Value of upper gastrointestinal endoscopy for gastric cancer surveillance in patients with Lynch syndrome.

In our study, we evaluated the effectiveness of upper gastrointestinal (GI) endoscopy as an instrument for early gastric cancer (GC) detection in Lynch syndrome (LS) patients by analyzing data from the registry of the German Consortium for Familial Intestinal Cancer. In a prospective, multicenter cohort study, 1128 out of 2009 registered individuals with confirmed LS underwent 5176 upper GI endoscopies. Compliance was good since 77.6% of upper GI endoscopies were completed within the recommended interval of 1 to 3 years. Forty-nine GC events were observed in 47 patients. MLH1 (n = 21) and MSH2 (n = 24) mutations were the most prevalent. GCs in patients undergoing regular surveillance were diagnosed significantly more often in an early-stage disease (UICC I) than GCs detected through symptoms (83% vs 25%; P = .0231). Thirty-two (68%) patients had a negative family history of GC. The median age at diagnosis was 51 years (range 28-66). Of all GC patients, 13 were diagnosed at an age younger than 45. Our study supports the recommendation of regular upper GI endoscopy surveillance for LS patients beginning no later than at the age of 30.

Correction: Cancer risks by gene, age, and gender in 6350 carriers of pathogenic mismatch repair variants: findings from the Prospective Lynch Syndrome Database.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cancer risks by gene, age, and gender in 6350 carriers of pathogenic mismatch repair variants: findings from the Prospective Lynch Syndrome Database.

PURPOSE: Pathogenic variants affecting MLH1, MSH2, MSH6, and PMS2 cause Lynch syndrome and result in different but imprecisely known cancer risks. This study aimed to provide age and organ-specific cancer risks according to gene and gender and to determine survival after cancer. METHODS: We conducted an international, multicenter prospective observational study using independent test and validation cohorts of carriers of class 4 or class 5 variants. After validation the cohorts were merged providing 6350 participants and 51,646 follow-up years. RESULTS: There were 1808 prospectively observed cancers. Pathogenic MLH1 and MSH2 variants caused high penetrance dominant cancer syndromes sharing similar colorectal, endometrial, and ovarian cancer risks, but older MSH2 carriers had higher risk of cancers of the upper urinary tract, upper gastrointestinal tract, brain, and particularly prostate. Pathogenic MSH6 variants caused a sex-limited trait with high endometrial cancer risk but only modestly increased colorectal cancer risk in both genders. We did not demonstrate a significantly increased cancer risk in carriers of pathogenic PMS2 variants. Ten-year crude survival was over 80% following colon, endometrial, or ovarian cancer. CONCLUSION: Management guidelines for Lynch syndrome may require revision in light of these different gene and gender-specific risks and the good prognosis for the most commonly associated cancers.

No Difference in Colorectal Cancer Incidence or Stage at Detection by Colonoscopy Among 3 Countries With Different Lynch Syndrome Surveillance Policies.

BACKGROUND & AIMS: Patients with Lynch syndrome are at high risk for developing colorectal cancer (CRC). Regular colonoscopic surveillance is recommended, but there is no international consensus on the appropriate interval. We investigated whether shorter intervals are associated with lower CRC incidence and detection at earlier stages by comparing the surveillance policies in Germany, which evaluates patients by colonoscopy annually, in the Netherlands (patients evaluated at 1-2-year intervals), and Finland (patients evaluated at 2-3-year intervals). METHODS: We collected data from 16,327 colonoscopic examinations (conducted from 1984 through 2015) of 2747 patients with Lynch syndrome (pathogenic variants in the MLH1, MSH2, or MSH6 genes) from the German HNPCC Consortium, the Dutch Lynch Syndrome Registry, and the Finnish Lynch Syndrome Registry. Our analysis included 23,309 person-years of cumulative observation time. Time from the index colonoscopy to incident CRC or adenoma was analyzed using the Kaplan-Meier method; groups were compared using the log-rank test. We performed multivariable Cox regression analyses to identify factors associated with CRC risk (diagnosis of CRC before the index colonoscopy, sex, mutation, age, and presence of adenoma at the index colonoscopy). RESULTS: The 10-year cumulative CRC incidence ranged from 4.1% to 18.4% in patients with low- and high-risk profiles, respectively, and varied with age, sex, mutation, and prior detection of CRC or adenoma. Observed colonoscopy intervals were largely in accordance with the country-specific recommendations. We found no significant differences in cumulative CRC incidence or CRC stage at detection among countries. There was no significant association between CRC stage and time since last colonoscopy. CONCLUSIONS: We did not find a significant reduction in CRC incidence or stage of detection in Germany (annual colonoscopic surveillance) than in countries with longer surveillance intervals (the Netherlands, with 1-2-year intervals, and Finland, with 2-3-year intervals). Overall, we did not find a significant association of the interval with CRC risk, although age, sex, mutation, and prior neoplasia were used to individually modify colonoscopy intervals. Studies are needed to develop and validate risk-adapted surveillance strategies and to identify patients who benefit from shorter surveillance intervals.

