The MLH1 c.1852_1853delinsGC (p.K618A) Variant in Colorectal Cancer: Genetic Association Study in 18,723 Individuals

Abstract

Colorectal cancer is one of the most frequent neoplasms and an important cause of mortality in the developed world. Mendelian syndromes account for about 5% of the total burden of CRC, being Lynch syndrome and familial adenomatous polyposis the most common forms. Lynch syndrome tumors develop mainly as a consequence of defective DNA mismatch repair associated with germline mutations in MLH1, MSH2, MSH6 and PMS2. A significant proportion of variants identified by screening these genes correspond to missense or noncoding changes without a clear pathogenic consequence, and they are designated as “variants of uncertain significance”, being the c.1852_1853delinsGC (p.K618A) variant in the MLH1 gene a clear example. The implication of this variant as a low-penetrance risk variant for CRC was assessed in the present study by performing a case-control study within a large cohort from the COGENT consortium-COST Action BM1206 including 18,723 individuals (8,055 colorectal cancer cases and 10,668 controls) and a case-only genotype-phenotype correlation with several clinical and pathological characteristics restricted to the Epicolon cohort. Our results showed no involvement of this variant as a low-penetrance variant for colorectal cancer genetic susceptibility and no association with any clinical and pathological characteristics including family history for this neoplasm or Lynch syndrome.

About the Authors:

Anna Abulí

Affiliations Department of Gastroenterology, Hospital Clínic, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Catalonia, Spain, Department of Gastroenterology, Hospital del Mar-IMIM (Hospital del Mar Medical Research Centre), Pompeu Fabra University, Barcelona, Catalonia, Spain

Luis Bujanda

Affiliation: Gastroenterology Department, Hospital Donostia, Networked Biomedical Research Centre for Hepatic and Digestive Diseases (CIBEREHD), Basque Country University, San Sebastián, Spain

Jenifer Muñoz

Affiliation: Department of Gastroenterology, Hospital Clínic, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Catalonia, Spain

Stephan Buch

Affiliation: Department of Medine I, University Hospital Dresden, Dresden, Germany

Clemens Schafmayer

Affiliation: Department of General and Thoracic Surgery, University Hospital Schleswig-Holstein, Kiel, Germany

Maria Valeria Maiorana

Affiliation: IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Italy

Silvia Veneroni

Affiliation: Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy

Tom van Wezel

Affiliation: Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands

Tao Liu

Affiliation: Department of Molecular Medicine and Surgery, Karolinska Institute, Stockholm, Sweden

Helga Westers

Affiliation: Department of Genetics, University Medical Center Groningen, Groningen, The Netherlands

Clara Esteban-Jurado

Affiliation: Department of Gastroenterology, Hospital Clínic, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Catalonia, Spain

Teresa Ocaña

Affiliation: Department of Gastroenterology, Hospital Clínic, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Catalonia, Spain

Josep M. Piqué

Affiliation: Department of Gastroenterology, Hospital Clínic, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Catalonia, Spain

Montserrat Andreu

Affiliation: Department of Gastroenterology, Hospital del Mar-IMIM (Hospital del Mar Medical Research Centre), Pompeu Fabra University, Barcelona, Catalonia, Spain

Rodrigo Jover

Affiliation: Department of Gastroenterology, Hospital General d'Alacant, Alicante, Spain

Angel Carracedo

Affiliations Galician Public Foundation of Genomic Medicine (FPGMX), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Genomics Medicine Group, Hospital Clínico, Santiago de Compostela, University of Santiago de Compostela, Galicia, Spain, Center of Excellence in Genomic Medicine Research, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia

Rosa M. Xicola

Affiliation: Section of Digestive Diseases and Nutrition, University of Illinois at Chicago, Chicago, Illinois, United States of America

Xavier Llor

Affiliation: Section of Digestive Diseases and Nutrition, University of Illinois at Chicago, Chicago, Illinois, United States of America

Antoni Castells

Affiliation: Department of Gastroenterology, Hospital Clínic, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Catalonia, Spain

The EPICOLON Consortium

¶Membership of the EPICOLON Consortium is provided in Appendix S1.

