Extending Genome-Wide Association Study Data Through Analysis of SNPs in the G6PC2 Gene
Baerenwald, Devin August
This project and the experiments discussed in this document sought to contribute a functional basis for SNPs within G6PC2 significantly associated with variations in fasting blood glucose by genome-wide association study. Through a combination of binding, reporter gene expression, and in vitro minigene splicing analyses, the roles of a number of SNPs were examined, including rs573225, rs13431652, rs2232316, and rs560887. It was demonstrated that rs13431652 and rs2232316 affect binding of NF-Y and Foxa2, respectively, with changes seen in fusion gene assays correlating these data with the genetic associations of the GWAS. Variations in Foxa2 binding were also observed with allelic variation at rs573225, although fusion gene analysis of this SNP failed to support the binding and genetic data, as well as the function of G6PC2. Because of this inconsistency, future utilization of cell lines and transient transfection for this nature of experiment must be heavily scrutinized. Additionally, splicing variation of G6PC2 exons, based on allelic variation at a number of SNPs, was assessed. By utilizing a minigene plasmid capable of forming a spliced RNA product in vitro, it was found that rs560887 significantly affects splicing of exon 4. The magnitude of this result was found to be based in part on the non-consensus 5’ exon 4 splice junction present in humans. These experiments as a whole serve to corroborate and lend a functional basis to the SNPs of G6PC2 associated with variations in fasting blood glucose.