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.