HUMAN MUTATION 29(3), 367^374, 2008

RESEARCH ARTICLE

Classification of Ambiguous Mutations in DNA Mismatch Repair Genes Identified in a Population- Based Study of Colorectal Cancer

Rebecca A. Barnetson,1Ã Nicola Cartwright,1 Annelot van Vliet,1 Naila Haq,1 Kate Drew,1 Susan Farrington,1 Nicola Williams,2 Jon Warner,2 Harry Campbell,3 Mary E. Porteous,2 and Malcolm G. Dunlop1 1Colon Cancer Genetics Group, University of Edinburgh Cancer Research Centre, School of Molecular and Clinical Medicine and Medical Research Council (MRC) Human Genetics Unit, Western General Hospital, Edinburgh, United Kingdom; 2South East (SE) Scotland Clinical Genetics Service, Western General Hospital, Edinburgh, United Kingdom; 3Public Health Sciences, University of Edinburgh, United Kingdom

Communicated by Albert de la Chapelle Identification of germline mutations in DNA mismatch repair genes in colorectal cancer probands without an extensive family history can be problematic when ascribing relevance to cancer causation. We undertook a structured assessment of the disease-causing potential of sequence variants identified in a prospective, population- based study of 932 colorectal cancer patients, diagnosed at o55 years of age. Patient samples were screened for germline mutations in MLH1, MSH2,andMSH6. Of 110 carriers, 74 (67%) had one of 33 rare variants of uncertain pathogenicity (12 MLH1,11MSH2,and10MSH6). Pathogenicity was assessed by determining segregation in families, allele frequency in large numbers of unaffected controls, effect on mRNA for putative splice- site mutations, effect on protein function by bioinformatic analysis and tumor microsatellite instability (MSI) status and DNA mismatch repair protein expression by immunohistochemistry. Because of the ambiguous nature of these variants and lack of concordance between functional assays and control allele frequency, we devised a scoring system torankthedegreeofsupportforapathogenicrole.MLH1 c.200G4Ap.G67E,MLH1 c.2041G4A p.A681T, and MSH2 c.263415G4C were categorized as pathogenic through assimilation of all available data, while 14 variants were categorized as benign (seven MLH1,threeMSH2, and four MSH6). Interestingly, there is tentative evidence suggesting a possible protective effect of three variants (MLH1 c.2066A4G pQ689R, c.2146G4A p.V716M, and MSH2 c.965G4A p.G322D). These findings support a causal link with colorectal cancer for several DNA mismatch repair gene variants. However, the majority of missense changes are likely to be inconsequential polymorphisms. Hum Mutat 29(3), 367–374, 2008. r 2007 Wiley-Liss, Inc.

KEY WORDS: DNA mismatch repair; Lynch syndrome; HNPCC; colorectal cancer; MLH1; MSH2; MSH6

INTRODUCTION et al., 1997; Quehenberger et al., 2005], it is often not possible to obtain an informative pedigree. Therefore, other criteria have Lynch syndrome or hereditary nonpolyposis colorectal cancer been proposed for the testing of probands for Lynch syndrome, (HNPCC) (MIM 120435) is an autosomal dominant syndrome that take into account early age at onset, gender, tumor site, and that accounts for about 1 to 3% of all colorectal cancer cases [Aaltonen et al., 1998; Samowitz et al., 2001]. The majority of Lynch syndrome cases have a germline mutation in one of the DNA mismatch repair genes MLH1, MSH2,orMSH6 [Akiyama The Supplementary Material referred to in this article can be et al., 1997; Bronner et al., 1994; Fishel et al., 1993; Leach et al., accessed at http://www.interscience.wiley.com/jpages/1059-7794/ suppmat. 1993; Papadopoulos et al., 1994]. In most cases, this results in Received 21February 2007; accepted revised manuscript 7 August tumor microsatellite instability (MSI) because of defective DNA 2007. mismatch repair. Mutation carriers are at a greatly increased risk of ÃCorrespondence to: Rebecca A. Barnetson, Colon Cancer Genet- developing colorectal cancer at an early age and are also ics Group, MRC Human Genetics Unit, Western General Hospital, predisposed to carcinomas of the endometrium, ovary, gastro- Edinburgh, EH42XU,United Kingdom. intestinal tract, and urinary tract [Vasen et al., 1996]. Recently, we E-mail: [email protected] have shown that 6% of males and 3% of females diagnosed with Grant sponsor: Cancer Research UK; Grant number: C348/A3758; colorectal cancer aged less than 55 years carry such mutations Grant sponsor: Scottish ExecutiveChief Scientist’sO⁄ce;Grant num- bers: CZB/4/11; and K/OPR/2/2/D333; Grant sponsor: Medical Re- [Barnetson et al., 2006]. The Amsterdam criteria have been search Council; Grant number: G0000657-53203. developed for the diagnosis of Lynch syndrome, based on the DOI 10.1002/humu.20635 patient’s family history of colon cancer [Vasen et al., 1991]. As Published online 21 November 2007 in Wiley InterScience (www. many families are small and penetrance is incomplete [Dunlop interscience.wiley.com). r 2007 WILEY-LISS, INC. 368 HUMAN MUTATION 29(3), 367^374, 2008 extracolonic carcinomas [Barnetson et al., 2006; Rodriguez-Bigas ambiguous variants identified in a series of colorectal cancer cases, et al., 1997]. using a number of complementary approaches. These included Many DNA mismatch repair mutations are short insertions, comparison