Figure 2. Morphological profiles of 1,767 LMNA variants identify variants that alter function, including aggregating and pathogenic variants.

(A) Morphological profile analysis workflow. (1) A matrix of ~6.6 million single cells each with 1,077 CellProfiler-derived features, a sequenced barcode and associated lamin A variant was generated. (2) Feature medians and earth-mover distances were computed among all cells expressing each variant; feature selection removed features that were highly correlated, had low variance, or were biologically irrelevant. (3) Two metrics were computed to quantify the overall effect of each variant. Morphological impact score was defined as the cosine distance between a variant morphological profile and the median synonymous profile. Distinguishability score was defined as the area under a receiver operating characteristic curve (AUROC) of a classifier trained to distinguish a variant from WT using single cell feature profiles. (4) Variant embeddings were visualized using UMAP following dimensionality reduction with PCA. Heatmaps of position by substitution effects were visualized for each feature.

(B) Boxplots of the number of profiled single cells per variant of the indicated class across both replicates.

(C) Median over cells of variant mean nuclear intensity in the mEGFP-lamin A channel for variants in both replicates colored by variant type as in (B), with Pearson’s r shown.

(D) Stacked barplot showing proportion of all features, selected features which generate variant profiles, and hit features (defined in (E)) by imaging channel, cellular compartment, or method of computing the feature. The number of features in each set is indicated. Color legends are shown below.

(E) Volcano plot of the median of LMNA feature median z-scores across all 1,767 variants (x-axis) against geometric-mean p-values (y-axis, Komologorov-Smirnov test against the WT distribution of feature values). Both the median effect (x-axis) and p-values (y-axis) are computed over all profiled variants. Red dashed lines show the Bonferroni-corrected p<0.01 threshold and median effect thresholds used to define hit features. Point area is proportional to the number of variants that pass thresholds. Coloring is by imaging channel, see (D) for color legend.

(F) Boxplots of morphological impact scores for variants of the indicated class. Aggregating controls indicate independently-measured variants that aggregated in >15% of HEK 293T cells⁴² (ClinVar variants of uncertain significance=VUS and likely pathogenic/pathogenic variants=P/LP). *** indicates Mann-Whitney p<0.001.

(G) Boxplots of distinguishability scores for variant classes from (F). *** indicates Mann-Whitney p<0.001.

(H) UMAP visualization of PCA-transformed morphological profiles for each variant. Circles indicate synonymous (green), missense (grey) and frameshift (purple) variants, triangles indicate ClinVar likely pathogenic/pathogenic (P/LP, red).

(I) UMAP visualization from (H). Colored symbols indicate independently measured fraction of cells (“+” symbols, >15%; “x” symbols, <15%) with lamin A aggregates in HEK 293T cells.