Role of germline aberrations affecting CTNNA1, MAP3K6 and MYD88 in gastric cancer susceptibility.

BACKGROUND: In approximately 10% of all gastric cancer (GC) cases, a heritable cause is suspected. A subset of these cases have a causative germline CDH1 mutation; however, in most cases the cause remains unknown. Our objective was to assess to what extent these remaining cases may be explained by germline mutations in the novel candidate GC predisposing genes CTNNA1, MAP3K6 or MYD88. METHODS: We sequenced a large cohort of unexplained young and/or familial patients with GC (n=286) without a CDH1germline mutation for germline variants affecting CTNNA1, MAP3K6 and MYD88 using a targeted next-generation sequencing approach based on single-molecule molecular inversion probes. RESULTS: Predicted deleterious germline variants were not encountered in MYD88, but recurrently observed in CTNNA1 (n=2) and MAP3K6 (n=3) in our cohort of patients with GC. In contrast to deleterious variants in CTNNA1, deleterious variants in MAP3K6 also occur frequently in the general population. CONCLUSIONS: Based on our results MAP3K6 should no longer be considered a GC predisposition gene, whereas deleterious CTNNA1 variants are confirmed as an infrequent cause of GC susceptibility. Biallelic MYD88 germline mutations are at most a very rare cause of GC susceptibility as no additional cases were identified.

Identification of Novel Candidate Genes for Early-Onset Colorectal Cancer Susceptibility.

Approximately 25-30% of colorectal cancer (CRC) cases are expected to result from a genetic predisposition, but in only 5-10% of these cases highly penetrant germline mutations are found. The remaining CRC heritability is still unexplained, and may be caused by a hitherto-undefined set of rare variants with a moderately penetrant risk. Here we aimed to identify novel risk factors for early-onset CRC using whole-exome sequencing, which was performed on a cohort of CRC individuals (n = 55) with a disease onset before 45 years of age. We searched for genes that were recurrently affected by rare variants (minor allele frequency ≤ 0.001) with potentially damaging effects and, subsequently, re-sequenced the candidate genes in a replication cohort of 174 early-onset or familial CRC individuals. Two functionally relevant genes with low frequency variants with potentially damaging effects, PTPN12 and LRP6, were found in at least three individuals. The protein tyrosine phosphatase PTP-PEST, encoded by PTPN12, is a regulator of cell motility and LRP6 is a component of the WNT-FZD-LRP5-LRP6 complex that triggers WNT signaling. All variants in LRP6 were identified in individuals with an extremely early-onset of the disease (≤30 years of age), and two of the three variants showed increased WNT signaling activity in vitro. In conclusion, we present PTPN12 and LRP6 as novel candidates contributing to the heterogeneous susceptibility to CRC.

Copy number variation analysis and targeted NGS in 77 families with suspected Lynch syndrome reveals novel potential causative genes.

