Fig. 5: Endogenous scanning mutations in MFN2 simulate variants of uncertain significance.

a Scatterplot of genotyped cells. x and y axes are the number of models placing that cell in the top 5% or 10% of all cells, respectively. A subset of cells are shown with an inset of the fluorescent image taken during the screen and the gRNA present within the cell (labeled by the nucleotide position before the start codon [B], within the coding region [C], or after the stop codon [P], also see Supplementary Data 1). b Scatterplot of predicted frameshift frequency vs. mitochondrial anomaly score for all the gRNAs recovered from the screen. c Screening results from 357 gRNAs across MFN2. The heatmap has 3 sections, each showing half a dozen examples of models constructed with logistic regression, ridge classifiers, or linear SVMs. The color indicates the prediction score of the model where green means a low value (WT-like) and red means a high value (mutant-MFN2 like). Significance of the gRNA is indicated by the height of the blue bar. d Established isogenic lines of U2OS cells with the specific MFN2 variant(s) rank ordered based on their phenotypes. More “WT-like” phenotypes (likely benign) are shown to the left while more “mutant-like” phenotypes (likely pathogenic) are shown on the right. Bars show average with 95% confidence intervals (n = 18–32 well/plate replicates, L76P mutant is shown in yellow and the parental control clone WT 3E5 is shown in black. *p < 0.05, **p < 0.01, ***p < 0.001). e Example images of isogenic U2OS cells with either a control gRNA that did not cut, 5’UTRins A04 isogenic line, or H20fs/WT isogenic line. The frameshift-mutant (H20fs/WT) has similar mitochondrial aggregation to the L76P mutant (Fig. 1), while the UTR mutant has a distinct but subtle mitochondrial morphology. Scale bar 50 µm. f Metabolic analysis of OCR (oxygen consumption rate) for WT, L76P, and the H20fs/WT lines. OCR is measured in four phases, first basal, then mitochondrial toxins Oligomycin (ATP synthase inhibitor), FCCP (uncoupler), and Rotenone (electron transport inhibitor). OCR is normalized by nuclei count. The asterisk in c are V459fs/WT cells spiked into the screen.