A Pharmacogenetic Approach to the Treatment of Patients With PPARG Mutations

Abstract

Loss-of-function mutations in PPARG cause familial partial lipodystrophy type 3 (FPLD3) and severe metabolic disease in many patients. Missense mutations in PPARG are present in ∼1 in 500 people. Although mutations are often binarily classified as benign or deleterious, prospective functional classification of all missense PPARG variants suggests that their impact is graded. Furthermore, in testing novel mutations with both prototypic endogenous (e.g., prostaglandin J2 [PGJ2]) and synthetic ligands (thiazolidinediones, tyrosine agonists), we observed that synthetic agonists selectively rescue function of some peroxisome proliferator-activated receptor-γ (PPARγ) mutants. We report on patients with FPLD3 who harbor two such PPARγ mutations (R308P and A261E). Both PPARγ mutants exhibit negligible constitutive or PGJ2-induced transcriptional activity but respond readily to synthetic agonists in vitro, with structural modeling providing a basis for such differential ligand-dependent responsiveness. Concordant with this finding, dramatic clinical improvement was seen after pioglitazone treatment of a patient with R308P mutant PPARγ. A patient with A261E mutant PPARγ also responded beneficially to rosiglitazone, although cardiomyopathy precluded prolonged thiazolidinedione use. These observations indicate that detailed structural and functional classification can be used to inform therapeutic decisions in patients with PPARG mutations.

1A. Schematic representation of the three major domains of PPARγ, showing the locations of the two mutations and the conservation of the mutated residues between species (A261, R308 – PPARγ2 nomenclature). 1B. Transcriptional responses of empty vector (pcDNA), R308P or A261E mutant PPARγ2 to PGJ2 and Rosiglitazone, Pioglitazone and Farglitazar (doses in nM on x-axis) when tested with a (PPARE)₃TKLUC reporter construct and Bos-β-gal internal control plasmid. Results are expressed as a percentage of the maximum activation achieved with wild type (WT) PPARγ2 and represent the mean ± SEM of at least three independent experiments in triplicate. 1C. Transcriptional responses of empty vector (pcDNA), R308P or A261E mutant PPARγ2 to PGJ2 and Rosiglitazone, Pioglitazone and Farglitazar (doses in nM on x-axis) when tested with a hFABP4-Luc reporter construct and Bos-β-gal internal control plasmid. Results are expressed as a percentage of the maximum activation achieved with wild type (WT) PPARγ2 and represent the mean ± SEM of at least three independent experiments in triplicate.

2. A-I. Crystallographic modelling based on structures of unliganded PPARγ (1PRG) or bound to PGJ2 (2ZK1) or pioglitazone (2XKW). One mutated residue (A261) is in the proximity of PGJ2 (A) whereas the other amino acid (R308) is in the vicinity of pioglitazone (B). Substitution of glutamic acid for alanine at residue 261 (A261E) can interfere with PGJ2 binding via steric hindrance (C). The side-chain of arginine 308 (R308) participates in a network of intrahelical (H3) and interhelical (e.g. E287 in H2) hydrogen bonds in unliganded (D) and liganded (E,F) PPARγ. Mutation of this residue to Proline likely disrupts this hydrogen bond network (G). PGJ2, which binds elsewhere in the ligand binding cavity, is unable to prevent loss of such interactions (H), whereas pioglitazone, which binds in the vicinity, hydrogen bonds with E287 and could preserve receptor conformation (I). H, helix.