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Prospective functional classification of all possible missense variants in PPARG

2016; Nature Portfolio; Volume: 48; Issue: 12 Linguagem: Inglês

10.1038/ng.3700

1546-1718

Amit R. Majithia, Ben Tsuda, Maura Agostini, Keerthana Gnanapradeepan, Robert H. Rice, Gina M. Peloso, Kashyap Patel, Xiaolan Zhang, Marjoleine F. Broekema, Nick Patterson, Marc Duby, Ted Sharpe, Eric Kalkhoven, Evan D. Rosen, Inês Barroso, Sian Ellard, Sekar Kathiresan, Stephen O’Rahilly, Krishna Chatterjee, José C. Florez, Tarjei S. Mikkelsen, David B. Savage, David Altshuler,

RNA modifications and cancer

Amit Majithia and colleagues employ a pooled assay in human macrophages to assess the functional effects of all possible missense variants in PPARG. Their study shows the value of saturation mutagenesis and prospective experimental characterization to support diagnostic interpretation of newly discovered missense variants in disease-related genes. Clinical exome sequencing routinely identifies missense variants in disease-related genes, but functional characterization is rarely undertaken, leading to diagnostic uncertainty1,2. For example, mutations in PPARG cause Mendelian lipodystrophy3,4 and increase risk of type 2 diabetes (T2D)5. 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.