Fig. 1: Quantifying readthrough of thousands of pathogenic PTCs.
From: Genome-scale quantification and prediction of pathogenic stop codon readthrough by small molecules
a, Readthrough drugs stimulate full-length protein synthesis and decrease NMD-mediated transcript degradation. b, Experimental design, ~5,800 nonsense variants in human genetic diseases and cancer were retrieved from ClinVar, TCGA and MSK-IMPACT datasets, cloned in a readthrough reporter, integrated into the genome of HEK293T_LP human cell line and treated with eight readthrough compounds. A readthrough efficiency value was obtained for each variant–drug pair. c, Sort-sequencing overview. Each cell integrates one copy of one variant, cells are sorted based on mCherry fluorescence (x-axis), bins are sequenced and readthrough percentages are calculated from the mCherry distribution of reads of each variant normalized to the distribution of a no-nonsense variant. d, Deep mutational scanning (DMS) versus individual measurements Pearson’s correlation (r = 0.95), where 15 variants spanning the whole readthrough range under SRI treatment were individually measured (Spearman correlation (ρ) = 0.86). e, The same 15 variants shown in d were episomally transfected in MCF7 and HeLa cells, and their readthrough percentages were correlated with HEK293T_LP’s. Pearson’s correlation and P values are shown. f, DMS Pearson’s correlation (and corresponding P values) with measurements from previous studies14,16,20,21,22,23,24 (Spearman’s correlation (ρ) = 0.56, 0.93, 0.71, 0.59, 1, 0.94, from top-left to bottom-right plots). Titles indicate the gene for which nonsense variants were tested and the drug used to stimulate readthrough. The bottom-right plot does not show DMS estimates, but measurements of individual variants also tested in refs. 23,24, which were used to validate the readthrough reporter. Note that the readthrough scales differ across some of the studies, illustrating how differences in the assay, conditions and reporter influence the absolute readthrough.
