A novel POLE mutation associated with cancers of colon, pancreas, ovaries and small intestine
In some families there is an increased risk for colorectal cancer, caused by heritable, but often unidentified genetic mutations predisposing to the disease. We have identified the likely genetic cause for disease predisposition in a large family with high burden of colorectal adenomas and carcinomas, in addition to extra-colonic cancers. This family had previously been tested for known cancer susceptibility genes, with negative results. Exome sequencing was used to identify a novel mutation, c.1373A>T (p.Tyr458Phe), in the gene for DNA polymerase epsilon catalytic subunit (POLE). This mutation is located in the active site of the exonuclease domain of the enzyme, and affects a residue that has previously been shown to be important for exonuclease activity. The first predisposing mutation identified in POLE (c.1270C>G, p.Leu424Val) was associated with colorectal cancer only, but another mutation with a broader tumour spectrum (c.1089C>A, p.Asn363Lys) has recently been reported. In the family described in the present study, carriers generally have multiple colorectal adenomas and cancer of colon, pancreas, ovaries and small intestine which represents an important broadening of the tumour spectrum of POLE mutation carriers. We also observe a large phenotypic variation among the POLE mutation carriers in this family, most likely explained by modifying variants in other genes. One POLE mutation carrier has a novel variant in EXO1 (c.458C>T, p.Ala153Val), which may contribute to a more severe phenotype. The findings in this study will have important implications for risk assessment and surveillance of POLE mutation carriers.
KeywordsColorectal cancer Polymerase epsilon POLE Germline mutation
About one-third of all colorectal cancer (CRC) cases are presumed to be caused by hereditary factors [1, 2, 3]. The genetic basis for predisposition is unknown in the majority of familial CRC cases, and only about 5 % of all CRC cases are associated with CRC syndromes caused by highly penetrant mutations in known CRC predisposing genes . Lynch Syndrome is the most commonly occurring CRC syndrome and is caused by a germline mutation in one of the DNA mismatch repair (MMR) genes MLH1 (MIM *120436), MSH2 (MIM *609309), MSH6 (MIM *600678) or PMS2 (MIM *600259). Polyposis is rare in Lynch Syndrome, but affected individuals develop colonic adenomas and carcinomas with higher frequency compared to the general population . Familial adenomatous polyposis (FAP) is the second most common hereditary CRC syndrome and is caused by a germline mutation in the APC gene (MIM *611731). Classical FAP is characterized by hundreds to thousands of colonic adenomas starting to appear in adolescence. Attenuated FAP is a less severe form of the condition with fewer adenomas and later onset of disease . MutYH-associated polyposis (MAP) is caused by biallelic mutations of the MUTYH gene (MIM *604933). Adenomatous polyps predominate in MAP but hyperplastic polyps are also common. Peutz–Jeghers Syndrome, Juvenile Polyposis Syndrome and Cowden Syndrome are conditions characterized by hamartomatous polyposis. Peutz–Jeghers Syndrome and Cowden Syndrome are caused by a mutation in STK11 (MIM *602216) and PTEN (MIM +601728), respectively, whereas Juvenile Polyposis Syndrome is caused by mutations in either BMPR1A (MIM *601299) or SMAD4 (MIM *600993). Recently a new CRC predisposing syndrome named polymerase proofreading-associated polyposis (PPAP) was described . This syndrome is caused by germline mutations in POLE (MIM *174762) or POLD1 (MIM *174761), encoding the catalytic and proofreading subunit of the DNA polymerase ε and δ enzyme complexes, respectively.
Currently, clinical presentation of CRC patients is used to guide genetic testing. Although there are some distinct clinical features associated with each CRC syndrome, the phenotypes overlap extensively and this can complicate phenotype-guided genetic testing and counselling. For several of the above mentioned syndromes, affected individuals can present with varying number of adenomas (typically 10–100) at a young age, which can develop into CRC if left untreated. The extra-colonic tumour spectrum may also be somewhat overlapping for several of the CRC syndromes involving endometrium, stomach, ovaries, pancreas, small bowel and brain .
