A Pharmacogenetic Approach to the Treatment of Patients With PPARG Mutations

Peroxisome proliferator–activated receptor-γ (PPARγ) is a nuclear receptor originally identified in adipocytes (1). Although widely expressed, cell-based loss-of-function studies attest to its primary role in regulating adipogenesis and adipocyte function, with rodent knockout studies robustly corroborating these data (2,3). Heterozygous, dominant-negative, loss-of-function mutations in human PPARG were first described in 1999 (4), with subsequent identification of many more receptor defects (5–9). Clinical findings in such patients have refined the phenotype (now known as familial partial lipodystrophy type 3 [FPLD3]) characterized by a paucity of limb fat; preserved abdominal fat; insulin resistant diabetes; dyslipidemia with particularly labile, diet-sensitive hypertriglyceridemia; polycystic ovarian syndrome; and hypertension.

Like many nuclear receptors, PPARγ has an amino-terminal activation domain (AF1), a central DNA-binding domain, and a carboxy-terminal ligand-binding domain. PPARγ heterodimerizes with retinoid X receptor α, and transcriptional activation is triggered by ligand binding, resulting in the release of a corepressor complex and recruitment of a coactivator complex. Fatty acids and eicosanoids can activate PPARγ, with prostaglandin J2 (PGJ2) considered prototypic of such putative endogenous PPARγ ligands (10,11). Structural studies have suggested that the ligand-binding pocket of PPARγ is promiscuous and can accommodate several different fatty acids (3). Thiazolidinediones (TZDs), a class of synthetic PPARγ agonists, promote adipogenesis and improve insulin sensitivity, underpinning their therapeutic use as insulin sensitizers in patients with type 2 diabetes (12).

That as many as 1 in 500 people may have missense mutations in PPARG prompted Majithia et al. (7) to generate and functionally characterize all possible missense PPARG mutations to expedite clinical interpretation of the growing number of missense variants identified in patients. This resource should aid prompt functional classification of novel PPARG variants. For individuals with established loss-of-function mutations and a disease phenotype, therapeutic possibilities are limited. Current options include strict dietary fat and calorie restriction, metformin, insulin, and glucagon-like peptide 1 agonists. Leptin has been tried in patients with very low leptin levels (13). Isolated reports of TZD use also exist (9,14,15), but responses were variable (summarized in Supplementary Table 1).

In characterizing the properties of all possible PPARG missense mutations (7), we were struck by two observations. First, the spectrum of functional scores exhibited by the range of all missense PPARG variants suggested that even mutations associated with a monogenic disease are likely to perturb protein function to a variable degree, predisposing to a similarly variable phenotype rather than fitting an arbitrary designation as disease-causing or benign. Such gradation of PPARγ dysfunction is also likely to translate into differential, graded responses to metabolic stress and to molecularly targeted therapeutic interventions. Second, we noted that a few variants, like R308P, manifested a clearly abnormal transcriptional response to prototypic natural ligand (e.g., PGJ2), whereas their function, when tested with a synthetic agonist, was near normal (7). These in vitro observations suggest that patients harboring such receptor mutants might respond to treatment with synthetic PPARγ agonists. Thus, we report the dramatic clinical response of a patient who harbors the Arg308Pro (R308P) PPARγ variant after treatment with rosiglitazone. We also describe a novel Ala261Glu (A261E) PPARγ mutation, present in two apparently unrelated families, with similarly discordant responses to PGJ2 versus synthetic PPARγ agonists.

Participants provided informed written consent, and investigations were approved by local (Cambridge, U.K., and Cape Town, Africa) research ethics committees and conducted in accordance with the Declaration of Helsinki.

Characterization of transcriptional activity of PPARγ variants was undertaken as described previously (16). In brief, HEK293 Epstein-Barr nuclear antigen cells cultured in DMEM and 10% FCS were transfected with Lipofectamine 2000 in 96-well plates and assayed for luciferase and β-galactosidase activity after a 36-h incubation with or without ligand. Results represented the mean ± SEM of at least three independent experiments in triplicate.

Crystallographic modeling of PPARγ mutants was undertaken by using PPARγ structures (1PRG, 2PRG, 1FM9, 2ZK1, 3DZY, 2XKW) with different ligands. Results were illustrated by using the molecular graphics system MacPyMOL (Schrödinger, New York, NY). Additional methodological details are available in the Supplementary Data.

Two different heterozygous missense mutations in the ligand-binding pocket of PPARγ were identified in patients who presented with typical features of FPLD3. An R308P mutation was detected in a New Zealand woman, and an A261E mutation was identified in two unrelated women from South Africa (see Supplementary Data for clinical details and Table 1 for biochemical results).

