Characterization of POLE c.1373A > T p.(Tyr458Phe), causing high cancer risk.

The cancer syndrome polymerase proofreading-associated polyposis results from germline mutations in the POLE and POLD1 genes. Mutations in the exonuclease domain of these genes are associated with hyper- and ultra-mutated tumors with a predominance of base substitutions resulting from faulty proofreading during DNA replication. When a new variant is identified by gene testing of POLE and POLD1, it is important to verify whether the variant is associated with PPAP or not, to guide genetic counseling of mutation carriers. In 2015, we reported the likely pathogenic (class 4) germline POLE c.1373A > T p.(Tyr458Phe) variant and we have now characterized this variant to verify that it is a class 5 pathogenic variant. For this purpose, we investigated (1) mutator phenotype in tumors from two carriers, (2) mutation frequency in cell-based mutagenesis assays, and (3) structural consequences based on protein modeling. Whole-exome sequencing of two tumors identified an ultra-mutator phenotype with a predominance of base substitutions, the majority of which are C > T. A SupF mutagenesis assay revealed increased mutation frequency in cells overexpressing the variant of interest as well as in isogenic cells encoding the variant. Moreover, exonuclease repair yeast-based assay supported defect in proofreading activity. Lastly, we present a homology model of human POLE to demonstrate structural consequences leading to pathogenic impact of the p.(Tyr458Phe) mutation. The three lines of evidence, taken together with updated co-segregation and previously published data, allow the germline variant POLE c.1373A > T p.(Tyr458Phe) to be reclassified as a class 5 variant. That means the variant is associated with PPAP.

Role of POLE and POLD1 in familial cancer.

PURPOSE: Germline pathogenic variants in the exonuclease domain (ED) of polymerases POLE and POLD1 predispose to adenomatous polyps, colorectal cancer (CRC), endometrial tumors, and other malignancies, and exhibit increased mutation rate and highly specific associated mutational signatures. The tumor spectrum and prevalence of POLE and POLD1 variants in hereditary cancer are evaluated in this study. METHODS: POLE and POLD1 were sequenced in 2813 unrelated probands referred for genetic counseling (2309 hereditary cancer patients subjected to a multigene panel, and 504 patients selected based on phenotypic characteristics). Cosegregation and case-control studies, yeast-based functional assays, and tumor mutational analyses were performed for variant interpretation. RESULTS: Twelve ED missense variants, 6 loss-of-function, and 23 outside-ED predicted-deleterious missense variants, all with population allele frequencies <1%, were identified. One ED variant (POLE p.Met294Arg) was classified as likely pathogenic, four as likely benign, and seven as variants of unknown significance. The most commonly associated tumor types were colorectal, endometrial and ovarian cancers. Loss-of-function and outside-ED variants are likely not pathogenic for this syndrome. CONCLUSIONS: Polymerase proofreading-associated syndrome constitutes 0.1-0.4% of familial cancer cases, reaching 0.3-0.7% when only CRC and polyposis are considered. ED variant interpretation is challenging and should include multiple pieces of evidence.

Cmk2 kinase is essential for survival in arsenite by modulating translation together with RACK1 orthologue Cpc2 in Schizosaccharomyces pombe.

Different studies have demonstrated multiple effects of arsenite on human physiology. However, there are many open questions concerning the mechanism of response to arsenite. Schizosaccharomyces pombe activates the Sty1 MAPK pathway as a common response to several stress conditions. The specificity of the response is due to the activation of different transcription factors and specific targets such the Cmk2 MAPKAP kinase. We have previously shown that Cmk2 is phosphorylated and activated by the MAPK Sty1 in response to oxidative stress. Here, we report that Cmk2 kinase is specifically necessary to overcome the stress caused by metalloid agents, in particular arsenite. Deletion of cmk2 increases the protein level of various components of the MAPK pathway. Moreover, Cmk2 negatively regulates translation through the Cpc2 kinase: the RACK1 orthologue in fission yeast. RACK1 is a receptor for activated C-kinase. Interestingly, RACK1 is a constituent of the eukaryotic ribosome specifically localized in the head region of the 40 S subunit. Cmk2 controls arsenite response through Cpc2 and it does so through Cpc2 ribosomal function, as observed in genetic analysis using a Cpc2 mutant unable to bind to ribosome. These findings suggest a role for Cmk2 in regulating translation and facilitating adaptation to arsenite stress in the ribosome.