of variant frequency in cases and large numbers of deletions, or nucleotide changes that create premature stop population-based controls, tumor MSI and DNA mismatch repair codons, or nucleotide changes at the intron–exon boundaries that protein expression analysis, cosegregation with cancer in families, affect mRNA splicing. Such mutations are readily categorized as mRNA analysis, and bioinformatic assessment of the functional pathogenic, because they encode a truncated protein, resulting in consequence of the amino acid change. The findings have defective DNA mismatch repair. However, the majority of putative considerable relevance to the clinical management of colorectal mutations identified in this study were missense changes that alter cancer probands and their relatives, as well as to the investigation a single amino acid or are close to splice junctions and were of DNA mismatch repair function. therefore of ambiguous relevance to pathogenicity. Where tumor material is available, microsatellite analysis can be MATERIALS AND METHODS a useful indicator of DNA mismatch repair proficiency. However, in studies that select cases on the basis of family history or very A population-based series of patients from throughout Scotland, early age at onset, the likelihood of MSI is considerable because who were diagnosed with colorectal cancer when they were less such criteria enrich for mutation carriers. This does not necessarily than 55 years of age, were recruited to the study between February mean that every variant identified will be disease-causing, as it 1999 and June 2004. During the same period, unaffected controls may be another DNA mismatch repair mutation that is involved. were ascertained from a population-based register (community This is particularly an issue for variants identified in cases that health index) and were invited to participate. Blood samples for have been ‘‘pre-screened’’ by MSI analysis of tumor samples. In DNA analysis were obtained and a family history was taken from addition, MSI is not a universal indicator of pathogenicity as the all cases and controls recruited to the study. majority of tumors from MSH6 mutation carriers are microsatellite Sequence analysis of MLH1, MSH2, and MSH6 was undertaken stable (MSS) or have low-level microsatellite instability (MSI-L), using DNA samples from all patients recruited to the study, and with only one out of five markers affected [Barnetson et al., 2006; primer sequences and mutation nomenclature were based on Lagerstedt Robinson et al., 2007; Wu et al., 1999]. Furthermore, NCBI reference sequences U40960.1 (MLH1), U04045.1 84% of colorectal carcinomas with high-level microsatellite (MSH1), and U73732.1 (MSH6), respectively, with 11 as the instability (MSI-H) lack MLH1 expression as a result of promoter A of the ATG initiation codon. Mutation analysis comprised PCR hypermethylation [Herman et al., 1998]. Immunohistochemistry amplification of all exons of MLH1, MSH2, and MSH6 and can be useful but the presence of a DNA mismatch repair protein analysis of products by dHPLC on the WAVE (Transgenomics, does not unequivocally establish that the expressed protein is Omaha, NE, USA) or by direct sequencing as described previously functional. [Barnetson et al., 2006]. Variant alleles were confirmed by Segregation of the variant with the disease within a family can sequencing (ABI 3730 DNA Analyser; Applied Biosystems, provide evidence of pathogenicity. However, DNA is not always Warrington, UK). MLH1 and MSH2 were also screened for large available from all family members, many carriers are from small genomic deletions by multiplex ligation-dependent probe ampli- nuclear families, and Lynch syndrome mutations are not fication (MLPA; MRC-Holland, Amsterdam, The Netherlands) completely penetrant [Dunlop et al., 1997; Quehenberger et al., and samples were run on an AB3100 Genetic Analyser and 2005]. Even if a variant does segregate with the disease, it may be analyzed with Genotyper (Applied Biosystems) and custom linked to another mutation that is pathogenic. Determination of software (www.ngrl.org.uk/Manchester/Publications.htm]MLPA). the variant frequency in unaffected controls to assess pathogeni- Allelic discrimination assays were designed to determine whether city requires large sample sizes, as most are found at low thesequencevariants,MLH1:c.65G4C p.G22A, c.200G4A frequencies. Many published studies have only screened 50–100 p.G67E, c.277A4G p.S93G, c.1217G4A p.S406N, c.1852AA4GC controls and therefore lack definitive statistical power. p.K618A, c.2041G4A p.A681T, and c.2066A4G p.Q689R; MSH2: A number of functional assays have been devised to assess the c.1A4C, c.965G4A p.G322D, and c.1787A4G p.N596S; and effect of missense changes on DNA mismatch repair. One MSH6: c.431G4T p.S144I and c.1508C4G p.S503C, were present examines the ability of a human MLH1 variant cDNA to rescue in controls. DNA from mutation carriers was included as a positive the dominant negative effect that results in a mutator phenotype control and samples were run on the ABI PRISM HT7900 and in yeast [Shimodaira et al., 1998] but the mechanism is not fully analyzed using SDS2.0 software (Applied Biosystems). All