Abstract 200: Functional Mapping of the 8q24 Noncoding Region Reveals Enhancer Elements and Association Between GWAS SNPs and TRIB1 Gene Expression

Abstract

Single nucleotide polymorphisms (SNPs) in the human 8q24 locus have repeatedly been associated by genome-wide association studies (GWAS) with multiple human metabolic traits such as plasma lipids and coronary artery disease. These SNPs lie in a non-coding region ~30kb downstream of the gene Tribbles-1 ( TRIB1 ). While a large body of in vivo evidence from Trib1 gain- and loss-of-function mouse models strongly supports TRIB1 as the gene of interest at this locus, there has been no demonstrated association between SNPs in the GWAS region and the expression of the neighboring TRIB1 gene to date. To address this, we performed RNA-seq and genome-wide genotyping on 42 human cadaveric liver samples, and next performed allele-specific expression (ASE) analysis of the TRIB1 locus in 23 samples that harbored heterozygous coding SNPs in the TRIB1 gene, allowing for allelic discrimination. While subjects homozygous for the major allele of the lead GWAS SNP (rs2954029) had even allelic expression ( p =0.29 by non-parametric t-test), samples heterozygous for the minor allele at rs2954029 exhibited imbalanced allelic expression ( p <0.0005). This finding suggests that SNPs in the TRIB1 locus do affect TRIB1 gene expression; however, it remains unclear which SNP(s) is causal. To that end, we used ENCODE data from primary hepatocytes and HepG2 cells to identify 10 genomic regions of interest that contained SNPs with significant GWAS p-values and epigenetic markers consistent with enhancer activity. Two regions (R2, R9) exhibited strong enhancer activity when cloned in front of a minimal promoter driving a luciferase reporter (pGL4.23), increasing luciferase activity 2-5-fold as compared to empty vector ( p <0.05). Introduction of the minor alleles at three separate candidate SNPs in R2 reduced enhancer activity. In summary, we show that common variation in the 8q24 GWAS locus does affect TRIB1 gene expression, as measured by ASE in human livers. We also identified multiple enhancer elements that exhibit reduced activity when the minor alleles of GWAS SNPs are introduced into them. We are currently pursuing modification of the endogenous locus in HepG2 cells via CRISPR/Cas genome editing to further elucidate the roles of both these enhancer elements and the SNPs contained in them.