title: Prospective functional classification of all possible missense variants in PPARG.
abstract: 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.
authors.affiliation: Program in Medical & Population Genetics, Broad Institute of Harvard and MIT, Cambridge, MA, USA.|Diabetes Research Center, Diabetes Unit, Department of Medicine, Massachusetts General Hospital, Boston, MA, USA.|Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA, USA.|Department of Medicine, Harvard Medical School, Boston, MA, USA.
authors.forename: Amit R
authors.initials: AR
authors.lastname: Majithia
authors.affiliation: Program in Medical & Population Genetics, Broad Institute of Harvard and MIT, Cambridge, MA, USA.
authors.forename: Ben
authors.initials: B
authors.lastname: Tsuda
authors.affiliation: University of Cambridge Metabolic Research Laboratories, Wellcome Trust-Medical Research Council Institute of Metabolic Science, Cambridge CB2 0QQ, United Kingdom.
authors.forename: Maura
authors.initials: M
authors.lastname: Agostini
authors.affiliation: Program in Medical & Population Genetics, Broad Institute of Harvard and MIT, Cambridge, MA, USA.
authors.forename: Keerthana
authors.initials: K
authors.lastname: Gnanapradeepan
authors.affiliation: Program in Medical & Population Genetics, Broad Institute of Harvard and MIT, Cambridge, MA, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Rice
authors.affiliation: Program in Medical & Population Genetics, Broad Institute of Harvard and MIT, Cambridge, MA, USA.|Department of Biostatistics, Boston University School of Public Health, Boston, MA, USA.
authors.forename: Gina
authors.initials: G
authors.lastname: Peloso
authors.affiliation: Institute of Biomedical and Clinical Science, University of Exeter Medical School, Exeter, UK.
authors.forename: Kashyap A
authors.initials: KA
authors.lastname: Patel
authors.affiliation: Program in Medical & Population Genetics, Broad Institute of Harvard and MIT, Cambridge, MA, USA.
authors.forename: Xiaolan
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Molecular Cancer Research and Center for Molecular Medicine, University Medical Centre Utrecht, Universiteitsweg 100, 3584 CG, Utrecht, The Netherlands.
authors.forename: Marjoleine F
authors.initials: MF
authors.lastname: Broekema
authors.affiliation: Program in Medical & Population Genetics, Broad Institute of Harvard and MIT, Cambridge, MA, USA.
authors.forename: Nick
authors.initials: N
authors.lastname: Patterson
authors.affiliation: Program in Medical & Population Genetics, Broad Institute of Harvard and MIT, Cambridge, MA, USA.
authors.forename: Marc
authors.initials: M
authors.lastname: Duby
authors.affiliation: Program in Medical & Population Genetics, Broad Institute of Harvard and MIT, Cambridge, MA, USA.
authors.forename: Ted
authors.initials: T
authors.lastname: Sharpe
authors.affiliation: Molecular Cancer Research and Center for Molecular Medicine, University Medical Centre Utrecht, Universiteitsweg 100, 3584 CG, Utrecht, The Netherlands.
authors.forename: Eric
authors.initials: E
authors.lastname: Kalkhoven
authors.affiliation: Department of Medicine, Harvard Medical School, Boston, MA, USA.|Division of Endocrinology and Metabolism, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02115, USA.
authors.forename: Evan D
authors.initials: ED
authors.lastname: Rosen
authors.affiliation: University of Cambridge Metabolic Research Laboratories, Wellcome Trust-Medical Research Council Institute of Metabolic Science, Cambridge CB2 0QQ, United Kingdom.
authors.forename: Inês
authors.initials: I
authors.lastname: Barroso
authors.affiliation: Institute of Biomedical and Clinical Science, University of Exeter Medical School, Exeter, UK.|Department of Molecular Genetics, Royal Devon and Exeter National Health Service Foundation Trust, Exeter, UK.
authors.forename: Sian
authors.initials: S
authors.lastname: Ellard
authors.affiliation: Program in Medical & Population Genetics, Broad Institute of Harvard and MIT, Cambridge, MA, USA.|Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA, USA.|Department of Medicine, Harvard Medical School, Boston, MA, USA.|Cardiovascular Research Center, Department of Medicine, Massachusetts General Hospital, Boston, MA, USA.
authors.forename: Sekar
authors.initials: S
authors.lastname: Kathiresan
authors.affiliation: University of Cambridge Metabolic Research Laboratories, Wellcome Trust-Medical Research Council Institute of Metabolic Science, Cambridge CB2 0QQ, United Kingdom.
authors.forename: Stephen
authors.initials: S
authors.lastname: O'Rahilly
authors.affiliation: University of Cambridge Metabolic Research Laboratories, Wellcome Trust-Medical Research Council Institute of Metabolic Science, Cambridge CB2 0QQ, United Kingdom.
authors.forename: Krishna
authors.initials: K
authors.lastname: Chatterjee
authors.affiliation: Program in Medical & Population Genetics, Broad Institute of Harvard and MIT, Cambridge, MA, USA.|Diabetes Research Center, Diabetes Unit, Department of Medicine, Massachusetts General Hospital, Boston, MA, USA.|Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA, USA.|Department of Medicine, Harvard Medical School, Boston, MA, USA.
authors.forename: Jose C
authors.initials: JC
authors.lastname: Florez
authors.affiliation: Program in Medical & Population Genetics, Broad Institute of Harvard and MIT, Cambridge, MA, USA.
authors.forename: Tarjei
authors.initials: T
authors.lastname: Mikkelsen
authors.affiliation: University of Cambridge Metabolic Research Laboratories, Wellcome Trust-Medical Research Council Institute of Metabolic Science, Cambridge CB2 0QQ, United Kingdom.
authors.forename: David B
authors.initials: DB
authors.lastname: Savage
authors.affiliation: Program in Medical & Population Genetics, Broad Institute of Harvard and MIT, Cambridge, MA, USA.|Diabetes Research Center, Diabetes Unit, Department of Medicine, Massachusetts General Hospital, Boston, MA, USA.|Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA, USA.|Department of Medicine, Harvard Medical School, Boston, MA, USA.
authors.forename: David
authors.initials: D
authors.lastname: Altshuler
mesh_terms: D019943:Amino Acid Substitution; D016022:Case-Control Studies; D003924:Diabetes Mellitus, Type 2 / Q000235:genetics*; D005260:Female; D006801:Humans; D008060:Lipodystrophy / Q000235:genetics*; D008264:Macrophages / Q000378:metabolism / Q000473:pathology; D008297:Male; D020125:Mutation, Missense / Q000235:genetics*; D009203:Myocardial Infarction / Q000235:genetics*; D047495:PPAR gamma / Q000235:genetics*; D011446:Prospective Studies
chemical_list: D047495:PPAR gamma