Identification of MYH Mutation Carriers in Colorectal Cancer: A Multicenter, Case-Control, Population-Based Study
2007; Elsevier BV; Volume: 5; Issue: 3 Linguagem: Inglês
10.1016/j.cgh.2006.12.025
ISSN1542-7714
AutoresFrancesc Balaguer, Sergi Castellví–Bel, Antoni Castells, Montserrat Andreu, Jenifer Muñoz, Javier P. Gisbert, Xavier Llor, Rodrigo Jover, Rafael de Cid, Victòria Gonzalo, Xavier Bessa, Rosa M. Xicola, Elisenda Pons, Cristina Alenda, Artemio Payá, Josep M. Piqué,
Tópico(s)Colorectal and Anal Carcinomas
ResumoBackground & Aims: Whereas it has conclusively been demonstrated that biallelic MutY human homolog (MYH) mutations confer a significant risk for colorectal cancer (CRC), the influence of monoallelic mutations remains controversial. Characterization of MYH-associated CRC is critical to identify individuals who might benefit from preventive strategies. This prospective, multicenter, case-control, population-based study was aimed at (1) establishing the CRC risk associated with specific germline MYH mutations and (2) devising a set of clinical criteria to identify MYH carriers among newly diagnosed CRC. Methods: Genotyping for Y165C and G382D was performed by TaqMan technology. Single-stranded conformation polymorphism analysis was performed in heterozygotes to screen for mutations in the entire gene. All individuals were re-screened for any additional pathogenic variant. Results: Biallelic and monoallelic MYH mutations were found in 8 (0.7%) and 19 (1.7%) of 1116 CRC patients, respectively. None of the 934 control subjects carried biallelic mutations, whereas 22 (2.3%) of them were monoallelic carriers. In a meta-analysis including all previous case-control studies, monoallelic MYH carriers were not at increased risk for CRC (odds ratio, 1.11; 95% confidence interval, 0.90–1.37), although a significant association was found with the Y165C mutation in either homozygotes or heterozygotes (odds ratio, 1.67; 95% confidence interval, 1.17–2.40). Furthermore, presence of more than 15 synchronous colorectal adenomas or CRC diagnosed before the age of 50 years was the most effective set of criteria for the identification of biallelic MYH mutation carriers. Conclusions: This study proposes the first set of clinical criteria designed to identify CRC patients with biallelic MYH mutations, and it argues against an increased risk for monoallelic carriers. Background & Aims: Whereas it has conclusively been demonstrated that biallelic MutY human homolog (MYH) mutations confer a significant risk for colorectal cancer (CRC), the influence of monoallelic mutations remains controversial. Characterization of MYH-associated CRC is critical to identify individuals who might benefit from preventive strategies. This prospective, multicenter, case-control, population-based study was aimed at (1) establishing the CRC risk associated with specific germline MYH mutations and (2) devising a set of clinical criteria to identify MYH carriers among newly diagnosed CRC. Methods: Genotyping for Y165C and G382D was performed by TaqMan technology. Single-stranded conformation polymorphism analysis was performed in heterozygotes to screen for mutations in the entire gene. All individuals were re-screened for any additional pathogenic variant. Results: Biallelic and monoallelic MYH mutations were found in 8 (0.7%) and 19 (1.7%) of 1116 CRC patients, respectively. None of the 934 control subjects carried biallelic mutations, whereas 22 (2.3%) of them were monoallelic carriers. In a meta-analysis including all previous case-control studies, monoallelic MYH carriers were not at increased risk for CRC (odds ratio, 1.11; 95% confidence interval, 0.90–1.37), although a significant association was found with the Y165C mutation in either homozygotes or heterozygotes (odds ratio, 1.67; 95% confidence interval, 1.17–2.40). Furthermore, presence of more than 15 synchronous colorectal adenomas or CRC diagnosed before the age of 50 years was the most effective set of criteria for the identification of biallelic MYH mutation carriers. Conclusions: This study proposes the first set of clinical criteria designed to identify CRC patients with biallelic MYH mutations, and it argues against an increased risk for monoallelic carriers. Although it is assumed that up to 20%–25% of colorectal cancer (CRC) cases develop as a result of inherited genetic factors, known genes predisposing to this malignancy account for less than 5%.1Burt R. Neklason D.W. Genetic testing for inherited colon cancer.Gastroenterology. 