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STUDY QUESTION Is there a relationship between karyotype abnormalities in fetuses and children conceived by ICSI and their father’s semen parameters? SUMMARY ANSWER The de novo chromosomal abnormality rate in pre- and postnatal karyotypes of ICSI offspring was higher than in the general population and related to fathers’ sperm parameters. WHAT IS KNOWN ALREADY Several studies have reported a higher rate of de novo chromosomal anomalies in ICSI fetuses but recent data from large cohorts are limited. Overall, reported prevalences of non-inherited karyotype aberrations are increased in fetuses conceived after ICSI and vary between 1.6% and 4.2%. Only a few studies focus on the relation between karyotype anomalies in ICSI offspring and semen parameters of their fathers. Furthermore, an increased incidence of abnormal karyotypes in ICSI neonates has been described, but the rates vary widely across studies. STUDY DESIGN, SIZE, DURATION We report on karyotype results from prenatal testing by means of chorionic villus sampling and amniocentesis and results from postnatal blood sampling in offspring conceived by ICSI in a single center. Ongoing pregnancies resulting from an oocyte retrieval between January 2004 and December 2012 and after transfer of fresh ICSI embryos obtained using ejaculated or non-ejaculated sperm (fresh or frozen-thawed) were considered. Pregnancies following frozen embryo transfer, oocyte or sperm donation, IVF, preimplantation genetic testing and IVM were excluded. All abnormal prenatal results after sampling are reported irrespective of the outcome of the pregnancy. PARTICIPANTS/MATERIALS, SETTING, METHODS From the 4816 ongoing ICSI pregnancies, information on pregnancy outcome was available for 4267 pregnancies. Prenatal testing was performed in 22.3% of the pregnancies, resulting in a diagnosis in 1114 fetuses. A postnatal karyotype was obtained in 29.4% of the pregnancies in which no invasive prenatal diagnosis was performed, resulting in a total of 1391 neonates sampled. The prevalence of chromosomal anomalies according to maternal age and semen quality was analyzed with logistic regression. For definitions of normal semen quality, the World Health Organization reference values for human semen characteristics were adopted. MAIN RESULTS AND THE ROLE OF CHANCE An abnormal fetal karyotype was found in 29 singletons and 12 multiples (41/1114; 3.7%; 95% CI 2.7–4.9%): 36 anomalies were de novo (3.2%; 95% CI 2.3–4.4), either numerical (n = 25), sex (n = 6) or structural (n = 5), and five were inherited. Logistic regression analysis did not show a significant association between maternal age and a de novo chromosomal fetal abnormality (odds ratio (OR) 1.05; 95% CI 0.96–1.15; P = 0.24). In all but one case, fetuses with an abnormal karyotype were conceived by ICSI using ejaculated sperm. Abnormal karyotypes were found in 14 (1.0%; 95% CI 0.6–1.7) out of 1391 postnatal samples of children born after ICSI who were not tested prenatally: 12 were de novo anomalies and two were inherited balanced karyotypes. The 14 abnormal karyotypes were all found in children born after ICSI using ejaculated sperm. The odds of a de novo karyotype aberration increased with maternal age when combining pre- and postnatal data (OR 1.11; 95% CI 1.04–1.19). A higher rate of de novo chromosomal abnormalities was found in fetuses and children of couples with men having a sperm concentration LIMITATIONS, REASONS FOR CAUTION We cannot exclude that the observation of a higher prevalence of karyotype anomalies in ICSI offspring compared to literature data in the general population is due to enhanced surveillance after ART given the lack of a control group. Although we did not find more chromosomal anomalies after ICSI with non-ejaculated sperm, the small numbers do not allow firm conclusions. WIDER IMPLICATIONS OF THE FINDINGS The observed increased risk of a de novo karyotype anomaly after ICSI conception in couples with poor sperm warrants continued counseling toward prenatal testing. The current and widespread use of innovative non-invasive prenatal testing will result in larger datasets, adding to a balanced estimation of the prevalence of karyotype anomalies in ICSI offspring. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by the Methusalem grants issued by the Vrije Universiteit Brussel. All authors declared no conflict of interest related to this study. TRIAL REGISTRATION NUMBER N/A

International audience; Purpose: Juvenile polyposis syndrome (JPS) is a rare, autosomal-dominantly inherited cancer predisposition caused in approximately 50% of cases by pathogenic germline variants in SMAD4 and BMPR1A. We aimed to gather detailed clinical and molecular genetic information on JPS disease expression to provide a basis for management guidelines and establish open access variant databases.Methods: We performed a retrospective, questionnaire-based European multicenter survey on and established a cohort of SMAD4/BMPR1A pathogenic variant carriers from the medical literature.Results: We analyzed questionnaire-based data on 221 JPS patients (126 kindreds) from ten European centers and retrieved literature-based information on 473 patients. Compared with BMPR1A carriers, SMAD4 carriers displayed anemia twice as often (58% vs. 26%), and exclusively showed overlap symptoms with hemorrhagic telangiectasia (32%) and an increased prevalence (39% vs. 13%) of gastric juvenile polyps. Cancer, reported in 15% of JPS patients (median age 41 years), mainly occurred in the colorectum (overall: 62%, SMAD4: 58%, BMPR1A: 88%) and the stomach (overall: 21%; SMAD4: 27%, BMPR1A: 0%).Conclusion: This comprehensive retrospective study on genotype-phenotype correlations in 694 JPS patients corroborates previous observations on JPS in general and SMAD4 carriers in particular, facilitates recommendations for clinical management, and provides the basis for open access variant SMAD4 and BMPR1A databases.