Both R308 and A261 in PPARγ were highly conserved (Fig. 1A). In transfection assays that used reporter constructs containing either synthetic [(PPARE)₃TKLUC] or natural (human FABP4-LUC) enhancer/promoter elements, both R308P and A261E mutants exhibited negligible basal transcriptional activity and minimal responsiveness with PGJ2 (Fig. 1B and C). However, moderate (100 nmol/L farglitazar) or higher concentrations (1 μmol/L rosiglitazone; 10 μmol/L pioglitazone) of synthetic agonists restored transcriptional activity comparable to wild-type receptor (Fig. 1B and C). Of note, the R308P variant returned an intermediate, nondiagnostic functional score when tested in a high-throughput cellular assay (7) (Table 1), reflecting a similar discordance between failure to respond to PGJ2 and activation with rosiglitazone. These results suggest that the R308P mutant is transcriptionally resistant to both natural ligands present endogenously within transfected cells and PGJ2, with such loss-of-function likely contributing to the patient’s lipodystrophic phenotype.

In the crystal structure of the PPARγ ligand-binding domain, A261 and R308 are situated on different sides of the ligand-binding pocket (Fig. 2A). R308, located close to the amino terminus of helix 3 (Fig. 2A and B), participates in an extensive hydrogen-bond network (Fig. 2D–F) involving E287 in helix 2 and residues in the loop between helix 2 and 3. In the unliganded receptor, R308 also makes hydrogen bonds within helix 3 (Fig. 2D). Upon ligand binding, the loop between helix 2 and 3 adopts varying conformations, depending on the nature of the ligand (Supplementary Fig. 1). Mutation R308P would completely disrupt both the intra- and interhelical hydrogen-bond networks (Fig. 2G). Although PGJ2 does not alter the structural architecture of this region (Fig. 2H), binding of farglitazar, rosiglitazone, and pioglitazone can potentially alter the conformation of the loop between helix 2 and 3, thereby providing a mechanism that counteracts the destabilizing effect of the R308P mutation and preserving transcriptional responsiveness to these synthetic ligands (Fig. 2F and I and Supplementary Fig. 1B–D). In keeping with this prediction, proton nuclear magnetic resonance spectral analysis confirmed that pioglitazone can bind effectively to the R308P mutant (Supplementary Fig. 2).

A261 is located in helix 2a (Fig. 2A–C), and the size and charge difference of the A261E mutation will cause displacement of helix 2a and the loop to helix 2b, thereby destabilizing the ligand-binding pocket. This was confirmed by using circular dichroism studies showing a lower thermal denaturation temperature compared with the wild-type receptor (Supplementary Table 1). Because PGJ2 docks in this part of the ligand-binding cavity, its binding to receptor is expected to be impaired (Fig. 2C). In contrast, receptor occupancy by rosiglitazone, farglitazar, and pioglitazone is not structurally dependent on this region, correlating with preservation of transcriptional activation of the A261E mutant (Fig. 2B and Supplementary Fig. 1).

The R308P proband had previously been treated with dietary advice and metformin, but her metabolic control remained suboptimal, so pioglitazone 30 mg/day was commenced, resulting in dramatic improvements in glycemic control and dyslipidemia (Table 2). Her hirsutism, hyperandrogenism, and acanthosis nigricans also improved. These changes were largely sustained over a 3-year period without a substantial change in BMI (23.7–22.0 kg/m² at 12 months and 23.0 kg/m² at 24 months).

One of the patients with A261E was treated twice with rosiglitazone (4 mg twice daily) when her glycemic and triglyceride control deteriorated significantly. On each occasion, this intervention was accompanied by substantial falls in her HbA_1c as well as improvements in fasting triglyceride levels, although these remained labile (Supplementary Fig. 3). However, therapy was discontinued because it exacerbated the patient’s severe congestive heart failure, which ultimately caused her death at age 26 years.

The remarkable increase in access to and use of next-generation sequencing has accelerated the discovery of novel Mendelian disorders and detection of mutations in genes known to cause monogenic disorders like FPLD3, where ∼1 in 500 people harbor missense mutations (7). Although most are benign or mild in their impact, others are pathogenic but likely in a graded rather than a binary categorical fashion. Because synthetic PPARγ ligands are licensed treatments and given the severity of the metabolic complications seen in patients with FPLD3, use of TZDs is the obvious therapeutic option. Theoretically, patients with FPLD3 could be 1) resistant to TZDs because of the extreme deleterious nature of the underlying PPARγ defect; 2) responsive to therapy with mutations that are unresponsive to low-affinity, endogenous ligands yet activated by higher-affinity synthetic agonists; or 3) potentially hyperresponsive to specific designer ligands that can overcome the molecular defect particular to a specific receptor mutation.