2005; 128: 1696-1716Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar Besides the APC gene causing familial adenomatous polyposis and the mismatch repair genes responsible for Lynch syndrome, recent studies have identified a new gene involved in hereditary CRC, MutY human homolog (MYH), which encodes a member of the base excision repair system.2Al-Tassan N. Chmiel N.H. Maynard J. et al.Inherited variants of MYH associated with somatic G:C→T:A mutations in colorectal tumors.Nat Genet. 2002; 30: 227-232Crossref PubMed Scopus (1094) Google Scholar This system is constituted by 3 enzymes (MYH, OGG1, and MTH1) that contribute to protect cells against the mutagenic effects of aerobic metabolism.3Cheadle J.P. Sampson J.R. Exposing the MYtH about base excision repair and human inherited disease.Hum Mol Genet. 2003; 12: R159-R165Crossref PubMed Scopus (79) Google Scholar MYH is a DNA glycosylase that acts at a third level of defense, being responsible for the removal of adenines mispaired with 8-oxoguanine, one of the most mutagenic DNA products of oxidative DNA damage. Failure to correct these mispairs leads to G:C→T:A transversions in target genes, including APC.2Al-Tassan N. Chmiel N.H. Maynard J. et al.Inherited variants of MYH associated with somatic G:C→T:A mutations in colorectal tumors.Nat Genet. 2002; 30: 227-232Crossref PubMed Scopus (1094) Google Scholar Up to now, pathogenic variants in the base excision repair system have been limited to the MYH gene, with no unambiguous mutations found in the MTH1 and OGG1 genes.2Al-Tassan N. Chmiel N.H. Maynard J. et al.Inherited variants of MYH associated with somatic G:C→T:A mutations in colorectal tumors.Nat Genet. 2002; 30: 227-232Crossref PubMed Scopus (1094) Google Scholar, 4Sieber O.M. Lipton L. Crabtree M. et al.Multiple colorectal adenomas, classic adenomatous polyposis, and germ-line mutations in MYH.N Engl J Med. 2003; 348: 791-799Crossref PubMed Scopus (745) Google Scholar, 5Jones S. Emmerson P. Maynard J. et al.Biallelic germline mutations in MYH predispose to multiple colorectal adenoma and somatic G:C→T:A mutations.Hum Mol Genet. 2002; 11: 2961-2967Crossref PubMed Google Scholar Since the first description of the association between the MYH gene and CRC in a British family by Al-Tassan et al,2Al-Tassan N. Chmiel N.H. Maynard J. et al.Inherited variants of MYH associated with somatic G:C→T:A mutations in colorectal tumors.Nat Genet. 2002; 30: 227-232Crossref PubMed Scopus (1094) Google Scholar biallelic MYH mutations have been consistently found to predispose to an attenuated form of familial adenomatous polyposis. Indeed, germline MYH mutations are responsible for as much as 40% of attenuated familial adenomatous polyposis without mutations in the APC gene, especially in those cases with a recessive family history.4Sieber O.M. Lipton L. Crabtree M. et al.Multiple colorectal adenomas, classic adenomatous polyposis, and germ-line mutations in MYH.N Engl J Med. 2003; 348: 791-799Crossref PubMed Scopus (745) Google Scholar, 6Sampson J.R. Dolwani S. Jones S. et al.Autosomal recessive colorectal adenomatous polyposis due to inherited mutations of MYH.Lancet. 2003; 362: 39-41Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar The 2 most common variants in the MYH gene are Y165C and G382D missense mutations, both accounting for more than 80% of all MYH variants reported in white populations.3Cheadle J.P. Sampson J.R. Exposing the MYtH about base excision repair and human inherited disease.Hum Mol Genet. 2003; 12: R159-R165Crossref PubMed Scopus (79) Google Scholar, 4Sieber O.M. Lipton L. Crabtree M. et al.Multiple colorectal adenomas, classic adenomatous polyposis, and germ-line mutations in MYH.N Engl J Med. 2003; 348: 791-799Crossref PubMed Scopus (745) Google Scholar, 5Jones S. Emmerson P. Maynard J. et al.Biallelic germline mutations in MYH predispose to multiple colorectal adenoma and somatic G:C→T:A mutations.Hum Mol Genet. 2002; 11: 2961-2967Crossref PubMed Google Scholar Other pathogenic variants have been found in different ethnic populations,6Sampson J.R. Dolwani S. Jones S. et al.Autosomal recessive colorectal adenomatous polyposis due to inherited mutations of MYH.Lancet. 