In the present study we describe a large family with high burden of colorectal adenomas and carcinomas in addition to extra-colonic cancers. Initially, three separate families were identified, but they were later found to have shared ancestry. Genetic examinations of family members started in 1995 and since then several CRC predisposing genes have been analysed without identification of a causal mutation. Due to the striking dominant inheritance in this family, we strongly suspected a highly penetrant mutation as the cause of cancer predisposition. By exome sequencing we identified a novel mutation in POLE which seems to explain the cancer predisposition. Further, we discuss whether modifying effects of variants in other genes may explain the phenotypic variation observed among the POLE mutation carriers.
Materials and methods
Recruitment of participants
The power of family-based studies can be optimized by careful selection of candidates for sequencing. However, because of ethical and legal constraints, recruitment of individuals to this study had to be done through members of the family who had previously received genetic counselling based on their personal concern for developing cancer. We asked this initial group of family members to distribute our invitation letter to additional relatives. This broadened the possibility to recruit participants but gave no guarantee of reaching specific important individuals. Although we were able to recruit enough informative participants to identify a likely causal mutation for predisposition in this family, it is clear that too strict legal constraints for recruitment to family-based studies can hamper such efforts.
Description of pedigree
Exome capture and sequencing
We exome sequenced DNA samples from 14 family members (III:16, IV:3, IV:9, IV:10, IV:12, IV:17, IV:21, V:2, V:4, V:5, V:7, V:8, V:9, V:10), both affected and unaffected, to identify the causative mutation predisposing to CRC in this family (see Fig. 1 and Online Resource 1 for overview of sequenced individuals). Exome capture was performed according to manufacturer’s protocol, using SureSelectXT Human All Exon V5+UTRs (Protocol version 1.6, Agilent Technologies, Santa Clara, CA). Briefly, the samples were quantified using Qubit® 2.0 Fluorometer (Life Technologies, Carlsbad, CA). Genomic DNA was fragmented to approximately 170 bp by sonication using Covaris M220 Focused-ultrasonicator™ (Covaris, Woburn, MA). Fragment sizes were determined on Agilent 2100 Bioanalyzer (Agilent Technologies). Library concentrations were measured using Qubit® 2.0 Fluorometer and StepOnePlus™ Real-Time PCR System (Life Technologies). The libraries were sequenced on Illumina HiSeq 2500 (Illumina, San Diego, CA) with 2 × 100 bp paired end sequencing.
Data analysis, filtering and annotation
Variants identified by sequencing with potential functional impact
III:2, IV:8, IV:9, IV:10, IV:13, IV:15, IV:17, IV:20, IV:21, V:4, V:5, V:8
All variants identified in the present study and reported here have been submitted to LOVD 3.0 shared installation (http://databases.lovd.nl/shared/genes).
Confirmatory Sanger sequencing
DNA from EDTA-preserved whole blood or paraffin-embedded tissue was analysed to confirm the variants c.1373A>T (POLE), c.1739T>C (BMPR1A), c.458C>T (EXO1), c.1100del (CHEK2) and c.5265del (LAMB4) detected by exome sequencing, and to test additional family members for the respective variants. PCR was performed using AmpliTaq Gold® 360 MasterMix and 360 GC Enhancer (Life Technologies). Cycle sequencing reaction was performed with BigDye® Terminator v3.1 (Life Technologies) and subsequent capillary electrophoresis was performed by the 3130xl Genetic Analyzer (Life Technologies). Sanger sequencing data was analysed using SeqScape Software v2.5 (Life Technologies).
Sequencing data from 95 CRC patients fulfilling the Amsterdam criteria but without identified germline mutation (previously tested for MLH1, PMS2, MSH6, MSH2, APC and MUTYH) was investigated for the POLE mutation. The library was prepared according to the manufacturer’s instructions using a custom Haloplex kit (Agilent Technologies) and was subsequently sequenced on a HiSeq 2500 (Illumina) with 2 × 100 bp paired end sequencing.
