Delineating genotype and parent-of-origin effect on the phenotype in MSH6-associated Lynch syndrome.

BACKGROUND: This study investigates the potential influence of genotype and parent-of-origin effects (POE) on the clinical manifestations of Lynch syndrome (LS) within families carrying (likely) disease-causing MSH6 germline variants. PATIENTS AND METHODS: A cohort of 1615 MSH6 variant carriers (310 LS families) was analyzed. Participants were categorized based on RNA expression and parental inheritance of the variant. Hazard ratios (HRs) were calculated using weighted Cox regression, considering external information to address ascertainment bias. The findings were cross-validated using the Prospective Lynch Syndrome Database (PLSD) for endometrial cancer (EC). RESULTS: No significant association was observed between genotype and colorectal cancer (CRC) risk (HR = 1.06, 95% confidence interval [CI]: 0.77-1.46). Patients lacking expected RNA expression exhibited a reduced risk of EC (Reference Cohort 1: HR = 0.68, 95% CI: 0.43-1.03; Reference Cohort 2: HR = 0.63, 95% CI: 0.46-0.87). However, these results could not be confirmed in the PLSD. Moreover, no association was found between POE and CRC risk (HR = 0.78, 95% CI: 0.52-1.17) or EC risk (Reference Cohort 1: HR = 0.93, 95% CI: 0.65-1.33; Reference Cohort 2: HR = 0.8, 95% CI: 0.64-1.19). DISCUSSION AND CONCLUSION: No evidence of POE was detected in MSH6 families. While RNA expression may be linked to varying risks of EC, further investigation is required to explore this observation.

Discordant Staining Patterns and Microsatellite Results in Tumors of MSH6 Pathogenic Variant Carriers.

Diagnosis of Lynch syndrome (LS) caused by a pathogenic germline MSH6 variant may be complicated by discordant immunohistochemistry (IHC) and/or by a microsatellite stable (MSS) phenotype. This study aimed to identify the various causes of the discordant phenotypes of colorectal cancer (CRC) and endometrial cancer (EC) in MSH6-associated LS. Data were collected from Dutch family cancer clinics. Carriers of a (likely) pathogenic MSH6 variant diagnosed with CRC or EC were categorized based on an microsatellite instability (MSI)/IHC test outcome that might fail to result in a diagnosis of LS (eg, retained staining of all 4 mismatch repair proteins, with or without an MSS phenotype, and other staining patterns). When tumor tissue was available, MSI and/or IHC were repeated. Next-generation sequencing (NGS) was performed in cases with discordant staining patterns. Data were obtained from 360 families with 1763 (obligate) carriers. MSH6 variant carriers with CRC or EC (n = 590) were included, consisting of 418 CRCs and 232 ECs. Discordant staining was reported in 77 cases (36% of MSI/IHC results). Twelve patients gave informed consent for further analysis of tumor material. Upon revision, 2 out of 3 MSI/IHC cases were found to be concordant with the MSH6 variant, and NGS showed that 4 discordant IHC results were sporadic rather than LS-associated tumors. In 1 case, somatic events explained the discordant phenotype. The use of reflex IHC mismatch repair testing, the current standard in most Western countries, may lead to the misdiagnosis of germline MSH6 variant carriers. The pathologist should point out that further diagnostics for inheritable colon cancer, including LS, should be considered in case of a strong positive family history. Germline DNA analysis of the mismatch repair genes, preferably as part of a larger gene panel, should therefore be considered in potential LS patients.

Clinical value of a screening tool for tumor predisposition syndromes in childhood cancer patients (TuPS): a prospective, observational, multi-center study.