This study aimed to determine the prevalence of APC-associated familial adenomatous polyposis (FAP) and MUTYH-associated polyposis (MAP) in a large cohort, taking into account factors as adenoma count and year of diagnosis. All application forms used to send patients in for APC and MUTYH variant analysis between 1992 and 2017 were collected (n = 2082). Using the data provided on the application form, the APC and biallelic MUTYH prevalence was determined and possible predictive factors were examined using multivariate multinomial logistic regression analysis in SPSS. The prevalence of disease causing variants in the APC gene significantly increases with adenoma count while MAP shows a peak prevalence in individuals with 50–99 adenomas. Logistic regression analysis shows significant odds ratios for adenoma count, age at diagnosis, and, interestingly, a decline in the chance of finding a variant in either gene over time. Moreover, in 22% (43/200) of patients with FAP-related extracolonic manifestations a variant was identified. The overall detection rates are above 10% for patients with >10 adenomas aged 20 adenomas aged T variant in the tumor or a first-degree relative with >10 adenomas. Therefore, APC and MUTYH testing in patients with >10 adenomas aged 20 adenomas aged

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We report a case of a 22-year-old female patient who was diagnosed with a cribriform-morular variant of papillary thyroid carcinoma (CMV-PTC). While at early ages this thyroid cancer variant is highly suggestive for familial adenomatous polyposis (FAP), there was no family history of FAP. In the tumor biallelic, inactivating APC variants were identified. The patient tested negative for germline variants based on analysis of genomic DNA from peripheral blood leukocytes. Somatic mosaicism was excluded by subsequent deep sequencing of leukocyte and normal thyroid DNA using next generation sequencing (NGS). This report presents a rare sporadic case of CMV-PTC, and to the best of our knowledge the first featuring two somatic APC mutations underlying the disease, with an overview of CMV-PTC cases with detected APC and CTNNB1 pathogenic variants from the literature. Electronic supplementary material The online version of this article (10.1007/s10689-019-00146-4) contains supplementary material, which is available to authorized users.

Findings 5585 families with Lynch syndrome from 22 countries were eligible for the analysis. Of these, there were insufficient numbers to estimate penetrance for Asia and South America, and for those with EPCAM variants. Therefore, we used data (collected between July 11, 2014, and Dec 31, 2018) from 5255 families (1829 MLH1, 2179 MSH2, 798 MSH6, and 449 PMS2), comprising 79 809 relatives, recruited in 15 countries in North America, Europe, and Australasia. There was strong evidence of the existence of unknown familial risk factors modifying colorectal cancer risk for Lynch syndrome carriers (p 0 center dot 0001 for each of the three three continents). These familial risk factors resulted in a wide within-gene variation in the risk of colorectal cancer for men and women from each continent who all carried pathogenic variants in the same gene or the MSH2 c.942+3A T variant. The variation was especially prominent for MLH1 and MSH2 variant carriers, depending on gene, sex and continent, with 7-56% of carriers having a colorectal cancer penetrance of less than 20%, 9-44% having a penetrance of more than 80%, and onlyBackground Existing clinical practice guidelines for carriers of pathogenic variants of DNA mismatch repair genes (Lynch syndrome) are based on the mean age-specific cumulative risk (penetrance) of colorectal cancer for all carriers of pathogenic variants in the same gene. We aimed to estimate the variation in the penetrance of colorectal cancer between carriers of pathogenic variants in the same gene by sex and continent of residence. Methods In this retrospective cohort study, we sourced data from the International Mismatch Repair Consortium, which comprises 273 members from 122 research centres or clinics in 32 countries from six continents who are involved in Lynch syndrome research. Families with at least three members and at least one confirmed carrier of a pathogenic or likely pathogenic variant in a DNA mismatch repair gene (MLH1, MSH2, MSH6, or PMS2) were included. The families of probands with known de-novo pathogenic variants were excluded. Data were collected on the method of ascertainment of the family, sex, carrier status, cancer diagnoses, and ages at the time of pedigree collection and at last contact or death. We used a segregation analysis conditioned on ascertainment to estimate the mean penetrance of colorectal cancer and modelled unmeasured polygenic factors to estimate the variation in penetrance. The existence of unknown familial risk factors modifying colorectal cancer risk for Lynch syndrome carriers was tested by use of a Wald p value for the null hypothesis that the polygenic SD is zero. Findings 5585 families with Lynch syndrome from 22 countries were eligible for the analysis. Of these, there were insufficient numbers to estimate penetrance for Asia and South America, and for those with EPCAM variants. Therefore, we used data (collected between July 11, 2014, and Dec 31, 2018) from 5255 families (1829 MLH1, 2179 MSH2, 798 MSH6, and 449 PMS2), comprising 79 809 relatives, recruited in 15 countries in North America, Europe, and Australasia. There was strong evidence of the existence of unknown familial risk factors modifying colorectal cancer risk for Lynch syndrome carriers (pT variant. The variation was especially prominent for MLH1 and MSH2 variant carriers, depending on gene, sex and continent, with 7-56% of carriers having a colorectal cancer penetrance of less than 20%, 9-44% having a penetrance of more than 80%, and only 10-19% having a penetrance of 40-60%. Interpretation Our study findings highlight the important role of risk modifiers, which could lead to personalised risk assessments for precision prevention and early detection of colorectal cancer for people with Lynch syndrome. Funding National Health and Medical Research Council, Australia. Copyright (c) 2021 Elsevier Ltd. All rights reserved.Methods In this retrospective cohort study, we sourced data from the International Mismatch Repair Consortium, which comprises 273 members from 122 research centres or clinics in 32 countries from six continents who are involved in Lynch syndrome research. Families with at least three members and at least one confirmed carrier of a pathogenic or likely pathogenic variant in a DNA mismatch repair gene (MLH1, MSH2, MSH6, or PMS2) were included. The families of probands with known de-novo pathogenic variants were excluded. Data were collected on the method of ascertainment of the family, sex, carrier status, cancer diagnoses, and ages at the time of pedigree collection and at last contact or death. We used a segregation analysis conditioned on ascertainment to estimate the mean penetrance of colorectal cancer and modelled unmeasured polygenic factors to estimate the variation in penetrance. The existence of unknown familial risk factors modifying colorectal cancer risk for Lynch syndrome carriers was tested by use of a Wald p value for the null hypothesis that the polygenic SD is zero.

