Screening Concept Aims to Aid Diagnosis for VUS

Fascinating new research headed by a team in Utrecht has applied the strategies now aiding drug discovery to inform and direct clinical investigations of variants of unknown significance (VUS) that are a product of the genetic revolution in medical diagnosis.

With the emergence of VUS that result from their detection by DNA sequencing but which may nonetheless confound a diagnosis or, for example, genetic counselling it has been, thus far, impractical and uneconomical to consider the enormous battalion of tests that might be required to provide a definitive diagnosis, with no certainty of an effective outcome.

They authors of this new work demonstrate that by creating a screening approach they can provide a morphological- and organelle-based feature set for cluster analysis to better direct the clinical investigations for patients with VUS.

They chose the Imagestream imaging flow cytometer to meet statistical sampling demands and to collect image information, from both the assay-designed and inherent image parameters.

Imaging Flow Cytometry and DRAQ5 have long been combined as a solid foundation for assays (500+ co-citations) and this work is no different.  The authors chose DRAQ5 as the DNA counterstain here and it shows its many capabilities: for the fixed cell assays, for the live cell assays, to confirm cytoplasmic-to-nucleus translocations, to show nuclear morphology (and changes thereof) and nuclear staining intensity (i.e. DNA content as an indicator of altered proliferation) compared to controls.

Patient -derived fibroblasts were chosen due to the accessibility of samples via skin biopsies and also given that fibroblasts have a large cytoplasm.

They chose six assays that reflected changes in important organelles and pathways that would allow tractability to downstream investigations. From these they were able to correlate the VUS of well-known genes with abberancies in the relevant positive controls while in those cases where there was a gene of uncertain significance, differences in one or more of the assay readouts gave direction for onwards investigation. 

What also transpired was that a broader, untargeted approach combining all the ca. 300 features that could be extracted from each assay, including those of the nuclear and brightfield images, proved able to separate samples into defined clusters, despite the susceptibility identified being non-tractable directly from any of the 6 assays - akin to phenotypic screening "hits" in drug discovery, and perhaps specifically the "cell painting" assay. These diagnostic "hits" were then confirmed by orthogonal analysis, for example increased intensity of a nucleus correlating to de novo DNA synthesis in the S phase of the cell cycle and therefore indicating different cell proliferation compared to healthy controls, shown by simple DNA histograms.  These correlated to a common genetic variant.

This work may signal a route to a screening-type strategy as a powerful yet relatively low-cost intermediate gateway to aid the conclusion of a detailed diagnosis with affected pathways that can assist clinicians and patients uncover the landscape of the VUS in question.

Reference:

Muffels et al. Imaging flow cytometry‑based cellular screening elucidates pathophysiology in individuals with Variants of Uncertain Significance.  Genome Medicine (2025) 17:12

DOI:10.1186/s13073-025-01433-9