ENG 436X and ENG 93X comparisons. (A) ATF4::nanoLuc reporter system to detect induction of the ISR/eIF2α activation, using ATF4 expression as an ISR readout.67 P value calculated by Dunn's test after Kruskal-Wallis test. Shown are results from 3 biological replicate transfections on separate days, each with 4 technical replicates. ∗∗∗∗P < .001. (B-E) Analysis of benign (green), ambiguous (blue in panels B,C,E; brown in panel D) and pathogenic (red) missense substitution predictions by AlphaMissense. (B) Comparison of benign/ambiguous/pathogenic predictions for all 19 alternative amino acids at ENG R93 and ENG R436. Identity and metrics for all predicted pathogenic missense substitutions are shown. P value calculated by Fisher's exact test. (C) Distribution of AlphaMissense metrics for all 19 possible substitutions at ENG R93 and Q436, ∗∗∗∗P < .001 calculated by Mann-Whitney U test. (D) AlphaMissense predictions for the amino acids most likely to be substituted if translational (ribosomal) readthrough occurs at R93X and Q436X. Displayed are the stop codon sequences, the specific amino acids encoded by near-cognate codons distinguishing those with experimental evidence, and the AlphaMissense pathogenicity scores and predictions. (E) Comparison of AlphaMissense metrics for the amino acids most likely to be substituted if translational (ribosomal) readthrough occurs at ENG R93 and Q436; ∗P < .05 calculated by Mann-Whitney U test. RLU, relative luminescence units.