BackgroundPathogenic germline variants in subunits of succinate dehydrogenase (SDHB, SDHC and SDHD) are broadly associated with disease subtypes of phaeochromocytoma–paraganglioma (PPGL) syndrome. Our objective was to investigate the role of variant type (ie, missense vs truncating) in determining tumour phenotype.MethodsThree independent datasets comprising 950 PPGL and head and neck paraganglioma (HNPGL) patients were analysed for associations of variant type with tumour type and age-related tumour risk. All patients were carriers of pathogenic germline variants in the SDHB, SDHC or SDHD genes.ResultsTruncating SDH variants were significantly over-represented in clinical cases compared with missense variants, and carriers of SDHD truncating variants had a significantly higher risk for PPGL (pSDHB truncating variants displayed a trend towards increased risk of PPGL, and all three SDH genes showed a trend towards over-representation of missense variants in HNPGL cases. Overall, variant types conferred PPGL risk in the (highest-to-lowest) sequence SDHB truncating, SDHB missense, SDHD truncating and SDHD missense, with the opposite pattern apparent for HNPGL (pConclusionsSDHD truncating variants represent a distinct group, with a clinical phenotype reminiscent of but not identical to SDHB. We propose that surveillance and counselling of carriers of SDHD should be tailored by variant type. The clinical impact of truncating SDHx variants is distinct from missense variants and suggests that residual SDH protein subunit function determines risk and site of disease.

BackgroundPathogenic variants in the CDKN2A gene are generally associated with the development of melanoma and pancreatic ductal adenocarcinoma (PDAC), but specific genotype-phenotype correlations might exist and the extent of PDAC risk is not well established for many variants.MethodsUsing the Dutch national familial melanoma database, we identified all families with a pathogenic CDKN2A variant and investigated the occurrence of PDAC within these families. We also estimated the standardised incidence ratio and lifetime PDAC risk for carriers of a highly prevalent variant in these families.ResultsWe identified 172 families in which 649 individuals carried 15 different pathogenic variants. The most prevalent variant was the founder mutation c.225_243del (p16-Leiden, 484 proven carriers). Second most prevalent was c.67G>C (55 proven carriers). PDAC developed in 95 of 163 families (58%, including 373 of 629 proven carriers) harbouring a variant with an effect on the p16INK4a protein, whereas PDAC did not occur in the 9 families (20 proven carriers) with a variant affecting only p14ARF. In the c.67G>C families, PDAC occurred in 12 of the 251 (5%) persons at risk. The standardised incidence ratio was 19.1 (95% CI 8.3 to 33.6) and the cumulative PDAC incidence at age 75 years (lifetime risk) was 19% (95% CI 7.5% to 30.1%).ConclusionsOur results support the notion that pathogenic CDKN2A variants affecting the p16INK4a protein, including c.67G>C, are associated with increased PDAC risk and carriers of such variants should be offered pancreatic cancer surveillance. There is no clinical evidence that impairment of only the p14ARF protein leads to an increased PDAC risk.

Although duodenal adenomas and cancer appear to occur significantly less frequently in autosomal recessive MUTYH-associated polyposis (MAP) than in autosomal dominant familial adenomatous polyposis (FAP),1 current guidelines recommend similar endoscopic surveillance for both disorders.2-4 This involves gastro-duodenoscopy starting at 25 to 35 years of age and repeated at intervals determined by Spigelman staging based on the number, size, histological type and degree of dysplasia of adenomas, and by ampullary staging. Case reports of duodenal cancers in MAP suggest that they may develop in the absence of advanced Spigelman stage benign disease and even without coexisting adenomas.1 Recent molecular analyses suggest thatMAPduodenal adenomashave a higher mutational burden than FAP adenomas and are more likely to harbor oncogenic drivermutations, such as those in KRAS.5 These apparent differences in the biology and natural history of duodenal polyposis in FAP and MAP challenge the assumption that the same surveillance should be applied in both conditions.

