Multiplex Assessment of Protein Variant Abundance by Massively Parallel Sequencing

Matreyek, Kenneth A.; Stephany, Jason J.; Martin, Beth; Chiasson, Melissa A; Gray, Vanessa E.; Kircher, Martin; Khechaduri, Arineh; Dines, Jennifer N.; Hause, Ronald J.; Bhatia, Smita; Evans, William E.; Relling, Mary V.; Yang, Wenjian; Shendure, Jay; Fowler, Douglas M.

doi:10.1101/211011

Cited by 25 publications

(30 citation statements)

References 85 publications

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“…As outlined above, many missense variants in PAH have been linked to PKU. Although some missense variants may directly ablate protein function, for example, by changes in the active or cofactor‐binding sites, it has been shown that, in general, most missense variant proteins are less structurally stable than the corresponding wild‐type protein (Matreyek et al., ; Tokuriki, Stricher, Schymkowitz, Serrano, & Tawfik, ). Thus, a likely mechanism for the loss of function of many missense variants is loss of stability (Casadio, Vassura, Tiwari, Fariselli, & Luigi, ; Yue, Li, & Moult, ), leading to increased proteasomal degradation and thus insufficient amounts of PAH protein.…”

Section: Resultsmentioning

confidence: 99%

Toward mechanistic models for genotype–phenotype correlations in phenylketonuria using protein stability calculations

et al. 2019

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Phenylketonuria (PKU) is a genetic disorder caused by variants in the gene encoding phenylalanine hydroxylase (PAH), resulting in accumulation of phenylalanine to neurotoxic levels. Here, we analyzed the cellular stability, localization, and interaction with wild‐type PAH of 20 selected PKU‐linked PAH protein missense variants. Several were present at reduced levels in human cells, and the levels increased in the presence of a proteasome inhibitor, indicating that proteins are proteasome targets. We found that all the tested PAH variants retained their ability to associate with wild‐type PAH, and none formed aggregates, suggesting that they are only mildly destabilized in structure. In all cases, PAH variants were stabilized by the cofactor tetrahydrobiopterin (BH4), a molecule known to alleviate symptoms in certain PKU patients. Biophysical calculations on all possible single‐site missense variants using the full‐length structure of PAH revealed a strong correlation between the predicted protein stability and the observed stability in cells. This observation rationalizes previously observed correlations between predicted loss of protein destabilization and disease severity, a correlation that we also observed using new calculations. We thus propose that many disease‐linked PAH variants are structurally destabilized, which in turn leads to proteasomal degradation and insufficient amounts of cellular PAH protein.

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Section: Resultsmentioning

confidence: 99%

Toward mechanistic models for genotype–phenotype correlations in phenylketonuria using protein stability calculations

et al. 2019

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“…For instance, almost all the top‐performing methods adjusted their scores to the variant solvent accessibility. This feature was indeed found to be the most correlated with assay scores in the recently published study on the PTEN‐TPMT data set (Matreyek et al, ) and perhaps provided these methods with a predictive advantage. We note that it is also possible that methods were similar to each other in their deviation from the requirements of the prediction challenge, that is, there was a mismatch between method objectives and the nature of the experimental data (see Section 4.4 for more details).…”

Section: Discussionmentioning

confidence: 62%

“…Some issues are important to consider here: First, note that VSP assays measure the steady‐state abundance of mutated proteins as a reflection of stability. In fact, protein abundance may be affected by other means, for example, altered posttranscriptional regulation, translational speeds, and disrupted trafficking (Matreyek et al, ). Similarly, note that the experiment‐specific (here, VSP assay‐specific) resolution limits and variation between replicates complicate the use of individual experimental data sets as a fixed gold standard to evaluate computational method performance.…”

Section: Discussionmentioning

confidence: 99%

“…Participating groups were allowed up to six submissions, and were asked to provide brief descriptions of the methods underlying each of their submissions (Supporting Information Methods). We note that although the true variant stability scores are now published (Matreyek et al, ), they were not made available to the participating groups until after the challenge was closed.…”

