Determining the pathogenicity of human genetic variants is a critical challenge, and functional assessment is often the only option. Experimentally characterizing millions of possible missense variants in thousands of disease-related genes requires generalizable, scalable assays. We developed Variant Abundance by Massively Parallel Sequencing (VAMP-seq), which measures the effect of thousands of missense variants of a protein on intracellular abundance in a single experiment. Using VAMP-seq, we quantified the abundance of 7,801 variants of two disease-related proteins, PTEN and TPMT. We identified 1,138 low-abundance PTEN variants that could be pathogenic and 777 low-abundance TPMT variants that could alter drug metabolism. We observed selection for low-abundance PTEN variants in cancer, and identified a novel dominant negative PTEN variant found in ~10% of melanomas. Finally, we showed that VAMP-seq can be applied to other genes, highlighting its potential as a generalizable way to characterize missense variants.
A mixed population of cells each expressing one protein variant fused to EGFP is created. The variant dictates the abundance of the variant-EGFP fusion protein, resulting in a range of cellular EFGP fluorescence levels. Cells are then sorted into bins based on their level of fluorescence, and high throughput sequencing is used to quantify every variant in each bin. VAMP-seq scores are calculated from the scaled, weighted average of variants across bins. The resulting sequence-function maps describe the relative intracellular abundance of thousands of protein variants.
TPMT/PTEN Protein Variant Abundance Scores
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Matreyek KA*, Starita LM*, Stephany JJ, Martin B, Chiasson MA, Gray VE, Kircher M, Khechaduri A, Dines JN, Hause RJ, Bhatia S, Evans WE, Relling MV, Yang W, Shendure J, Fowler DM. *contributed equally
Nature Genetics. In Press.Link to journal website