European Journal of Human Genetics (2008) 16, 1477–1486 & 2008 Macmillan Publishers Limited All rights reserved 1018-4813/08 $32.00 www.nature.com/ejhg ARTICLE Deciphering the genetics of hereditary non-syndromic colorectal cancer Eli Papaemmanuil1,13, Luis Carvajal-Carmona2,13, Gabrielle S Sellick1,13, Zoe Kemp2,13, Emily Webb1, Sarah Spain2, Kate Sullivan1, Ella Barclay2, Steven Lubbe1, Emma Jaeger2, Jayaram Vijayakrishnan1, Peter Broderick1, Maggie Gorman2,3, Lynn Martin4,5, Anneke Lucassen6, D Timothy Bishop7, D Gareth Evans4, Eamonn R Maher5, Verena Steinke8, Nils Rahner8, Hans K Schackert9, Timm O Goecke10, Elke Holinski-Feder10, Peter Propping8, Tom Van Wezel11, Juul Wijnen11, Jean-Baptiste Cazier12, Huw Thomas3, Richard S Houlston*,1,14 and Ian Tomlinson*,2,14, The CORGI Consortium 1Section of Cancer Genetics, Institute of Cancer Research, Sutton, Surrey, UK; 2Molecular and Population Genetics Laboratory, Cancer Research UK, London, UK; 3Colorectal Cancer Unit, Cancer Research UK, St Mark’s Hospital, Harrow, UK; 4Medical Genetics, St Mary’s Hospital, Manchester, UK; 5Department of Medical and Molecular Genetics, University of Birmingham School of Medicine and West Midlands Genetics Service, Birmingham Women’s Hospital, Edgbaston, Birmingham, UK; 6Wessex Clinical Genetics Service, Princess Anne Hospital, Southampton, UK; 7Genetic Epidemiology Laboratory, Cancer Research UK, St James’s University Hospital, Leeds, UK; 8Institute of Human Genetics, University of Bonn, Bonn, Germany; 9Department of Surgical Research, Technische Universita¨t Dresden, Dresden, Germany; 10Institute of Human Genetics, University of Du¨sseldorf, Germany; 11Department of Pathology, Leiden Medical Centre, Leiden, The Netherlands; 12Bioinformatics and Biostatistics, London Research Institute, Cancer Research UK, London, UK Previously we have localized to chromosome 3q21–q24, a predisposition locus for colorectal cancer (CRC), through a genome-wide linkage screen (GWLS) of 69 families without familial adenomatous polyposis or hereditary non-polyposis CRC. To further investigate Mendelian susceptibility to CRC, we extended our screen to include a further GWLS of an additional 34 CRC families. We also searched for a disease gene at 3q21–q24 by linkage disequilibrium mapping in 620 familial CRC cases and 960 controls by genotyping 1676 tagging SNPs and sequencing 30 candidate genes from the region. Linkage analysis was conducted using the Affymetrix 10K SNP array. Data from both GWLSs were pooled and multipoint linkage statistics computed. The maximum NPL score (3.01; P ¼ 0.0013) across all families was at 3q22, maximal evidence for linkage coming from families segregating rectal CRC. The same genomic position also yielded the highest multipoint heterogeneity LOD (HLOD) score under a dominant model (HLOD ¼ 2.79; P ¼ 0.00034), with an estimated 43% of families linked. In the case–control analysis, the strongest association was obtained at rs698675 (P ¼ 0.0029), but this was not significant after adjusting for multiple testing. Analysis of candidate gene mapping to the region of maximal linkage on 3q22 failed to identify a causal mutation. There was no evidence for linkage to the previously reported 9q CRC locus (NPL ¼ 0.95, P ¼ 0.23; HLODdominant ¼ 0.40, HLODrecessive ¼ 0.20). Our findings are consistent with the hypothesis that variation *Correspondence: Dr Professor R Houlston, Section of Cancer Genetics, Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5 NG, UK. Tel: þ 44 208 722 4175; Fax: þ 44 208 722 4359; E-mail: [email protected] or Professor I Tomlinson Molecular and Population Genetics Laboratory, London Research Institute, Cancer Research UK, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK. Tel: þ 44 207 269 2884; Fax: þ 44 207 269 3093; E-mail: [email protected] 13These authors contributed equally to this work 14These authors have joint PI status Received 26 March 2008; revised 29 May 2008; accepted 29 May 2008; published online 16 July 2008 Deciphering the genetics of HNSCRC E Papaemmanuil et al 1478 at 3q22 contributes to the risk of CRC, but this is unlikely to be mediated through a restricted set of alleles. European Journal of Human Genetics (2008) 16, 1477–1486; doi:10.1038/ejhg.2008.129; published online 16 July 2008 Keywords: colorectal cancer; linkage; chromosome 3q22 Introduction pedigrees as phase 2. As before, familial CRC cases were Family history is well established to be one of the strongest ascertained through the COloRectal tumour Gene Identi- risk factors for the development of colorectal cancer (CRC).1 fication (CORGI) Study Consortium.8 Briefly, pedigrees Although germline mutations in APC,themismatchrepair informative for linkage had at least three affected indivi- (MMR) genes, MUTYH/MYH, SMAD4, ALK3 