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Pooled image-base screening of mitochondria with microraft isolation distinguishes pathogenic mitofusin 2 mutations.

ABSTRACT: Most human genetic variation is classified as 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 method termed "Raft-Seq". We highlight the efficacy of our platform by showing its ability to distinguish pathogenic point mutations of the mitochondrial regulator Mitofusin 2, 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 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.

SUBMITTER: Yenkin AL

PROVIDER: S-EPMC9596453 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Pooled image-base screening of mitochondria with microraft isolation distinguishes pathogenic mitofusin 2 mutations.

Yenkin Alex L AL Bramley John C JC Kremitzki Colin L CL Waligorski Jason E JE Liebeskind Mariel J MJ Xu Xinyuan E XE Chandrasekaran Vinay D VD Vakaki Maria A MA Bachman Graham W GW Mitra Robi D RD Milbrandt Jeffrey D JD Buchser William J WJ

Communications biology 20221025 1

Most human genetic variation is classified as 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 method termed ...[more]

PMID: 36284160

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Project description:ObjectiveDominantly inherited PSTPIP1 mutations cause a spectrum of autoinflammatory manifestations epitomized by PAPA syndrome (pyogenic sterile arthritis, pyoderma gangrenosum, and acne (PAPA) syndrome.). The connections between PSTPIP1 and PAPA syndrome are poorly understood, although evidence suggests involvement of pyrin inflammasome activation. Interleukin-18 (IL-18) is an inflammasome-activated cytokine associated with susceptibility to macrophage activation syndrome (MAS). This study was undertaken to investigate an association of IL-18 with PAPA syndrome.MethodsClinical and genetic data and serum samples were obtained from patients referred to institutions due to symptoms indicative of PAPA syndrome. Serum IL-18, IL-18 binding protein (IL-18BP), and CXCL9 levels were assessed by bead-based assay, and free IL-18 levels were assessed by enzyme-linked immunosorbent assay.ResultsThe symptoms of PSTPIP1-positive patients with PAPA syndrome overlapped with those of mutation-negative patients with PAPA-like conditions, but mutation-positive patients had earlier onset and a greater proportion had a history of arthritis. We found uniform elevation of total serum IL-18 in treated PAPA syndrome patients at levels nearly as high as those seen in NLRC4-associated autoinflammation with infantile enterocolitis patients, and well above levels found in most familial Mediterranean fever patients. Serum IL-18 elevation in PAPA syndrome patients persisted despite fluctuations in disease activity. Levels of the soluble IL-18 antagonist IL-18BP were modestly elevated, and PAPA syndrome patients had detectable free IL-18. PAPA syndrome was rarely associated with elevation of CXCL9, an indicator of interferon-γ activity, but no PAPA syndrome patients had a history of MAS.ConclusionPAPA syndrome is a refractory and often disabling monogenic autoinflammatory disease associated with chronic and unopposed elevation of serum IL-18 levels but not with risk of MAS. These findings affect our understanding of the diseases in which IL-18 is overproduced and suggest a link between pyrin inflammasome activation, IL-18, and autoinflammation, without susceptibility to MAS.

Dataset Information

Pooled image-base screening of mitochondria with microraft isolation distinguishes pathogenic mitofusin 2 mutations.

Publications

Pooled image-base screening of mitochondria with microraft isolation distinguishes pathogenic mitofusin 2 mutations.

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