2003; 362: 39-41Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar, 7Gismondi V. Meta M. Bonelli L. et al.Prevalence of the Y165C, G382D and 1395delGGA germline mutations of the MYH gene in Italian patients with adenomatous polyposis coli and colorectal adenomas.Int J Cancer. 2004; 109: 680-684Crossref PubMed Scopus (137) Google Scholar thus being consistent with founder effects. Recent studies have demonstrated that biallelic MYH gene mutations also predispose to CRC with an autosomal recessive pattern, accounting for up to 1% of these neoplasms.8Jenkins M.A. Croitoru M.E. Monga N. et al.Risk of colorectal cancer in monoallelic and biallelic carriers of MYH mutations: a population-based case-family study.Cancer Epidemiol Biomarkers Prev. 2006; 15: 312-314Crossref PubMed Scopus (146) Google Scholar, 9Croitoru M.E. Cleary S.P. Di Nicola N. et al.Association between biallelic and monoallelic germline MYH gene mutations and colorectal cancer risk.J Natl Cancer Inst. 2004; 96: 1631-1634Crossref PubMed Scopus (221) Google Scholar, 10Peterlongo P. Mitra N. Chuai S. et al.Colorectal cancer risk in individuals with biallelic or monoallelic mutations of MYH.Int J Cancer. 2005; 114: 505-507Crossref PubMed Scopus (51) Google Scholar, 11Farrington S.M. Tenesa A. Barnetson R. et al.Germline susceptibility to colorectal cancer due to base-excision repair gene defects.Am J Hum Genet. 2005; 77: 1Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 12Enholm S. Hienonen T. Suomalainen A. et al.Proportion and phenotype of MYH-associated colorectal neoplasia in a population-based series of Finnish colorectal cancer patients.Am J Pathol. 2003; 163: 827-832Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar In this setting, biallelic MYH mutations have been found to be associated with a 93-fold excess risk of CRC, with an almost complete penetrance at 60 years of age.11Farrington S.M. Tenesa A. Barnetson R. et al.Germline susceptibility to colorectal cancer due to base-excision repair gene defects.Am J Hum Genet. 2005; 77: 1Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 13Tenesa A. Campbell H. Barnetson R. et al.Association of MUTYH and colorectal cancer.Br J Cancer. 2006; 95: 239-242Crossref PubMed Scopus (75) Google Scholar In contrast, the influence of monoallelic MYH mutations on CRC risk remains controversial. Although no single study has found statistically significant evidence,9Croitoru M.E. Cleary S.P. Di Nicola N. et al.Association between biallelic and monoallelic germline MYH gene mutations and colorectal cancer risk.J Natl Cancer Inst. 2004; 96: 1631-1634Crossref PubMed Scopus (221) Google Scholar, 10Peterlongo P. Mitra N. Chuai S. et al.Colorectal cancer risk in individuals with biallelic or monoallelic mutations of MYH.Int J Cancer. 2005; 114: 505-507Crossref PubMed Scopus (51) Google Scholar, 11Farrington S.M. Tenesa A. Barnetson R. et al.Germline susceptibility to colorectal cancer due to base-excision repair gene defects.Am J Hum Genet. 2005; 77: 1Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 12Enholm S. Hienonen T. Suomalainen A. et al.Proportion and phenotype of MYH-associated colorectal neoplasia in a population-based series of Finnish colorectal cancer patients.Am J Pathol. 2003; 163: 827-832Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 13Tenesa A. Campbell H. Barnetson R. et al.Association of MUTYH and colorectal cancer.Br J Cancer. 2006; 95: 239-242Crossref PubMed Scopus (75) Google Scholar, 14Fleischmann C. Peto J. Cheadle J. et al.Comprehensive analysis of the contribution of germline MYH variation to early-onset colorectal cancer.Int J Cancer. 2004; 109: 554-558Crossref PubMed Scopus (99) Google Scholar a recent meta-analysis of published data has demonstrated an almost significant heterozygous gene effect (relative risk [RR], 1.3; 95% confidence interval [CI], 1.0–1.7; P = .09).13Tenesa A. Campbell H. Barnetson R. et al.Association of MUTYH and colorectal cancer.Br J Cancer. 2006; 95: 239-242Crossref PubMed Scopus (75) Google Scholar In agreement with this putative association, Farrington et al,11Farrington S.M. Tenesa A. Barnetson R. et al.Germline susceptibility to colorectal cancer due to base-excision repair gene defects.Am J Hum Genet. 