variants understood. In a second assay, the equivalent missense MLH1 identified were confirmed by sequencing. The population frequency of variant is generated in yeast MLH1 and repair proficiency is MLH1: c.925C4T p.P309S, c.1820T4A p.L607H, c.2146G4A assessed [Shcherbakova and Kunkel, 1999] but this technique is p.V716M, c.2152C4T p.H718Y, and c.2252A4G p.K751R; MSH2: restricted to assessment of evolutionarily conserved amino acid c.138C4G p.H46Q, c.317G4A p.R106K, c.2009C4T p.P670L, residues. This problem can be overcome by using a hybrid human- c.2337G4A p.M779I, c.2420C4G p.T807S, c.2503A4C yeast MLH1 gene to assess mismatch repair function [Ellison et al., p.N835H, c.263415G4C, and c.2732T4G p.L911R; and MSH6: 2001] or by the stable expression of the variant human cDNA in a c.38A4C p.K13T, c.194C4T p.S65L, c.1403G4A p.R468H, mismatch repair deficient ovarian cancer cell line [Blasi et al., c.1739C4T p.S580L, c.2633T4C p.V878A, c.3556–3A4T, 2006]. However, functional assays are technically demanding and, c.3694G4C p.V1232L, and c.3961A4G p.R1321G were deter- as illustrated in Table 1, findings between assays do not always mined by automated sequence analysis of control DNA samples. All concur. Furthermore, the pathogenicity of a variant may not primer and probe sequences are listed in Supplementary Table S1 correlate completely with the effect observed in an experimental (available online at http://www.interscience.wiley.com/jpages/1059- system. 7794/suppmat). MSI analysis was performed on tumor samples using A combination of approaches seems to offer the best strategy for the recommended panel of microsatellite markers (BAT25, BAT26, determining whether a sequence variant is cancer-causing. We D2S123, D5S346, and D17S250) [Boland et al., 1998]. DNA undertook a structured assessment of the pathogenicity of all extracted from microdissected fixed tissue sections and corresponding à TABLE 1. Mutations Identi¢ed in Colorectal Cancer Cases and Evidence for Pathogenicity

Tu m o r I H C a

No. Tu m o r No. of cases Cases frequency No. of E¡ect Amstii Tu m o r s MSI Conserved SIFT PolyPhen In vitro MMR Category of Mutation identi¢ed (n 5 932) (95% CI) controls Controls frequency (95% CI) size 95%CI cases assessed statusa MLH1 MSH2 MSH6 (ClustalW) tolerated assessmentb (References)c pathogenicity Scoref

MLH1 c.65G4C p.G22A 1 0.0011 (0.0002^0.006) 2/1051 0.002 (0.0006^0.007) 0.58 0.06^5.58d 0 1 MSS 111 Ye s N o 3 B e n i g n 0 c.200G4A p.G67E 1 0.0011 (0.0002^0.006) 0/1688 0 (0^0.002) 16.63 0.28^997.5d 1 1 MSI^H ^ N/A N/A Yes No 3 Pathogenic 19 c.277 A 4G p.S93G 1 0.0011 (0.0002^0.006) 0/1167 0 (0^0.003) 9.51 0.18^491.25d 0 1 MSS 111 No Yes 3 1 (1,2,3) Unde¢ned 2 c.925C4Tp.P309S 1 0.0011 (0.0002^0.006) 0/1000 0 (0^0.004) 7.95 0.16^401.57d 0 1 MSS 111 Ye s N o 3 U n d e ¢ n e d 5 c.1217G4A p.S406N 1 0.0011 (0.0002^0.006) 1/1022 0.001 (00002^0.005) 1.1 0.07^17.60 0 1 MSS 111 No Yes 2 1 (3,4) Benign 0 c.1820T4A p.L607H 1 0.0011 (0.0002^0.006) 0/1003 0 (0^0.004) 7.97 0.16^402.95d 0 1 MSS 11^NoYes1 Unde¢ned0 c.1852AA4GC p.K618A 16 0.017 (0.011^0.028) 14/1553 0.009 (0.005^0.016) 1.92 0.93^3.95 0 6 MSS 111 No Yes 2 À/1 (2,3,5^7) Benign 0 c.2041G4A p.A681T 2 0.002 (0.0004^0.008) 0/1430 0 (0^0.003) 12.6 0.74^215.21d 2 2 MSI^H ^ 11 Ye s N o 2 À/1(2^4,6,8,9) Pathogenic 19 c.2066A4G pQ689R 2 0.002 (0.0004^0.008) 5/1066 0.005 (0.002^0.012) 0.46 0.09^2.36 0 2 MSS 111 No Yes 1 1(3) Benign 0 c.2146G4A p.V716M 1 0.0011 (0.0002^0.006) 6/1000 0.008 (0.005^0.015) 0.18 0.002^1.48 0 1 MSS 111 No Yes 1 À/1 (2,3,7) Benign 0 c.2152C4T p.H718Y 1 0.0011 (0.0002^0.006) 1/1000 0.001 (0.0002^0.006) 1.07 0.07^17.18 0 0 N/A N/A N/A N/A Yes No 3 À/1 (3,4) Benign N/A c.2252A4G p.K751R 2 0.002 (0.0004^0.008) 1/1000 0.001 (0.0002^0.006) 2.15 0.19^23.7 0 2 MSS 111 Ye s Ye s 1 À/1 (3,10) Benign 0 MSH2 c.1A4C 1 0.0011 (0.0002^0.006) 0/1422 0 (0^0.003) 12.50 0.23^687.8d 01MSI^L111 No No 1 Unde¢ned 4 c.138C4G p.H46Q 1 0.0011 (0.0002^0.006) 0/1006 0 (0^0.004) 8.0 0.16^404.34d 0 1 MSS 111 No No 3 Unde¢ned 4 c.317G4Ap.R106K 1 0.0011 (0.0002^0.006) 1/1023 0.001 (0.0002^0.005) 1.09 0.07^17.62 0 1 MSS 111 No Yes 1 Benign 0 c.965G4A p.G322D 17 0.018 (0.011^0.029) 13/406 0.03 (0.018^0.056) 0.56 0.27^1.17 1 7 MSS 111 Ye s Ye s 2 1/^ (8,11) Benign 0 c.1787A 4G p.N596S 1 0.0011 (0.0002^0.006) 0/1017 0 (0^0.004) 8.09 0.16^409.5d 01MSI^L111 No Yes 1 Unde¢ned 2 c.2009C4Tp.P670L 1 0.0011 (0.0002^0.006) 0/1072 0 (0^0.003) 8.59 0.17^436.95d 0 1 MSS 111 Ye s N o 3 U n d e ¢ n e d 5 c.2337G4Ap.M779I 1 0.0011 (0.0002^0.006) 0/1062 0 (0^0.003) 8.5 0.17^431.75d 0 1 MSS 1 ^^ NoYes2 Unde¢ned5 c.2420C4Gp.T807S 1 0.0011 (0.0002^0.006) 0/1062 0 (0^0.003) 8.5 0.17^431.75d 0 1 MSI^H ^ N/S 1 No Yes 1 Unde¢ned N/A c.2503A4Cp.N835H 1 0.0011 (0.0002^0.006) 1/1011 0.001 (0.0002^0.005) 1.08 0.07^17.40 0 1 MSI^L 111 No Yes 1 Benign 0 c.263415G4C 1 0.0011 (0.0002^0.006) 0/1017 0 (0^0.004) 8.09 0.16^409.5d 1 1 MSI^H 1 ^ ^ N/R N/R N/R Pathogenice N/A d