In many families with suspected Lynch syndrome (LS), no germline mutation in the causative mismatch repair (MMR) genes is detected during routine diagnostics. To identify novel causative genes for LS, the present study investigated 77 unrelated, mutation-negative patients with clinically suspected LS and a loss of MSH2 in tumor tissue. An analysis for genomic copy number variants (CNV) was performed, with subsequent next generation sequencing (NGS) of selected candidate genes in a subgroup of the cohort. Genomic DNA was genotyped using Illumina's HumanOmniExpress Bead Array. After quality control and filtering, 25 deletions and 16 duplications encompassing 73 genes were identified in 28 patients. No recurrent CNV was detected, and none of the CNVs affected the regulatory regions of MSH2. A total of 49 candidate genes from genomic regions implicated by the present CNV analysis and 30 known or assumed risk genes for colorectal cancer (CRC) were then sequenced in a subset of 38 patients using a customized NGS gene panel and Sanger sequencing. Single nucleotide variants were identified in 14 candidate genes from the CNV analysis. The most promising of these candidate genes were: (i) PRKCA, PRKDC, and MCM4, as a functional relation to MSH2 is predicted by network analysis, and (ii) CSMD1, as this is commonly mutated in CRC. Furthermore, six patients harbored POLE variants outside the exonuclease domain, suggesting that these might be implicated in hereditary CRC. Analyses in larger cohorts of suspected LS patients recruited via international collaborations are warranted to verify the present findings.

Prevalence of germline mutations in the spindle assembly checkpoint gene BUB1B in individuals with early-onset colorectal cancer.

Germline mutations in BUB1B, encoding BUBR1, one of the crucial components of the spindle assembly checkpoint (SAC), have been shown to cause variable phenotypes, including the recessive mosaic variegated aneuploidy (MVA) syndrome, which predisposes to cancer. Reduced levels of the wild-type BUBR1 protein have been linked to the development of gastrointestinal neoplasms. To determine whether mutations in BUB1B are enriched in individuals with colorectal cancer (CRC), we performed amplicon-based targeted next-generation sequencing of BUB1B on germline DNA of 192 individuals with early-onset CRC (≤50 years). None of the individuals was found to be homozygous or compound heterozygous for mutations in BUB1B. However, we did identify two rare heterozygous variants, p.Glu390del and p.Cys945Tyr, in patients who developed CRC at the ages of 41 and 43 years, respectively. Both variants were shown not to affect BUBR1 protein expression levels and protein localization. Since the p.Glu390del variant is located in the BUB3-binding domain, we also performed immunoprecipitation to examine whether this variant affects the binding of BUB1 or BUB3 to BUBR1 but, compared to wild-type BUBR1, no difference was observed. Our data suggest that mutations in BUB1B do not occur frequently in the germline of individuals with CRC and that BUB1B unlikely plays a major role in the predisposition to early-onset CRC. Whether carriers of pathogenic BUB1B mutations, such as the parents of MVA syndrome patients, have an increased risk for cancer remains of interest, as studies in mice have suggested that haploinsufficiency of BUB1B may cause an increase in carcinogen-induced tumors. © 2016 Wiley Periodicals, Inc.

Germline deletions in the tumour suppressor gene FOCAD are associated with polyposis and colorectal cancer development.

Heritable genetic variants can significantly affect the lifetime risk of developing cancer, including polyposis and colorectal cancer (CRC). Variants in genes currently known to be associated with a high risk for polyposis or CRC, however, explain only a limited number of hereditary cases. The identification of additional genetic causes is, therefore, crucial to improve CRC prevention, detection and treatment. We have performed genome-wide and targeted DNA copy number profiling and resequencing in early-onset and familial polyposis/CRC patients, and show that deletions affecting the open reading frame of the tumour suppressor gene FOCAD are recurrent and significantly enriched in CRC patients compared with unaffected controls. All patients carrying FOCAD deletions exhibited a personal or family history of polyposis. RNA in situ hybridization revealed FOCAD expression in epithelial cells in the colonic crypt, the site of tumour initiation, as well as in colonic tumours and organoids. Our data suggest that monoallelic germline deletions in the tumour suppressor gene FOCAD underlie moderate genetic predisposition to the development of polyposis and CRC.