Results and discussion
Filtering of variants
The family included in this study show an autosomal dominant inheritance pattern with colorectal adenomas, carcinomas and other extra-colonic cancers detected in every successive generation (Fig. 1). Several family members had previously been tested for mutations in APC, MUTYH, MLH1, PMS2, MSH2, MSH6, BRCA1 and BRCA2, with negative results. We therefore exome sequenced samples from 14 family members, both affected and unaffected, to detect any cancer predisposing mutation in this family. Average coverage across all sample was 152× (see Online Resource 1 for average coverage and the percentage of target regions covered in each sample) and approximately 25,000 variants were initially detected in each individual. These variants were first filtered against the 1000 Genomes Project and dbSNP, in order to focus on rare variants. Further, all synonymous variants and variants that did not pass quality filters were removed. This reduced the list to approximately 200 variants for each individual. Because of the broad spectrum of cancers and the varying phenotypes in this family, we used two complementary strategies for further variant filtering. The filtering strategy based on disease status identified 4 variants shared by the 7 affected individuals, none of which were assessed to be likely causative in terms of gene function or functional impact of the variants. The number of variants increased to 8, 15, 24, 42 and 105 if the number of affected individuals was reduced to at least 6, 5, 4, 3 or 2, respectively. This corresponds to assuming that at least one individual may have developed CRC by an alternative pathway, which is not unreasonable in a large family (>100 individuals). The gene panel strategy resulted in 8 variants in 8 different genes. Of these, 1 variant was shared by 6 affected individuals, 1 variant was present in two affected individuals and the remaining 6 variants were private. A novel mutation, c.1373A>T (p.Tyr458Phe), in POLE (NM_006231.2), present in 6 of the 7 patients classified as “affected” (V:4, V:5, V:8, IV:9, IV:10 and IV:17) and not present in the “unaffected” individual (III:16) was assessed to be the most likely causative mutation. This mutation was identified by both filtering methods. The POLE mutation was subsequently also found in 6 additional affected individuals (III:2, IV:8, IV:13, IV:15, IV:20 and IV:21). Samples from III:2, IV:8, IV:13, IV:15 and IV:20 were not available for exome sequencing, but were sequenced by the Sanger method. Individual IV:21 was exome sequenced, but could not be confidently classified as “affected” prior to filtering as only one adenoma had been detected in this patient, and the latest performed colonoscopy was 8 years ago. However, colonoscopy performed after exome sequencing of IV:21 revealed CRC and several adenomas. The individual classified as “affected” without POLE mutation (V:7) was found to harbour a novel variant in BMPR1A. See Fig. 1 and Online Resource 1 for overview of POLE mutation carriers. Only variants that are likely to have functional impact at the protein level and relevance to cancer predisposition are presented here (Table 1, see further discussion below). All these variants have been confirmed by Sanger sequencing.
The POLE mutation c.1373A>T (p.Tyr458Phe)
95 additional samples from CRC patients fulfilling the Amsterdam criteria but without identified germline mutation (previously tested for MLH1, PMS2, MSH6, MSH2, APC and MUTYH) were analysed for the POLE mutation encoding the p.Tyr458Phe alteration. One index patient diagnosed with CRC at 44 years of age, and cancer duodenum at 59 years of age, was found to harbour the POLE mutation. His deceased brother got CRC at 42 years of age, and his son has removed several adenomas at age 34. Analysis of archived paraffin-embedded tissue material from the brother and DNA from wole blood from his son revealed that both of them carried the POLE mutation. The parents of the index patient died in their early fifties and sixties, however, no material from them were available for testing. We could not find common ancestors in the two families described here, although this cannot be completely ruled out. Consequently, we have identified two apparently unrelated families with history of CRC with the same POLE mutation.
Data from The Cancer Genome Atlas (TCGA), The International Cancer Genome Consortium (ICGC) cancer genome projects and other cancer genomics studies was accessed through the Catalogue of Somatic Mutations in Cancer (COSMIC)  and cBioPortal for Cancer Genomics  to find somatic POLE alterations encoding a change at position p.Tyr458, with negative results (Table 1). The codon next to p.Tyr458 is identified as a mutational hotspot with p.Ser459Phe found in 4 different hyper-mutated CRCs without microsatellite instability [44, 45, 46]. Another study of samples from microsatellite stable CRCs identified the somatic mutations p.Ser459Phe and p.Thr457Met in two cases each . The exonuclease domain was also checked against Exome Aggregation Consortium (ExAC) . As shown in Fig. 2, no previously known somatic or germline mutations in p.Tyr458 were found.