Context Pheochromocytomas and paragangliomas (PPGLs) are neuroendocrine tumors in which altered central metabolism appears to be a major driver of tumorigenesis, and many PPGL genes encode proteins involved in the tricarboxylic acid (TCA) cycle. Objective/design While about 40% of PPGL cases carry a variant in a known gene, many cases remain unexplained. In patients with unexplained PPGL showing clear evidence of a familial burden or multiple tumors, we aimed to identify causative factors using genetic analysis of patient DNA and functional analyses of identified DNA variants in patient tumor material and engineered cell lines. Patients and Setting Patients with a likely familial cancer burden of pheochromocytomas and/or paragangliomas and under investigation in a clinical genetic and clinical research setting in university hospitals. Results While investigating unexplained PPGL cases, we identified a novel variant, c.1151C>T, p.(Pro384Leu), in exon 14 of the gene encoding dihydrolipoamide S-succinyltransferase (DLST), a component of the multi-enzyme complex 2-oxoglutarate dehydrogenase. Targeted sequence analysis of further unexplained cases identified a patient carrying a tumor with compound heterozygous variants in DLST, consisting of a germline variant, c.1121G>A, p.(Gly374Glu), together with a somatic missense variant identified in tumor DNA, c.1147A>G, p.(Thr383Ala), both located in exon 14. Using a range of in silico and functional assays we show that these variants are predicted to be pathogenic, profoundly impact enzyme activity, and result in DNA hypermethylation. Conclusions The identification and functional analysis of these DLST variants further validates DLST as an additional PPGL gene involved in the TCA cycle.

Study question Does In vitro maturation (IVM) of immature oocytes affect health, including growth at 2 years of age, in singletons born to mothers with polycystic ovary syndrome (PCOS)? Summary answer This study of 92 singletons born after IVM in mothers with PCOS showed no significant differences in anthropometry and health outcome parameters in comparison with a cohort of 74 peers born after intracytoplasmic sperm injection (ICSI) and conventional controlled ovarian stimulation (COS) in mothers with PCOS. What is known already IVM has been used worldwide in women with PCOS. However, the paucity of available data related to children's health following IVM is an important impediment to a more widespread use of the technology. Although previous reports on the neonatal outcome after IVM are generally reassuring, these studies have flaws that hamper the interpretation of outcomes. Moreover, few studies have reported on health outcomes after IVM beyond infancy, and particularly growth data in children born after IVM of immature oocytes from mothers with PCOS are lacking. Study design size duration This single-center cohort study compared anthropometry and health outcomes in 92 singletons born after ICSI of in vitro matured oocytes with 74 singletons born after ICSI without IVM (COS). All participants were born to mothers who were diagnosed with PCOS phenotype A, B, C or D and reached the age of 2 years between November 2012 and June 2019. Singletons born after COS were randomly selected for follow-up until young adulthood. Participants/materials setting methods Anthropometric parameters and health status data were prospectively collected at birth, 4 months and 2 years in cohorts of singletons followed since birth. Results were adjusted for neonatal (birthweight z-score, birth order), treatment (day of transfer, number of embryos transferred, mode of transfer) and parental (maternal smoking, age, body mass index (BMI), anti-Mullerian hormone level, PCOS phenotype, gestational diabetes, hypertensive disorder and paternal BMI) characteristics. Main results and the role of chance Overall, no differences were found for bodyweight, height and head circumference z-score between IVM and COS children at birth, at 4 months or at 2 years (all P > 0.05). In addition, z-scores of waist and mid-upper arm circumference at 2 years were comparable in IVM and COS children. Adjustment for covariates did not change the conclusion. Surgical intervention rate as well as the hospital admission rate were comparable between the IVM and COS group (all P > 0.05). The proportion of children born to mothers with metabolically unfavorable PCOS phenotypes (A and C) was comparable in the two groups (52.1% in IVM and 45.9% in COS). Mothers giving birth to a child conceived using IVM were younger than mothers in the COS group but their BMI was comparable. Limitations reasons for caution Although our study describes the largest cohort to date of singletons born after IVM applied in mothers with well-defined PCOS phenotypes, the current sample size only allowed us to detect moderate differences in anthropometry. Also, follow-up of children born after IVM for indications other than PCOS, for example fertililty preservation after cancer diagnosis, is highly recommended. Wider implications of the findings We did not observe adverse effects of IVM on growth parameters in offspring ~2 years of age compared to COS, but future studies should focus on cardiovascular and metabolic outcomes in these children and adolescents given their mother's PCOS condition. Study funding/competing interests This study was supported by Methusalem grants and by grants from Wetenschappelijk Fonds Willy Gepts; all issued by the Vrije Universiteit Brussel (VUB). All co-authors, except M.B., M.D.V. and H.T. declared no conflict of interest. M.B. has received consultancy fees from MSD, Serono Symposia and Merck. M.D.V. has received fees for lectures from MSD, Ferring, Gedeon Richter and Cook Medical. H.T. has received consultancy fees from Gedeon Richter, Merck, Ferring, Abbott and ObsEva. The Universitair Ziekenhuis Brussel (UZ Brussel) and the Center for Medical Genetics have received several educational grants from IBSA, Ferring, MSD and Merck for establishing the database for follow-up research and organizing the data collection.

BackgroundIn 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.MethodsWe 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.ResultsPredicted 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.ConclusionsBased 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.

