Imaging flow cytometry-based cellular screening elucidates pathophysiology in individuals with Variants of Uncertain Significance

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. 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. 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. 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.