A simple and robust reporter-based framework for deep functional characterization of PPARγ mutants.

Missense mutations in nuclear receptor (NR) transcription factors cause a number of genetic disorders, including PPARG mutations that result in familial partial lipodystrophy type 3 (FPLD3). Experimental assessment is essential to establish a newly identified mutation as disease-causing, as accurately predicting the effect of a new mutation in silico remains challenging due to the multifunctional and modular nature of these proteins. However, deep structure-function characterization often requires specialized and technically demanding approaches, which may not be readily available. Therefore, we established a simple and robust experimental framework based on 4 complementary reporter assays that independently assess (1) ability of the full-length receptor to activate transcription; (2) integrity of the ligand-binding domain; (3) heterodimerization potential; and (4) DNA-binding capacity. As a proof of concept, we analyzed 3 uncharacterized FPLD3-associated loss-of-function variants and 2 bladder cancer-associated gain-of-function variants. Together, the 4 complementary assays showed unique functional phenotypes for all 5 mutants that were further supported by coregulator profiling. We therefore conclude that this framework provides a simple and robust first-line approach to identify functional alterations in peroxisome proliferator-activated receptor γ mutants with mechanistic resolution. This framework is broadly applicable across NRs and offers a scalable path to systematic variant interpretation both in research and clinical contexts.