Role of POLE and POLD1 in familial cancer

Abstract

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.

Keywords: PPAP; exonuclease domain; hereditary colorectal cancer; polymerase proofreading–associated polyposis; ultramutated phenotype.

Fig. 1. Schematic representation of the characteristics of the cohorts analyzed in this study, prevalence of (likely) pathogenic variants in cancer-predisposing genes, and results of the current study for exonuclease domain (ED) missense variants in POLE and POLD1.

BC breast cancer, BrC brain tumor, CRC colorectal cancer, EC endometrial cancer, GC gastric cancer, HBC hereditary breast cancer, HBOC hereditary breast and ovarian cancer, HOC hereditary ovarian cancer, HNPCC hereditary nonpolyposis colorectal cancer, LF Li–Fraumeni syndrome, LP likely pathogenic, OC ovarian cancer, PPAP polymerase proofreading–associated polyposis, VUS variant of unknown significance.

Fig. 2. Functional analysis carried out in Schizosaccharomyces pombe for variants located within (or close to) the POLE exonuclease domain (ED).

(a) Alignment of human POLE and their homolog in yeast (Pol2). The identified variants are highlighted in yellow (conserved residues) and the POLE p.Leu424Val, used as positive control, in green. ED is depicted in red (human: residues 268–471, yeast: residues 98–428) and its sequence motifs are shaded in gray. (b) Left panel: box plots showing mutation rates of ade6-485 S. pombe (number of colonies) for pol2 wildtype (WT, negative control), ED mutation-positive control (pol2-Leu425Val; C+) and the corresponding variants. Right panel: fold rate increase relative to the median number of revertants in the WT. Data obtained from two independent experiments performed in triplicate. *p value = 0.01, **p value = 0.01–0.001, and ***p value < 0.001 indicate the differences with the WT clone, and were calculated using the Mann–Whitney nonparametric test.

Fig. 3. Structural representation of human POLE and POLD1 and location of the ED variants identified in the current study.

(a) 3D structure of POLE. (b) 3D structure of POLD1. Single-stranded DNA from the aligned bacteriophage T4 polymerase complex (PDB ID: 1NOY) is shown in yellow. Variants in the DNA binding pocket are highlighted in red.

Fig. 4. Somatic analysis performed in tumors from POLE/POLD1 variant carriers.

(a) Tumor features including mismatch repair (MMR) deficiency status and mutational burden (hypermutation was considered when the tumor had more than 10 exonic Mut/Mb). ^aHigh-grade serous ovarian cancer. ^bIntestinal origin. ^cMicrosatellite instability (MSI) classification using Bethesda panel. C+, positive controls, i.e., tumors from carriers of variants affecting the POLE/POLD1 ED previously classified as pathogenic. (b) Mutational signature contribution (DeconstructSigs) for hyper/ultramutated tumors (>10 Mut/Mb). CRC colorectal cancer.