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Mitochondrial Phenotypes Distinguish Pathogenic MFN2 Mutations by Pooled Functional Genomics Screen

View ORCID ProfileAlex L. Yenkin, View ORCID ProfileJohn C. Bramley, View ORCID ProfileColin L. Kremitzki, View ORCID ProfileJason E. Waligorski, View ORCID ProfileMariel J. Liebeskind, View ORCID ProfileXinyuan E. Xu, View ORCID ProfileMaria A. Vakaki, View ORCID ProfileVinay D. Chandrasekaran, View ORCID ProfileRobi D. Mitra, View ORCID ProfileJeffrey D. Milbrandt, View ORCID ProfileWilliam J. Buchser
doi: https://doi.org/10.1101/2021.03.12.434746
Alex L. Yenkin
Department of Genetics, Washington University School of Medicine, St Louis MO 63110 Functional Imaging for Variant Elucidation at the McDonnell Genome Institute
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  • ORCID record for Alex L. Yenkin
John C. Bramley
Department of Genetics, Washington University School of Medicine, St Louis MO 63110 Functional Imaging for Variant Elucidation at the McDonnell Genome Institute
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Colin L. Kremitzki
Department of Genetics, Washington University School of Medicine, St Louis MO 63110 Functional Imaging for Variant Elucidation at the McDonnell Genome Institute
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Jason E. Waligorski
Department of Genetics, Washington University School of Medicine, St Louis MO 63110 Functional Imaging for Variant Elucidation at the McDonnell Genome Institute
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Mariel J. Liebeskind
Department of Genetics, Washington University School of Medicine, St Louis MO 63110 Functional Imaging for Variant Elucidation at the McDonnell Genome Institute
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Xinyuan E. Xu
Department of Genetics, Washington University School of Medicine, St Louis MO 63110 Functional Imaging for Variant Elucidation at the McDonnell Genome Institute
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Maria A. Vakaki
Department of Genetics, Washington University School of Medicine, St Louis MO 63110 Functional Imaging for Variant Elucidation at the McDonnell Genome Institute
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Vinay D. Chandrasekaran
Department of Genetics, Washington University School of Medicine, St Louis MO 63110 Functional Imaging for Variant Elucidation at the McDonnell Genome Institute
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Robi D. Mitra
Department of Genetics, Washington University School of Medicine, St Louis MO 63110 Functional Imaging for Variant Elucidation at the McDonnell Genome Institute
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Jeffrey D. Milbrandt
Department of Genetics, Washington University School of Medicine, St Louis MO 63110 Functional Imaging for Variant Elucidation at the McDonnell Genome Institute
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William J. Buchser
Department of Genetics, Washington University School of Medicine, St Louis MO 63110 Functional Imaging for Variant Elucidation at the McDonnell Genome Institute
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  • For correspondence: wbuchser@wustl.edu
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Abstract

Most human genetic variation is classified as VUS - variants of uncertain significance. While advances in genome editing have allowed innovation in pooled screening platforms, many screens deal with relatively simple readouts (viability, fluorescence) and cannot identify the complex cellular phenotypes that underlie most human diseases. In this paper, we present a generalizable functional genomics platform that combines high-content imaging, machine learning, and microraft isolation in a new method termed “Raft-Seq”. We highlight the efficacy of our platform by showing its ability to distinguish pathogenic point mutations of the mitochondrial regulator MFN2, even when the cellular phenotype is subtle. We also show that our platform achieves its efficacy using multiple cellular features, which can be configured on-the-fly. Raft-Seq enables a new way to perform pooled screening on sets of mutations in biologically relevant cells, with the ability to physically capture any cell with a perturbed phenotype and expand it clonally, directly from the primary screen.

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Here, we address the need to evaluate the impact of a multitude of genetic variants. This manuscript depicts the methods of using machine learning on a biologically relevant phenotype to predict specific point mutations, followed by physically capturing those mutated cells.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted March 12, 2021.
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Mitochondrial Phenotypes Distinguish Pathogenic MFN2 Mutations by Pooled Functional Genomics Screen
Alex L. Yenkin, John C. Bramley, Colin L. Kremitzki, Jason E. Waligorski, Mariel J. Liebeskind, Xinyuan E. Xu, Maria A. Vakaki, Vinay D. Chandrasekaran, Robi D. Mitra, Jeffrey D. Milbrandt, William J. Buchser
bioRxiv 2021.03.12.434746; doi: https://doi.org/10.1101/2021.03.12.434746
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Mitochondrial Phenotypes Distinguish Pathogenic MFN2 Mutations by Pooled Functional Genomics Screen
Alex L. Yenkin, John C. Bramley, Colin L. Kremitzki, Jason E. Waligorski, Mariel J. Liebeskind, Xinyuan E. Xu, Maria A. Vakaki, Vinay D. Chandrasekaran, Robi D. Mitra, Jeffrey D. Milbrandt, William J. Buchser
bioRxiv 2021.03.12.434746; doi: https://doi.org/10.1101/2021.03.12.434746

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