© American College of Medical Genetics and Genomics ORIGINAL RESEARCH ARTICLE Open POLE and POLD1 mutations in 529 kindred with familial colorectal cancer and/or polyposis: review of reported cases and recommendations for genetic testing and surveillance Fernando Bellido1, Marta Pineda, PhD1, Gemma Aiza1, Rafael Valdés-Mas2, Matilde Navarro, MD1, Diana A. Puente2, Tirso Pons, PhD3, Sara González1, Silvia Iglesias1, Esther Darder4, Virginia Piñol, MD, PhD5,6, José Luís Soto, PhD7, Alfonso Valencia, PhD3, Ignacio Blanco, MD, PhD1, Miguel Urioste, MD, PhD8, Joan Brunet, MD, PhD4,6, Conxi Lázaro, PhD1, Gabriel Capellá, MD, PhD1, Xose S. Puente, PhD2, and Laura Valle, PhD1 Purpose: Germ-line mutations in the exonuclease domains of with strong evidence for pathogenicity) were identified in nonpoly­ POLE and POLD1 have been recently associated with polyposis and posis CRC families. Phenotypic data from these and previously colorectal cancer (CRC) predisposition. Here, we aimed to gain a bet- reported POLE/POLD1 carriers point to an associated phenotype ter understanding of the phenotypic characteristics of this syndrome characterized by attenuated or oligo-adenomatous colorectal polypo- to establish specific criteria for POLE and POLD1 mutation screening sis, CRC, and probably brain tumors. In addition, POLD1 mutations and to help define the clinical management of mutation carriers. predispose to endometrial and breast tumors. Methods: The exonuclease domains of POLE and POLD1 were Conclusion: Our results widen the phenotypic spectrum of the studied in 529 kindred, 441 with familial nonpolyposis CRC and 88 POLE/POLD1-associated syndrome and identify novel pathogenic with polyposis, by using pooled DNA amplification and massively variants. We propose guidelines for genetic testing and surveillance parallel sequencing. recommendations. Results: Seven novel or rare genetic variants were identified. In Genet Med advance online publication 2 July 2015 addition to the POLE p.L424V recurrent mutation in a patient with Key Words: adenomatous polyposis; genetic testing; hereditary polyposis, CRC and oligodendroglioma, six novel or rare POLD1 nonpolyposis colorectal cancer; polymerase proofreading-associated­ variants (four of them, p.D316H, p.D316G, p.R409W, and p.L474P, polyposis INTRODUCTION POLE and POLD1 exonuclease mutation screening and to help Germ-line mutations in the exonuclease (proofreading) domain define the clinical management of mutation carriers. To fulfill of DNA polymerases Pol δ and Pol ε have been associated with this aim, here we study the complete exonuclease domains of a dominantly inherited syndrome that confers increased risk to POLE and POLD1 in 529 independent families characterized by colorectal cancer (CRC) and polyposis.1 Two recurrent patho- the presence of familial or early-onset mismatch repair (MMR), genic variants, POLE p.L424V and POLD1 p.S478N, have been proficient CRC, and/or APC-negative and ­MUTYH-negative identified in 21 and 3 families, respectively.1–4 A novel mutation polyposes. in POLD1, p.L474P, was found in a hereditary nonpolyposis CRC family.2 MATERIALS AND METHODS Patients carrying POLE and POLD1 exonuclease domain Study sample mutations show variable phenotypes including multiple adeno- A total of 544 CRC cases belonging to 529 families were included mas and CRC, and endometrial cancer in the case of female in the study: 456 familial CRC cases from 441 uncharacterized POLD1 mutation carriers.1–4 A better characterization of the MMR-proficient families, including 60 Amsterdam-positive syndrome is currently required to establish specific criteria for families, and 88 polyposis cases. Most of them, 526 cases (511 The first two authors contributed equally to this work. 1Hereditary Cancer Program, Catalan Institute of Oncology, IDIBELL, Hospitalet de Llobregat, Spain 2Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Oncología del Principado de Asturias, Universidad de Oviedo, Oviedo, Spain; 3Structural Biology and Biocomputing Program, Spanish National Cancer Research Center (CNIO), Madrid, Spain; 4Hereditary Cancer Program, Catalan Institute of Oncology, IDIBGi, Girona, Spain; 5Gastroenterology Department, Hospital Dr. Josep Trueta, Girona, Spain; 6Medical Science Department, School of Medicine, University of Girona, Girona, Spain; 7Molecular Genetics Laboratory, Elche University Hospital, Elche, Spain; 8Familial Cancer Clinical Unit, Human Cancer Genetics Program, Spanish National Cancer Research Centre (CNIO) and Center for Biomedical Network Research on Rare Diseases (CIBERER), Madrid, Spain. Correspondence: Laura Valle ([email protected]) Submitted 22 January 2015; accepted 23 April 2015; advance online publication 2 July 2015. doi:10.1038/gim.2015.75 Genetics