Eufrasio_et_al_Online_Supplemental_Material.pdf (4.86 MB)

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

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posted on 06.10.2020 by Ada Admin, Ana Eufrásio, Chiara Perrod, Marta Duque, Fábio J.Ferreira, Mafalda Galhardo, José Bessa
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. The risk variant of two sequences decreased enhancer activity, while in another two incremented it. 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, other type 2 diabetes associated SNPs that truncate SLC30A8, confer protection to this disease, contradicting this explanation. Here, we clarify this incongruence showing that rs13266634 boosts the activity of an overlapping enhancer, suggesting a SLC30A8 gain-of-function as the cause for the increased risk for the disease. We further dissected the functionality of this enhancer finding a single nucleotide mutation sufficient to impair its activity. Overall, this work assesses 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.


This study was supported by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement no. ERC-2015-StG-680156-ZPR). José Bessa acknowledges Fundação para a Ciência e a Tecnologia (FCT), for a FCT Scientific stimulus grant (Grant CEECIND/03482/2018). Ana Eufrásio and Marta Duque are PhD fellows from Fundação para a Ciência e a Tecnologia (FCT) (Grants: AE – SFRH/BD/147762/2019 and MD ­ SFRH/BD/135957/2018). NORTE 2020