The family members with the POLE mutation were all heterozygous for the mutation. Second hit by somatic mutation or loss of heterozygosity (LOH) was not investigated in this study. However, Palles et al.  tested for second hits by LOH in 39 tumours from 11 carriers of the POLE mutation encoding p.Leu424Val, and detected LOH in 10 of these tumours. Rohlin et al.  searched for second hit by somatic mutations or LOH of the wt allele in two tumours from two carriers of the POLE mutation encoding p.Asn363Lys, but no aberrations were identified. Results from these two studies indicate that a second hit might not be required, and that the increased error rate during replication from only one faulty copy of the POLE gene might be enough to drive cancer development in humans. However, model studies in mice reveal that although mutation frequency is increased in mice that are heterozygous for a POLE mutation, only the homozygous mice showed increased susceptibility to cancer . This indicates that additional factors may be important. Whether POLE acts as a classic tumour suppressor gene is still unclear and further research is needed to clarify this.
The tumour spectrum of POLE mutation carriers
The tumour spectrum of the patients with previously reported pathogenic mutations affecting the exonuclease domain of Polε differs substantially. Palles et al.  first reported a family with a POLE mutation encoding the p.Leu424Val alteration that was solely affected with colorectal carcinomas and adenomas, while POLD1 mutation carriers, in addition to CRC, were affected with endometrial cancers. Rohlin et al.  recently described a family with a POLE mutation encoding the p.Asn363Lys mutation that had a broader tumour spectrum, including cancer in colon, endometrium, ovaries, brain and one single case of late onset pancreatic cancer. Spier et al.  reports several POLE mutation carriers with duodenal adenomas and one case of duodenal cancer. The present family seems to be predisposed to adenomas and carcinomas not only in colon and rectum, but also in the pancreas, small intestine, stomach, and ovaries. There are three cases of early onset pancreatic cancer in this family. The first (IV:8) was found to have the POLE mutation encoding p.Tyr458Phe, the second (III:9) was indirectly found to harbour the mutation through genetic testing of his child, while the third (III:7) was unavailable for testing and has no descendants. All three developed pancreatic cancer in their forties which is a considerably younger age of onset than average (~70 years) [50, 51]. Individual IV:17 had, in addition to CRC, two cancers in the small intestine. This strongly suggests that cancer of pancreas and small intestine is a part of the PPAP tumour spectrum. As suggested by Rohlin et al.  there might be a genotype-to-phenotype correlation for this gene, relating to the effect the amino acid substitution has on the protein. However, the discrepancy in tumour spectrum may also be explained by the sizes of the families. The family with p.Leu424Val mutation  is smaller with fewer affected individuals than the other two families, [10 and the present study]. Since CRC is the predominant effect of POLE exonuclease mutations, the limited tumour spectrum of that family may have occurred by chance. Two of the family members in the present study (IV:12 and IV:3) were affected with cancer in the urinary bladder at age 54 and lung cancer at age 70, respectively. However, they did not harbour the pathogenic POLE mutation. The lung cancer was most likely caused by environmental factors related to the person’s workplace.