c.2732T4G p.L911R 1 0.0011 (0.0002^0.006) 0/1118 0 (0^0.004) 8.1 0.16^409.97 0 0 N/A N/A N/A N/A No No 2 Benign N/A 369 367^374, 29(3), MUTATION 2008 HUMAN MSH6 c.38A4Cp.K13T 1 0.0011 (0.0002^0.006) 0/1104 0 (0^0.003) 8.89 0.17^454.23d 0 1 MSS 111 Ye s N o 2 U n d e ¢ n e d 4 c.194C4Tp.S65L 1 0.0011 (0.0002^0.006) 0/1104 0 (0^0.003) 8.89 0.17^454.23d 0 1 MSS 111 No Yes 1 Unde¢ned 0 c.431G4Tp.S144I 3 0.003(0.0008^0.01) 5/1059 0.005(0.002^0.012) 0.68 0.16^2.86 0 2 MSS 111 No Yes 2 1/^ (12,13) Benign 0 c.1403G4Ap.R468H 1 0.0011 (0.0002^0.006) 0/1008 0 (0^0.004) 8.01 0.16^405.27d 0 1 MSS 111 No Yes 2 Unde¢ned 1 c.1508C4G p.S503C 2 0.002 (0.0004^0.008) 2/1380 0.001 (0.0005^0.006) 1.48 0.21^10.54 0 2 MSS 111 No No 2 Benign 0 c.1739C4Tp.S580L 1 0.0011 (0.0002^0.006) 0/1008 0 (0^0.004) 8.01 0.16^405.27d 0 1 MSS 111 No Yes 3 Unde¢ned 2 c.2633T4C p.V878A 5 0.005 (0.0002^0.013) 8/521 0.015 (0.007^0.03) 0.35 0.11^1.06 0 1 MSS 111 No Yes 1 Benign 0 c.3556^3A4T 1 0.0011 (0.0002^0.006) 0/1121 0 (0^0.003) 9.05 0.18^463.83d 0 1 MSS 111 N/R N/R N/R Benigne N/A c.3694G4Cp.V1232L 1 0.0011 (0.0002^0.006) 0/1230 0 (0^0.003) 10.17 0.19^532.47d 0 0 N/A N/A N/A N/A No Yes N/A Unde¢ned N/A c.3961A4Gp.R1321G 1 0.0011 (0.0002^0.006) 0/1230 0 (0^0.003) 10.17 0.19^532.47d 0 0 N/A N/A N/A N/A No Yes N/A Unde¢ned N/A

*Mutation nomenclature was based on GenBank reference sequences U40960.1 (MLH1), U04045.1 (MSH2), and U73732.1 (MSH6) with 11 as theA of theATG start codon. Novel variants identi¢ed in this study are in bold. aFor MSI and IHC: MSS 5 microsatellite stable; MSI-H 5high degree of microsatellite instability; MSI^L 5low degree of microsatellite instability; N/A 5 sample not available; N/S 5 not scoreable; 15normal expression pattern; À 5 lack of expres- sion; N/R 5 not relevant. bPolyPhen score:1 5benign; 2 5 possibly damaging; 3 5 probably damaging. In vitro MMR: À 5 MMR de¢cient; 15MMR pro¢cient. cReferences:1 5Nystrom-Lahti et al. [2002]; 2 5 Raevaara et al. [2005]; 3 5 Takahashi et al. [2007]; 4 5 Kondo et al. [2003],Takahashi et al. [2007]; 5 5 Belvederesi et al. [2006]; 6 5 Shimodaira et al. [1998]; 7 5 Syngal et al. [1999]; 8 5 Ellison et al. [2001]; 9 5 Guerrette et al. [1999];10 5 Pang et al. [1997]; 11 5Drotschmann et al. [1999];12 5 Kariola et al. [2002];13 5 Kolodner et al. [1999]. dThis is an approximation to the exact odds ratio since one of the cells is zero. eRNA analysis for exon skipping. fScoring system for de¢ning pathogenicity: present in controls 5 À6; segregates in two family members 5 2; segregates in three to ¢ve family members 5 4; segregates in more than 5 family members 5 6; MSS 5 0; MSI^L 5 2; MSI^H 5 4; lack of expression of relevant gene by IHC 5 4; Conserved 51; not tolerated (SIFT) 5 2; PolyPhen 3 52; PolyPhen 2 51; in vitro MMR 51. 370 HUMANMUTATION29(3),367^374,2008 constitutional DNA was amplified by multiplex PCR for the mutations, 36 (33%) were either frameshift mutations, truncating microsatellite sequences as described previously [Barnetson et al., mutations, large deletions, or mutations at the splice site and as 2006]. MLH1, MSH2, and MSH6 protein expression was examined these were predicted to encode a truncated protein they were in representative fixed sections of colorectal carcinoma using readily categorized as being pathogenic. However, 74 (67%) had immunohistochemistry. Monoclonal antibodies to MLH1 (Pharmin- variants that were of uncertain relevance to cancer causation and gen, San Diego, CA, USA), MSH2 (Calbiochem, Darmstadt, these are the focus of this report. Germany) and MSH6 (BD Biosciences, San Jose, CA), were used We used a structured approach to assess whether sequence at dilutions of 1:50, 1:100, and 1:100, respectively. Sections were variants were pathogenic. For all variants identified, we assessed treated with 1 mM EDTA, 0.05% Tween 20, pH 8 at 95–1001Cfor20 population control frequencies using a panel of up to 1,772 control minutes for epitope enhancement, then immunohistochemistry was samples (993 males, 779 females; mean age 60.9 years). We performed using the LSAB immunoperoxidase staining kit (DAKO, screened over 400 to 520 controls for variants with a case Glostrup, Denmark). Sections were counterstained with Harris’ frequency of Z0.005 and to give adequate statistical power for less hematoxylin, dehydrated to xylene, and mounted under coverslips. common variants with a case frequency of o0.005, we screened Staining was assessed independently by two microscopists. 1,000 control samples. The variants MLH1: c.65G4C p.G22A, Missense changes were assessed using three in silico approaches. c.1217G4A p.S406N, c.1852AA4GC p.K618A, c.2066A4G ClustalW software (www.ebi.ac.uk/clustalw) was used to align the p.Q689R, c.2146G4A p.V716M, c.2152C4T p.H718Y, and amino acid sequence of MLH1 and MSH2 in human, mouse, c.2252A4G p.K751R; MSH2: c.317G4A p.R106K, c.965G4A zebrafish, Drosophila, and yeast and MSH6 in human, mouse, p.G322D, and c.2503A4C p.N835H; and MSH6: c.431G4T zebrafish, and yeast, to assess whether the variant amino acids had p.S144I, c.1508C4G p.S503C, and c.2633T4C p.V878A were been evolutionarily conserved between a number of phylogeneti- all identified at similar frequencies in controls (Table 1). Interest- cally diverse species. The variants were also assessed using ‘‘sort ingly, three variants (MLH1 c.2066A4G pQ689R and intolerant from tolerant’’ (SIFT) scores (http://blocks.fhcrc.org/ c.2146G4A p.V716M, and MSH2 c.965G4A p.G322D) were sift/SIFT.html), which