An unusual case of Cowden syndrome associated with ganglioneuromatous polyposis.

BACKGROUND: Ganglioneuromatous polyposis (GP) is a very rare disorder which may be associated with other clinical manifestations and syndromes, such as Cowden syndrome, multiple endocrine neoplasia (MEN) type II and neurofibromatosis (NF) 1. The risk for malignant transformation of ganglioneuromas is unknown, and the combination of GP with colon cancer has been only very seldom reported. METHODS AND RESULTS: We report the case of a 60-year old male patient with adenocarcinoma, adenomas and lipomas of the colon and multiple gastroduodenal lesions combined with generalised lipomatosis and macrocephaly. Based on the initial endoscopic and histological findings, a (restorative) proctocolectomy was recommended but declined by the patient. Instead, a colectomy was performed. The histological examination revealed an unforeseen GP in addition to the colon cancer. Extensive molecular diagnostics allowed for the differential diagnosis of the causes of the clinical manifestations, and the clinical suspicion of Cowden syndrome could not be confirmed using Sanger Sequencing and MLPA for the analysis of PTEN. Finally, a pathogenic germline mutation in PTEN (heterozygous stop mutation in exon 2: NM_000314 (PTEN):c.138C > A; p.Tyr46*) could be detected by next-generation sequencing (NGS), confirming an unusual presentation of Cowden syndrome with GP. CONCLUSIONS: Cowden syndrome should be considered in cases of GP with extracolonic manifestation and verified by combined clinical and molecular diagnostics. Because GP may represent a premalignant condition, a surgical-oncological prophylactic procedure should be considered. Based on our experience, we recommend early implementation of Panel NGS rather than classical Sanger sequencing for genetic diagnostics, especially if various diagnoses are considered.

Genome-wide analysis associates familial colorectal cancer with increases in copy number variations and a rare structural variation at 12p12.3.

Colorectal cancer (CRC) is one of the most common cancer worldwide. However, a large number of genetic risk factors involved in CRC have not been understood. Copy number variations (CNVs) might partly contribute to the 'missing heritability' of CRC. An increased overall burden of CNV has been identified in several complex diseases, whereas the association between the overall CNV burden and CRC risk is largely unknown. We performed a genome-wide investigation of CNVs on genomic DNA from 384 familial CRC cases and 1285 healthy controls by the Affymetrix 6.0 array. An increase of overall CNV burden was observed in familial CRC patients compared with healthy controls, especially for CNVs larger than 50kb (case/control ratio = 1.66, P = 0.025). In addition, we discovered for the first time a novel structural variation at 12p12.3 and determined the breakpoints by strategic PCR and sequencing. This 12p12.3 structural variation was found in four of 2862 CRC cases but not in 6243 healthy controls (P = 0.0098). RERGL gene (RERG/RAS-like), the only gene influenced by the 12p12.3 structural variation, sharing most of the conserved regions with its close family member RERG tumor suppressor gene (RAS-like, estrogen-regulated, growth inhibitor), might be a novel CRC-related gene. In conclusion, this is the first study to reveal the contribution of the overall burden of CNVs to familial CRC risk and identify a novel rare structural variation at 12p12.3 containing RERGL gene to be associated with CRC.

Evaluating the performance of clinical criteria for predicting mismatch repair gene mutations in Lynch syndrome: a comprehensive analysis of 3,671 families.