2005; 77: 1Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar in the largest population-based case-control study evaluating CRC risk in MYH mutation carriers, found an excess risk only for heterozygotes older than the age of 55 years, suggesting that monoallelic MYH mutation carriers could be at increased risk later in life. Overall, these discrepancies underscore a weak gene effect, thus needing larger sample sizes, population-based studies, specific-mutation analysis, and proper methodologic approaches such as meta-analysis or kin-cohort studies to ascertain the effect of low-penetrance genes. Accurate characterization of patients with MYH-associated CRC is critical to identify patients who might benefit from genetic testing and, consequently, from specific screening and surveillance strategies. However, little is known about clinical characteristics of patients with MYH-associated CRC, especially regarding personal conditions and familial background. In that sense, it has been mentioned that the presence of multiple polyps could constitute a phenotypic marker of MYH-associated CRC, but up to 25%–33% of them do not have synchronous adenomas.9Croitoru M.E. Cleary S.P. Di Nicola N. et al.Association between biallelic and monoallelic germline MYH gene mutations and colorectal cancer risk.J Natl Cancer Inst. 2004; 96: 1631-1634Crossref PubMed Scopus (221) Google Scholar, 11Farrington S.M. Tenesa A. Barnetson R. et al.Germline susceptibility to colorectal cancer due to base-excision repair gene defects.Am J Hum Genet. 2005; 77: 1Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 15Wang L. Baudhuin L.M. Boardman L.A. et al.MYH mutations in patients with attenuated and classic polyposis and with young-onset colorectal cancer without polyps.Gastroenterology. 2004; 127: 9-16Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar In addition, patients with germline MYH mutations are significantly younger than non-mutation carriers.9Croitoru M.E. Cleary S.P. Di Nicola N. et al.Association between biallelic and monoallelic germline MYH gene mutations and colorectal cancer risk.J Natl Cancer Inst. 2004; 96: 1631-1634Crossref PubMed Scopus (221) Google Scholar, 12Enholm S. Hienonen T. Suomalainen A. et al.Proportion and phenotype of MYH-associated colorectal neoplasia in a population-based series of Finnish colorectal cancer patients.Am J Pathol. 2003; 163: 827-832Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 16Jo W.S. Bandipalliam P. Shannon K.M. et al.Correlation of polyp number and family history of colon cancer with germline MYH mutations.Clin Gastroenterol Hepatol. 2005; 3: 1022-1028Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar Finally, a family history of CRC has also been noticed in some studies9Croitoru M.E. Cleary S.P. Di Nicola N. et al.Association between biallelic and monoallelic germline MYH gene mutations and colorectal cancer risk.J Natl Cancer Inst. 2004; 96: 1631-1634Crossref PubMed Scopus (221) Google Scholar, 12Enholm S. Hienonen T. Suomalainen A. et al.Proportion and phenotype of MYH-associated colorectal neoplasia in a population-based series of Finnish colorectal cancer patients.Am J Pathol. 2003; 163: 827-832Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 16Jo W.S. Bandipalliam P. Shannon K.M. et al.Correlation of polyp number and family history of colon cancer with germline MYH mutations.Clin Gastroenterol Hepatol. 2005; 3: 1022-1028Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar but not confirmed in others.9Croitoru M.E. Cleary S.P. Di Nicola N. et al.Association between biallelic and monoallelic germline MYH gene mutations and colorectal cancer risk.J Natl Cancer Inst. 2004; 96: 1631-1634Crossref PubMed Scopus (221) Google Scholar, 12Enholm S. Hienonen T. Suomalainen A. et al.Proportion and phenotype of MYH-associated colorectal neoplasia in a population-based series of Finnish colorectal cancer patients.Am J Pathol. 2003; 163: 827-832Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 16Jo W.S. Bandipalliam P. Shannon K.M. et al.Correlation of polyp number and family history of colon cancer with germline MYH mutations.Clin Gastroenterol Hepatol. 