Carriers of the same POLE mutation have differing phenotypes
The POLE mutation carriers of the present family had differing phenotypes, most likely explained by modifying variants in other genes. Most of the p.Tyr458Phe carriers had a multiple-adenoma phenotype similar to MAP and attenuated FAP, while some had fewer adenomas or cancer of ovaries or pancreas more resembling Lynch Syndrome. Phenotypic variation among family members carrying the same POLE mutation is also observed in another study . In the present study, the POLE mutation carrier with the most severe phenotype (IV:17) was also found to harbour the novel variant c.458C>T (p.Ala153Val) in EXO1 (NM_003686.4) with predicted functional impact (Table 1). This variant was identified using the CRC genepanel strategy. Another SNP (rs143955774, c.458C>G, p.Ala153Gly) without reported frequency is located at the same position. EXO1 encodes the enzyme Exonuclease 1 which belongs to the RAD2/XPG family of endo- and exonucleases. It exhibits 5′-3′-exonuclease and 5′-flap endonuclease activity and is involved in DNA repair, recombination, replication, and telomere integrity (reviewed in ). The residue p.Ala153 is located in the highly conserved XPG_2 site (PS00842), which includes a conserved pentapeptide, E-A-[DE]-A-[QS] (the residue in bold corresponds to p.Ala153), and is located next to one of the acidic residues of the active site involved in the catalytic mechanism of nuclease activity . Studies of POLE mutant Saccharomyces cerevisiae strains deleted for EXO1 show a markedly increased mutator phenotype compared to either of the single mutant strains [54, 55, 56]. This suggests that Exonuclease 1 is involved in correcting mismatches created by Polε during replication. Consequently, we postulate that the POLE and EXO1 variants detected in DNA from patient IV:17 may have a combined effect leading to an increased mutation rate causing the even more severe phenotype observed in this patient. Identification of modifying loci causing discrepancy in the phenotypes of POLE mutations carriers obviously needs further research. It might be useful to investigate additional variants in genes coding for proteins involved in the same pathways as Polε, or look for variation in regulatory regions. It is also possible that common variants can have a modifying effect when combined with a pathogenic POLE mutation. Differences in phenotypes due to genetic modifiers have also been observed in Lynch Syndrome .
Phenocopies may be explained by additional variants
Using the CRC gene panel filtering strategy we also identified other variants with potential functional impact (Table 1) in three family members without the POLE mutation. Initially these patients seemed to phenocopy POLE mutation carriers to some extent, but there were also clear differences.
Individual V:7, who was classified as “affected” but did not carry the POLE mutation, was found to have the mutation c.1379T>C (p.Met460Thr) in BMPR1A (NM_004329.2). He was initially thought to have a phenotype similar to his sister (V:8), who was found to carry the pathogenic POLE mutation, with hyperplastic polyps and adenomas from their twenties. Individual V:7 had previously only one tubular adenoma and one hyperplastic polyp detected, and during this project he developed a rectal neuroendocrine tumour. This type of tumour is not observed for any of the other family members, suggesting that this patient is affected with something other than PPAP. The BMPR1A variant has previously been found as a somatic change in a CRC analysed by the TCGA project, but prediction tools were inconsistent regarding functional impact (Table 1). In addition, considering that the BMPR1A variant was inherited from this person’s healthy mother (IV:18), who has not been examined with colonoscopy, and as neuroendocrine tumours are not associated with Juvenile Polyposis Syndrome, the variant was evaluated to be of uncertain clinical significance at this stage. A panel of genes related to endocrine tumours was also applied to the exome data of this individual, with negative results.
Individual V:2, who had a single adenoma detected at age 42, had the variants c.1100del in CHEK2 (NM_007194.3), and c.5265del in LAMB4 (NM_007356.2). The CHEK2 variant is a well-known, low penetrant founder mutation mainly associated with breast cancer, but also CRC and prostate cancer [58, 59, 60, 61, 62, 63]. A germline LAMB4 variant has recently been reported in another CRC patient with somatic loss of the wild-type allele in the tumour . LAMB4 was consequently implicated to be a possible tumour-suppressor gene where mutations may predispose to CRC. Both the CHEK2 and LAMB4 variant were present in TCGA data, but the CHEK2 variant as a germline mutation (Table 1). In the present study, the LAMB4 variant, but not the CHEK2 variant, was also found in the person’s father (IV:6) who was affected with prostate cancer at age 54 and two colorectal adenomas at age 60 and 67.
