Germline mutations affecting the proofreading domains of POLE and POLD1 predispose to colorectal adenomas and carcinomas

Abstract

Many individuals with multiple or large colorectal adenomas or early-onset colorectal cancer (CRC) have no detectable germline mutations in the known cancer predisposition genes. Using whole-genome sequencing, supplemented by linkage and association analysis, we identified specific heterozygous POLE or POLD1 germline variants in several multiple-adenoma and/or CRC cases but in no controls. The variants associated with susceptibility, POLE p.Leu424Val and POLD1 p.Ser478Asn, have high penetrance, and POLD1 mutation was also associated with endometrial cancer predisposition. The mutations map to equivalent sites in the proofreading (exonuclease) domain of DNA polymerases ɛ and δ and are predicted to cause a defect in the correction of mispaired bases inserted during DNA replication. In agreement with this prediction, the tumors from mutation carriers were microsatellite stable but tended to acquire base substitution mutations, as confirmed by yeast functional assays. Further analysis of published data showed that the recently described group of hypermutant, microsatellite-stable CRCs is likely to be caused by somatic POLE mutations affecting the exonuclease domain.

Figure 1. Pedigrees of the POLE L424V and POLD1 S478N families in the Discovery Phase

Families (a) SM2702 (POLE), (b) SM1645 (POLD1) and (c) SM1412 (POLD1) are shown. [•]=affected, [+]=mutation carrier and [−]=wildtype. S=whole genome-sequenced, L=genome-wide linkage analysis. For colorectal adenomas (ads), we show the cumulative tumour numbers from age at first presentation or screening colonoscopy to age at last contact. Diameter of the largest adenoma is also given where reported. Hyperplastic polyp (HP) numbers are also shown. For colorectal carcinomas (CRCs), endometrial carcinomas (ECs) and brain tumours, age at first presentation is given. Location of the CRC (colon, caecum, rectum) is also given where reported.

Figure 1. Pedigrees of the POLE L424V and POLD1 S478N families in the Discovery Phase

Families (a) SM2702 (POLE), (b) SM1645 (POLD1) and (c) SM1412 (POLD1) are shown. [•]=affected, [+]=mutation carrier and [−]=wildtype. S=whole genome-sequenced, L=genome-wide linkage analysis. For colorectal adenomas (ads), we show the cumulative tumour numbers from age at first presentation or screening colonoscopy to age at last contact. Diameter of the largest adenoma is also given where reported. Hyperplastic polyp (HP) numbers are also shown. For colorectal carcinomas (CRCs), endometrial carcinomas (ECs) and brain tumours, age at first presentation is given. Location of the CRC (colon, caecum, rectum) is also given where reported.

Figure 1. Pedigrees of the POLE L424V and POLD1 S478N families in the Discovery Phase

Families (a) SM2702 (POLE), (b) SM1645 (POLD1) and (c) SM1412 (POLD1) are shown. [•]=affected, [+]=mutation carrier and [−]=wildtype. S=whole genome-sequenced, L=genome-wide linkage analysis. For colorectal adenomas (ads), we show the cumulative tumour numbers from age at first presentation or screening colonoscopy to age at last contact. Diameter of the largest adenoma is also given where reported. Hyperplastic polyp (HP) numbers are also shown. For colorectal carcinomas (CRCs), endometrial carcinomas (ECs) and brain tumours, age at first presentation is given. Location of the CRC (colon, caecum, rectum) is also given where reported.

Figure 2. Modelling of the germline and exonuclease domain mutations

a) Composite model of the catalytic subunit of the yeast DNA polymerase δ (in ternary complex with DNA and an incoming nucleotide [PDB ID: 3IAY]) and the ssDNA component of the T4 polymerase complex [PDBID: 1NOY], modeled into the exonuclease active site. The polymerase is coloured blue, the exonuclease domain is shown in green, the dsDNA in orange and magenta, and the ssDNA in the exonulease active site in yellow. Mutations map to the active site of the exonuclease domain. b) Germline mutations POLE L424V (in red) and POLD1 S478N (magenta) map to a helix (478-487) and pack against another helix, forming part of the base of the exonuclease active site. Mutations will disrupt this packing of helices and distort the active site. The active site is defined by the ssDNA substrate shown in yellow. c) Mapping of the possibly pathogenic germline and somatic mutations to the exonuclease domain. All the POLE mutations (exonuclease domain somatic changes from the TCGA colorectal cancer data), and POLD1 P327L (germline variant from our patient, same location as POLE P286H) cluster around the active site (in red), whilst the POLD1 mutations S370R and G426S (germline variants from two other patients, shown in magenta) are more peripheral.