2005; 3: 1022-1028Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar Although all these characteristics have been suggested as potentially useful criteria to select out for MYH genetic testing, there is no study evaluating their performance to identify MYH mutation carriers among CRC patients in a general population setting. Here we present a prospective, multicenter, case-control, population-based study, performed within the Epicolon project,17Piñol V. Castells A. Andreu M. et al.Accuracy of revised Bethesda guidelines, microsatellite instability, and immunohistochemistry for the identification of patients with hereditary nonpolyposis colorectal cancer.JAMA. 2005; 293: 1986-1994Crossref PubMed Scopus (454) Google Scholar, 18Piñol V. Andreu M. Castells A. et al.Frequency of hereditary non-polyposis colorectal cancer and other colorectal cancer familial forms in Spain: a multicenter, prospective, nation-wide study—Gastrointestinal Oncology Group of the Spanish Gastroenterological Association.Eur J Gastroenterol Hepatol. 2004; 16: 39-45Crossref PubMed Scopus (72) Google Scholar which was aimed at (1) establishing the CRC risk associated with specific germline MYH mutations and (2) characterizing patients with MYH-associated tumors to define a set of recommendations to identify MYH mutation carriers among newly diagnosed CRC cases. Between November 2000–October 2001, all patients with newly diagnosed CRC in 25 Spanish hospitals were included in the Epicolon project, a clinical epidemiology survey aimed at establishing the incidence and characteristics of hereditary and familial CRC forms in Spain.17Piñol V. Castells A. Andreu M. et al.Accuracy of revised Bethesda guidelines, microsatellite instability, and immunohistochemistry for the identification of patients with hereditary nonpolyposis colorectal cancer.JAMA. 2005; 293: 1986-1994Crossref PubMed Scopus (454) Google Scholar, 18Piñol V. Andreu M. Castells A. et al.Frequency of hereditary non-polyposis colorectal cancer and other colorectal cancer familial forms in Spain: a multicenter, prospective, nation-wide study—Gastrointestinal Oncology Group of the Spanish Gastroenterological Association.Eur J Gastroenterol Hepatol. 2004; 16: 39-45Crossref PubMed Scopus (72) Google Scholar Nineteen of these 25 centers also agreed to participate in a nested case-control study aimed at evaluating the incidence and characteristics of MYH-associated CRC. Patients with familial adenomatous polyposis, germline APC, MSH2 and MLH1 mutation carriers, or personal history of inflammatory bowel disease, and those who refused to participate in the study were excluded from this analysis. The study was approved by the institutional ethics committee of each participating hospital, and written informed consent was obtained from all patients. Demographic, clinical, and tumor-related characteristics of probands, as well as a detailed family history, were obtained.17Piñol V. Castells A. Andreu M. et al.Accuracy of revised Bethesda guidelines, microsatellite instability, and immunohistochemistry for the identification of patients with hereditary nonpolyposis colorectal cancer.JAMA. 2005; 293: 1986-1994Crossref PubMed Scopus (454) Google Scholar Pedigrees were traced backward and laterally as far as possible or at least up to second-degree relatives, regarding cancer history. Age at cancer diagnosis, type, location, and tumor stage of the neoplasm, and current status were registered for each affected family member. Tissue samples from tumor and normal colonic mucosa were obtained from each patient, immediately frozen in liquid nitrogen, and stored at −70°C until use. In cases without frozen tissue, formalin-fixed, paraffin-embedded samples were used. Genomic DNA was isolated by using the QiaAmp Tissue Kit (Qiagen, Courtaboeuf, France). As part of the Epicolon project, microsatellite instability testing and immunostaining for DNA mismatch repair proteins were performed in all patients regardless of age, personal and family history, and tumor characteristics, as described elsewhere.17Piñol V. Castells A. Andreu M. et al.Accuracy of revised Bethesda guidelines, microsatellite instability, and immunohistochemistry for the identification of patients with hereditary nonpolyposis colorectal cancer.JAMA. 2005; 293: 1986-1994Crossref PubMed Scopus (454) Google Scholar Patients found to have tumors with microsatellite