Abstract
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.
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Acknowledgements
This work was supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (1K08DK102877-01, to A.R.M.; 1R01DK097768-01, to D.A.), NIH/Harvard Catalyst (1KL2TR001100-01, to A.R.M.), the Broad Institute (SPARC award, to A.R.M. and T.M.), and the Wellcome Trust (095564, to K.C.; 107064, to D.B.S.).
We thank J. Doench, C. Zhu, D. O'Connell, G. Cowley, M. Sullender, D. MacArthur, E. Minkel, B. Bulik-Sullivan, and J. Avruch for helpful discussions, laboratory assistance, and manuscript review.
This paper is dedicated to the memory of Promila Nandi (30 April 1933–27 December 2013).
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A.R.M., T.M., and D.A. designed the study. A.R.M., B.T., M.A., and K.G. performed experiments with help from R.R., X.Z., M.F.B., and E.K. A.R.M. and N.P. analyzed the data with help from B.T., T.S., G.P., K.A.P., M.D., and T.M. I.B., S.E., S.K., S.O'R., K.C., and D.B.S. contributed clinical data and genotypes. A.R.M. and D.A. wrote the manuscript. D.B.S., S.O'R., K.C., E.D.R., and J.C.F. revised the manuscript.
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Majithia, A., Tsuda, B., Agostini, M. et al. Prospective functional classification of all possible missense variants in PPARG. Nat Genet 48, 1570–1575 (2016). https://doi.org/10.1038/ng.3700
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DOI: https://doi.org/10.1038/ng.3700
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