Carriers of mismatch repair (MMR) gene mutations have a high lifetime risk for colorectal and endometrial cancers, as well as other malignancies. As mutation analysis to detect these patients is expensive and time-consuming, clinical criteria and tumor-tissue analysis are widely used as pre-screening methods. The aim of our study was to evaluate the performance of commonly applied clinical criteria (the Amsterdam I and II Criteria, and the original and revised Bethesda Guidelines) and the results of tumor-tissue analysis in predicting MMR gene mutations. We analyzed 3,671 families from the German HNPCC Registry and divided them into nine mutually exclusive groups with different clinical criteria. A total of 680 families (18.5%) were found to have a pathogenic MMR gene mutation. Among all 1,284 families with microsatellite instability-high (MSI-H) colorectal cancer, the overall mutation detection rate was 53.0%. Mutation frequencies and their distribution between the four MMR genes differed significantly between clinical groups (p < 0.001). The highest frequencies were found in families fulfilling the Amsterdam Criteria (46.4%). Families with loss of MSH2 expression had higher mutation detection rates (69.5%) than families with loss of MLH1 expression (43.1%). MMR mutations were found significantly more often in families with at least one MSI-H small-bowel cancer (p < 0.001). No MMR mutations were found among patients under 40-years-old with only colorectal adenoma. Familial clustering of Lynch syndrome-related tumors, early age of onset, and familial occurrence of small-bowel cancer were clinically relevant predictors for Lynch syndrome.

Germline mutations in the spindle assembly checkpoint genes BUB1 and BUB3 are risk factors for colorectal cancer.

The spindle assembly checkpoint controls proper chromosome segregation during mitosis and prevents aneuploidy-an important feature of cancer cells. We performed genome-wide and targeted copy number and mutation analyses of germline DNA from 208 patients with familial or early-onset (40 years of age or younger) colorectal cancer; we identified haploinsufficiency or heterozygous mutations in the spindle assembly checkpoint genes BUB1 and BUB3 in 2.9% of them. Besides colorectal cancer, these patients had variegated aneuploidies in multiple tissues and variable dysmorphic features. These results indicate that mutations in BUB1 and BUB3 cause mosaic variegated aneuploidy and increase the risk of colorectal cancer at a young age.

An American founder mutation in MLH1.

Mutations in the mismatch repair genes cause Lynch syndrome (LS), conferring high risk of colorectal, endometrial and some other cancers. After the same splice site mutation in the MLH1 gene (c.589-2A>G) had been observed in four ostensibly unrelated American families with typical LS cancers, its occurrence in comprehensive series of LS cases (Mayo Clinic, Germany and Italy) was determined. It occurred in 10 out of 995 LS mutation carriers (1.0%) diagnosed in the Mayo Clinic diagnostic laboratory. It did not occur among 1,803 cases tested for MLH1 mutations by the German HNPCC consortium, while it occurred in three probands and an additional five family members diagnosed in Italy. In the U.S., the splice site mutation occurs on a large (∼4.8 Mb) shared haplotype that also harbors the variant c.2146G>A, which predicts a missense change in codon 716 referred to here as V716M. In Italy, it occurs on a different, shorter shared haplotype (∼2.2 Mb) that does not carry V716M. The V716M variant was found to be present by itself in the U.S., German and Italian populations with individuals sharing a common haplotype of 280 kb, allowing us to calculate that the variant arose around 5,600 years ago (225 generations; 95% confidence interval 183-272). The splice site mutation in America arose or was introduced some 450 years ago (18 generations; 95% confidence interval 14-23); it accounts for 1.0% all LS in the Unites States and can be readily screened for.

A variant allele of Growth Factor Independence 1 (GFI1) is associated with acute myeloid leukemia.

The GFI1 gene encodes a transcriptional repressor, which regulates myeloid differentiation. In the mouse, Gfi1 deficiency causes neutropenia and an accumulation of granulomonocytic precursor cells that is reminiscent of a myelodysplastic syndrome. We report here that a variant allele of GFI1 (GFI1(36N)) is associated with acute myeloid leukemia (AML) in white subjects with an odds ratio of 1.6 (P < 8 x 10(-5)). The GFI1(36N) variant occurred in 1806 AML patients with an allele frequency of 0.055 compared with 0.035 in 1691 healthy control patients in 2 independent cohorts. We observed that both GFI1 variants maintain the same activity as transcriptional repressors but differ in their regulation by the AML1/ETO (RUNX1/RUNX1T1) fusion protein produced in AML patients with a t(8;21) translocation. AML1/ETO interacts and colocalizes with the more common GFI1(36S) form in the nucleus and inhibits its repressor activity. However, the variant GFI1(36N) protein has a different subnuclear localization than GFI1(36S). As a consequence, AML1/ETO does not colocalize with GFI1(36N) and is unable to inhibit its repressor activity. We conclude that both variants of GFI1 differ in their ability to be regulated by interacting proteins and that the GFI1(36N) variant form exhibits distinct biochemical features that may confer a predisposition to AML.