Recognizing a tumor predisposition syndrome (TPS) in a child with cancer is of clinical relevance. Earlier we developed a screening tool to increase diagnostic accuracy and clinical efficiency of identifying TPSs in children with cancer. Here we report on the value of this tool in clinical practice. TuPS is a prospective, observational, multi-center study including children newly diagnosed with cancer from 2016 to 2019 in the Netherlands. Children in whom a TPS had been diagnosed before the cancer diagnosis were excluded. The screening tool consists of a checklist, 2D and 3D photographic series and digital assessment of these by a clinical geneticist. If a TPS was suspected, the patient was assessed positive and referred for routine genetic consultation. Primary aim was to assess the clinical value of this new screening tool. Of the 363 included patients, 57% (208/363) were assessed positive. In 15% of patients (32/208), the 2D photographic series with (n = 12) or without (n = 20) 3D photographs were decisive in the positive assessment. In 2% (4/208) of positive assessed patients, a TPS was diagnosed, and in an additional 2% (4/208) a germline variant of uncertain significance was found. Thirty-five negatively assessed patients were evaluated through routine genetic consultation as controls, in none a TPS was detected. Using the screening tool, 57% of the patients were assessed as suspected for having a TPS. No false negative results were identified in the negative control group in the clinical care setting. The observed prevalence of TPS was lower than expected, due to selection bias in the cohort.

The Apparent Genetic Anticipation in PMS2-Associated Lynch Syndrome Families Is Explained by Birth-cohort Effect.

BACKGROUND: PMS2-associated Lynch syndrome is characterized by a relatively low colorectal cancer penetrance compared with other Lynch syndromes. However, age at colorectal cancer diagnosis varies widely, and a strong genetic anticipation effect has been suggested for PMS2 families. In this study, we examined proposed genetic anticipation in a sample of 152 European PMS2 families. METHODS: The 152 families (637 family members) that were eligible for analysis were mainly clinically ascertained via clinical genetics centers. We used weighted Cox-type random effects model, adjusted by birth cohort and sex, to estimate the generational effect on the age of onset of colorectal cancer. Probands and young birth cohorts were excluded from the analyses. Weights represented mutation probabilities based on kinship coefficients, thus avoiding testing bias. RESULTS: Family data across three generations, including 123 colorectal cancers, were analyzed. When compared with the first generation, the crude HR for anticipation was 2.242 [95% confidence interval (CI), 1.162-4.328] for the second generation and 2.644 (95% CI, 1.082-6.464) for the third generation. However, after correction for birth cohort and sex, the effect vanished [HR = 1.302 (95% CI, 0.648-2.619) and HR = 1.074 (95% CI, 0.406-2.842) for second and third generations, respectively]. CONCLUSIONS: Our study did not confirm previous reports of genetic anticipation in PMS2-associated Lynch syndrome. Birth-cohort effect seems the most likely explanation for observed younger colorectal cancer diagnosis in subsequent generations, particularly because there is currently no commonly accepted biological mechanism that could explain genetic anticipation in Lynch syndrome. IMPACT: This new model for studying genetic anticipation provides a standard for rigorous analysis of families with dominantly inherited cancer predisposition.

Cancer Risks for PMS2-Associated Lynch Syndrome.

PURPOSE: Lynch syndrome due to pathogenic variants in the DNA mismatch repair genes MLH1, MSH2, and MSH6 is predominantly associated with colorectal and endometrial cancer, although extracolonic cancers have been described within the Lynch tumor spectrum. However, the age-specific cumulative risk (penetrance) of these cancers is still poorly defined for PMS2-associated Lynch syndrome. Using a large data set from a worldwide collaboration, our aim was to determine accurate penetrance measures of cancers for carriers of heterozygous pathogenic PMS2 variants. METHODS: A modified segregation analysis was conducted that incorporated both genotyped and nongenotyped relatives, with conditioning for ascertainment to estimates corrected for bias. Hazard ratios (HRs) and corresponding 95% CIs were estimated for each cancer site for mutation carriers compared with the general population, followed by estimation of penetrance. RESULTS: In total, 284 families consisting of 4,878 first- and second-degree family members were included in the analysis. PMS2 mutation carriers were at increased risk for colorectal cancer (cumulative risk to age 80 years of 13% [95% CI, 7.9% to 22%] for males and 12% [95% CI, 6.7% to 21%] for females) and endometrial cancer (13% [95% CI, 7.0%-24%]), compared with the general population (6.6%, 4.7%, and 2.4%, respectively). There was no clear evidence of an increased risk of ovarian, gastric, hepatobiliary, bladder, renal, brain, breast, prostate, or small bowel cancer. CONCLUSION: Heterozygous PMS2 mutation carriers were at small increased risk for colorectal and endometrial cancer but not for any other Lynch syndrome-associated cancer. This finding justifies that PMS2-specific screening protocols could be restricted to colonoscopies. The role of risk-reducing hysterectomy and bilateral salpingo-oophorectomy for PMS2 mutation carriers needs further discussion.

