Making sense of sequence variation in PPARG: a comprehensive experimental approach. A. Majithia, J. Flannick, T. Mikkelsen, D. Altshuler Broad Institute, Cambridge, MA.
Discriminating benign and functional genetic variants is a key challenge in clinical interpretation of exome sequencing. The current gold standard for functional evaluation requires that variants identified in sequencing be recreated and tested in vitro, a resource intensive process that is not feasible on clinical time scales. We present a prospective experimental approach and demonstrate its application to interpret non-synonymous variants in PPARG, a master regulator of adipocyte differentiation. To generate a complete functional catalog, we synthesized a comprehensive library of 9576 PPARG variants consisting of every single amino acid substitution at every position and performed pooled screens for protein function in human pre-adipocytes. We validate this functional catalog by re-identifying previously known loss-of-function mutations in PPARG that cause human lipodystrophy and clinical insulin resistance. Subsequently, we apply it to prospectively evaluate the consequence of 50 novel non-synonymous PPARG variants of unknown function identified from exome sequencing of 20,000 type 2 diabetes (T2D) cases and controls. As a control, we also generated these newly discovered PPARG variants by conventional means, tested their function individually in an adipocyte differentiation assay, and compare results with our comprehensive functional catalog. When taken together, these variants showed no association with T2D (OR=1.35 p=0.17). However, of the 50 variants, 9 experimentally demonstrated reduced function, ranging from mild to complete inactivation of PPARG in the adipocyte differentiation assay. These functional variants substantially increased risk of T2D (OR=7.22 p=0.005). Our findings establish that rare, loss-of-function variants in PPARG increase T2D risk in the general population, and that they must be distinguished from a majority of benign variants. Our comprehensive functional catalog serves as a resource for interpreting the clinical consequence of novel PPARG variants as they are discovered in future exome sequencing.
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