Imaging flow cytometry-based cellular screening elucidates pathophysiology in individuals with Variants of Uncertain Significance
Abstract Background Deciphering variants of uncertain significance (VUS) represents a major diagnostic challenge, partially due to the lack of easy-to-use and versatile cellular readouts that aid the interpretation of pathogenicity and pathophysiology. To address this challenge, we propose a high-th...
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2025-02-01
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| Online Access: | https://doi.org/10.1186/s13073-025-01433-9 |
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| author | Irena Josephina Johanna Muffels Hans R. Waterham Giuseppina D’Alessandro Guido Zagnoli-Vieira Michael Sacher Dirk J. Lefeber Celine Van der Vinne Chaim M. Roifman Koen L. I. Gassen Holger Rehmann Desiree Y. Van Haaften-Visser Edward S. S. Nieuwenhuis Stephen P. Jackson Sabine A. Fuchs Femke Wijk Peter van Hasselt |
| author_facet | Irena Josephina Johanna Muffels Hans R. Waterham Giuseppina D’Alessandro Guido Zagnoli-Vieira Michael Sacher Dirk J. Lefeber Celine Van der Vinne Chaim M. Roifman Koen L. I. Gassen Holger Rehmann Desiree Y. Van Haaften-Visser Edward S. S. Nieuwenhuis Stephen P. Jackson Sabine A. Fuchs Femke Wijk Peter van Hasselt |
| author_sort | Irena Josephina Johanna Muffels |
| collection | DOAJ |
| description | Abstract Background Deciphering variants of uncertain significance (VUS) represents a major diagnostic challenge, partially due to the lack of easy-to-use and versatile cellular readouts that aid the interpretation of pathogenicity and pathophysiology. To address this challenge, we propose a high-throughput screening of cellular functionality through an imaging flow cytometry (IFC)-based platform. Methods Six assays to evaluate autophagic-, lysosomal-, Golgi- health, mitochondrial function, ER stress, and NF-κβ activity were developed in fibroblasts. Assay sensitivity was verified with compounds (N = 5) and positive control patients (N = 6). Eight healthy controls and 20 individuals with VUS were screened. Results All molecular compounds and positive controls showed significant changes on their cognate assays, confirming assay sensitivity. Simultaneous screening of positive control patients on all six assays revealed distinct phenotypic profiles. In addition, individuals with VUS(es) in well-known disease genes showed distinct – but similar—phenotypic profiles compared to patients with pathogenic variants in the same gene.. For all individuals with VUSes in Genes of Uncertain Significance (GUS), we found one or more of six assays were significantly altered. Broadening the screening to an untargeted approach led to the identification of two clusters that allowed for the recognition of altered cell cycle dynamics and DNA damage repair defects. Experimental follow-up of the ‘DNA damage repair defect cluster’ led to the discovery of highly specific defects in top2cc release from double-strand DNA breaks in one of these individuals, harboring a VUS in the RAD54L2 gene. Conclusions Our high-throughput IFC-based platform simplifies the process of identifying VUS pathogenicity through six assays and allows for the recognition of useful pathophysiological markers that structure follow-up experiments, thereby representing a novel valuable tool for precise functional diagnostics in genomics. |
| format | Article |
| id | doaj-art-747a21a83de04efc947fd0a0bedd076f |
| institution | Kabale University |
| issn | 1756-994X |
| language | English |
| publishDate | 2025-02-01 |
| publisher | BMC |
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| series | Genome Medicine |
| spelling | doaj-art-747a21a83de04efc947fd0a0bedd076f2025-02-09T12:48:41ZengBMCGenome Medicine1756-994X2025-02-0117112110.1186/s13073-025-01433-9Imaging flow cytometry-based cellular screening elucidates pathophysiology in individuals with Variants of Uncertain SignificanceIrena Josephina Johanna Muffels0Hans R. Waterham1Giuseppina D’Alessandro2Guido Zagnoli-Vieira3Michael Sacher4Dirk J. Lefeber5Celine Van der Vinne6Chaim M. Roifman7Koen L. I. Gassen8Holger Rehmann9Desiree Y. Van Haaften-Visser10Edward S. S. Nieuwenhuis11Stephen P. Jackson12Sabine A. Fuchs13Femke Wijk14Peter van Hasselt15Department of Metabolic Diseases, Division Pediatrics, Wilhelmina Children’s Hospital University Medical Centre Utrecht, Utrecht UniversityUnited For Metabolic Diseases (UMD)Cancer Research UK Cambridge Institute, University of CambridgeThe Gurdon Institute and Department of Biochemistry, University of CambridgeDepartment of Biology, Concordia UniversityTranslational Metabolic Laboratory, Department of Neurology, Department of Human Genetics, Donders Institute for Brain, Cognition and Behavior, Radboud University Medical CenterDepartment of Metabolic Diseases, Division Pediatrics, Wilhelmina Children’s Hospital University