Germline mutations in succinate dehydrogenase B (SDHB) predispose to hereditary paraganglioma (PGL) syndrome type 4. The risk of developing PGL or pheochromocytoma (PHEO) in SDHB mutation carriers is subject of recent debate. In the present nationwide cohort study of SDHB mutation carriers identified by the clinical genetics centers of the Netherlands, we have calculated the penetrance of SDHB associated tumors using a novel maximum likelihood estimator. This estimator addresses ascertainment bias and missing data on pedigree size and structure. A total of 195 SDHB mutation carriers were included, carrying 27 different SDHB mutations. The 2 most prevalent SDHB mutations were Dutch founder mutations: a deletion in exon 3 (31% of mutation carriers) and the c.423+1G>A mutation (24% of mutation carriers). One hundred and twelve carriers (57%) displayed no physical, radiological or biochemical evidence of PGL or PHEO. Fifty-four patients had a head and neck PGL (28%), 4 patients had a PHEO (2%), 26 patients an extra-adrenal PGL (13%). The overall penetrance of SDHB mutations is estimated to be 21% at age 50 and 42% at age 70 when adequately corrected for ascertainment. These estimates are lower than previously reported penetrance estimates of SDHB-linked cohorts. Similar disease risks are found for different SDHB germline mutations as well as for male and female SDHB mutation carriers.

Objective Succinate dehydrogenase B subunit (SDHB) gene germline mutations predispose to pheochromocytomas, sympathetic paragangliomas, head and neck paragangliomas and non-paraganglionic tumors (e.g. renal cell carcinoma, gastrointestinal stromal tumor and pituitary neoplasia). The aim of this study was to determine phenotypical characteristics of a large Dutch cohort of SDHB germline mutation carriers and assess differences in clinical phenotypes related to specific SDHB mutations. Design Retrospective descriptive study. Methods Retrospective descriptive study in seven academic centers. Results We included 194 SDHB mutation carriers consisting 65 (33.5%) index patients and 129 (66.5%) relatives. Mean age was 44.8 ± 16.0 years. Median duration of follow-up was 2.6 years (range: 0–36). Sixty persons (30.9%) carried the exon 3 deletion and 46 (23.7%) the c.423 + 1G > A mutation. Fifty-four mutation carriers (27.8%) had one or multiple head and neck paragangliomas, 4 (2.1%) had a pheochromocytoma and 26 (13.4%) had one or more sympathetic paragangliomas. Fifteen patients (7.7%) developed metastatic paraganglioma and 17 (8.8%) developed non-paraganglionic tumors. At study close, there were 111 (57.2%) unaffected mutation carriers. Statistical analyses showed no significant differences in the number and location of head and neck paragangliomas, sympathetic paragangliomas or pheochromocytomas, nor in the occurrence of metastatic disease or other tumors between carriers of the two founder SDHB mutations (exon 3 deletion vs c.423 + 1G > A). Conclusions In this nationwide study of disease-affected and unaffected SDHB mutation carriers, we observed a lower rate of metastatic disease and a relatively high number of head and neck paragangliomas compared with previously reported referral-based cohorts.

Objective Treatment for head and neck paragangliomas (HNGPL) can be more harmful than the disease. After diagnosis, an initial period of surveillance is often indicated, and surgery or radiotherapy is reserved for progressive disease. With the aim to optimize this “wait and scan” strategy, we studied growth and possible predictors. Design A retrospective cohort study was conducted. Setting This study was conducted at a tertiary referral center for patients with HNGPL. Methods Tumor volume was estimated for 184 SDHD-related carotid and vagal body paragangliomas using sequential magnetic resonance imaging. Cox regression was used to study predictors of tumor growth. Results The estimated fraction of growing tumors ranged from 0.42 after 1 year of follow-up to 0.85 after 11 years. A median growth rate of 10.4 and 12.0% per year was observed for carotid and vagal body tumors, respectively. Tumor location, initial volume, and age (p Conclusions We created a prediction model (available online), enabling a more individualized “wait and scan” strategy. The favorable natural course of carotid and vagal body paragangliomas was confirmed; although with long follow-up growth will be observed in most cases.

Key Clinical Message We report a novel KRT13 germ line variant that causes white sponge nevus (WSN) with mucosal dysplasia. Genital, vaginal, and cervical WSN were observed in four female patients, of whom two had premalignant cervical lesions at young age. Two of the 12 patients with oral WSN developed oral squamous cell carcinoma.

