Looking beyond GWAS: allele-specific transcription factor binding drives the association of GALNT2 to HDL-C plasma levels

Marco Cavalli, Gang Pan, Helena Nord, Claes Wadelius

Plasma levels of high-density lipoprotein cholesterol (HDL-C) have been associated to cardiovascular disease. The high heritability of HDL-C plasma levels has been an incentive for several genome wide association studies (GWASs) which identified, among others, variants in the first intron of the GALNT2 gene strongly associated to HDL-C levels. However, the lead GWAS SNP associated to HDL-C levels in this genomic region, rs4846914, is located outside of transcription factor (TF) binding sites defined by chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) experiments in the ENCODE project and is therefore unlikely to be functional. In this study we apply a bioinformatics approach which rely on the premise that ChIP-seq reads can identify allele specific binding of a TF at cell specific regulatory elements harboring allele specific SNPs (AS-SNPs). EMSA and luciferase assays were used to validate the allele specific binding and to test the enhancer activity of the regulatory element harboring the AS-SNP rs4846913 as well as the neighboring rs2144300 which are in high LD with rs4846914. Using luciferase assays we found that rs4846913 and the neighboring rs2144300 displayed allele specific enhancer activity. We propose that an inhibitor binds preferentially to the rs4846913-C allele with an inhibitory boost from the synergistic binding of other TFs at the neighboring SNP rs2144300. These events influence the transcription level of GALNT2. The results suggest that rs4846913 and rs2144300 drive the association to HDL-C plasma levels through an inhibitory regulation of GALNT2 rather than the reported lead GWAS SNP rs4846914.