Section: Methodsmentioning

confidence: 99%

“…The recently developed multiplexed variant stability profiling (VSP) assay (Matreyek et al, ) uses the fluorescent reporter system (EGFP; to measure the protein variant abundance) and mCherry (expressed cotranscriptionally or cotranslationally from the same construct) on cells carrying mutated proteins. The per‐cell EGFP/mCherry ratios are calibrated with respect to cells carrying the wild‐type protein or known destabilized proteins to define a (high to low, stable to unstable) range.…”

Section: Introductionmentioning

confidence: 99%

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Assessment of methods for predicting the effects of PTEN and TPMT protein variants

et al. 2019

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Thermodynamic stability is a fundamental property shared by all proteins. Changes in stability due to mutation are a widespread molecular mechanism in genetic diseases. Methods for the prediction of mutation‐induced stability change have typically been developed and evaluated on incomplete and/or biased data sets. As part of the Critical Assessment of Genome Interpretation, we explored the utility of high‐throughput variant stability profiling (VSP) assay data as an alternative for the assessment of computational methods and evaluated state‐of‐the‐art predictors against over 7,000 nonsynonymous variants from two proteins. We found that predictions were modestly correlated with actual experimental values. Predictors fared better when evaluated as classifiers of extreme stability effects. While different methods emerging as top performers depending on the metric, it is nontrivial to draw conclusions on their adoption or improvement. Our analyses revealed that only 16% of all variants in VSP assays could be confidently defined as stability‐affecting. Furthermore, it is unclear as to what extent VSP abundance scores were reasonable proxies for the stability‐related quantities that participating methods were designed to predict. Overall, our observations underscore the need for clearly defined objectives when developing and using both computational and experimental methods in the context of measuring variant impact.

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VariCarta: A Comprehensive Database of Harmonized Genomic Variants Found in Autism Spectrum Disorder Sequencing Studies

et al. 2019

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Recent years have seen a boom in the application of the next‐generation sequencing technology to the study of human disorders, including Autism Spectrum Disorder (ASD), where the focus has been on identifying rare, possibly causative genomic variants in ASD individuals. Because of the high genetic heterogeneity of ASD, a large number of subjects is needed to establish evidence for a variant or gene ASD‐association, thus aggregating data across cohorts and studies is necessary. However, methodological inconsistencies and subject overlap across studies complicate data aggregation. Here we present VariCarta, a web‐based database developed to address these challenges by collecting, reconciling, and consistently cataloging literature‐derived genomic variants found in ASD subjects using ongoing semi‐manual curation. The careful manual curation combined with a robust data import pipeline rectifies errors, converts variants into a standardized format, identifies and harmonizes cohort overlaps, and documents data provenance. The harmonization aspect is especially important since it prevents the potential double counting of variants, which can lead to inflation of gene‐based evidence for ASD‐association. The database currently contains 170,416 variant events from 10,893 subjects, collected across 61 publications, and reconciles 16,202 variants that have been reported in literature multiple times. VariCarta is freely accessible at http://varicarta.msl.ubc.ca. Autism Res 2019, 12: 1728–1736. © 2019 International Society for Autism Research, Wiley Periodicals, Inc. Lay Summary The search for genetic factors underlying Autism Spectrum Disorder (ASD) yielded numerous studies reporting potentially causative genomic variants found in ASD individuals. However, methodological differences and subject overlap across studies complicate the assembly of these data, diminishing its utility and accessibility. We developed VariCarta, a web‐based database that aggregates carefully curated, annotated, and harmonized literature‐derived variants identified in individuals with ASD using ongoing semi‐manual curation.

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Multiplex Assessment of Protein Variant Abundance by Massively Parallel Sequencing

Cited by 25 publications

References 85 publications

Toward mechanistic models for genotype–phenotype correlations in phenylketonuria using protein stability calculations

Toward mechanistic models for genotype–phenotype correlations in phenylketonuria using protein stability calculations

Assessment of methods for predicting the effects of PTEN and TPMT protein variants

VariCarta: A Comprehensive Database of Harmonized Genomic Variants Found in Autism Spectrum Disorder Sequencing Studies

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