Massively parallel characterization of CYP2C9 variant enzyme activity and abundance

Amorosi, Clara J.; Chiasson, Melissa A; McDonald, Matthew; Wong, Lai Hong; Sitko, Katherine A.; Boyle, Gabriel; Kowalski, John P.; Rettie, Allan E.; Fowler, Douglas M.; Dunham, Maitreya J.

doi:10.1016/j.ajhg.2021.07.001

Cited by 67 publications

(75 citation statements)

References 66 publications

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“…High throughput DNA sequencing is used to quantify the distribution of each variant across the four bins, and this distribution is analyzed to yield an abundance score for each variant. VAMP-seq has thus far been applied to five proteins: PTEN [ 9 ], TPMT [ 9 ], NUDT15 [ 24 ], VKOR [ 25 ], and CYP2C9 [ 26 ].…”

Section: Resultsmentioning

confidence: 99%

“…For different proteins and assays, each analogous plot will likely differ, and thus, care must be taken when integrating multiple large-scale variant effect datasets and interpreting what they mean for protein-specific relationship between abundance and activity. In fact, a recent comparative analysis of yeast-based enzymatic activity and mammalian intracellular abundance of CYP2C9 revealed the opposite pattern, where there were many abundant variants with little detectable enzymatic activity, suggesting that the sensitivities of the two assays had flipped in this case [ 26 ].…”

Section: Discussionmentioning

confidence: 99%

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Integrating thousands of PTEN variant activity and abundance measurements reveals variant subgroups and new dominant negatives in cancers

et al. 2021

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Background PTEN is a multi-functional tumor suppressor protein regulating cell growth, immune signaling, neuronal function, and genome stability. Experimental characterization can help guide the clinical interpretation of the thousands of germline or somatic PTEN variants observed in patients. Two large-scale mutational datasets, one for PTEN variant intracellular abundance encompassing 4112 missense variants and one for lipid phosphatase activity encompassing 7244 variants, were recently published. The combined information from these datasets can reveal variant-specific phenotypes that may underlie various clinical presentations, but this has not been comprehensively examined, particularly for somatic PTEN variants observed in cancers. Methods Here, we add to these efforts by measuring the intracellular abundance of 764 new PTEN variants and refining abundance measurements for 3351 previously studied variants. We use this expanded and refined PTEN abundance dataset to explore the mutational patterns governing PTEN intracellular abundance, and then incorporate the phosphatase activity data to subdivide PTEN variants into four functionally distinct groups. Results This analysis revealed a set of highly abundant but lipid phosphatase defective variants that could act in a dominant-negative fashion to suppress PTEN activity. Two of these variants were, indeed, capable of dysregulating Akt signaling in cells harboring a WT PTEN allele. Both variants were observed in multiple breast or uterine tumors, demonstrating the disease relevance of these high abundance, inactive variants. Conclusions We show that multidimensional, large-scale variant functional data, when paired with public cancer genomics datasets and follow-up assays, can improve understanding of uncharacterized cancer-associated variants, and provide better insights into how they contribute to oncogenesis.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Integrating thousands of PTEN variant activity and abundance measurements reveals variant subgroups and new dominant negatives in cancers

et al. 2021

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“…This particular process uses yeast as a surrogate to distinguish human gene variants based on the activity of their respective protein products. One such example, developed by Amorosi and colleagues (2021) , is Click-Seq. Click-Seq uses enzymatic activity as a readout for massively parallel characterization of human gene variants in yeast.…”

Section: Expanding the Scope Of Studying Human Genes In Yeastmentioning

confidence: 99%

“…NGS of plasmids from non-fluorescing yeast cells reveals catalytically inactive human variants. A yeast Click-Seq screen for human CYP2C9 , a highly polymorphic gene encoding a cytochrome P450 monooxygenase that metabolizes many molecules, including pharmaceuticals, identified variants that aberrantly metabolize drugs and are thus responsible for adverse drug effects ( Amorosi et al, 2021 ) ( Fig. 6 B).…”

Section: Expanding the Scope Of Studying Human Genes In Yeastmentioning

confidence: 99%

Humanized yeast to model human biology, disease and evolution

Kachroo

Vandeloo

Greco

et al. 2022

Disease Models &Amp; Mechanisms

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For decades, budding yeast, a single-cellular eukaryote, has provided remarkable insights into human biology. Yeast and humans share several thousand genes despite morphological and cellular differences and over a billion years of separate evolution. These genes encode critical cellular processes, the failure of which in humans results in disease. Although recent developments in genome engineering of mammalian cells permit genetic assays in human cell lines, there is still a need to develop biological reagents to study human disease variants in a high-throughput manner. Many protein-coding human genes can successfully substitute for their yeast equivalents and sustain yeast growth, thus opening up doors for developing direct assays of human gene function in a tractable system referred to as ‘humanized yeast’. Humanized yeast permits the discovery of new human biology by measuring human protein activity in a simplified organismal context. This Review summarizes recent developments showing how humanized yeast can directly assay human gene function and explore variant effects at scale. Thus, by extending the ‘awesome power of yeast genetics’ to study human biology, humanizing yeast reinforces the high relevance of evolutionarily distant model organisms to explore human gene evolution, function and disease.

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“…Phage display systems display proteins on the surface of a phage, and the selection is made with the use of specific antibodies or ligands [ 49 , 50 ]. Selection based on fluorescence-activated cell sorting (FACS) is useful for investigating variants that affect enzyme activity, protein stability, or protein abundance [ 51 , 52 , 53 ], where the fluorescence intensity is proportional to the features of proteins. In addition, luciferase reporter assay was reported to evaluate the impact of variants on splicing [ 54 ] and variants within the non-coding regions [ 39 ].…”

Section: The Emergence Of High-throughput Functional Assaysmentioning

confidence: 99%

Current and Future Approaches to Classify VUSs in LGMD-Related Genes

Haller

Weihl

2022

Genes

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Next-generation sequencing (NGS) has revealed large numbers of genetic variants in LGMD-related genes, with most of them classified as variants of uncertain significance (VUSs). VUSs are genetic changes with unknown pathological impact and present a major challenge in genetic test interpretation and disease diagnosis. Understanding the phenotypic consequences of VUSs can provide clinical guidance regarding LGMD risk and therapy. In this review, we provide a brief overview of the subtypes of LGMD, disease diagnosis, current classification systems for investigating VUSs, and a potential deep mutational scanning approach to classify VUSs in LGMD-related genes.

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Massively parallel characterization of CYP2C9 variant enzyme activity and abundance

Cited by 67 publications

References 66 publications

Integrating thousands of PTEN variant activity and abundance measurements reveals variant subgroups and new dominant negatives in cancers

Integrating thousands of PTEN variant activity and abundance measurements reveals variant subgroups and new dominant negatives in cancers

Humanized yeast to model human biology, disease and evolution

Current and Future Approaches to Classify VUSs in LGMD-Related Genes

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