206. rs398206 mediates the binding of the transcription factor, YY1, to increase MX2 levels, consistent with the cis-eQTL of MX2 in primary human melanocytes. Melanocyte-specific expression of human MX2 in a zebrafish model demonstrates accelerated melanoma formation in a BRAFV600E background. Our integrative approach streamlines GWAS follow-up studies and highlights a pleiotropic function of MX2 in melanoma susceptibility.
Highlights
Genome-wide association studies (GWAS) have identified ~20 melanoma susceptibility loci, most of which are not functionally characterized
Incorporation of this approach is attractive for GWAS functional follow-up studies, as (1) linkage disequilibrium (LD) limits statistical fine-mapping and leaves numerous variants as potential functional candidates, and (2) many trait-associated variants are hypothesized to contribute to allelic gene expression through cis-regulatory mechanisms that can be tested by reporter assays
To prioritize melanoma-associated variants to test by massively-parallel reporter assays (MPRA), we first selected 2748 variants that are in LD (r2 > 0.4) with these 46 primary and secondary lead SNPs (Methods; Supplementary Fig. 1; Supplementary Table 2)
Summary
Genome-wide association studies (GWAS) have identified ~20 melanoma susceptibility loci, most of which are not functionally characterized. Parallel Reporter Assays (MPRA) scale up conventional luciferase reporter assays for testing transcriptional activities of DNA elements, facilitating evaluation of tens of thousands of different short sequences at the same time in cells, which are deconvoluted by massively parallel sequencing[20,21,22] Incorporation of this approach is attractive for GWAS functional follow-up studies, as (1) linkage disequilibrium (LD) limits statistical fine-mapping and leaves numerous variants as potential functional candidates, and (2) many trait-associated variants are hypothesized to contribute to allelic gene expression through cis-regulatory mechanisms that can be tested by reporter assays. Using our approach we identify a functional risk variant that increases the level of an HIV-1 restriction gene, MX2, in cells of melanocytic lineage; subsequent expression of MX2 in melanocytes of a zebrafish melanoma model accelerates melanoma formation
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