Molecular Background of Colorectal Tumors From Patients With Lynch Syndrome Associated With Germline Variants in PMS2.

BACKGROUND & AIMS: Germline variants in mismatch repair genes MLH1, MSH2 (EPCAM), MSH6, or PMS2 cause Lynch syndrome. Patients with these variants have an increased risk of developing colorectal cancers (CRCs) that differ from sporadic CRCs in genetic and histologic features. It has been a challenge to study CRCs associated with PMS2 variants (PMS2-associated CRCs) because these develop less frequently and in older patients than CRCs with variants in other mismatch repair genes. METHODS: We analyzed 20 CRCs associated with germline variants in PMS2, 22 sporadic CRCs, 18 CRCs with germline variants in MSH2, and 24 CRCs from patients with germline variants in MLH1. Tumor tissue blocks were collected from Dutch pathology departments in 2017. After extraction of tumor DNA, we used a platform designed to detect approximately 3,000 somatic hotspot variants in 55 genes (including KRAS, APC, CTNNB1, and TP53). Somatic variant frequencies were compared using the Fisher exact test. RESULTS: None of the PMS2-associated CRCs contained any somatic variants in the catenin-ß1 gene (CTNNB1), which encodes ß-catenin, whereas 14 of 24 MLH1-associated CRCs (58%) contained variants in CTNNB1. Half the PMS2-associated CRCs contained KRAS variants, but only 20% of these were in hotspots that encoded G12D or G13D. These hotspot variants occurred more frequently in CRCs associated with variants in MLH1 (37.5%; P = .44) and MSH2 (71.4%; P = .035) than in those associated with variants in PMS2. CONCLUSIONS: In a genetic analysis of 84 colorectal tumors, we found tumors from patients with PMS2-associated Lynch syndrome to be distinct from colorectal tumors associated with defects in other mismatch repair genes. This might account for differences in development and less frequent occurrence.

SNP association study in PMS2-associated Lynch syndrome.

Lynch syndrome (LS) patients are at high risk of developing colorectal cancer (CRC). Phenotypic variability might in part be explained by common susceptibility loci identified in Genome Wide Association Studies (GWAS). Previous studies focused mostly on MLH1, MSH2 and MSH6 carriers, with conflicting results. We aimed to determine the role of GWAS SNPs in PMS2 mutation carriers. A cohort study was performed in 507 PMS2 carriers (124 CRC cases), genotyped for 24 GWAS SNPs, including SNPs at 11q23.1 and 8q23.3. Hazard ratios (HRs) were calculated using a weighted Cox regression analysis to correct for ascertainment bias. Discrimination was assessed with a concordance statistic in a bootstrap cross-validation procedure. Individual SNPs only had non-significant associations with CRC occurrence with HRs lower than 2, although male carriers of allele A at rs1321311 (6p21.31) may have increased risk of CRC (HR = 2.1, 95% CI 1.2-3.0). A polygenic risk score (PRS) based on 24 HRs had an HR of 2.6 (95% CI 1.5-4.6) for the highest compared to the lowest quartile, but had no discriminative ability (c statistic 0.52). Previously suggested SNPs do not modify CRC risk in PMS2 carriers. Future large studies are needed for improved risk stratification among Lynch syndrome patients.

Sending family history questionnaires to patients before a colonoscopy improves genetic counseling for hereditary colorectal cancer.

