Fig. 4

Imaging flow cytometry results for individuals with VUS in GUS. A UMAP showing clustering based on six assays for healthy controls (HC), positive control patients, and 13 individuals with VUS in GUS. Normalized percentage values as shown in Table 3 were scaled and used as input for UMAP. B Bar graph showing the Z-scores of individuals 106 and 211 and patient with pathogenic ERCC1 variants, for the Golgi, LAMP1, and mitochondrial assay. For the 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. C UMAP of the healthy controls (HC), positive control patients, and the individuals with VUS in GUS. To create the plot, all 1800 features were used, including those quantifying nuclear and brightfield intensity and morphology. Each patient was normalized against three healthy controls taken along in the same experiment. The circles were drawn using the ggforce package. D Showing the cluster characteristics of the cluster with the individuals with LIMK1 genetic variants (093 and 140). The dot plot on the left shows the features with foldchange < 0.5 and > 1.5 in both individuals compared to healthy controls. The median foldchange values for the two individuals is shown. Highly correlating features were removed from the graph (Pearson correlation coefficient > 0.9) to enhance graph readability. The increased nuclear intensity observed in both individuals with LIMK1 genetic variants suggests increased numbers of cells in S/M phase. The histogram plot shows the distribution of nuclear intensity for single cells. For this plot, healthy controls (N = 3) were merged, and individuals with LIMK1 VUS were merged (N = 2). The purple square indicates the gating that was used to determine the percentage of cells that were in the S/M phase. The bar chart shows the percentage of cells in S/M phase for each patient. Each dot represents the percentage of cell in S/M phase per donor. On the right, two representative examples of the autophagy staining are shown. For the images, the intensity threshold was set at 50%. E Showing the cluster characteristics of the cluster of the patient with pathogenic ERCC1 variants and individuals 106, 211, and 216. The dot plot on the left shows the features with foldchange < 0.5 and > 1.5 in both individuals compared to healthy controls. The median foldchange values for the individuals is shown. Features with Pearson correlation coefficient > 0.9 were removed to enhance graph readability. The histogram plot shows the distribution of the Contrast feature for healthy controls (N = 3) and individuals 106, 211, and 216. On the right, representative examples of IFC images of healthy control fibroblasts and individual 106 and 211 are shown. The histogram plot on the far right shows the distribution of the H Energy Std_5 of the autophagy staining for healthy controls (N = 3) and individuals 106 and 211. On the right, representative examples of IFC images of healthy control fibroblasts and individuals 106 and 211 are shown