A Massively Parallel Fluorescence Assay to Characterize the Effects of Synonymous Mutations on TP53 Expression

Although synonymous mutations can affect gene expression, they have generally not been considered in genomic studies that focus on mutations that increase the risk of cancer. However, mounting evidence implicates some synonymous mutations as driver mutations in cancer. Here, a massively parallel assay, based on cell sorting of a reporter containing a segment of p53 fused to GFP, was used to measure the effects of nearly all synonymous mutations in exon 6 of TP53 In this reporter context, several mutations within the exon caused strong expression changes including mutations that may cause potential gain or loss of function. Further analysis indicates that these effects are largely attributed to errors in splicing, including exon skipping, intron inclusion, and exon truncation, resulting from mutations both at exon-intron junctions and within the body of the exon. These mutations are found at extremely low frequencies in healthy populations and are enriched a few-fold in cancer genomes, suggesting that some of them may be driver mutations in TP53 This assay provides a general framework to identify previously unknown detrimental synonymous mutations in cancer genes.Implications: Using a massively parallel assay, this study demonstrates that synonymous mutations in the TP53 gene affect protein expression, largely through their impact on splicing.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/15/10/1301/F1.large.jpg Mol Cancer Res; 15(10); 1301-7. ©2017 AACR.