uses sequence homology between related more frequent in the control population, suggesting a possible genes and domains across species to predict the impact of all 19 protective effect against developing colorectal cancer. Carcinomas amino acids that could be substituted at a given position [Ng and from 20 carriers were MSS and one was MSI-L, indicating that Henikoff, 2001]. We also used Polymorphism Phenotype (Poly- 95% of these tumors were likely to be DNA mismatch repair Phen; www.bork.embl-heidelberg.de/PolyPhen), which, in addition proficient and nuclear expression of the affected gene was to sequence conservation, utilizes information from protein observed in all cases. In addition, MLH1 c.2732T4G p.L911R structure databases and three-dimensional structure databases to did not segregate with the cancer phenotype within the proband’s predict effects on protein secondary structure, interchain contacts, family. SIFT analysis predicted that 10 out of 14 (71%) of these and functional sites. Putative splice site variants were assessed missense changes would be tolerated. However, PolyPhen assess- using the Genscan (http://genes.mit.edu/GENSCAN), NetGene2 ment predicted that only 6 out of 14 (43%) of these changes were (www.cbs.dtu.dk/services/NetGene2), and Berkeley Drosophila likely to be benign. Our scoring system, which takes into account Genome Project (www.fruitfly.org/seq_tools/splice.html) splice all these variables and calculates the degree of support for a prediction programs. pathogenic role, gave a value of zero for all the missense variants On the basis of our findings, the missense changes were then listed above that could be calculated. scored using a simple arbitrary scoring system that we devised for MLH1 p.G67E was identified in one case but also in two other defining the likelihood of a variant being pathogenic. In order to unrelated Lynch syndrome families. Therefore, we assessed the calculate this, the following values were assigned for each variant: population frequency and did not identify this variant in 1,688 variant present in controls 5 À6; variant segregates in two family controls (Table 1). Substantial additional evidence supports a members 5 2, in three to five family members 5 4, in more than pathogenic role. The male carrier had been affected with five family members 5 6; tumor from carrier MSI-L 5 2, MSI-H colorectal, breast and prostate cancer and a musculoskeletal 5 4, lacked expression of relevant gene by immunohistochemistry sarcoma by the age of 50 years. In all, seven family members had (IHC) 5 4; variant conserved between species 5 1, not tolerated been diagnosed with early-onset Lynch syndrome–related carci- (SIFT) 5 2, PolyPhen 3 5 2, PolyPhen 2 5 1; variant found to lack nomas (two tumors tested were MSI-H) and the five affected function in in vitro mismatch repair (MMR) assays 5 1. individuals tested are p.G67E carriers. In addition, the mutation To determine whether MSH2 c.263415G4C and MSH6 segregates with colorectal cancer in two members of a second c.3556–3A4T had an effect on splicing, mRNA was extracted family and seven members of a third family. Tumors from carriers from carrier peripheral blood leukocytes using Tri-Reagent (Sigma- have also been found to lack MLH1 expression (Ian Frayling, Aldrich, St. Louis, MO) and reverse transcribed using First Strand personal communication) and SIFT predicted that this amino acid cDNA Synthesis Kit (Roche, Basel, Switzerland). Primers in exon change would not be tolerated. 14 and 16 were used to amplify cDNA from the MSH2 c.26341 We found strong evidence supporting a pathogenic role for 5G4C carrier and primers in exon 5 and 9 were used to amplify the MLH1 p.A681T missense change, which was also predicted cDNA from the MSH6 c.3556-3A4T carrier (Supplementary not to be tolerated using SIFT analysis. This variant was Table S1). Products were electrophoresed on a 2% agarose gel, not identified in 1,441 controls. Furthermore, tumors from both excised, and purified using a gel extraction kit (Qiagen, Hilden, index cases exhibited MSI-H and lacked MLH1 expression. At Germany), then sequenced. presentation, the only relevant family history was that both fathers had been diagnosed with colorectal cancer at 48 and 52 years, respectively. However, the pedigrees were confirmed and extended RESULTS through the General Registrar of Scotland and the Scottish Cancer Mutation analysis of MLH1, MSH2, and MSH6 was completed Registry, which subsequently showed that the index cases were for 932 colorectal cancer patients (508 males, 424 females; mean related through a common ancestor four generations previously. age 48.1 years). Of the 110 patients (12%) who carried germline The extended pedigree included 16 individuals with Lynch HUMANMUTATION29(3),367^374,2008 371 syndrome–related cancers and p.A681T is known to segregate controls. However, tumor samples were analyzed from 12 cases and with Lynch syndrome–related cancer in 11 affected family 10 were MSS, one showed low-level MSI (MSI-L) and only one members. was MSI-H. In addition, 10 of 11 tumors expressed the relevant Although MSH2 c.1A4C was identified in a single index case mismatch repair protein (Table 1). These