// Attje S. Hoekstra 1 , Erik F. Hensen 2 , Ekaterina S. Jordanova 3 , Esther Korpershoek 4 , Anouk N.A. van der Horst-Schrivers 5 , Cees Cornelisse 3 , Eleonora P.M. Corssmit 6 , Frederik J. Hes 7 , Jeroen C. Jansen 8 , Henricus P.M. Kunst 9 , Henri J.L.M. Timmers 10 , Adrian Bateman 11 , Diana Eccles 12 , Judith V.M.G. Bovee 3 , Peter Devilee 1, 3 , Jean-Pierre Bayley 1 1 Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands 2 Department of Otolaryngology/Head and Neck Surgery, VU University Medical Center, Amsterdam, The Netherlands 3 Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands 4 Department of Pathology, Josephine Nefkens Institute, Erasmus Medical Center Rotterdam, Rotterdam, The Netherlands 5 Department of Endocrinology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands 6 Department of Endocrinology and Metabolic Diseases, Leiden University Medical Center, Leiden, The Netherlands 7 Department of Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands 8 Department of Otorhinolaryngology, Leiden University Medical Center, Leiden, The Netherlands 9 Department of Otorhinolaryngology, Head and Neck Surgery, Radboud University Medical Centre, Nijmegen, The Netherlands 10 Department of Medicine, Division of Endocrinology, Radboud University Medical Centre, Nijmegen, The Netherlands 11 Department of Cellular Pathology, University Hospital Southampton, Southampton, UK 12 University of Southampton School of Medicine, Cancer Sciences Division, Somers Cancer Research Building, Southampton, UK Correspondence to: Jean-Pierre Bayley, email: J.P.L.Bayley@lumc.nl Peter Devilee, email: P.Devilee@lumc.nl Keywords: paraganglioma, pheochromocytoma, succinate dehydrogenase, Von Hippel-Lindau, loss of heterozygosity Received: September 06, 2016 Accepted: January 04, 2017 Published: January 14, 2017 ABSTRACT Germline mutations in the succinate dehydrogenase (SDHA, SDHB, SDHC, SDHD, SDHAF2) or Von Hippel-Lindau (VHL) genes cause hereditary paraganglioma/pheochromocytoma. While SDHB (1p36) and VHL (3p25) are associated with autosomal dominant disease, SDHD (11q23) and SDHAF2 (11q13) show a remarkable parent-of-origin effect whereby tumor formation is almost completely dependent on paternal transmission of the mutant allele. Loss of the entire maternal copy of chromosome 11 occurs frequently in SDHD -linked tumors, and has been suggested to be the basis for this typical inheritance pattern. Using fluorescent in situ hybridization, microsatellite marker and SNP array analysis, we demonstrate that loss of the entire copy of chromosome 11 is also frequent in SDHAF2 -related PGLs, occurring in 89% of tumors. Analysis of two imprinted differentially methylated regions (DMR) in 11p15, H19-DMR and KvDMR, showed that this loss always affected the maternal copy of chromosome 11. Likewise, loss of maternal chromosome 11p15 was demonstrated in 85% of SDHD and 75% of VHL -related PGLs/PCCs. By contrast, both copies of chromosome 11 were found to be retained in 62% of SDHB -mutated PGLs/PCCs, while only 31% showed loss of maternal chromosome 11p15. Genome-wide copy number analysis revealed frequent loss of 1p in SDHB mutant tumors and show greater genomic instability compared to SDHD and SDHAF2 . These results show that loss of the entire copy of maternal chromosome 11 is a highly specific and statistically significant event in SDHAF2 , SDHD and VHL -related PGLs/PCCs, but is less significant in SDHB -mutated tumors, suggesting that these tumors have a distinct genetic etiology.

Context: Heterozygous pathogenic germline variants in CDC73 predispose to the development of primary hyperparathyroidism (pHPT) and, less frequently, ossifying fibroma of the jaw and renal and uterine tumors. Clinical information on CDC73-related disorders has so far been limited to small case series.Objective: To assess the clinical manifestations and penetrance in CDC73-related disorders and to improve case detection in pHPT.Design: Nationwide retrospective Dutch cohort study.Setting: Tertiary referral center.Patients: We studied 89 patients with pHPT referred for germline CDC73 analysis and 43 subsequently tested relatives who proved to be mutation carriers.Investigation: Germline CDC73 mutation analysis.Mean Outcome: CDC73 mutation detection yield, referral rate, and CDC73-related disease penetrance.Results: Pathogenic germline CDC73 variants were identified in 11 of the 89 referred pHPT patients (12.4%), with (suspected) hyperparathyroidism-jaw tumor (HPT-JT) syndrome (n = 3), familial isolated pHPT (n = 5), apparently sporadic parathyroid carcinoma (n = 2), and apparently sporadic parathyroid adenoma (n = 1). The estimated penetrance of CDC73-related disorders was 65% at age 50 years (95% confidence interval, 48% to 82%) in 43 nonindex mutation carriers.Conclusions: Germline CDC73 analysis is recommended in individuals with (suspected) HPT-JT syndrome, familial isolated pHPT, atypical or malignant parathyroid histology, and young individuals with pHPT. These criteria would increase germline CDC73 mutation detection, enabling optimal clinical management of pHPT as well as genetic counseling and surveillance for family members at risk for developing CDC73-related disorders.

ObjectiveGene alterations leading to activation of the MAPK pathway are of interest for targeted therapy in patients with advanced radioactive iodine refractory (RAI-R) thyroid carcinoma. Due to technical reasons gene fusion analysis in RNA isolated from formalin-fixed tumor tissues has till now been limited. The objective of the present study was to identify targetable gene rearrangements in RNA isolated from formalin-fixed RAI-R thyroid carcinomas.DesignRetrospective study in 132 patients with RAI-R thyroid carcinoma (59 papillary-, 24 follicular-, 35 Hürthle cell- and 14 anaplastic thyroid carcinoma).MethodsTotal nucleic acid (undivided DNA and RNA) was isolated from formalin-fixed tissue. Extensive gene fusion analysis was performed in all samples that tested negative for pathogenicBRAF,NRAS,HRASandKRASvariants.ResultsSeven targetable gene fusions were identified in the remaining 60 samples without known DNA variants. This includes frequently reported gene fusions such asCCDC6/RET(PTC1),PRKAR1A/RET(PTC2) andETV6/NTRK3, and gene fusions that are less common in thyroid cancer (TPM3/NTRK1,EML4/ALKandEML4/NTRK3). Of note, most gene fusions were detected in papillary thyroid carcinoma and MAPK-associated alterations in Hürthle cell carcinomas are rare (2/35).ConclusionTargetable gene fusions were found in 12% of RAI-R thyroid carcinoma without DNA variants and can be effectively identified in formalin-fixed tissue. These gene fusions might provide a preclinical rationale to include specific kinase inhibitors in the treatment regimen for these patients. The latter intends to restore iodine transport and/or take advantage of the direct effect on tumor cell vitality once progressive disease is seen.