in medicine | Volume 18 | Number 4 | April 2016 325 ORIGINAL RESEARCH ARTICLE BELLIDO et al | Polymerase proofreading-associated syndrome families), were previously genotyped for POLE p.L424V and Informed consent was obtained from all subjects and the study POLD1 p.S478N (included in series no. 1 in the work by Valle received the approval of the Ethics Committee of the Institut et al.2). All of them were referred to the Genetic Counseling d'Investigació Biomèdica de Bellvitge (IDIBELL) (PR073/12). Units of the Catalan Institute of Oncology in the Spanish region of Catalonia between 1999 and 2012. Referral was based on Germ-line mutation identification in pooled samples family history of CRC or polyps, presence of early-onset CRC, Mutation screening of the exonuclease coding regions of POLE and/or personal history of polyposis. Eighteen additional CRC and POLD1 was performed by using a combination of pooled patients belonging to unrelated Amsterdam I MMR-proficient samples, PCR amplification (POLE exons 9–14 and POLD1 families were included in the study. These were recruited exons 6–12), and high-throughput sequencing, as previously through the Human Genetics Program of the Spanish National described.5 Amplification of the DNA pools was performed Cancer Research Center (CNIO). using Phusion High-Fidelity DNA Polymerase (New England Among the 456 MMR-proficient cases (441 families), 49 Biolabs, Ipswich, MA) and custom-designed primers cover- (10.7%) fulfilled Amsterdam I, 11 (2.4%) fulfilled Amsterdam ing the exons and intron–exon boundaries (Supplementary II, and 390 (85.5%) fulfilled the Bethesda criteria. No specific Table S1 online). Next-generation sequencing was performed information on family history was available for six patients. on a HiSeq-2000 at the Centro Nacional de Análisis Genómico The mean age at cancer diagnosis was 48.98 (SD: 12.54) (CNAG, Barcelona, Spain). for the tested individuals. Nonpolyposis cases were MMR- proficient, i.e., their tumors showed microsatellite stability Direct automated sequencing and expression of the MMR proteins MLH1, MSH2, MSH6, Direct automated (Sanger) sequencing was used to validate the and PMS2. results obtained by massive parallel sequencing, to identify the Clinical features of polyposis cases, which included ade- mutated cases within pools, and to perform the co-segregation nomatous polyposes (17.0%), attenuated adenomatous poly- studies. Sequencing was performed on an ABI Sequencer poses (47.7%), and nonadenomatous polyposes (15.9%), 3730 (Applied Biosystems, Life Technologies, Foster City, CA) were detailed by Valle et al.2 For these cases, screening of using a standard protocol. Data were analyzed using Mutation MUTYH and APC mutations was performed as previously Surveyor (version 3.10) (Softgenetics, State College, PA, USA). described.2 The primers used were the same as those used for germ-line Table 1 Germ-line variants in the exonuclease domain (amino acids R311 to L526) and adjacent regions of POLD1 identified in 456 familial or early-onset nonpolyposis CRC and 88 polyposis cases 3D structure Protein function prediction prediction Population Protein domain/ Evolutionary PPH2 Mutator MAF secondary conservation (HumDiv/ Mutation CUPSAT/ phenotypef Variant Variantsa (dbSNP/ESP) structure element (PhyloP)d HumVar) SIFT taster Condel I-Mutant/ERIS (S. cerevisiae) classification c.883G>A; n.a./0.0003 N-terminal/loop 1.096 0.410/ 0.09 N 0.371 Destabilize n.a. Uncertain p.(V295M) (rs199545019) 0.103 (N) (N) (N) (all) significance c.946G>C; 0/0c Exonucl. 6.977 1/1 0 D 0.690 No effect/ Yes 13,14 Pathogenic p.(D316H) (catalytic res.)/ (PbD) (D) (D) destabilize β-strand (I-Mut, ERIS)e c.947A>G; 0/0c Exonucl. (catalytic 6.549 1/1 0 D 0.688 Destabilize Yes 13,14 Pathogenic p.(D316G) res.)/β-strand (PbD) (D) (D) (all) c.1225C>T; 0/0c Exonucl./α-helix 1.115 1/1 0 D 0.610 Destabilize n.a. Probably p.(R409W) (PbD) (D) (D) (all) pathogenic c.1421T>Cb; 0/0c Exonucl./α-helix 5.593 1/1 0 D 0.702 Destabilize Yes 14 Pathogenic p.(L474P) (PbD) (D) (D) (all) c.1562G>A; n.a./0.00035 Exonucl./α-helix 8.762 0.816/ 0.05 D 0.435 No effect/ n.a. Uncertain p.(R521Q) (rs143076166) 0.195 (D) (N) destabilize significance (PsD/N) (I-Mut, ERIS)e Indicated in bold is the evidence that supports the damaging nature of the variants. In silico algorithm NNSplice did not predict splice site effects for any of the variants. Loss of POLD1 wild-type allele could not be identified in five of six tumors analyzed (Supplementary Table S3 online), in agreement with the haploinsufficiency model previously proposed.1 D, damaging or deleterious; ESP, NHLBI Exome Sequencing Project; Exonucl., exonuclease domain; MAF, minor allele frequency; N, neutral; n.a., not available; PbD, probably damaging;