Rare variants in PPARG with decreased activity in adipocyte differentiation are associated with increased risk of type 2 diabetes

Abstract

Peroxisome proliferator-activated receptor gamma (PPARG) is a master transcriptional regulator of adipocyte differentiation and a canonical target of antidiabetic thiazolidinedione medications. In rare families, loss-of-function (LOF) mutations in PPARG are known to cosegregate with lipodystrophy and insulin resistance; in the general population, the common P12A variant is associated with a decreased risk of type 2 diabetes (T2D). Whether and how rare variants in PPARG and defects in adipocyte differentiation influence risk of T2D in the general population remains undetermined. By sequencing PPARG in 19,752 T2D cases and controls drawn from multiple studies and ethnic groups, we identified 49 previously unidentified, nonsynonymous PPARG variants (MAF < 0.5%). Considered in aggregate (with or without computational prediction of functional consequence), these rare variants showed no association with T2D (OR = 1.35; P = 0.17). The function of the 49 variants was experimentally tested in a novel high-throughput human adipocyte differentiation assay, and nine were found to have reduced activity in the assay. Carrying any of these nine LOF variants was associated with a substantial increase in risk of T2D (OR = 7.22; P = 0.005). The combination of large-scale DNA sequencing and functional testing in the laboratory reveals that approximately 1 in 1,000 individuals carries a variant in PPARG that reduces function in a human adipocyte differentiation assay and is associated with a substantial risk of T2D.

Fig. 1.

High-throughput quantification of adipocyte differentiation in response to exogenous PPARγ. (A) Preadipocytes are cultured in 96-well plates, differentiated for 8 d, and stained for lipid (BODIPY) and nuclei (DAPI). Each well is imaged in a high-content microscope for lipid and nuclei. Adipocytes and undifferentiated cells are identified by the overlay of lipid and nuclei from automated image analysis. (B) Preadipocytes were plated at increasing density and differentiated. Parallel samples were subjected to image-based differentiation measurement or Oil Red O staining followed by lipid extraction and spectrophotometric quantification. (C) Preadipocyte differentiation in response to increasing doses of exogenous PPARγ with and without 3-isobutyl-1-methylxanthine (IBMX). (D) Gene expression levels in preadipocytes of endogenous and exogenous PPARγ in response to increasing doses of exogenous PPARγ. Error bars indicate ±1 SEM.

Fig. 2.

Experimental characterization of rare PPARγ variants identified from population sequencing. (A) Each PPARγ variant was generated and tested for its ability to rescue adipocyte differentiation in vitro. From left to right PPARγ variants are sorted by in vitro function in three groups: (i) WT from independent experiments, (ii) previously identified synthetic and human mutations, and (iii) variants identified in population based exon resequencing. Blue dashed lines denote the 95% confidence interval of WT function. (B) Roziglitazone (rozi) dose–response of PPARγ variants identified as LOF. The amino acid position along the PPARγ protein is shown. EC₅₀ WT denotes the rozi dose required to achieve 50% of maximal WT response. AD, activation domain; DBD, DNA-binding domain; LBD, ligand-binding domain. Error bars indicate ±1 SEM. Significant differences compared with WT are noted: *P < 0.05; **P < 0.005; ***P < 0.0001. ^‡Variants identified in families with partial lipodystrophy.

Fig. 3.

T2D case/control status in multiethnic individuals harboring nonsynonymous PPARG variants, according to PPARG function in vitro. Each point represents an individual variant; point size denotes the number of individuals carrying that variant. Function in vitro was determined by the ability of each variant to rescue adipocyte differentiation in comparison with WT PPARG. The blue dashed line indicates the threshold for a one-tailed t test below which variants are classified as LOF compared with WT PPARG (P < 0.05). Odds ratios and P values for T2D case status among individuals carrying benign and LOF variants were calculated as described in Methods. *Variants observed only in a single case or control individual.