variants were not with an MSI-L tumor and a father affected with stomach cancer, categorized on the basis that there was not enough evidence to this change was previously identified in three early-onset colorectal ascertain whether these variants are pathogenic. cancer cases. The variant is predicted to be pathogenic since it disrupts the initiation start site, although there is a second DISCUSSION methionine 26 residues into the protein sequence that appears to initiate translation of an unstable product [Farrington et al., 1998]. Variants of uncertain significance accounted for 67% of the 110 This mutation was of interest because tumors from carriers were mutations identified in DNA mismatch repair genes in this series MSI-L or MSS and expressed MSH2. There was a limited family of 932 early onset colorectal cancer cases. Therefore, there is a real history of cancer for three of the probands but the fourth carrier need to ascertain whether such alterations are causally implicated had a first-degree relative who was diagnosed with two primary in cancer predisposition. Here, we present a structured assessment colorectal carcinomas at 66 years and two second-degree relatives of each sequence variant identified, to determine whether or not who had been diagnosed with colorectal carcinoma. In addition, we they have a role in cancer causation. did not find this variant in 1,431 controls analyzed. Unlike MLH1 MLH1 p.S406N has previously been identified in a population p.G67E and p.A681T, which scored high values of 19 using our control and has consistently been shown to be MMR proficient in scoring system for calculating the degree of support for a pathogenic functional assays [Genuardi et al., 1999; Kondo et al., 2003; role, this missense change only scored a value of 4. Thus, we were Takahashi et al., 2007]. p.K618A and p.Q689R are in a region of unable to conclusively say whether this variant was pathogenic. MLH1 that interacts with PMS2. In vitro studies have demon- The tumor from the patient with the putative splice variant strated that the p.K618A substitution results in an 85% reduction MSH2 c.263415G4C was MSI-H and immunohistochemistry in efficiency of binding to PMS2 and that it was unable to reverse indicated loss of MSH2 expression. In addition, the variant the mutator phenotype in an MLH1 deficient ovarian cancer cell segregated with the disease in five affected family members and line [Blasi et al., 2006; Guerrette et al., 1999]. Despite this, was predicted to alter splicing using the Berkeley Drosophila previous studies have identified p.K618A and p.Q689R in patients Genome Project program. Therefore, we analyzed cDNA from the with MSS colorectal carcinomas that expressed MLH1 [Farrington carrier and identified a truncated cDNA product following et al., 1998; Liu et al., 1999; Mauillon et al., 1996; Muller-Koch amplification of exons 14–16. Sequence analysis revealed that et al., 2001; Samowitz et al., 2001]. MLH1 p.V716M has been exon 15 was skipped, which confirms that c.263415G4Cin reported in four patients with carcinomas that exhibited MSI. In intron 15 disrupts the splice-donor site (Fig. 1). addition, this change was not identified in three studies There was conflicting evidence as to whether MSH6 investigating 100–200 controls [Dieumegard et al., 2000; Hampel c.3556–3A4T would affect splicing. NetGene2 predicted that et al., 2005; Kowalski et al., 1997; Muller-Koch et al., 2001]. this change would disrupt the splice-donor site, while the Berkeley However, p.V716M has also been reported in patients with MSS Drosophila Genome Project program predicted that this variant carcinomas [Genuardi et al., 1998; Muller-Koch et al., 2001]. would not affect mRNA splicing. On analysis of carrier cDNA, we MLH1 p.H718Y has previously been identified in nine of 28 observed no difference in the wild-type and carrier coding unaffected African-Americans and on the basis of this has been sequence. Although it is possible that any product of aberrant reported to be a polymorphism [Kowalski et al., 1997]. MLH1 splicing may have been degraded by nonsense mediated decay, the p.K751R was previously reported in a colorectal cancer case that patient’s tumor was MSS and expressed MSH6. We therefore fulfilled the modified Amsterdam criteria and was not found in 50 concluded that this variant was unlikely to be pathogenic. controls [Jakubowska et al., 2001]. While not conclusive, we Missense changes were identified at low frequencies (0.001) in provide evidence by comparing variant frequencies in patients and an additional 14 cases, but were not found in 1,000 population controls and tumor analysis to suggest that none of these MLH1

