Experimental Characterization of SLC30A8 Variants for Protection Against Type 2 diabetes

Naturally occurring genetic variants that protect from disease may suggest novel therapeutic targets. We recently identified a series of loss-of-function variants in SLC30A8, which encodes the pancreatic islet zinc transporter ZnT8, collectively associated with a three-fold decrease in type 2 diabetes (T2D) risk. However, the molecular or cellular mechanism of the protective association remains unclear, in light of non-confirmatory animal and cellular models. In the proposed work, we hypothesize that a range of SLC30A8 variants – of intermediate effect between wild type and loss-of-function – could lend insight into the mechanism of action relating SLC30A8 perturbation to disease risk. Specifically, we aim to (a) identify additional missense SLC30A8 variants from large-scale exome sequencing studies; (b) adapt established HeLa cellular models and zinc transport assays to quantify the efficiency of ZnT8 zinc transport; (c) measure the effects of a series of (10-20) ZnT8 missense variants on zinc transport; and (d) assess the extent to which molecular alterations in zinc transport correlate with physiological effects on risk of T2D or related glycemic and cardiometabolic traits. Compared to previous studies to understand the connection between SLC30A8 and T2D, and most studies to dissect the biology behind genetic associations in general, this proposal is innovative in using genetic variation – assayed via both large-scale sequencing and large-scale molecular characterizations – to link physiological, molecular, and cellular mechanisms. We will extract variants and their measured phenotypic effects from the Type 2 Diabetes Knowledge Portal, which today includes 17,000 exome sequences with plans to expand to 55,000 sequences within a year. We will characterize these variants in HeLa cells, which have successfully been used to assess ZnT8 function in the past, via previously established optical fluorescence assays and a more quantitative label-free X-ray fluorescence approach (the XRpro® assay from Icagen, Inc.) We expect that variants of more severe molecular effect will be associated with stronger protection from T2D; indeed, preliminary analysis of missense SLC30A8 variants suggest that computationally predicted damaging mutations are also associated with T2D protection, albeit with lesser effects than full loss-of-function variants. If successful, this work will provide a series of molecular “handles” to interrogate SLC30A8 function with effects that can be physiologically assessed via genetic studies – overcoming the limitations of previous approaches to validate the relevance of a cellular assay or animal model to human pathophysiology. These handles in turn could be used to assess whether inhibition of ZnT8 may be an effective therapeutic strategy for T2D.