and STK11/LKB1 duals (confirmed by pathology reports). Individuals were are associated with Mendelian susceptibility to CRC, classed as affected if they had CRC at age r75 years or collectively, these do not account for the observed familial ‘significant’ adenomas (three or more synchronous or aggregation of the disease.2,3 Direct evidence for unchar- metachronous, and/or villous morphology, and/or severe acterized high/moderate-penetrance CRC genes is provided dysplasia, and/or diameter 41 cm and/or presentation at by CRC families showing evidence against linkage to known r45 years of age). No patient had clinical features of loci and by kindreds that fulfil the clinical (Amsterdam) Peutz-Jeghers syndrome (PJS), juvenile polyposis (JPS), criteria for a diagnosis of hereditary non-polyposis colo- hereditary mixed polyposis (HMPS) or inflammatory bowel rectal cancer (HNPCC) but whose CRCs do not display MMR disease. Germline MMR gene mutations were excluded by deficiency.4 Familial CRC risks in relatives of colorectal microsatellite instability (MSI) testing of BAT25 and BAT26 adenoma (CRA) cases parallel those seen in relatives of CRC in two CRCs from each family; kindreds in which both cases.1 It is therefore likely that a significant proportion of cancers were unstable were excluded. Where one MSI þ inherited predisposition to CRC is mediated through tumour was found or if the only available CRC was susceptibility to CRAs. These observations have provided a unstable, direct mutation screening of all coding regions strong rationale for searching of novel predisposition genes of MSH2 and MLH1 was undertaken. In addition, all through genome-wide linkage screens (GWLSs) of heredi- families with an attenuated familial adenomatous poly- tary non-FAP/HNPCC CRC families (hereditary non- posis (FAP)-like phenotype (45 adenomas) were tested for syndromic colorectal cancer – HNSCRC). mutations in APC, and the entire data set was tested for the Using a high-density SNP array, we have previously two common MYH variants (Y165C and G382D) using performed a GWLS of 69 HNSCRC families in which fluorescence-SSCP analysis. involvement of known predisposition genes had been The case–control study was based on 620 CRC cases (280 excluded and a novel CRC susceptibility locus at 3q21– male, 340 female) with at least one affected first-degree q24 was identified.5 Other workers have proposed relative ascertained through CORGI. As with the linkage additional loci for CRC susceptibility genes on the basis families, none had clinical features of FAP, PJS, JPS, HMPS of linkage, most notably on 9q22.2–31.2.6,7 or inflammatory bowel disease. In addition, none had a To further examine the impact of unknown moderate/ family history compatible with a diagnosis of HNPCC. high-penetrance genes on CRC risk, we conducted a Samples collected from unaffected spouses/partners of further GWLS on an additional 34 HNSCRC families using cases served as a source of 965 controls (439 male, 526 the same analytical platform employed in our first analysis. female). We then pooled data from scans. In an effort to predict the Genomic DNA was extracted from whole blood using the most likely location of the putative CRC gene on 3q21– Chemagic Magnetic Separation Module 1t. All samples q24, we undertook linkage disequilibrium (LD) mapping at were obtained with informed consent and local ethical 1676 tagging SNPs in 620 HNSCRC cases and 961 controls. review board approval in accordance with the tenets of the In addition, we have sought to identify disease-causing Declaration of Helsinki. variants by direct mutational analysis of candidate genes localizing to the region of maximal linkage on 3q22. Genome-wide linkage screen The current analysis was based on 34 families segregating CRC and/or CRA in which involvement of known loci had Materials and methods been excluded. A GWLS of families was undertaken using Ascertainment and collection of families and cases the GeneChips Mapping 10K Xba 142 Array (Affymetrix For clarity, we refer to our previously reported GWLS of 69 Inc., Santa Clara, CA). SNP genotypes were obtained by pedigrees as phase 18 and the current analysis of 34 following the Affymetrix protocol. Briefly, for each sample, European Journal of Human Genetics Deciphering the genetics of HNSCRC E Papaemmanuil et al 1479 250 ng of genomic DNA was digested with the restriction and 0.638 with corresponding phenocopy rates of 0.00004, endonuclease XbaI for 2.5 h, mixed with Xba adapters and 0.0003, 0.0026 and 0.023. ligated using T4 DNA ligase for 2.5 h. Ligated DNA was A second analysis was undertaken in which individuals added to four separate PCRs, cycled, pooled and purified were classed as affected if they had either CRC or to