OBJECTIVES: To investigate whether sending a family history questionnaire to patients prior to undergoing colonoscopy results in an increased availability of family history and better genetic counseling. METHODS: A questionnaire was mailed to patients before they underwent outpatient colonoscopy at a university hospital in 2013. These patients' additional characteristics and referral for genetic evaluation were retrieved from the electronic medical records. Patients undergoing inpatient coloboscopy, with confirmed hereditary colorectal cancer (CRC) or inflammatory bowel disease were excluded. All study patients from 2010 to 2013 were matched with the database of the genetics department to determine who consulted a geneticist. RESULTS: A total of 6163 patients underwent colonoscopy from 2010 to 2013. Of 1421 who underwent colonoscopy in 2013, 53 (3.7%) consulted a geneticist, while 75 (1.6%) of 4742 patients undergoing colonoscopy between 2010 and 2012 did so (P < 0.01). A total of 974 patients undergoing colonoscopy in 2013 were included to evaluate the completed questionnaire. Of these, 282 (29.0%) completed the questionnaire. Family history was not recorded in the electronic medical records of 393 (40.3%). In 129 (32.8%), family history was obtained from the completed questionnaire. In 2013, 49 (60.5%) out of 81 patients referred for genetic counseling were referred based on their family history. Eight (9.9%) patients were referred based on the completed questionnaire. CONCLUSION: Screening for hereditary CRC in a population undergoing outpatient colonoscopy with a questionnaire sent by mail resulted in an increased availability of family histories and genetic counseling.

Childhood tumours with a high probability of being part of a tumour predisposition syndrome; reason for referral for genetic consultation.

INTRODUCTION: Recognising a tumour predisposition syndrome (TPS) in childhood cancer patients is of major clinical relevance. The presence of a TPS may be suggested by the type of tumour in the child. We present an overview of 23 childhood tumours that in themselves should be a reason to refer a child for genetic consultation. METHODS: We performed a PubMed search to review the incidence of TPSs in children for 85 tumour types listed in the International Classification of Childhood Cancer third edition (ICCC-3). The results were discussed during a national consensus meeting with representative clinical geneticists from all six academic paediatric oncology centres in The Netherlands. A TPS incidence of 5% or more was considered a high probability and therefore in itself a reason for referral to a clinical geneticist. RESULTS: The literature search resulted in data on the incidence of a TPS in 26 tumours. For 23/26 tumour types, a TPS incidence of 5% or higher was reported. In addition, during the consensus meeting the experts agreed that children with any carcinoma should always be referred for clinical genetic consultation as well, as it may point to a TPS. CONCLUSION: We present an overview of 23 paediatric tumours with a high probability of a TPS; this will facilitate paediatric oncologists to decide which patients should be referred for genetic consultation merely based on type of tumour.

Validation of a clinical screening instrument for tumour predisposition syndromes in patients with childhood cancer (TuPS): protocol for a prospective, observational, multicentre study.

INTRODUCTION: Recognising a tumour predisposition syndrome (TPS) in patients with childhood cancer is of significant clinical relevance, as it affects treatment, prognosis and facilitates genetic counselling. Previous studies revealed that only half of the known TPSs are recognised during standard paediatric cancer care. In current medical practice it is impossible to refer every patient with childhood cancer to a clinical geneticist, due to limited capacity for routine genetic consultation. Therefore, we have developed a screening instrument to identify patients with childhood cancer with a high probability of having a TPS. The aim of this study is to validate the clinical screening instrument for TPS in patients with childhood cancer. METHODS AND ANALYSIS: This study is a prospective nationwide cohort study including all newly diagnosed patients with childhood cancer in the Netherlands. The screening instrument consists of a checklist, two- and three-dimensional photographic series of the patient. 2 independent clinical geneticists will assess the content of the screening instrument. If a TPS is suspected based on the instrument data and thus further evaluation is indicated, the patient will be invited for full genetic consultation. A negative control group consists of 20% of the patients in whom a TPS is not suspected based on the instrument; they will be randomly invited for full genetic consultation. Primary outcome measurement will be sensitivity of the instrument. ETHICS AND DISSEMINATION: The Medical Ethical Committee of the Academic Medical Centre stated that the Medical Research Involving Human Subjects Act does not apply to this study and that official approval of this study by the Committee was not required. The results will be offered for publication in peer-reviewed journals and presented at International Conferences on Oncology and Clinical Genetics. The clinical data gathered in this study will be available for all participating centres. TRIAL REGISTRATION NUMBER: NTR5605.

Comprehensive Mutation Analysis of PMS2 in a Large Cohort of Probands Suspected of Lynch Syndrome or Constitutional Mismatch Repair Deficiency Syndrome.