Since CRC is one of the most common malignancies in Norway it is likely that a large family like this also will have sporadic, non-hereditary cases of colorectal adenomas and cancer. There may also be additional genetic factors leading to a small increase in cancer susceptibility, like c.1100del in CHEK2, which together with environmental factors may lead to formation of adenomas or CRC. This clearly demonstrates the challenge of using phenotype-guided genetic testing combined with Sanger sequencing of single genes to find the genetic predisposition in familial CRC. Exome sequencing has successfully been applied to find the genetic cause for a wide range of Mendelian disorders (reviewed in ), but only a few studies have interrogated familial CRC [7, 32, 34, 65, 66]. It has previously been discussed that phenocopies and incomplete penetrance might hamper analysis of exome sequencing data when studying familial CRC . In the current study we show that it is possible to identify the mutation causing the main burden of CRC in a family with multiple affected family members by using both “affected” and “unaffected” individuals, even in the presence of phenocopies. This clearly demonstrates the power of exome sequencing in genetic diagnostics of hereditary predisposition to cancer, and we anticipate that future studies will bring new insight in the molecular genetics of still unexplained cases of familial CRC.
Exome sequencing of members of a family with high burden of colorectal adenomas and carcinomas, in addition to extra-colonic cancers, has identified the novel mutation c.1373A>T (p.Tyr458Phe) in POLE as a likely predisposing mutation. Previous functional and structural studies have shown that the position p.Tyr458 in Polε is important for exonuclease activity, and that the tumorigenic effect of p.Tyr458Phe is increased mutation rate due to reduced exonuclease activity, and consequently also reduced replication fidelity. The role of POLE in predisposition to cancer is consistent with previous studies where other mutations affecting the Polε exonuclease domain have been associated with CRC. Including the present study, POLE mutations have been associated with lesions in colon, rectum, small intestine, stomach, ovaries, endometrium, pancreas and brain. The overall evidence clearly suggests that extra-colonic cancers need to be taken into consideration in risk management and follow up of patients with POLE mutation. The varying phenotypes among POLE carriers are likely to be caused by modifying effects of other alleles, and further studies are necessary to provide personalized risk assessment. PPAP is a fairly recently described cancer susceptibility syndrome and guidelines regarding management of POLE and POLD1 mutation carriers do not yet exist. It is important for this group of patients that such guidelines are implemented, incorporating the new knowledge on POLE mutations.
We thank the family members who participated in this research and are grateful for their contribution. The sequencing service was provided by the Genomics Core Facility, Norwegian University of Science and Technology, and NMC—a national technology platform supported by the functional genomics program (FUGE) of the Research Council of Norway. The bioinformatics analyses were performed at the Bioinformatics core facility, Norwegian University of Science and Technology (NTNU). We thank Dr. Bente Talseth-Palmer for comments on the manuscript and Dr. Berge Solberg for fruitful discussions and advice. This work was supported by grants from the Liaison Committee between the Central Norway Regional Health Authority (RHA) and the Norwegian University of Science and Technology (NTNU); and travel support for scientific meetings and courses from COST Action BM1206.
Conflict of interest
The authors declare that they have no conflict of interest.
All procedures performed in studies involving human participants were in accordance with the ethical standards of the research committee (Regional Committee for Medical and Health Research Ethics of Central Norway, approval 2012/1707) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Informed consent was obtained from all individual participants included in the study.
- 10.Broad Institute Picard Tools. http://broadinstitute.github.io/picard/. Accessed 22 Jan 2015
- 13.Van der Auwera GA, Carneiro MO, Hartl C et al (2013) From FastQ data to high-confidence variant calls: the genome analysis toolkit best practices pipeline. In: Bateman A, Pearson WR, Stein LD et al (eds) Current protocols in bioinformatics, 43rd edn. Wiley, Hoboken, pp 1–11Google Scholar
- 15.How-to/exome analysis—SEQwiki. http://seqanswers.com/wiki/How-to/exome_analysis. Accessed 22 Jan 2015
- 17.Vigeland MD Filtus. http://folk.uio.no/magnusv/filtus.html. Accessed 22 Jan 2015
- 29.Cambridge M Exome Aggregation Consortium (ExAC). http://exac.broadinstitute.org/. Accessed 1 Dec 2014
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