Germline truncating-mutations in BRCA1 and MSH6 in a patient with early onset endometrial cancer.

BACKGROUND: Hereditary Breast and Ovarian Cancer Syndrome (HBOCS) and Hereditary Non-Polyposis Colorectal Cancer Syndrome (HNPCC, Lynch Syndrome) are two tumor predisposition syndromes responsible for the majority of hereditary breast and colorectal cancers. Carriers of both germline mutations in breast cancer genes BRCA1 or BRCA2 and in mismatch repair (MMR) genes MLH1, MSH2, MSH6 or PMS2 are very rare. CASE PRESENTATION: We identified germline mutations in BRCA1 and in MSH6 in a patient with increased risk for HBOC diagnosed with endometrial cancer at the age of 46 years. CONCLUSIONS: Although carriers of mutations in both MMR and BRCA genes are rare in Caucasian populations and anamnestical and histopathological findings may guide clinicians to identify these families, both syndromes can only be diagnosed through a complete gene analysis of the respective genes.

Aberrant protein expression and frequent allelic loss of MSH3 in colorectal cancer with low-level microsatellite instability.

PURPOSE: High level of microsatellite instability (MSI-H) in colorectal cancer (CRC) is caused by the inactivation of mismatch repair (MMR) genes; however, it is unknown for tumors with low level MSI (MSI-L). The protein complex involving MSH3 preferentially recognizes insertion/deletion loops (IDLs) of two to eight bases and di- and tetranucleotide repeats are affected in the majority of MSI-L CRC. METHODS: We selected 10 and eight MSI-L CRCs from 228 and 204 patients with sporadic and hereditary disease, respectively. The tumors were analyzed for protein expression of MSH3, MSH2, MSH6, MLH1, and PMS2, and for mutations and loss of heterozygosity (LOH) in MSH3. RESULTS: Four tumors showed a markedly reduced MSH3 expression, whereas all 18 tumors had normal expression of the remaining MMR proteins. Twenty-five different sequence variants were identified. None of these results in a truncated protein, though L902W represents the first constitutional missense mutation in MSH3 predicted to be functional based on conservation among mutS homologues. All variants have also been found in normal DNA of the patients and in controls. LOH intragenic to MSH3 was evident for 12 of 16 (75%) informative tumors. CONCLUSIONS: Occurrence of sequence variants in normal DNA of the patients and in controls excludes somatic mutations and mutations specific to the CRC patient population, respectively. In contrast, the high frequency of LOH as well as the aberrant protein expression in some tumors indicates an involvement of MSH3 impairment in MSI-L CRC.

Risk of colorectal and endometrial cancers in EPCAM deletion-positive Lynch syndrome: a cohort study.