We report on two FPLD3-associated PPARγ mutations (A261E, R308P) whose properties fall into the latter categories (2 and 3 above). Despite its transcriptional efficacy with wild-type PPARγ, PGJ2, a ligand that is prototypic of the various endogenous fatty acid and eicosanoid PPARγ activators, was unable to fully activate transcription mediated by A261E or R308P mutants (Fig. 1B), whereas exposure to high-affinity synthetic ligands like rosiglitazone, pioglitazone, and farglitazar achieved full transcriptional activity. Crystal structures of PPARγ bound to either farglitazar, rosiglitazone, pioglitazone, or PGJ2 show differences between these ligands in the nature of their occupancy of the binding cavity (Supplementary Fig. 1), and structural modeling provides a plausible basis for differential mutant PPARγ responses to prototypic endogenous versus synthetic ligands. Although synthetic agonists do not occupy the region of the pocket where A261 is situated, this residue is in proximity to PGJ2 and other fatty acid ligands (16). Modeling of the A261E mutation suggests that the alanine to glutamic acid change is likely to perturb PGJ2 binding directly through steric hindrance, whereas receptor interaction with rosiglitazone, pioglitazone, and farglitazar would be preserved, correlating with the observed transcriptional responses (Fig. 1B and C).

The R308P mutation involves a different part of the ligand-binding cavity, and this residue does not make direct contact with ligands. Structural modeling suggests that the arginine to proline change would disrupt local hydrogen-bond networks, with deleterious conformational consequences affecting transcriptional function of the receptor. Rosiglitazone, pioglitazone, and farglitazar (but not PGJ2), bind in proximity to R308, possibly stabilizing receptor structure. In particular, pioglitazone, which we have shown to bind effectively to the R308P mutant receptor, makes a hydrogen bond with E287 in helix 2, as does R308, counteracting the effect of the R308P mutation, which is predicted to disrupt this interaction (Fig. 2F–I).

In vitro studies with R308P mutant PPARγ mirrored the patient’s dramatic and sustained response to pioglitazone therapy. Thus, her case highlights the importance of recognizing and then establishing the genetic basis for severe, early-onset, metabolic disease. Identification of a PPARG mutation enabled early treatment with pioglitazone in preference to other standard glucose-lowering therapies, resulting in substantial clinical improvements in all metabolic abnormalities paralleled by specific redistribution of body fat away from visceral and with expansion of subcutaneous depots. Moreover, structural modeling and verification with studies of mutant receptor function in vitro provide a plausible explanation for in vivo observations. Specifically, impaired receptor activation by endogenous ligands presumably mediates diminished adipogenesis and the FPLD3 phenotype; the subsequent profound therapeutic response to pioglitazone likely reflects the ability of this synthetic agonist to bypass or overcome the molecular consequences of this mutation. In patients harboring A261E mutant PPARγ, we have documented similar discordant transcriptional responses to prototypic endogenous ligand versus synthetic agonists. We have shown that this translates into a beneficial therapeutic response to rosiglitazone in one patient with this receptor defect.

Although our observations are based on prismatic case studies, they appear to be supported by structural analyses of the isolated reports of TZD use in patients with FPLD3 (Supplementary Table 1). Such concordance among structural modeling of PPARγ mutations, transcriptional responses of mutant PPARγ to ligands in vitro, and clinical responses to treatment with synthetic agonists in vivo highlight the potential for this approach to inform individualized therapeutic choices.

Acknowledgments. The authors are grateful to the participating patients.

Funding. J.W.R.S., K.C., and D.B.S. are supported by the Wellcome Trust (grants WT100237, WT095564, and WT107064, respectively), the MRC Metabolic Disease Unit, the National Institute for Health Research (NIHR) Cambridge Biomedical Research Centre, and the NIHR Rare Disease Translational Research Collaboration. J.W.R.S. holds a Royal Society Wolfson Research Merit Award.

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. M.A. and E.S. undertook functional studies of the mutants, analyzed data, and wrote parts of the manuscript. E.S., L.F., F.W.M., and J.W.R.S. undertook structural modeling of mutations, circular dichroism, and nuclear magnetic resonance studies and reviewed the manuscript. J.B. and R.M. characterized and treated proband 1 and wrote parts of the manuscript. I.S., S.O., R.K.S., L.N., and A.R.M. analyzed data and reviewed the manuscript. O.R. performed sequencing studies, identified the PPARG mutations, and reviewed the manuscript. C.A. coordinated studies and reviewed the manuscript. A.D.M. and D.J.B. characterized probands 2 and 3, analyzed data, and wrote parts of the manuscript. R.M., K.C., and D.B.S. planned studies, analyzed data, and wrote the manuscript. D.B.S. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.