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.

Genome‐wide approaches applied for the identification of new hereditary colorectal cancer (CRC) genes, identified several potential causal genes, including RPS20, IL12RB1, LIMK2, POLE2, MRE11, POT1, FAN1, WIF1, HNRNPA0, SEMA4A, FOCAD, PTPN12, LRP6, POLQ, BLM, MCM9, and the epigenetic inactivation of PTPRJ. Here we attempted to validate the association between variants in these genes and nonpolyposis CRC by performing a mutational screening of the genes and PTPRJ promoter methylation analysis in 473 familial/early‐onset CRC cases, a systematic review of the published cases, and assessment of allele frequencies in control population. In the studied cohort, 24 (5%) carriers of (predicted) deleterious variants in the studied genes and no constitutional PTPRJ epimutations were identified. Assessment of allele frequencies in controls compared with familial/early‐onset patients with CRC showed association with increased nonpolyposis CRC risk of disruptive variants in RPS20, IL12RB1, POLE2, MRE11 and POT1, and of FAN1 c.149T>G (p.Met50Arg). Lack of association was demonstrated for LIMK2, PTPN12, LRP6, PTPRJ, POLQ, BLM, MCM9 and FOCAD variants. Additional studies are required to provide conclusive evidence for SEMA4A, WIF1, HNRNPA0 c.−110G>C, and FOCAD large deletions.

Technological advances have allowed the identification of new adenomatous and serrated polyposis genes, and of several candidate genes that require additional supporting evidence of causality. Through an exhaustive literature review and mutational screening of 177 unrelated polyposis patients, we assessed the involvement of MCM9, FOCAD, POLQ, and RNF43 in the predisposition to (nonserrated) colonic polyposis, as well as the prevalence of NTHL1 and MSH3 mutations among genetically unexplained polyposis patients. Our results, together with previously reported data and mutation frequency in controls, indicate that: MCM9 and POLQ mutations are not associated with polyposis; germline RNF43 mutations, with a prevalence of 1.5–2.5% among serrated polyposis patients, do not cause nonserrated polyposis; MSH3 biallelic mutations are highly infrequent among European polyposis patients, and the prevalence of NTHL1 biallelic mutations among unexplained polyposes is ~2%. Although nonsignificant, FOCAD predicted deleterious variants are overrepresented in polyposis patients compared to controls, warranting larger studies to provide definite evidence in favor or against their causal association with polyposis predisposition.