Malcolm Dunlop

Affiliation: Colon Cancer Genetics Group, Institute of Genetics and Molecular Medicine, University of Edinburgh and MRC Human Genetics Unit, Edinburgh, United Kingdom

Robert Hofstra

Affiliation: Department of Genetics, University Medical Center Groningen, Groningen, The Netherlands

Annika Lindblom

Affiliation: Department of Molecular Medicine and Surgery, Karolinska Institute, Stockholm, Sweden

Juul Wijnen

Affiliation: Departments of Human Genetics and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands

Paolo Peterlongo

Affiliation: IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Italy

Jochen Hampe

Affiliation: Department of Medine I, University Hospital Dresden, Dresden, Germany

Clara Ruiz-Ponte

Affiliation: Galician Public Foundation of Genomic Medicine (FPGMX), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Genomics Medicine Group, Hospital Clínico, Santiago de Compostela, University of Santiago de Compostela, Galicia, Spain

Sergi Castellví-Bel

* E-mail: [email protected]

Affiliation: Department of Gastroenterology, Hospital Clínic, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Catalonia, Spain

Introduction

Colorectal cancer (CRC) is one of the most frequent neoplasms and an important cause of mortality in the developed world. This cancer is caused by both genetic and environmental factors although 35% of the variation in CRC susceptibility involves inherited genetic differences. Mendelian syndromes account for about 5% of the total burden of CRC, being Lynch syndrome and familial adenomatous polyposis the most common forms. Lynch syndrome tumors develop mainly as a consequence of defective DNA mismatch repair (MMR) associated with germline mutations in the MLH1, MSH2, MSH6 and PMS2 genes [1]. Once clinical criteria for this syndrome are complied, genetic screening of these genes is performed when a MMR defect is detected in the patient's tumor. When a pathogenic variant is detected, management of this disease can be significantly improved by identifying carriers that will benefit from specific screening, preventive, and therapeutic measures. Also, identifying non-carriers in additional family members permit to release these individuals from intensive surveillance. Noteworthy, a significant proportion of variants identified in the MMR genetic screening correspond to missense or noncoding changes without a clear pathogenic consequence, and they are designated as “variants of uncertain significance” (VUS). Therefore, differentiating pathogenic and neutral genetic variants is still a major challenge in clinical genetics [2].

The c.1852_1853delinsGC (p.K618A) variant in the MLH1 gene corresponds to a clear example of VUS in Lynch syndrome. When consulting the Leiden Open Variation Database (LOVD v.2.0), there are 120 entries for this variant [3]. Available past studies reached contradictory conclusions about its pathogenicity reporting harmful in silico predictions [4], absence of splicing or mRNA alteration [5], presence in patients with a defective MMR tumor [6], co-occurrence with clearly pathogenic MMR mutations [7], apparent segregation with disease [8], and a majority of non-altered in vitro functional studies [9], [10]. All previous data permitted to categorize it in LOVD as a class 1 variant (non-pathogenic/low clinical significance) [11]. Therefore, it should be considered as a neutral variant in terms of its implication with Lynch syndrome.

Recently, genome-wide association studies (GWAS) successfully identified so far 30 common, low-penetrance susceptibility variants in 25 risk loci for CRC [12]–[19]. Some genetic variants in hereditary CRC genes labeled as VUS could constitute low-penetrance risk alleles for CRC. Indeed, this hypothesis has been previously tested for some variants in those genes [20]. In agreement with this rationale, the main aim of the present study was to assess the implication of the c.1852_1853delinsGC (p.K618A) variant in the MLH1 gene as a low-penetrance risk variant for CRC by performing a case-control study within a large cohort from the COGENT consortium-COST Action BM1206, an international effort to facilitate the study of inherited genetic predisposition to CRC [21], [22].

Materials and Methods

Study population

The current genetic association study totalized 8,055 CRC cases and 10,668 controls from 7 different cohorts (Edinburgh, Epicolon, Groningen, Kiel, Leiden, Milano, Stockholm) and recruitment details are summarized below. The study was approved by the institutional ethical committee of each participating hospital and written informed consent was obtained from all patients.

Edinburgh cohort (1,553 CRC cases and 932 controls).