Monoallelic PMS2 germline mutations cause 5%-15% of Lynch syndrome, a midlife cancer predisposition, whereas biallelic PMS2 mutations cause approximately 60% of constitutional mismatch repair deficiency (CMMRD), a rare childhood cancer syndrome. Recently improved DNA- and RNA-based strategies are applied to overcome problematic PMS2 mutation analysis due to the presence of pseudogenes and frequent gene conversion events. Here, we determined PMS2 mutation detection yield and mutation spectrum in a nationwide cohort of 396 probands. Furthermore, we studied concordance between tumor IHC/MSI (immunohistochemistry/microsatellite instability) profile and mutation carrier state. Overall, we found 52 different pathogenic PMS2 variants explaining 121 Lynch syndrome and nine CMMRD patients. In vitro mismatch repair assays suggested pathogenicity for three missense variants. Ninety-one PMS2 mutation carriers (70%) showed isolated loss of PMS2 in their tumors, for 31 (24%) no or inconclusive IHC was available, and eight carriers (6%) showed discordant IHC (presence of PMS2 or loss of both MLH1 and PMS2). Ten cases with isolated PMS2 loss (10%; 10/97) harbored MLH1 mutations. We confirmed that recently improved mutation analysis provides a high yield of PMS2 mutations in patients with isolated loss of PMS2 expression. Application of universal tumor prescreening methods will however miss some PMS2 germline mutation carriers.

Whole Gene Capture Analysis of 15 CRC Susceptibility Genes in Suspected Lynch Syndrome Patients.

BACKGROUND AND AIMS: Lynch Syndrome (LS) is caused by pathogenic germline variants in one of the mismatch repair (MMR) genes. However, up to 60% of MMR-deficient colorectal cancer cases are categorized as suspected Lynch Syndrome (sLS) because no pathogenic MMR germline variant can be identified, which leads to difficulties in clinical management. We therefore analyzed the genomic regions of 15 CRC susceptibility genes in leukocyte DNA of 34 unrelated sLS patients and 11 patients with MLH1 hypermethylated tumors with a clear family history. METHODS: Using targeted next-generation sequencing, we analyzed the entire non-repetitive genomic sequence, including intronic and regulatory sequences, of 15 CRC susceptibility genes. In addition, tumor DNA from 28 sLS patients was analyzed for somatic MMR variants. RESULTS: Of 1979 germline variants found in the leukocyte DNA of 34 sLS patients, one was a pathogenic variant (MLH1 c.1667+1delG). Leukocyte DNA of 11 patients with MLH1 hypermethylated tumors was negative for pathogenic germline variants in the tested CRC susceptibility genes and for germline MLH1 hypermethylation. Somatic DNA analysis of 28 sLS tumors identified eight (29%) cases with two pathogenic somatic variants, one with a VUS predicted to pathogenic and LOH, and nine cases (32%) with one pathogenic somatic variant (n = 8) or one VUS predicted to be pathogenic (n = 1). CONCLUSIONS: This is the first study in sLS patients to include the entire genomic sequence of CRC susceptibility genes. An underlying somatic or germline MMR gene defect was identified in ten of 34 sLS patients (29%). In the remaining sLS patients, the underlying genetic defect explaining the MMRdeficiency in their tumors might be found outside the genomic regions harboring the MMR and other known CRC susceptibility genes.

Combined mismatch repair and POLE/POLD1 defects explain unresolved suspected Lynch syndrome cancers.

Many suspected Lynch Syndrome (sLS) patients who lack mismatch repair (MMR) germline gene variants and MLH1 or MSH2 hypermethylation are currently explained by somatic MMR gene variants or, occasionally, by germline POLE variants. To further investigate unexplained sLS patients, we analyzed leukocyte and tumor DNA of 62 sLS patients using gene panel sequencing including the POLE, POLD1 and MMR genes. Forty tumors showed either one, two or more somatic MMR variants predicted to affect function. Nine sLS tumors showed a likely ultramutated phenotype and were found to carry germline (n=2) or somatic variants (n=7) in the POLE/POLD1 exonuclease domain (EDM). Six of these POLE/POLD1-EDM mutated tumors also carried somatic MMR variants. Our findings suggest that faulty proofreading may result in loss of MMR and thereby in microsatellite instability.