FIGURE 1. Primers in MSH2 exons14 and16 were used to amplify cDNA from an individual with an MSH2 c.263415G4C change (A) and wild-type cDNA (B). Products were isolated and sequenced and it was found that exon15 was deleted. [Color ¢gure can be viewed in the online issue, which is available at www.interscience.wiley.com.] 372 HUMANMUTATION29(3),367^374,2008 missense changes confer a greatly increased risk of colorectal provide for determining whether a variant was pathogenic or cancer (Table 1). benign was consistent with the SIFT assessment or ClustalW The glycine residue altered in the MSH2 p.G322D variant is alignment for 12 out of 16 (75%) amino acid changes compared highly conserved between species and independent in vitro studies with only 7 out of 16 (44%) amino acid changes using PolyPhen. demonstrated that the equivalent amino acid change in yeast Although the nucleotides immediately adjacent to intron–exon (p.G317D) substantially impairs DNA mismatch repair proficiency boundaries are required for splicing, the flanking sequences are less [Drotschmann et al., 1999; Ellison et al., 2001; Schmutte et al., well conserved. Therefore, it is less clear whether variants in this 2001]. However, a previous study identified this common variant region will affect splicing. A number of algorithms have been in 13 of 406 unaffected individuals, suggesting a major excess risk devised to predict whether a sequence is likely to be involved in of cancer was unlikely [Cravo et al., 2002; Liu et al., 1998]. exonic splicing. Splice predictions vary between programs and do Consistent with these findings, we show here that the allele not always correlate with RNA analysis so variants predicted to frequency in controls was the same as that in cases and carriers affect splicing should be confirmed. MSH2 c.263415G4C had MSS carcinomas. This leads us to conclude that the mutation resulted in the skipping of exon 15, which, along with segregation is not pathogenic and demonstrates definitively that in vitro studies, MSI analysis, and immunohistochemistry confirmed that assessment does not necessarily translate into measurable cancer this variant was disease-causing. However, MSH6 c.3556–3A4T risk (Table 1). is unlikely to be pathogenic as it did not appear to affect mRNA MSH6 p.S144I and p.V878A were previously identified in splicing or protein expression. familial colorectal and endometrial cancer cases [Berends et al., There are considerable difficulties in categorizing whether a 2002; Charames et al., 2000; de Abajo et al., 2005; Kolodner et al., rare variant is pathogenic, when it does not appear to dramatically 1999; Wijnen et al., 1999; Wu et al., 1999]. p.S144I was affect the coding sequence. Functional assays, tumor microsatellite considered pathogenic on the basis that: 1) carcinomas were MSI- analysis and immunohistochemistry, segregation analysis, and case/ H or MSI-L; 2) no carriers were identified in 399 controls; and 3) control frequency comparisons all have their limitations. Here, loss of DNA mismatch repair function was observed in a yeast- we have systematically assessed the evidence for a causal role in based assay (although an in vitro assay, using a human DNA colorectal cancer of sequence variants identified in 74 cases in a mismatch repair system did not observe loss of function) [Kariola series of 932 colorectal cancer cases. We present data that support et al., 2002]. p.V878A was reported to segregate in four affected a pathogenic role for variants in four cases, bringing the family members and tumors were MSI-H [Charames et al., 2000; total number of causal mutations in this series to 41 (4.4%). Wijnen et al., 1999; de Abajo et al., 2005]. Despite these prior More importantly we were able to determine that the majority observations, we show that these variants are present at similar (55/74) of carriers had variants that are likely to be incon- frequencies in cases and controls and tumors from carriers sequential polymorphisms. It is possible that some variants, expressed MSH6. Our findings indicate that these MSH6 variants for example MLH1 p.K618A (Table 1) have a small effect have no relevance to cancer risk. as reported for MLH1 p.D132H [Lipkin et al., 2004]. However, MLH1 pG67E segregated with the disease in three families and the variants discussed here are not frequent enough to assess tumors from carriers are MSI-H and lack MLH1 expression. This is this, and such a low risk would not affect clinical management for consistent with reports suggesting that MLH1 pG67R is patho- carriers or be important from a public health perspective. Three genic [Heinimann et al., 1999; Herfarth et al., 1997; Sasaki et al., missense variants (MLH1 pQ689R and p.V716M, and MSH2 1997; Wang et al., 1999]. MLH1 p.A681T was reported in a p.G322D) were found to be more frequent in the unaffected patient with an MSI-H colorectal carcinoma and four other control population and although not conclusive, it is possible that affected family members [Froggatt et al., 1996]. This amino acid these may have a protective effect against developing colorectal change has been demonstrated to cause a reduction in MLH1- cancer. As these variants occur at such low frequencies, this PMS2 binding and a 480% reduction in interaction between finding would need to be verified in other large case/control MLH1 and Exo1 [Schmutte et al., 2001]. However, functional sample sets. assays provide conflicting evidence as to whether DNA mismatch Sequence variants that are of uncertain significance to disease repair proficiency is affected [Ellison et al., 2001; Guerrette et al., causation present a real challenge to the genetic counselor, 1999; Kondo et al., 2003; Shimodaira et al., 1998]. Consistent