Fig. 3

Imaging flow cytometry (IFC) results of individuals with VUS in well-known disease genes. A UMAP plot (Manhattan distance, neighbors = 8, minimal distance = 0.01) showing the healthy controls (HC), individuals with VUS(es), and positive control counterparts matching the individuals with VUS in well-known disease genes. Normalized percentage values as shown in Table 2 were first scaled and used as input for UMAP. Most individuals with VUS clustered together with their positive control counterparts on UMAP, suggesting similar underlying mechanisms of disease. Only the individual with EPG5 VUS clustered with healthy controls. B Bar graph showing aberrancies for two patients with pathogenic variants in CLN3 and individuals with CLN3 VUSes (CLN3 VUS). C Bar graph showing the Z-scores of the individual with ACAD9 VUS and its positive control counterpart (one patient with pathogenic ACAD9 variants) for Golgi, NF-κβ, and mitochondrial assays. D Bar graph showing aberrancies for one patient with a pathogenic variant in TAZ and one individual with TAZ VUS (TAZ VUS) for Golgi and mitochondrial assays. E Bar graph showing the Z-scores of the individuals with DLP1 VUS and their positive control counterpart (one patient with a pathogenic DLP1 variant) for the NF-κβ and the mitochondrial assay. F Bar graph showing the Z-scores for the individual with EPG5 VUS and two patients harboring pathogenic EPG5 variants for the autophagy assay (LC3—Spot Count) and LAMP1 assay (LAMP1—Internalization). For all bar charts, the raw values for each experiment were converted to Z-scores. The mean of all single cell Z-scores was calculated for each individual. The bars represent the mean and the error bars represent the range for healthy controls, individuals with VUS or patients with pathogenic variants. Each dot represents the mean Z-score per donor. Each experiment was performed once, and at least three healthy controls were included for each experiment, except for the mitochondrial experiment, where only one healthy control was included