Medical Centre Utrecht, Utrecht UniversityThe Hospital for Sick Children and Research Institute, The University of TorontoDepartment of Genetics, University Medical Center UtrechtDepartment of Energy and Biotechnology, Flensburg University of Applied SciencesDepartment of Pediatrics, Center for Lysosomal and Metabolic Diseases, Erasmus University Medical CenterDepartment of Metabolic Diseases, Division Pediatrics, Wilhelmina Children’s Hospital University Medical Centre Utrecht, Utrecht UniversityCancer Research UK Cambridge Institute, University of CambridgeDepartment of Metabolic Diseases, Division Pediatrics, Wilhelmina Children’s Hospital University Medical Centre Utrecht, Utrecht UniversityCenter for Translational Immunology (CTI), University Medical Center Utrecht (UMC), Utrecht University (UU)Department of Metabolic Diseases, Division Pediatrics, Wilhelmina Children’s Hospital University Medical Centre Utrecht, Utrecht UniversityAbstract Background Deciphering variants of uncertain significance (VUS) represents a major diagnostic challenge, partially due to the lack of easy-to-use and versatile cellular readouts that aid the interpretation of pathogenicity and pathophysiology. To address this challenge, we propose a high-throughput screening of cellular functionality through an imaging flow cytometry (IFC)-based platform. Methods Six assays to evaluate autophagic-, lysosomal-, Golgi- health, mitochondrial function, ER stress, and NF-κβ activity were developed in fibroblasts. Assay sensitivity was verified with compounds (N = 5) and positive control patients (N = 6). Eight healthy controls and 20 individuals with VUS were screened. Results All molecular compounds and positive controls showed significant changes on their cognate assays, confirming assay sensitivity. Simultaneous screening of positive control patients on all six assays revealed distinct phenotypic profiles. In addition, individuals with VUS(es) in well-known disease genes showed distinct – but similar—phenotypic profiles compared to patients with pathogenic variants in the same gene.. For all individuals with VUSes in Genes of Uncertain Significance (GUS), we found one or more of six assays were significantly altered. Broadening the screening to an untargeted approach led to the identification of two clusters that allowed for the recognition of altered cell cycle dynamics and DNA damage repair defects. Experimental follow-up of the ‘DNA damage repair defect cluster’ led to the discovery of highly specific defects in top2cc release from double-strand DNA breaks in one of these individuals, harboring a VUS in the RAD54L2 gene. Conclusions Our high-throughput IFC-based platform simplifies the process of identifying VUS pathogenicity through six assays and allows for the recognition of useful pathophysiological markers that structure follow-up experiments, thereby representing a novel valuable tool for precise functional diagnostics in genomics.https://doi.org/10.1186/s13073-025-01433-9Imaging flow cytometryVariant of uncertain significancePrecision genomic diagnosticsDNA damage repair defectGenetic diseasesMetabolic disorders |
| spellingShingle | Irena Josephina Johanna Muffels Hans R. Waterham Giuseppina D’Alessandro Guido Zagnoli-Vieira Michael Sacher Dirk J. Lefeber Celine Van der Vinne Chaim M. Roifman Koen L. I. Gassen Holger Rehmann Desiree Y. Van Haaften-Visser Edward S. S. Nieuwenhuis Stephen P. Jackson Sabine A. Fuchs Femke Wijk Peter van Hasselt Imaging flow cytometry-based cellular screening elucidates pathophysiology in individuals with Variants of Uncertain Significance Genome Medicine Imaging flow cytometry Variant of uncertain significance Precision genomic diagnostics DNA damage repair defect Genetic diseases Metabolic disorders |
| title | Imaging flow cytometry-based cellular screening elucidates pathophysiology in individuals with Variants of Uncertain Significance |
| title_full | Imaging flow cytometry-based cellular screening elucidates pathophysiology in individuals with Variants of Uncertain Significance |
| title_fullStr | Imaging flow cytometry-based cellular screening elucidates pathophysiology in individuals with Variants of Uncertain Significance |
| title_full_unstemmed | Imaging flow cytometry-based cellular screening elucidates pathophysiology in individuals with Variants of Uncertain Significance |
| title_short | Imaging flow cytometry-based cellular screening elucidates pathophysiology in individuals with Variants of Uncertain Significance |
| title_sort | imaging flow cytometry based cellular screening elucidates pathophysiology in individuals with variants of uncertain significance |
| topic | Imaging flow cytometry Variant of uncertain significance Precision genomic diagnostics DNA damage repair defect Genetic diseases Metabolic disorders |
| url | https://doi.org/10.1186/s13073-025-01433-9 |
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