284-OR: An Alternative Downstream Translational Start Site in PPARG Produces a Novel Protein That Rescues Loss-of-Function Mutations in Humans

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PPARG is the target of thiazolidinediones (TZD) and plays a central role in glucose and lipid metabolism. Loss-of-function (LOF) mutations in PPARG increase the risk of type 2 diabetes (T2D) and cause Mendelian lipodystrophy. In population-scale sequencing studies, we identified protein-truncating variants in PPARG expected to cause lipodystrophy, but surprisingly showing no burden of metabolic disease in their human carriers. To further understand this finding, we use CRISPR/CAS9 to systematically target deletions to every exon of PPARG revealing that mutations causing deleterious frameshifts in exons 1 and 2 resulted in cells with intact PPARG responses. We subsequently constructed a library of PPARG transgenes encoding termination sequences at every possible codon (1-505) in the PPARG2 cDNA and tested them for transactivation function. We found that PPARG transgenes encoding termination sequences prior to codon 135 (c.ATG402-404) corresponding to exon 2 in the genomic DNA (chr3:12381414 Hg38) retained transactivation activity. We hypothesized that c.ATG402-404 served as an alternative translational start site that generates a PPARG isoform lacking the N-terminus but retaining DNA-binding, ligand-binding, and transactivation properties. Indeed, cells engineered to disrupt PPARG prior to chr3:12381414 retained TZD responsiveness and produced a 40 kDa protein recognized by antibodies targeting the C-terminus but not the N-terminus of PPARG. Analysis of the global transcriptional activity of the PPARG p.M135-505 compared to full-length PPARG revealed that p.M135-505 produced a similar TZD-induced upregulation of known PPARG targets and gene sets. In summary, PPARG p.M135-505 is a truncated but fully functional protein that responds to PPARG agonists and may rescue disruptive mutations that would otherwise cause lipodystrophy. Disclosure K.Mendez lara: None. X.Du: None. K.Glass: None. R.Hernandez: None. C.De arruda saldanha: None. S.Heinz: Research Support; Hologic Inc., Stock/Shareholder; Leash Laboratories. A.R.Majithia: None. Funding National Institutes of Health (RDK01DK123422)

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