Background Germline mutations affecting the exonuclease domains of POLE and POLD1 predispose to colorectal adenomas and carcinoma. Here, we aimed to screen the exonuclease domains to find the genetic causes of multiple colorectal polyps in unexplained cases. Methods Using a custom next‐generation sequencing panel, we sequenced the exonuclease domains of POLE and POLD1 in 332 index patients diagnosed with multiple colorectal polyps without germline alteration in colorectal polyposis predisposing genes. Results We identified two variants of unknown significance. One germline POLD1 c.961G>A, p.(Gly321Ser) variant was found in two cases. The first patient was diagnosed with multiple polyps at age 35 and colorectal cancer (CRC) at age 37, with no known family history of CRC. The second patient was diagnosed with CRC at age 44 and cumulatively developed multiple polyps; this patient had two sisters with endometrial cancer who did not carry the variant. Furthermore, we identified a novel POLD1 c.955 T>G, p.(Cys319Gly) variant in a patient diagnosed with multiple colorectal adenomas at age 40. Co‐segregation analysis showed that one sister who cumulatively developed multiple adenomas from age 34, and another sister who developed CRC at age 38 did not carry the variant. We did not identify pathogenic variants in POLE and POLD1. Conclusion This study confirms the low frequency of causal variants in these genes in the predisposition for multiple colorectal polyps, and also establishes that these genes are a rare cause of the disease.

Background: This study aimed to determine the prevalence of APC-associated familial adenomatous polyposis (FAP) and MUTYH-associated polyposis (MAP) in a large cohort, taking into account several factors including adenoma count and year of diagnosis. Methods: All application forms used to send patients in for APC and MUTYH mutation analysis between 1992 and 2017 were collected (n=2082). Using this data, possible predictive factors were examined using multivariate multinomial logistic regression analysis in SPSS. Findings: The prevalence of APC mutations significantly increases with adenoma count while MAP shows a peak prevalence in individuals with 50-99 adenomas. Logistic regression analysis shows significant odds ratios for adenoma count, age at diagnosis and having a first degree family member with more than 10 polyps. Moreover, the chance of finding an APC or MUTYH mutation steadily declined over the past 23 years. Mutations in APC and MUTYH were found in 22% (43/200) of patients with FAP-related extracolonic manifestations, such as duodenal polyps and desmoid tumors. Mutation detection rate were 40% and 10% for respectively patients with >10 adenomas under age 60 and >20 adenomas under age 70. Mutation carriers not falling into these criteria, had FAP-related extracolonic manifestations, colorectal cancer (CRC) below the age of 40, somatic KRAS c.34G>T variant in the tumor or a first-degree family member with >10 adenomas. Interpretation: Adenoma count, age at diagnosis of the adenomas and year of analysis are important predictive factors for APC and MUTYH mutations. Based on our results, APC and MUTYH testing is advised in patients >10 adenomas under age 60 and >20 adenomas under age 70. Almost all FAP and MAP patients who did not meet these criteria showed other characteristics that can be used as an indication to prompt genetic testing. Funding Statement: The authors declare: "N/A." Declaration of Interests: The authors state: "No competing interest to declare." Ethics Approval Statement: The study was approved by local ethics review boards (P01.019).

Context DICER1 syndrome is a rare autosomal-dominantly inherited disorder that predisposes to a variety of cancerous and noncancerous tumors of mostly pediatric and adolescent onset, including differentiated thyroid carcinoma (DTC). DTC has been hypothesized to arise secondarily to the increased prevalence of thyroid hyperplastic nodules in syndromic patients. Objective To determine somatic alterations in DICER1-associated DTC and to study patient outcomes. Design Retrospective series. Setting Tertiary referral centers. Patients Ten patients with germline pathogenic DICER1 variants and early-onset DTC. Methods Somatic DICER1 mutation analysis, extensive somatic DNA variant and gene fusion analyses were performed on all tumors. Results Median age at DTC diagnosis was 13.5 years and there was no recurrent or metastatic disease (median follow-up, 8 years). All thyroid specimens showed diffuse nodular hyperplasia with at least one focus suspicious of DTC but without infiltrative growth, extrathyroidal extension, vascular invasion, or lymph node metastasis. Most of the individual nodules (benign and malignant) sampled from the 10 tumors harbored distinct DICER1 RNase IIIb hotspot mutations, indicating a polyclonal composition of each tumor. Furthermore, nine of 10 DICER1-related DTCs lacked well-known oncogenic driver DNA variants and gene rearrangements. Conclusion On the basis of our clinical, histological, and molecular data, we consider that most DICER1-related DTCs form a low-risk subgroup. These tumors may arise within one of multiple benign monoclonal nodules; thus, hemi-thyroidectomy or, more likely, total thyroidectomy may often be required. However, radioiodine treatment may be unnecessary given the patients’ ages and the tumors’ low propensity for metastases.

Monoallelic PMS2 germline mutations cause 5-15% of Lynch syndrome, a midlife cancer predisposition, whereas biallelic PMS2 mutations cause approximately 60% of constitutional MMR 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 MMR 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 pre-screening methods will however miss some PMS2 germline mutation carriers. This article is protected by copyright. All rights reserved.