BACKGROUND: Lynch syndrome is caused by germline mutations in MSH2, MLH1, MSH6, and PMS2 mismatch-repair genes and leads to a high risk of colorectal and endometrial cancer. We previously showed that constitutional 3' end deletions of EPCAM can cause Lynch syndrome through epigenetic silencing of MSH2 in EPCAM-expressing tissues, resulting in tissue-specific MSH2 deficiency. We aim to establish the risk of cancer associated with such EPCAM deletions. METHODS: We obtained clinical data for 194 carriers of a 3' end EPCAM deletion from 41 families known to us at the Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands and compared cancer risk with data from a previously described cohort of 473 carriers from 91 families with mutations in MLH1, MSH2, MSH6, or a combined EPCAM-MSH2 deletion. FINDINGS: 93 of the 194 EPCAM deletion carriers were diagnosed with colorectal cancer; three of the 92 women with EPCAM deletions were diagnosed with endometrial cancer. Carriers of an EPCAM deletion had a 75% (95% CI 65-85) cumulative risk of colorectal cancer before the age of 70 years (mean age at diagnosis 43 years [SD 12]), which did not differ significantly from that of carriers of combined EPCAM-MSH2 deletion (69% [95% CI 47-91], p=0·8609) or mutations in MSH2 (77% [64-90], p=0·5892) or MLH1 (79% [68-90], p=0·5492), but was higher than noted for carriers of MSH6 mutation (50% [38-62], p<0·0001). By contrast, women with EPCAM deletions had a 12% [0-27] cumulative risk of endometrial cancer, which was lower than was that noted for carriers of a combined EPCAM-MSH2 deletion (55% [20-90], p<0·0001) or of a mutation in MSH2 (51% [33-69], p=0·0006) or MSH6 (34% [20-48], p=0·0309), but did not differ significantly from that noted for MLH1 (33% [15-51], p=0·1193) mutation carriers. This risk seems to be restricted to deletions that extend close to the MSH2 gene promoter. Of 194 carriers of an EPCAM deletion, three had duodenal cancer and four had pancreatic cancer. INTERPRETATION: EPCAM deletion carriers have a high risk of colorectal cancer; only those with deletions extending close to the MSH2 promoter have an increased risk of endometrial cancer. These results underscore the effect of mosaic MSH2 deficiency, leading to variable cancer risks, and could form the basis of an optimised protocol for the recognition and targeted prevention of cancer in EPCAM deletion carriers.

Recurrence and variability of germline EPCAM deletions in Lynch syndrome.

Recently, we identified 3' end deletions in the EPCAM gene as a novel cause of Lynch syndrome. These truncating EPCAM deletions cause allele-specific epigenetic silencing of the neighboring DNA mismatch repair gene MSH2 in tissues expressing EPCAM. Here we screened a cohort of unexplained Lynch-like families for the presence of EPCAM deletions. We identified 27 novel independent MSH2-deficient families from multiple geographical origins with varying deletions all encompassing the 3' end of EPCAM, but leaving the MSH2 gene intact. Within The Netherlands and Germany, EPCAM deletions appeared to represent at least 2.8% and 1.1% of the confirmed Lynch syndrome families, respectively. MSH2 promoter methylation was observed in epithelial tissues of all deletion carriers tested, thus confirming silencing of MSH2 as the causative defect. In a total of 45 families, 19 different deletions were found, all including the last two exons and the transcription termination signal of EPCAM. All deletions appeared to originate from Alu-repeat mediated recombination events. In 17 cases regions of microhomology around the breakpoints were found, suggesting nonallelic homologous recombination as the most likely mechanism. We conclude that 3' end EPCAM deletions are a recurrent cause of Lynch syndrome, which should be implemented in routine Lynch syndrome diagnostics.

Identification of candidate predisposing copy number variants in familial and early-onset colorectal cancer patients.

In the majority of colorectal cancers (CRCs) under clinical suspicion for a hereditary cause, the disease-causing genetic factors are still to be discovered. To identify such genetic factors we stringently selected a discovery cohort of 41 CRC index patients with microsatellite-stable tumors. All patients were below 40 years of age at diagnosis and/or exhibited an overt family history. We employed genome-wide copy number profiling using high-resolution SNP arrays on germline DNA, which resulted in the identification of novel copy number variants (CNVs) in six patients (15%) encompassing, among others, the cadherin gene CDH18, the bone morphogenetic protein antagonist family gene GREM1, and the breakpoint cluster region gene BCR. In addition, two genomic deletions were encountered encompassing two microRNA genes, hsa-mir-491/KIAA1797 and hsa-mir-646/AK309218. None of these CNVs has previously been reported in relation to CRC predisposition in humans, nor were they encountered in large control cohorts (>1,600 unaffected individuals). Since several of these newly identified candidate genes may be functionally linked to CRC development, our results illustrate the potential of this approach for the identification of novel candidate genes involved in CRC predisposition.