Interpretation of variants identified during genetic testing is a significant clinical challenge. In this study, we developed a high-throughput CDKN2A functional assay and characterized all possible human CDKN2A missense variants. We found that 17.7% of all missense variants were functionally deleterious. We also used our functional classifications to assess the performance of in silico models that predict the effect of variants, including recently reported models based on machine learning. Notably, we found that all in silico models performed similarly when compared to our functional classifications with accuracies of 39.5–85.4%. Furthermore, while we found that functionally deleterious variants were enriched within ankyrin repeats, we did not identify any residues where all missense variants were functionally deleterious. Our functional classifications are a resource to aid the interpretation of CDKN2A variants and have important implications for the application of variant interpretation guidelines, particularly the use of in silico models for clinical variant interpretation.

Missense ‘hotspot’ mutations localized in six p53 codons account for 20% of TP53 mutations in human cancers. Hotspot p53 mutants have lost the tumor suppressive functions of the wildtype protein, but whether and how they may gain additional functions promoting tumorigenesis remain controversial. Here, we generated Trp53^Y217C, a mouse model of the human hotspot mutant TP53^Y220C. DNA damage responses were lost in Trp53^Y217C/Y217C (Trp53^YC/YC) cells, and Trp53^YC/YC fibroblasts exhibited increased chromosome instability compared to Trp53^-/- cells. Furthermore, Trp53^YC/YC male mice died earlier than Trp53^-/- males, with more aggressive thymic lymphomas. This correlated with an increased expression of inflammation-related genes in Trp53^YC/YC thymic cells compared to Trp53^-/- cells. Surprisingly, we recovered only one Trp53^YC/YC female for 22 Trp53^YC/YC males at weaning, a skewed distribution explained by a high frequency of Trp53^YC/YC female embryos with exencephaly and the death of most Trp53^YC/YC female neonates. Strikingly, however, when we treated pregnant females with the anti-inflammatory drug supformin (LCC-12), we observed a fivefold increase in the proportion of viable Trp53^YC/YC weaned females in their progeny. Together, these data suggest that the p53^Y217C mutation not only abrogates wildtype p53 functions but also promotes inflammation, with oncogenic effects in males and teratogenic effects in females.