Multidimensional PTEN missense variant analysis reveals variant subgroups including potential dominant negatives

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 clinically important genes will likely require generalizable, scalable assays. We previously developed Variant Abundance by Massively Parallel Sequencing (VAMP-seq), which measures the effects of thousands of missense variants of a protein on intracellular abundance in a single experiment. Here, we reapplied VAMP-seq to quantify the abundances of additional PTEN missense variants which were missing from the original experiment. Overall design: Barcoded missense variant libraries of EGFP-fused PTEN were recombined into HEK293T cells previously engineered to contain a Bxb1 recombination site at the AAVS1 locus. Upon sorting cells for EGFP expression, genomic DNA was extracted. Barcodes were amplified and counted with Illumina sequencing. Barcodes were associated back to the corresponding PTEN variant by comparison with a barcode-variant map previously created by sequencing the variant library plasmids using PacBio sequencing. Copyright 2018 University of Washington. Data and Scores are owned by the University of Washington. Permission is hereby granted to use, reproduce, and distribute the Data and Scores for noncommercial academic research purposes only, provided that (i) credit for source and copyright are included with each copy and (ii) a link to the original material is provided whenever the material is published elsewhere on the Web. For questions regarding use by non-profit entities or commercial purposes contact: Douglas Fowler, [e-mail]