The effect of genotypes and parent of origin on cancer risk and age of cancer development in PMS2 mutation carriers.

PURPOSE: Lynch syndrome (LS), a heritable disorder with an increased risk of primarily colorectal cancer (CRC) and endometrial cancer (EC), can be caused by mutations in the PMS2 gene. We wished to establish whether genotype and/or parent-of-origin effects (POE) explain (part of) the reported variability in severity of the phenotype. METHODS: European PMS2 mutation carriers (n = 381) were grouped and compared based on RNA expression and whether the mutation was inherited paternally or maternally. RESULTS: Mutation carriers with loss of RNA expression (group 1) had a significantly lower age at CRC diagnosis (51.1 years vs. 60.0 years, P = 0.035) and a lower age at EC diagnosis (55.8 years vs. 61.0 years, P = 0.2, nonsignificant) compared with group 2 (retention of RNA expression). Furthermore, group 1 showed slightly higher, but nonsignificant, hazard ratios (HRs) for both CRC (HR: 1.31, P = 0.38) and EC (HR: 1.22, P = 0.72). No evidence for a significant parent-of-origin effect was found for either CRC or EC. CONCLUSIONS: PMS2 mutation carriers with retention of RNA expression developed CRC 9 years later than those with loss of RNA expression. If confirmed, this finding would justify a delay in surveillance for these cases. Cancer risk was not influenced by a parent-of-origin effect.Genet Med 18 4, 405-409.

Genetic variation in the functional ENG allele inherited from the non-affected parent associates with presence of pulmonary arteriovenous malformation in hereditary hemorrhagic telangiectasia 1 (HHT1) and may influence expression of PTPN14.

HHT shows clinical variability within and between families. Organ site and prevalence of arteriovenous malformations (AVMs) depend on the HHT causative gene and on environmental and genetic modifiers. We tested whether variation in the functional ENG allele, inherited from the unaffected parent, alters risk for pulmonary AVM in HHT1 mutation carriers who are ENG haploinsufficient. Genetic association was found between rs10987746 of the wild type ENG allele and presence of pulmonary AVM [relative risk = 1.3 (1.0018-1.7424)]. The rs10987746-C at-risk allele associated with lower expression of ENG RNA in a panel of human lymphoblastoid cell lines (P = 0.004). Moreover, in angiogenically active human lung adenocarcinoma tissue, but not in uninvolved quiescent lung, rs10987746-C was correlated with expression of PTPN14 (P = 0.004), another modifier of HHT. Quantitative TAQMAN expression analysis in a panel of normal lung tissues from 69 genetically heterogeneous inter-specific backcross mice, demonstrated strong correlation between expression levels of Eng, Acvrl1, and Ptpn14 (r2 = 0.75-0.9, P < 1 × 10(-12)), further suggesting a direct or indirect interaction between these three genes in lung in vivo. Our data indicate that genetic variation within the single functional ENG gene influences quantitative and/or qualitative differences in ENG expression that contribute to risk of pulmonary AVM in HHT1, and provide correlative support for PTPN14 involvement in endoglin/ALK1 lung biology in vivo. PTPN14 has been shown to be a negative regulator of Yap/Taz signaling, which is implicated in mechanotransduction, providing a possible molecular link between endoglin/ALK1 signaling and mechanical stress. EMILIN2, which showed suggestive genetic association with pulmonary AVM, is also reported to interact with Taz in angiogenesis. Elucidation of the molecular mechanisms regulating these interactions in endothelial cells may ultimately provide more rational choices for HHT therapy.

Lynch syndrome caused by germline PMS2 mutations: delineating the cancer risk.

