The standard genetic test for Lynch syndrome (LS) frequently reveals an absence of pathogenic mutations in DNA mismatch repair genes known to be associated with LS. It was recently shown that germ line deletions in the last exons of EPCAM are involved in the etiology of LS. The aim of this study was to evaluate the prevalence of EPCAM deletions in a Spanish population and the clinical implications of deletion. Probands from 501 families suspected of having LS were enrolled in the study. Twenty-five cases with MSH2 loss were identified: 10 had mutations of MSH2, five had mutations of MSH6 , and 10 did not show MSH2/MSH6 mutations. These 25 cases were analyzed for EPCAM deletions using multiplex ligation-dependent probe amplification , and deletions were mapped using long-range PCR analysis. One subject with no MSH2/MSH6 mutations had a large deletion in the EPCAM locus that extended for 8.7 kb and included exons 8 and 9. The tumor exhibited MSH2 promoter hypermethylation. EPCAM deletion analysis followed by MSH2 methylation testing of the tumor is a fast low-cost procedure that can be used to identify mutations that cause LS. We propose that this procedure be incorporated into clinical genetic analysis strategies and present a decision-support flow diagram for the diagnosis of

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BackgroundTGF-β receptor type I is a mediator of growth inhibitory signals. TGFBR1*6A (rs11466445) is a common polymorphic variant of the TGF-β receptor I gene and has been associated with tumour susceptibility. Nevertheless, the role of this polymorphism as a risk factor for colorectal cancer is controversial. The aim of this study was to assess the association between TGFBR1*6A and colorectal cancer, age, sex, tumour location and tumour stage in a Spanish population.MethodsThe case-control study involved 800 Spanish subjects: 400 sporadic colorectal cancer patients and 400 age-, sex-, and ethnic-matched controls. The odds ratio (OR) and 95% confidence interval (95% CI) for the TGFBR1*6A polymorphism were calculated using unconditional logistic regression adjusted for age and sex. Analysis of somatic mutations at the GCG repeat of TGFBR1 exon 1 and germline allele-specific expression were also conducted to obtain further information on the contribution of the TGFBR1*6A allele to CRC susceptibility.ResultsThere was no statistically significant association between the TGFBR1*6A allele and CRC (p > 0.05). The OR was 1.147 (95% CI: 0.799–1.647) for carriers of the TGFBR1*6A allele and 0.878 (95% CI: 0.306–2.520) for homozygous TGFBR1*6A individuals compared with the reference. The frequency of the polymorphism was not affected by age, sex or tumour stage. The TGFBR1*6A allele was more prevalent among colon tumour patients than among rectal tumour patients. Tumour somatic mutations were found in only two of 69 cases (2.9%). Both cases involved a GCG deletion that changed genotype 9A/9A in normal DNA to genotype 9A/8A. Interestingly, these two tumours were positive for microsatellite instability, suggesting that these mutations originated because of a deficient DNA mismatch repair system.Allele-specific expression of the 9A allele was detected in seven of the 14 heterozygous 9A/6A tumour cases. This could have been caused by linkage disequilibrium of the TGFBR1*6A allele with mutations that cause allele-specific expression, as was recently suggested.ConclusionOur results suggest that the TGFBR1*6A allele does not confer an increased risk of colorectal cancer in the Spanish population.

BackgroundLynch syndrome (LS) is an autosomal dominant inherited cancer syndrome characterized by early onset cancers of the colorectum, endometrium and other tumours. A significant proportion of DNA variants in LS patients are unclassified. Reports on the pathogenicity of the c.1852_1853AA>GC (p.Lys618Ala) variant of the MLH1 gene are conflicting. In this study, we provide new evidence indicating that this variant has no significant implications for LS.MethodsThe following approach was used to assess the clinical significance of the p.Lys618Ala variant: frequency in a control population, case-control comparison, co-occurrence of the p.Lys618Ala variant with a pathogenic mutation, co-segregation with the disease and microsatellite instability in tumours from carriers of the variant. We genotyped p.Lys618Ala in 1034 individuals (373 sporadic colorectal cancer [CRC] patients, 250 index subjects from families suspected of having LS [revised Bethesda guidelines] and 411 controls). Three well-characterized LS families that fulfilled the Amsterdam II Criteria and consisted of members with the p.Lys618Ala variant were included to assess co-occurrence and co-segregation. A subset of colorectal tumour DNA samples from 17 patients carrying the p.Lys618Ala variant was screened for microsatellite instability using five mononucleotide markers.ResultsTwenty-seven individuals were heterozygous for the p.Lys618Ala variant; nine had sporadic CRC (2.41%), seven were suspected of having hereditary CRC (2.8%) and 11 were controls (2.68%). There were no significant associations in the case-control and case-case studies. The p.Lys618Ala variant was co-existent with pathogenic mutations in two unrelated LS families. In one family, the allele distribution of the pathogenic and unclassified variant was in trans, in the other family the pathogenic variant was detected in the MSH6 gene and only the deleterious variant co-segregated with the disease in both families. Only two positive cases of microsatellite instability (2/17, 11.8%) were detected in tumours from p.Lys618Ala carriers, indicating that this variant does not play a role in functional inactivation of MLH1 in CRC patients.ConclusionsThe p.Lys618Ala variant should be considered a neutral variant for LS. These findings have implications for the clinical management of CRC probands and their relatives.

In colorectal cancer (CRC), an inherited susceptibility risk affects about 35% of patients, whereas high-penetrance germline mutations account for <6% of cases. A considerable proportion of sporadic tumors could be explained by the coinheritance of multiple low-penetrance variants, some of which are common. We assessed the susceptibility to CRC conferred by genetic variants at the TGFBR1 locus. We analyzed 14 polymorphisms and the allele-specific expression (ASE) of TGFBR1 in 1025 individuals from the Spanish population. A case-control study was undertaken with 504 controls and 521 patients with sporadic CRC. Fourteen polymorphisms located at the TGFBR1 locus were genotyped with the iPLEX Gold (MassARRAY-Sequenom) technology. Descriptive analyses of the polymorphisms and haplotypes and association studies were performed with the SNPator workpackage. No relevant associations were detected between individual polymorphisms or haplotypes and the risk of CRC. The TGFBR1*9A/6A polymorphism was used for the ASE analysis. Heterozygous individuals were analyzed for ASE by fragment analysis using cDNA from normal tissue. The relative level of allelic expression was extrapolated from a standard curve. The cutoff value was calculated with Youden's index. ASE was found in 25.4% of patients and 16.4% of controls. Considering both bimodal and continuous types of distribution, no significant differences between the ASE values of patients and controls were identified. Interestingly, a combined analysis of the polymorphisms and ASE for the association with CRC occurrence revealed that ASE-positive individuals carrying one of the most common haplotypes (H2: 20.7%) showed remarkable susceptibility to CRC (RR: 5.25; 95% CI: 2.547–5.250; p<0.001) with a synergy factor of 3.7. In our study, 54.1% of sporadic CRC cases were attributable to the coinheritance of the H2 haplotype and TGFBR1 ASE. These results support the hypothesis that the allelic architecture of cancer genes, rather than individual polymorphisms, more accurately defines the CRC risk.