A population-based series of patients from throughout Scotland, who were diagnosed with colorectal cancer when they were less than 55 years of age, were recruited to the study between February 1999 and June 2004. During the same period, unaffected controls were ascertained from a population-based register (community health index) and were invited to participate.

Epicolon (2,001 CRC cases and 1,647 controls).

Cases and controls were recruited through the EPICOLON Consortium that is based on a prospective, multicenter and population-based epidemiology survey of the incidence and features of CRC in the Spanish population [23]. Briefly, cases were selected as patients with de novo histologically confirmed diagnosis of colorectal adenocarcinoma. Exclusion criteria were hereditary CRC forms, such as Lynch syndrome and familial adenomatous polyposis (FAP) and a personal history of inflammatory bowel disease. Controls were from the Spanish National DNA bank and were confirmed not to have cancer or history of neoplasm and no family history of CRC. All cases and controls were of Caucasian ethnicity.

Groningen (559 CRC cases and 501 controls).

Unselected CRC cases and hospital patient controls from the Netherlands included in the SCOPE project.

Kiel (1,768 CRC cases and 2.030 controls).

Cases and controls from population-based biobank projects including POPGEN (Population Genetic Cohort) from Schleswig-Holstein, north Germany, and SHIP (Survey of Health in Pommerania) from east and north-east Germany.

Leiden (505 CRC cases and 836 controls).

Cases and controls were recruited as previously described [24]. Briefly, most of the cases were recruited through the clinical genetics department. All cases were diagnosed with CRC and had early onset and/or positive family history for CRC. Known dominant polyposis syndromes, HNPCC/Lynch syndrome or bi-allelic MutYH mutation carriers were excluded. A single proband from each family was included in this study. Controls were healthy blood donors from the southwest region of the Netherlands. All cases and controls were of Caucasian ethnicity.

Milano (619 CRC cases and 2,526 controls).

Briefly, the cases were consecutive individuals affected with CRC who underwent surgery at the Fondazione IRCCS Istituto Nazionale Tumori in Milan (INT). The controls were blood donors recruited through the Immunohematology and Transfusion Medicine Service of INT the Associazione Volontari Italiani Sangue Comunale in Milan. All cases and controls were of Caucasian ethnicity.

Stockholm (1,729 CRC cases and 1,487 controls).

Unselected cases ascertained through 12 hospitals serving the Stockholm-Gotland and Uppsala-Örebro health-care regions in Sweden and blood donor controls.

Genotyping

DNA was obtained from peripheral blood by standard extraction procedures. Allelic discrimination to genotype the c.1852_1853delinsGC (p.K618A) variant in the MLH1 gene was performed by using a custom assay with the TaqMan allelic discrimination system (Life Technologies, Foster City, USA). As quality control, DNA from a known carrier of this variant was used as positive control, as well as duplicates and negative controls for amplification. Data could be available upon request. An example of allelic discrimination for this variant is shown in Figure 1.

[Figure omitted. See PDF.]

Figure 1. Allelic discrimination for c.1852_1853delinsGC (p.K618A) variant in the MLH1 gene by using the TaqMan system.

Red dots correspond to non-carriers (AA/AA genotype) and green dots to heterozygous carriers (AA/GC).

https://doi.org/10.1371/journal.pone.0095022.g001

Statistical analysis

To test the association between the c.1852_1853delinsGC (p.K618A) variant in the MLH1 gene and CRC risk, odds ratios (OR) and 95%CI were calculated for each genotype by using PLINK v1.07 [25], separately in each cohort and globally. No deviation of the genotype frequency in controls from those expected under Hardy-Weinberg equilibrium (HWE) was detected by χ2 test (1 df) (P-value = 0.6294) [26].

In order to explore if personal and/or familial characteristics were associated with the presence of the c.1852_1853delinsGC (p.K618A) variant in the MLH1 gene, univariate analysis was performed restricted to the CRC cases from the Epicolon cohort due to data availability in this cohort. The selected clinical variables to be evaluated were gender, age (dichotomized by 50 y.o.), location of CRC, previous neoplasm, previous/synchronous adenoma, CRC familiy history (any relative with CRC), Lynch syndrome family history (any relative affected), microsatellite instability (MSI) and TNM tumor stage. Categorical variables were compared by the χ2 test (1 df), applying the Yates' correction when needed. All P-values were two-sided, and a value less than 0.05 was considered statistically significant. Calculations were performed using the SPSS software version 18.0 (SPSS Inc, Chicago, Ill).