PURPOSE: The clinical consequences of PMS2 germline mutations are poorly understood compared with other Lynch-associated mismatch repair gene (MMR) mutations. The aim of this European cohort study was to define the cancer risk faced by PMS2 mutation carriers. METHODS: Data were collected from 98 PMS2 families ascertained from family cancer clinics that included a total of 2,548 family members and 377 proven mutation carriers. To adjust for potential ascertainment bias, a modified segregation analysis model was used to calculate colorectal cancer (CRC) and endometrial cancer (EC) risks. Standardized incidence ratios (SIRs) were calculated to estimate risks for other Lynch syndrome-associated cancers. RESULTS: The cumulative risk (CR) of CRC for male mutation carriers by age 70 years was 19%. The CR among female carriers was 11% for CRC and 12% for EC. The mean age of CRC development was 52 years, and there was a significant difference in mean age of CRC between the probands (mean, 47 years; range, 26 to 68 years) and other family members with a PMS2 mutation (mean, 58 years; range, 31 to 86 years; P < .001). Significant SIRs were observed for cancers of the small bowel, ovaries, breast, and renal pelvis. CONCLUSION: CRC and EC risks were found to be markedly lower than those previously reported for the other MMR. However, these risks embody the isolated risk of carrying a PMS2 mutation, and it should be noted that we observed a substantial variation in cancer phenotype within and between families, suggesting the influence of genetic modifiers and lifestyle factors on cancer risks.

Genetic variants of Adam17 differentially regulate TGFß signaling to modify vascular pathology in mice and humans.

Outcome of TGFß1 signaling is context dependent and differs between individuals due to germ-line genetic variation. To explore innate genetic variants that determine differential outcome of reduced TGFß1 signaling, we dissected the modifier locus Tgfbm3, on mouse chromosome 12. On a NIH/OlaHsd genetic background, the Tgfbm3b(C57) haplotype suppresses prenatal lethality of Tgfb1(-/-) embryos and enhances nuclear accumulation of mothers against decapentaplegic homolog 2 (Smad2) in embryonic cells. Amino acid polymorphisms within a disintegrin and metalloprotease 17 (Adam17) can account, at least in part, for this Tgfbm3b effect. ADAM17 is known to down-regulate Smad2 signaling by shedding the extracellular domain of TGFßRI, and we show that the C57 variant is hypomorphic for down-regulation of Smad2/3-driven transcription. Genetic variation at Tgfbm3 or pharmacological inhibition of ADAM17, modulates postnatal circulating endothelial progenitor cell (CEPC) numbers via effects on TGFßRI activity. Because CEPC numbers correlate with angiogenic potential, this suggests that variant Adam17 is an innate modifier of adult angiogenesis, acting through TGFßR1. To determine whether human ADAM17 is also polymorphic and interacts with TGFß signaling in human vascular disease, we investigated hereditary hemorrhagic telangiectasia (HHT), which is caused by mutations in TGFß/bone morphogenetic protein receptor genes, ENG, encoding endoglin (HHT1), or ACVRL1 encoding ALK1 (HHT2), and considered a disease of excessive abnormal angiogenesis. HHT manifests highly variable incidence and severity of clinical features, ranging from small mucocutaneous telangiectases to life-threatening visceral and cerebral arteriovenous malformations (AVMs). We show that ADAM17 SNPs associate with the presence of pulmonary AVM in HHT1 but not HHT2, indicating genetic variation in ADAM17 can potentiate a TGFß-regulated vascular disease.

Colorectal cancer risk variants on 11q23 and 15q13 are associated with unexplained adenomatous polyposis.

BACKGROUND: Colorectal adenomatous polyposis is associated with a high risk of colorectal cancer (CRC) and is frequently caused by germline mutations in APC or MUTYH. However, in about 20-30% of patients no underlying gene defect can be identified. In this study, we tested if recently identified CRC risk variants play a role in patients with >10 adenomas. METHODS: We analysed a total of 16 SNPs with a reported association with CRC in a cohort of 252 genetically unexplained index patients with >10 colorectal adenomas and 745 controls. In addition, we collected detailed clinical information from index patients and their first-degree relatives (FDRs). RESULTS: We found a statistically significant association with two of the variants tested: rs3802842 (at chromosome 11q23, OR=1.60, 95% CI 1.3 to 2.0) and rs4779584 (at chromosome 15q13, OR=1.50, 95% CI 1.2 to 1.9). The majority of index patients (84%) had between 10 and 100 adenomas and 15% had >100 adenomas. Only two index patients (1%), both with >100 adenomas, had FDRs with polyposis. Forty-one per cent of the index patients had one or more FDRs with CRC. CONCLUSIONS: These SNPs are the first common, low-penetrant variants reported to be associated with adenomatous polyposis not caused by a defect in the APC, MUTYH, POLD1 and POLE genes. Even though familial occurrence of polyposis was very rare, CRC was over-represented in FDRs of polyposis patients and, if confirmed, these relatives will therefore benefit from surveillance.