instability and/or lack of protein expression underwent germline genetic testing for MSH2 and MLH1. In addition, patients with more than 15 synchronous colorectal adenomas also underwent germline APC gene testing. As mentioned above, cases with mutations in these genes were excluded from the present analysis. Age- and sex-matched control subjects with no personal history of cancer at the time of ascertainment were recruited from a large cohort of individuals attending the outpatient clinics of orthopedic surgery departments of participating institutions. Information on family history of colorectal neoplasia or previous colonoscopy reports was not available for these subjects. Genomic DNA was obtained from peripheral blood samples. A 3-stage approach was performed to identify individuals carrying heterozygous or homozygous variants in the MYH gene (GenBank accession number NM_012222). First, we screened for the 2 most common mutations, Y165C and G382D, by allelic discrimination with allele-specific TaqMan probes and resolved on a 7300 Real Time PCR System (Applied Biosystems, Foster City, CA). Second, in heterozygotes for the Y165C or G382D mutations, the coding region and exon-intron boundaries of the entire MYH gene were screened by single-strand conformation polymorphism (SSCP) analysis. Primer details are available on request. Samples with abnormal band shifts were sequenced in forward and reverse orientations by using the BigDye terminator v31 cycle sequencing kit (Applied Biosystems). With this approach, we identified all homozygotes or compound heterozygotes of any subjects in whom at least one allele was either Y165C or G382D. In addition, to minimize the probability of underdetecting individuals with other mutations, CRC patients with more than 15 synchronous adenomas were also evaluated by SSCP, although no mutation was found by the previous approach. Third, all CRC patients and control subjects were re-screened for any pathogenic variant found in the SSCP analysis by allelic discrimination with allele-specific TaqMan probes. To allow comparison of our results, we used the MYH sequence used by previous authors (GenBank accession number U63329) for the G382D, Y165C, 1103delC, and 1186_1187insGG mutations, instead of the actual reference sequence (GenBank accession number NM_012222), which has 11 additional codons in exon 3, resulting in changes in numbering all variants (G393D, Y176C, 1138delC, and 1220_1221insGG, respectively). To test the association between the MYH gene and CRC risk, odds ratios (ORs) and 95% CIs were calculated for each genotype.19Fleiss J.L. Statistical methods for rates and proportions. John Wiley & Sons, New York1981Google Scholar Because of the low frequency of some variants, the analysis of individual allele effects was limited to the G382D and Y165C mutations. Analyses were done for biallelic, monoallelic, and any mutation carriers. A meta-analysis including all previous case-control studies evaluating the CRC risk associated to the Y165C and G382D MYH mutations was performed (Figure 1). The homogeneity of effects throughout studies was appraised by using a homogeneity test based on the χ2 test. Because of the low power of this test, a minimum cutoff P value of .1 was established as a threshold of homogeneity, lower values indicating heterogeneity. In addition, the I2 statistic was calculated to assess the impact of heterogeneity on the results. This statistic describes the percentage of the variability and in effect estimates that it is due to heterogeneity rather than sampling error (chance). A value >50% might be considered substantial heterogeneity. Meta-analysis was performed by combining OR of the individual studies in a global OR, under the assumption-free model (or fixed effects model). We chose to use the fixed effects model to obtain more precision on the estimates, because the CIs are narrower than with the random effects model. Significance and 95% CI were provided for the combined OR. The analysis was performed for monoallelic and any mutation carriers. All calculations were performed with the freeware program Review Manager 4.2.8 developed by the Cochrane Collaboration (Copenhagen, Denmark). To identify personal and/or familial characteristics associated with the presence of germline MYH