Results and Discussion

Genotyping for the c.1852_1853delinsGC (p.K618A) variant in the MLH1 gene was successful in 8,055 CRC cases and 10,668 controls from 7 independent cohorts. Percentage of carriers varied between 0.4–2.6% in CRC cases and 0.5–3.1% for controls in the different cohorts, being 1.4% and 1.5% in the entire cohort for CRC cases and controls, respectively. Genotypic association results are shown in Table 1 for each cohort and globally. No association of this variant with CRC risk was detected neither in a specific cohort nor globally.

[Figure omitted. See PDF.]

Table 1. Genotypic association results for the MLH1 c.1852_1853delinsGC (p.K618A) variant in 18,723 individuals from 7 cohorts.

https://doi.org/10.1371/journal.pone.0095022.t001

In order to further explore the putative implication of this MLH1 variant with CRC risk, we performed a case-only genotype-phenotype correlation restricted to the Epicolon cohort (2,001 CRC cases) with several clinical and pathological characteristics. Results are shown in Table 2. Again, none of the analyzed variables showed a distinct association with the presence of the c.1852_1853delinsGC (p.K618A) variant. Results for CRC family history and Lynch syndrome family history were statistically significant but the presence of any of these variables was linked with the wild-type genotype (AA/AA). The rest of variables showed a similar distribution between carriers and non-carriers.

[Figure omitted. See PDF.]

Table 2. Genotype-phenotype correlation of the MLH1 c.1852_1853delinsGC (p.K618A) variant with clinical and pathological characteristics in colorectal cancer cases from the Epicolon cohort.

https://doi.org/10.1371/journal.pone.0095022.t002

Obviously, genetic variants causing a missense mutation have a less clear pathogenic interpretation than those causing a premature termination of the protein. The c.1852_1853delinsGC (p.K618A) variant in the MLH1 gene is a prominent example of a VUS that has been controversial for many years in the context of Lynch syndrome genetic diagnosis. However, recent functional studies have permitted to characterize more thoroughly its real effect of the MLH1 protein and it can be concluded that its effect is neutral or with very subtle effect [5], [8]–[11].

Regarding its putative implication in CRC risk as a rare low-penetrance variant, previous studies were sparse and included a small number of CRC cases and controls [27], [28]. Consequently, it was justified to perform a case-control association study in a large cohort in order to reach more solid conclusions. Our results showed no involvement of this variant in CRC risk as a low-penetrance variant in the MLH1 gene.

Regarding its putative implication in familial CRC, this variant was also seen to be over-represented in families with suspected Lynch syndrome in a previous study [29]. Our results will be not in agreement with this previous observation since the K618A variant was not linked in the Epicolon cohort to the presence of CRC family history and Lynch syndrome family history. Therefore, our study is adding to the existing literature by showing that this variant is not linked to familial CRC.

Finally, we can conclude from our results and previous evidence that the c.1852_1853delinsGC (p.K618A) variant in the MLH1 gene should be regarded from now on as a polymorphism without functional effect on the MLH1 protein, no role in genetic predisposition to Lynch syndrome, as well as no apparent effect as a low-penetrance variant for CRC genetic susceptibility.

Supporting Information

[Figure omitted. See PDF.]

Appendix S1.

Members of the EPICOLON Consortium (Gastrointestinal Oncology Group of the Spanish Gastroenterological Association).

https://doi.org/10.1371/journal.pone.0095022.s001

(DOCX)

Acknowledgments

Epicolon: We are sincerely grateful to all patients participating in this study who were recruited in 25 (EPICOLON 1) and 14 (EPICOLON 2) Spanish hospitals as part of the EPICOLON project. We are also indebted to the USC and UPF nodes of Spanish National Genotyping Center (CEGEN-ISCIII), the Genomics Unit of the Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) and the Biobanks of Hospital Clínic – IDIBAPS and Hospital Donostia for technical help. The work was carried out (in part) at the Esther Koplowitz Centre, Barcelona.

