Comprehensive Assessment of Protein Variant Abundance by Massively Parallel Sequencing

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.

Overview of experimental workflow

VAMP-seq overview

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

Academic License Agreement

The University of Washington hereby allows User and Institution to search, download, copy or use TPMT/PTEN Variant Abundance Scores (TPMT/PTEN variant abundance scores) on the following conditions:

1. The scores are being made available here for non-profit, non-commercial uses only. If User wishes to use TPMT/PTEN variant abundance scores, derivatives of TPMT/PTEN variant abundance scores, or the data on which they are based, for any commercial or for-profit purpose, please contact the corresponding authors. Low-cost or no-cost licenses will be extended for commercial or for-profit uses, contingent on the engagement of the licensing entity in best practices specified by ClinGen for TPMT/PTEN variant data sharing.

2. The University of Washington owns the intellectual property for TPMT/PTEN variant abundance scores. In any reposting of the scores or their derivatives, User agrees to retain the copyright, trademark, or other notices pertaining to TPMT/PTEN variant abundance scores as provided by UW associated with the TPMT/PTEN variant abundance scores. Use of the copyright in any printed excerpts shall include the following notice (or the equivalent thereof): 'TPMT/PTEN variant abundance scores are the copyright of the University of Washington.'

3. Any risk associated with using TPMT/PTEN variant abundance scores by User is with User and Institution. TPMT/PTEN variant abundance scores are experimental in nature and is made available as a research courtesy 'AS IS,' without obligation by the University of Washington to provide accompanying services or support. The TPMT/PTEN variant abundance scores are for informational purposes only, and is not intended to be a substitute for professional medical advice, diagnosis, or treatment. UW AND THE DEVELOPER EXPRESSLY DISCLAIM ANY AND ALL WARRANTIES REGARDING THE TPMT/PTEN ABUNDANCE SCORES, WHETHER EXPRESS OR IMPLIED, INCLUDING BUT NOT LIMITED TO WARRANTIES PERTAINING TO NON-INFRINGEMENT, MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE.

4. TPMT/PTEN variant abundance scores may be accessed after providing the following information:



Multiplex Assessment of Protein Variant Abundance by Massively Parallel Sequencing.

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

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