PMC1368963_01
A Canadian multigenerational FPLD3 kindred. Darkened symbols indicate affected individuals, each of whom was later confirmed to be heterozygous for the respective PPARG nonsense mutation. Dots inside white symbols indicate normal PPARG genotype. Subject identification number and age in years are shown below the appropriate symbols.
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F1
Subject III-3 (Figure 1).
Selected imaging studies from the FPLD3 subject. Panel shows computed tomography cross-section of upper thigh of a normal individual and subject II-5, who had markedly decreased subcutaneous fat on both extremities.
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F2
Figure 2 shows a computed tomographic scan of the legs of the proband and a normal subject; the proband had a relative paucity of fat.
PMC1368963_02
A Canadian multigenerational FPLD3 kindred. Darkened symbols indicate affected individuals, each of whom was later confirmed to be heterozygous for the respective PPARG nonsense mutation. Dots inside white symbols indicate normal PPARG genotype. Subject identification number and age in years are shown below the appropriate symbols.
1471-2350-7-3-1.jpg
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F1
Subject III-3 (Figure 1).
Genomic DNA sequence electropherograms of heterozygous PPARG mutation. The bottom electropherogram tracing shows both alleles in subject II-5 from kindred 1 (Y355X), compared to sequence from a healthy subject. The position of the mutation is indicated by the arrow. Normal nucleotide and amino acid sequence is shown above the wild type electropherogram tracing.
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F3
Subjects II-5 and III-3 were each found to be heterozygous for a nonsense mutation (single nucleotide base change), that predicted a premature termination signal rather than the wild-type (WT) tyrosine at codon 355 (Figure 3).
Y355X mutation reduces PPARgamma protein stability and transcriptional activity. (A) Western blot of lysates from NIH 3T3 cells transfected with the indicated amount of plasmid expressing either FLAG tagged Y355X (fl355X) or wild-type (flWT) PPARgamma proteins. PPARgamma was visualized with anti-FLAG antibodies and ECFP produced from co-transfected plasmid (visualized with an anti-ECFP antibody) was utilized as a transfection and loading control. The numbers below the panel represent the density of each PPARgamma-FLAG band normalized to the ECFP band. Roughly equal amounts of mutant and WT protein expression were achieved by a 15 fold excess of transfected mutant over WT PPARgamma DNA. (B) Rosiglitazone dose-response curves for WT and Y355X PPARgamma in NIH 3T3 cells transfected with 1 ng of WT or 15 ng of Y355X expression plasmids (non-FLAG-tagged versions) or 15 ng of empty vector, together with a PPAR responsive reporter construct (pFATP-luc) and a beta-galactosidase reference plasmid. Data are normalized to the WT vehicle controls and are means +- SD (n = 3).
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WT and mutant PPARgamma clones were transfected into NIH3T3 cells. Western analysis of extracts from these cells demonstrated that much less Y355X mutant protein was produced from the same amount of transfected DNA suggesting that the mutant receptor was significantly less stable than the wild-type receptor (Figure 4A). Under these conditions the Y355X mutation did not display any transcriptional activity at any dose of the ligand rosiglitazone (Figure 4B).
The Y355X mutant receptor does not inhibit the activity of wild-type PPARgamma. (A) WT and Y355X receptors (1 and 15 ng respectively) were transfected into NIH 3T3 cells individually or in combination in the presence or absence of 5 muM rosiglitazone (Rosi). (B) NIH 3T3 cells were transfected with the combination of 1 ng of WT PPARgamma and increasing amounts of either the WT (1-4 ng), the dominant/negative mutant V290M (1-4 ng) or the Y355X mutant receptor (1-15 ng) as indicated. Data are normalized to the transcriptional activity of 1 ng of WT receptor and are presented as means +- SD (n = 3).
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F5
In the absence of ligand, the transcriptional activity of the combination of 1 ng of WT and 15 ng of Y355X DNA (equal protein amounts) was similar to 1 ng of WT receptor alone (Figure 5A). WT receptor was mixed with a fixed amount of WT PPARgamma (Figure 5B). While increasing the amount of the wild-type receptor caused a dose dependent increase in transcriptional activity (Figure 5B, open bars), the addition of increasing amounts of Y355X PPARgamma to a fixed amount of wild-type receptor had no significant effect on transcription (Figure 5B, filled bars). Increasing amounts of V290M PPARgamma caused a dose-dependent decrease in wild-type PPARgamma transcriptional activity in this assay (Figure 5B, grey bars).