Leiden: Shanti Jagmohan-Changur and Melanie Schrumpf are acknowledged for technical support.

Milano: We thank all individuals who agreed to participate in the study. We also thank the personnel of Tissue Bank of Fondazione IRCCS Istituto dei Tumori for sample collection and all pathologists for their contribution and collaboration.

Stockholm: We acknowledge the contribution to recruitment and data collection of the Swedish Low-Risk Colorectal Cancer Study Group.

Author Contributions

Conceived and designed the experiments: TW MD RH AL JW PP JH CRP SCB. Performed the experiments: AA JM SB MVM TW TL HW CEJ MD RH AL JW PP JH CRP SCB. Analyzed the data: AA JM SB MVM TW TL HW CEJ MD RH AL JW PP JH CRP SCB. Contributed reagents/materials/analysis tools: AA LB JM SB CS MVM SV TW TL HW CEJ TO JMP MA RJ AC RMX XL AC EC MD RH AL JW PP JH CRP SCB. Wrote the paper: AA TW TL HW MD RH AL JW PP JH CRP SCB.

Citation: Abulí A, Bujanda L, Muñoz J, Buch S, Schafmayer C, Valeria Maiorana M, et al. (2014) The MLH1 c.1852_1853delinsGC (p.K618A) Variant in Colorectal Cancer: Genetic Association Study in 18,723 Individuals. PLoS ONE 9(4): e95022. https://doi.org/10.1371/journal.pone.0095022

References

1. Jasperson KW, Tuohy TM, Neklason DW, Burt RW (2010) Hereditary and familial colon cancer. Gastroenterology 138: 2044–58.

2. Rasmussen LJ, Heinen CD, Royer-Pokora B, Drost M, Tavtigian S, et al. (2012) Pathological assessment of mismatch repair gene variants in Lynch syndrome: past, present, and future. Hum Mutat 33: 1617–25.

3. Fokkema IF, Taschner PE, Schaafsma GC, Celli J, Laros JF, et al. (2011) LOVD v.2.0: the next generation in gene variant databases. Hum Mutat 2011 32: 557–63.

4. Chao EC, Velasquez JL, Witherspoon MS, Rozek LS, Peel D, et al. (2008) Accurate classification of MLH1/MSH2 missense variants with multivariate analysis of protein polymorphisms-mismatch repair (MAPP-MMR). Hum Mutat 29: 852–60.

5. Tournier I, Vezain M, Martins A, Charbonnier F, Baert-Desurmont S, et al. (2008) A large fraction of unclassified variants of the mismatch repair genes MLH1 and MSH2 is associated with splicing defects. Hum Mutat 29: 1412–24.

6. Caldes T, Godino J, de la Hoya M, Garcia Carbonero I, Perez Segura P, et al. (2002) Prevalence of germline mutations of MLH1 and MSH2 in hereditary nonpolyposis colorectal cancer families from Spain. Int J Cancer 98: 774–9.

7. Liu T, Tannergård P, Hackman P, Rubio C, Kressner U, et al. (1999) Missense mutations in hMLH1 associated with colorectal cancer. Hum Genet 105: 437–41.

8. Pastrello C, Pin E, Marroni F, Bedin C, Fornasarig M, et al. (2011) Integrated analysis of unclassified variants in mismatch repair genes. Genet Med 13: 115–24.

9. Takahashi M, Shimodaira H, Andreutti-Zaugg C, Iggo R, Kolodner RD, et al. (2007) Functional analysis of human MLH1 variants using yeast and in vitro mismatch repair assays. Cancer Res 67: 4595–604.

10. Hinrichsen I, Brieger A, Trojan J, Zeuzem S, Nilbert M, Plotz G (2013) Expression defect size among unclassified MLH1 variants determines pathogenicity in Lynch syndrome diagnosis. Clin Cancer Res 19: 2432–41.

11. InSiGHT (2014) Application of a 5-tiered scheme for standardized classification of 2,360 unique mismatch repair gene variants in the InSiGHT locus-specific database. Nat Genet 46: 107–15.

12. Tenesa A, Dunlop MG (2009) New insights into the aetiology of colorectal cancer from genome-wide association studies. Nat Rev Genet 10: 353–8.