Polymorphisms in ACVRL1 and endoglin genes are not associated with sporadic and HHT-related brain AVMs in Dutch patients.

We aimed to replicate the association of the IVS3-35A>G polymorphism in the activin receptor-like kinase (ACVRL) 1 gene and the 207G>A polymorphism in the endoglin (ENG) gene with sporadic brain arteriovenous malformations (BAVM) in Dutch BAVM patients. In addition, we assessed whether these polymorphisms contribute to the risk of BAVM in patients with hereditary haemorrhagic telangiectasia type 1 (HHT1). We genotyped 143 Dutch sporadic BAVM patients and 360 healthy volunteers for four variants in the ACVRL1 gene including IVS3-35A>G and two variants in the ENG gene including 207G>A. Differences in allele and genotype frequencies between sporadic BAVM patients and controls and their combined effect were analysed with a likelihood ratio test. Furthermore, we compared the allele and genotype frequencies between 24 HHT1 patients with a BAVM with those of a relative with HHT1 without a BAVM in a matched pair analysis using Wilcoxon signed rank test. No significant differences in allele frequency were found between sporadic BAVM cases and controls or between HHT1 patients with and without BAVM for any of the polymorphisms or the combination of ACVRL1 and ENG polymorphisms. Meta-analysis of the current and the two previous studies for the ACVRL1 IVS3-35A polymorphism showed a persisting association between the ACVRL1 IVS3-35A polymorphism and risk of sporadic BAVM (odds ratio, 1.86; 95% CI: 1.32-2.61, p<0.001). We did not replicate the previously found association between a polymorphism in ACVRL1 IVS3-35A>G and BAVM in Dutch patients. However, meta-analysis did not rule out a possible effect.

Adherence to microsatellite instability testing in young-onset colorectal cancer patients.

BACKGROUND: In 1997, the Bethesda guidelines recommended microsatellite instability testing for colorectal cancer in patients younger than 45 years to screen for Lynch syndrome. In 2004, these guidelines were revised to set the screening age at younger than 50 years. OBJECTIVE: The aim of this study was to investigate to what extent these guidelines were followed in young patients with colorectal cancer in the Mid-Netherlands and to identify the predictors of nonadherence. DESIGN: This is a retrospective cohort study. SETTINGS: This study was conducted in 1 academic and 5 nonacademic hospitals. PATIENTS: All patients diagnosed with colorectal cancer younger than 45 years in the period 1999 to 2004 and younger than 50 years in the period 2005 to 2008 were included. Patients known to be affected by or at risk for Lynch syndrome before diagnosis were excluded. MAIN OUTCOME MEASURES: Patient and tumor characteristics, including microsatellite instability testing results, were collected from the database of the Comprehensive Cancer Center, the National Pathological Archive, participating hospitals, and the regional institute of clinical genetics. Logistic regression analysis was performed to detect a trend in adherence over the years and to identify the predictors of nonadherence. RESULTS: A total of 335 patients were identified. Microsatellite instability testing was performed in 130/335 (39%) patients. Adherence did not improve in the period 1999 to 2008. We found that older age at diagnosis (OR 0.96, 95% CI 0.92-1.00), male sex (OR 0.60, 95% CI 0.38-0.95), and stage IV colorectal cancer (OR 0.45, 95% CI 0.24-0.84) were independent predictors of nonadherence, whereas proximal tumor localization, poor differentiation, and mucinous histology were not. LIMITATIONS: This study was limited by its retrospective design. CONCLUSIONS: Adherence to the Bethesda guidelines in young-onset colorectal cancer is low, particularly in older and male patients and in patients with metastatic disease, which suggests that efforts to improve adherence are needed.