because decisions about long-term colonic and gynecological with the findings of Froggatt et al. [1996], we found that p.A681T surveillance are taken on the basis of the interpretation of also segregated with cancer in the family members in our study and pathogenicity. Since the majority of variants described here have associated carcinomas exhibited MSI-H and did not express been found in more than one proband and in other populations, MLH1. In addition, neither of these MLH1 variants was detected these findings are relevant for diagnostic purposes in genetic in controls (Table 1) and so these missense changes are likely to be clinics. These findings are also relevant in a research setting disease-causing. because mutation carrier frequency may be overestimated and MSH2 c.1A4C appears to be causally implicated in colorectal findings skewed by the inclusion of carriers of nonpathogenic cancer on the basis that it has been identified previously by variants. Large mutation databases reporting all mutations ourselves and others in a number of colorectal cancer cases identified in the DNA mismatch repair genes, including all the [Otway et al., 2000], but was not present in a large number of available evidence for or against pathogenicity provide a useful controls. However, tumors from carriers do not lose expression of tool for predicting the relevance of each sequence variant. Our MSH2 or exhibit MSI-H and it is possible that this initiation findings suggest that the majority but not all of the missense codon change is associated with an attenuated phenotype, as is changes evaluated in this study are not pathogenic. found in MSH6 mutation carriers. It will be of interest to investigate this further, as additional carriers are identified. ACKNOWLEDGMENTS SIFT analysis and ClustalW alignment were found to provide a more accurate indication of whether missense changes are disease The study has been a considerable effort over a long period of causing than PolyPhen analysis. The evidence we were able to time and we are grateful to everyone involved, if not named HUMAN MUTATION 29(3), 367^374, 2008 373 specifically here, especially participating patients and control Charames GS, Millar AL, Pal T, Narod S, Bapat B. 2000. Do MSH6 subjects. We are indebted to Therese Bradley, David Goudie, and mutations contribute to double primary cancers of the colorectum and all the scientists and clinicians in the Scottish Molecular Genetics endometrium? Hum Genet 107:623–629. Consortium for providing DNA samples, confirmation of muta- Cravo M, Afonso AJ, Lage P,Albuquerque C, Maia L, Lacerda C, Fidalgo P, tions, and testing of family members. We thank all nursing and Chaves P, Cruz C, Nobre-Leitao C. 2002. Pathogenicity of missense and splice site mutations in hMSH2 and hMLH1 mismatch repair genes: office staff employed by the COGS and SOCCS studies for their implications for genetic testing. Gut 50:405–412. work in recruitment, in particular the invaluable contributions of de Abajo AS, de la Hoya M, Tosar A, Godino J, Fernandez JM, Asenjo JL, Ruth Wilson, Maureen Edwards, and Roseanne Cetnarskyj. We Villamil BP,Segura PP,Diaz-Rubio E, Caldes T. 2005. Low prevalence of also acknowledge the Wellcome Trust Clinical Research Facility for germline hMSH6 mutations in colorectal cancer families from Spain. sample preparations, in particular Stuart Bayliss and Lee Murphy. World J Gastroenterol 11:5770–5776. We have enjoyed excellent collaborative interactions with Dieumegard B, Grandjouan S, Sabourin JC, Le Bihan ML, Lefrere I, National Health Service (NHS) consultant surgeons, oncologists, Bellefqih, Pignon JP, Rougier P, Lasser P, Benard J, Couturier D, Bressac- and nursing teams in every Scottish hospital. We are indebted to de Paillerets B. 2000. Extensive molecular screening for hereditary non- consultant pathologists and staff in every Pathology Department in polyposis colorectal cancer. Br J Cancer 82:871–880. Scotland, for tumor specimens and pathology data and to Paul Drotschmann K, Clark AB, Kunkel TA. 1999. Mutator phenotypes of Fineron and Alistair Lessells for their advice on immunohisto- common polymorphisms and missense mutations in MSH2. Curr Biol 9:907–910. chemistry. We also thank the relevant departments in central Dunlop MG, Farrington SM, Carothers AD, Wyllie AH, Sharp L, Burn J, Scottish NHS, including Alison Fordyce at the Cancer Registry, Liu B, Kinzler KW, Vogelstein B. 1997. Cancer risk associated with the Scottish Cancer Intelligence Unit of the Information Services germline DNA mismatch repair gene mutations. Hum Mol Genet Division (ISD), and the Practitioner Services Division of ISD for 6:105–110. invaluable help in recruiting population controls. This work is Ellison AR, Lofing J, Bitter GA. 2001. Functional analysis of human MLH1 subject to NHS management approval for every participating and MSH2 missense variants and hybrid human-yeast MLH1 proteins in hospital and ethical approval was granted by the MultiCentre Saccharomyces cerevisiae. Hum Mol Genet 10:1889–1900. Research Ethics Committee for Scotland. Farrington SM, Lin-Goerke J, Ling J, Wang Y, Burczak JD, Robbins DJ, Dunlop MG. 1998. 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