13. Houlston RS, Cheadle J, Dobbins SE, Tenesa A, Jones AM, et al. (2010) Meta-analysis of three genome-wide association studies identifies susceptibility loci for colorectal cancer at 1q41, 3q26.2, 12q13.13 and 20q13.33. Nat Genet 42: 973–977.

14. Tomlinson IP, Carvajal-Carmona LG, Dobbins SE, Tenesa A, Jones AM, et al. (2011) Multiple common susceptibility variants near BMP pathway loci GREM1, BMP4, and BMP2 explain part of the missing heritability of colorectal cancer. PLoS Genet 7: e1002105.

15. Dunlop MG, Dobbins SE, Farrington SM, Jones AM, Palles C, et al. (2012) Common variation near CDKN1A, POLD3 and SHROOM2 influences colorectal cancer risk. Nat Genet 44: 770–776.

16. Kinnersley B, Migliorini G, Broderick P, Whiffin N, Dobbins SE, et al. (2012) The TERT variant rs2736100 is associated with colorectal cancer risk. Br J Cancer 107: 1001–8.

17. Fernández-Rozadilla C, Cazier JB, Tomlinson IP, Carvajal-Carmona LG, Palles C, et al. (2013) A colorectal cancer genome-wide association study in a Spanish cohort identifies two variants associated with colorectal cancer risk at 1p33 and 8p12. BMC Genomics 26 14: 55.

18. Jia WH, Zhang B, Matsuo K, Shin A, Xiang YB, et al. (2013) Genome-wide association analyses in East Asians identify new susceptibility loci for colorectal cancer. Nat Genet 45: 191–196.

19. Peters U, Jiao S, Schumacher FR, Hutter CM, Aragaki AK, et al. (2013) Identification of Genetic Susceptibility Loci for Colorectal Tumors in a Genome-Wide Meta-analysis. Gastroenterology 144: 799–807.

20. Picelli S, Lorenzo Bermejo J, Chang-Claude J, Hoffmeister M, Fernández-Rozadilla C, et al. (2013) Meta-Analysis of Mismatch Repair Polymorphisms within the Cogent Consortium for Colorectal Cancer Susceptibility. PLoS One 8: e72091.

21. Tomlinson IP, Dunlop M, Campbell H, Zanke B, Gallinger S, et al. (2010) COGENT (COlorectal cancer GENeTics): an international consortium to study the role of polymorphic variation on the risk of colorectal cancer. Br J Cancer 102: 447–54.

22. Houlston RS; members of COGENT (2012) COGENT (COlorectal cancer GENeTics) revisited. Mutagenesis 27: 143–51.

23. Piñol V, Castells A, Andreu M, Castellví-Bel S, Alenda C, et al. (2005) Accuracy of revised Bethesda guidelines, microsatellite instability, and immunohistochemistry for the identification of patients with hereditary nonpolyposis colorectal cancer. JAMA 293: 1986–94.

24. Middeldorp A, Jagmohan-Changur S, van Eijk R, Tops C, Devilee P, et al. (2009) Enrichment of low penetrance susceptibility loci in a Dutch familial colorectal cancer cohort. Cancer Epidemiol Biomarkers Prev 18: 3062–7.

25. Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, et al. (2007) PLINK: a tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet 81: 559–575.

26. Wigginton, JE, Cutler DJ, Abecasis GR. A note on exact tests of Hardy-Weinberg equilibrium. Am J Hum Genet 76(5): 887–93.

27. Christensen LL, Madsen BE, Wikman FP, Wiuf C, Koed K, et al. (2008) The association between genetic variants in hMLH1 and hMSH2 and the development of sporadic colorectal cancer in the Danish population. BMC Med Genet 11 9: 52.

28. Castillejo A, Guarinos C, Martinez-Canto A, Barbera VM, Egoavil C, et al. (2011) Evidence for classification of c.1852_1853AA>GC in MLH1 as a neutral variant for Lynch syndrome. BMC Med Genet 12: 12.

29. Medeiros F, Lindor NM, Couch FJ, Highsmith WE Jr (2012) The germline MLH1 K618A variant and susceptibility to Lynch syndrome-associated tumors. J Mol Diagn 14: 264–73.