In vivo reporter assays uncover changes in enhancer activity caused by type 2 diabetes associated SNPs

Many single nucleotide polymorphisms (SNPs) associated to type 2 diabetes overlap with putative endocrine pancreatic enhancers, suggesting that these SNPs modulate enhancer activity and consequently, gene expression. We performed in vivo mosaic transgenesis assays in zebrafish to quantitatively test the enhancer activity of type 2 diabetes-associated loci. Six out of ten tested sequences are endocrine pancreatic enhancers, one overlapping with an exon of SLC30A8. In two endocrine pancreaticenhancers, the risk variant decreased enhancer activity, while in another two incremented. One of the latter (rs13266634) locates in a SLC30A8 exon, encoding a tryptophan-to-arginine substitution that decreases SLC30A8 function, being the canonical explanation for type 2 diabetes risk association. However, SLC30A8truncating SNPs associated to type 2 diabetes protection contradict this explanation. Here, we clarify this incongruence showing that rs13266634 boosts the activity of an overlapping enhancer, suggesting SLC30A8 gain-of-function as the cause for theincreased risk for the disease. We further dissected the functionality of this enhancer finding a single nucleotide mutation sufficient to impair its activity. Overall, this workassesses in vivo the importance of disease-associated SNPs in the activity of endocrine pancreatic enhancers, including a poorly explored case where a coding SNP modulates the activity of an enhancer.