mutations, univariate and multivariate analyses were performed with respect to biallelic, monoallelic, and any mutation carriers. For the univariate analysis, categorical variables were compared by the χ2 test, applying the Yates correction when needed, and continuous variables by the Student t test. Variables achieving a P value of less than .1 were subsequently included in the multivariate analysis by using a stepwise backward logistic regression procedure to identify independent predictors of germline MYH mutations. Performance characteristics (sensitivity, specificity, positive and negative predictive value, and overall accuracy) of each independent variable selected in the multivariate analysis and their combination were calculated with respect to the presence of MYH mutations. Continuous variables were expressed as mean ± standard deviation. All P values were two sided, and a value less than .05 was considered statistically significant. Calculations were performed with the SPSS software version 11.0 (SPSS Inc, Chicago, IL). During the study period, 1978 patients with newly diagnosed CRC were included in 25 centers. One hundred seventeen patients were excluded because tumor developed in the context of familial adenomatous polyposis (n = 11) or inflammatory bowel disease (n = 14), germline MSH2 and MLH1 gene mutations (n = 11), patients did not consent to participate in the study (n = 32), or incomplete family history (n = 49). Of the remaining 1861 eligible patients, 1116 were diagnosed in centers agreeing to participate in the nested case-control study aimed at evaluating the incidence and characteristics of MYH-associated CRC. Characteristics of this group of patients (Table 1) did not differ from the whole series.18Piñol V. Andreu M. Castells A. et al.Frequency of hereditary non-polyposis colorectal cancer and other colorectal cancer familial forms in Spain: a multicenter, prospective, nation-wide study—Gastrointestinal Oncology Group of the Spanish Gastroenterological Association.Eur J Gastroenterol Hepatol. 2004; 16: 39-45Crossref PubMed Scopus (72) Google Scholar Simultaneously, 934 control subjects were also included.Table 1Characteristics of the 1116 Patients With CRCCharacteristicsAge (y)aExpressed as mean ± standard deviation.70.0 ± 11.4Gender, n (%) Female461 (41.3)Site of tumor, n (%) Proximal to splenic flexure313 (28.0)TNM tumor stage,bReferred to 1078 CRC patients. n (%) I132 (11.8) II464 (41.6) III297 (26.6) IV185 (16.6)Synchronous CRC, n (%)58 (5.2)Synchronous colorectal adenomas, n (%)298 (26.7)Past history of CRC, n (%)14 (1.3)Past history of any non-colorectal neoplasia, n (%)96 (8.6)Family history of CRC,cReferred to first- and/or second-degree relatives. n (%)184 (16.5)a Expressed as mean ± standard deviation.b Referred to 1078 CRC patients.c Referred to first- and/or second-degree relatives. Open table in a new tab Biallelic MYH mutations were found in 8 (0.7%) CRC patients. In addition to the G382D and Y165C missense mutations, we identified 2 additional, previously described, pathogenic mutations4Sieber O.M. Lipton L. Crabtree M. et al.Multiple colorectal adenomas, classic adenomatous polyposis, and germ-line mutations in MYH.N Engl J Med. 2003; 348: 791-799Crossref PubMed Scopus (745) Google Scholar, 20Aretz S. Uhlhaas S. Goergens H. et al.MUTYH-associated polyposis: 70 of 71 patients with biallelic mutations present with an attenuated or atypical phenotype.Int J Cancer. 2006; 119: 807-814Crossref PubMed Scopus (166) Google Scholar, 21Isidro G. Laranjeira F. Pires A. et al.Germline MUTYH (MYH) mutations in Portuguese individuals with multiple colorectal adenomas.Hum Mutat. 2004; 24: 353-354Crossref PubMed Scopus (81) Google Scholar (1103delC and 1186_1187insGG) in 3 patients (Table 2). Monoallelic MYH mutations were identified in 19 (1.7%) CRC patients, 15 for the G382D mutation and 4 for the Y165C mutation.Table 2Risk of CRC Associated With Germline MYH MutationsCases (n = 1116)Controls (n = 934)OR (95% CI)Non-mutation carriers, n (%)1089 (97.5)913 (97.8)1 (reference)Biallelic mutation carriers, n (%)8 (0.7)0 (–)NA Y165C/Y165C2 (0.2)0 (–)NA Y165C/G382D2 (0.2)0 (–)NA G382D/G382D1 (0.1)0 (–)NA G382D/1103delC1 (0.1)0 (–)NA 1186_1187insGG/1186_1187insGG2 (0.2)0 (–)NAMono
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