Prospective functional classification of all possible missense variants in PPARG

Nat Genet. 2016 Dec;48(12):1570-1575. doi: 10.1038/ng.3700. Epub 2016 Oct 17.


Clinical exome sequencing routinely identifies missense variants in disease-related genes, but functional characterization is rarely undertaken, leading to diagnostic uncertainty. For example, mutations in PPARG cause Mendelian lipodystrophy and increase risk of type 2 diabetes (T2D). Although approximately 1 in 500 people harbor missense variants in PPARG, most are of unknown consequence. To prospectively characterize PPARγ variants, we used highly parallel oligonucleotide synthesis to construct a library encoding all 9,595 possible single-amino acid substitutions. We developed a pooled functional assay in human macrophages, experimentally evaluated all protein variants, and used the experimental data to train a variant classifier by supervised machine learning. When applied to 55 new missense variants identified in population-based and clinical sequencing, the classifier annotated 6 variants as pathogenic; these were subsequently validated by single-variant assays. Saturation mutagenesis and prospective experimental characterization can support immediate diagnostic interpretation of newly discovered missense variants in disease-related genes.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Case-Control Studies
  • Diabetes Mellitus, Type 2 / genetics*
  • Female
  • Humans
  • Lipodystrophy / genetics*
  • Macrophages / metabolism
  • Macrophages / pathology
  • Male
  • Mutation, Missense / genetics*
  • Myocardial Infarction / genetics*
  • PPAR gamma / genetics*
  • Prospective Studies


  • PPAR gamma