Although it is well established that paternally transmitted germline variants in SDHD are associated with multifocal paragangliomas and lifelong follow-up is generally advised, the risk of metachronous lesions is presently unknown. In a large Dutch cohort of SDHD variant carriers, we studied the development of new paragangliomas, and the evolution of symptoms and cranial nerve impairment. Recurrent event analysis and the Kaplan-Meier product limit estimator were used to study the risk of new lesions. The relation between several predictors and development of new symptoms was assessed using logistic regression. Of the 222 SDHD variant carriers included, 65% presented with symptoms and 11% with cranial nerve dysfunction. Over a median period of 8 years, 42% reported new symptoms, and new cranial nerve impairment was observed in 11% of subjects. The estimated fraction of subjects that developed new HNPGL increased to 73% (95% CI: 52-85%) after 22 years of follow-up. Males were more likely to develop new HNPGL compared to females (HR: 1.63, 95% CI: 1.10-2.40), as were subjects that presented with symptoms, compared to subjects that were asymptomatic at baseline (HR: 1.61, 95% CI: 1.01-2.55). In addition, the risk of new lesions decreased with number of HNPGL present at first diagnosis (HR: 0.68 and 95% CI: 0.56-0.82). Carriers of a paternally inherited SDHD variant face a considerable risk for new HNPGL. In addition, nearly 50% of subjects reported new symptoms. However, new cranial nerve deficits were observed in only 11%, which is less than reported in surgical series. These risks should be taken into account when considering treatment strategies and counseling.

Patients synchronously or metachronously presenting with ovarian and colon cancer can pose diagnostic challenges. A primary colon carcinoma can metastasize to one or both ovaries, two independent primary tumors can arise or an ovarian carcinoma can metastasize to the colon. Clinical and immunohistochemical characterization can aid the diagnosis. Recently, we reported that in difficult cases finding pathogenic APC variants supports a colonic origin. In this case report we describe the clinical history of a female patient suspected for Lynch syndrome. She was diagnosed with a bilateral ovarian cancer at age 44, followed by the detection of a colon carcinoma 12.5 months later. Lesions of both sites showed a DNA mismatch repair deficiency with immunohistochemical loss of MLH1 and PMS2 expression without MLH1 promoter hypermethylation. In absence of germline MMR gene variants identical somatic MLH1 and CTNNB1 gene variants were found, indicating a clonal relation. MMR germline mosaicism was made unlikely by ultra deep sequencing of the MLH1 variant in DNA isolated from normal mucosa, blood, urine and saliva. Although initially being suspect for Lynch syndrome it was eventually concluded that a metachronously diagnosed colon carcinoma that metastasized to both ovaries was most likely.

Context Paraganglioma (PGL) has the highest degree of heritability among human neoplasms. Current clinical understanding of germline SDHA mutation carriers is limited. Objective To estimate the contribution of SDHA mutations in PGL and to assess clinical manifestations and age-related penetrance. Design Nationwide retrospective cohort study. Setting Tertiary referral centers in the Netherlands (multicenter). Patients Germline SDHA analysis was performed in 393 patients with genetically unexplained PGL. Subsequently, 30 index SDHA mutation carriers and 56 nonindex carriers were studied. Main Outcome Measures SDHA mutation detection yield, clinical manifestations, and SDHA-related disease penetrance. Results Pathogenic germline SDHA variants were identified in 30 of the 393 referred patients with PGL (7.6%), who had head and neck PGL (21 of 174 [12%]), pheochromocytoma (4 of 191 [2%]), or sympathetic PGL (5 of 28 [18%]). The median age at diagnosis was 43 years (range, 17 to 81 years) in index SDHA mutation carriers compared with 52 years (range, 7 to 90 years) in nonmutation carriers (P = 0.002). The estimated penetrance of any SDHA-related manifestation was 10% at age 70 years (95% confidence interval, 0% to 21%) in nonindex mutation carriers. Conclusion Germline SDHA mutations are relatively common (7.6%) in patients with genetically unexplained PGL. Most index patients presented with apparently sporadic PGL. In this SDHA series, the largest assembled so far, we found the lowest penetrance of all major PGL predisposition genes. This suggests that recommendations for genetic counseling of at-risk relatives and stringency of surveillance for SDHA mutation carriers might need to be reassessed.

Biallelic germline mutations affecting NTHL1 predispose to adenomatous polyposis and colorectal cancer, but the complete phenotype is unknown, which hampers patient recognition. We describe 29 individuals from 17 families, of which 26 developed one (n=10) or multiple (n=16) malignancies in 14 different tissues. An unexpected high breast cancer incidence was observed in women with biallelic NTHL1 mutations (60%). Mutational signature analysis of 14 tumors from seven organs revealed that deficient NTHL1 repair underlies the main mutational process in all but one of the tumors (93%). These results reveal NTHL1 as a multi-tumor predisposition gene with a high lifetime risk for extracolonic cancers, and a typical mutational signature observed across tumor types, which can assist in the recognition of this syndrome.

Hartmut P.H. Neumann is supported by grants from the Deutsche Krebshilfe (Grant 107995 to H.P.H.N.). Stephane Richard is supported by grants from the Direction Generale de l’Organisation des Soins (French Department of Health), the Institut National du Cancer (INCa; French NCI), and the Ligue Nationale contre le Cancer (Comites du Cher et de l’Indre; French League against Cancer).

Introduction Germline pathogenic variants in the canonical TP53 gene cause Li-Fraumeni, a severe cancer predisposition syndrome characterised by early onset of multiple tumours, particularly breast cancer, brain tumours, soft-tissue sarcomas, osteosarcomas and leukaemia. Genetic variation in the alternatively spliced TP53 exons 9β and 9γ, each encoding a different carboxy-termini, have been poorly studied. Therefore, the clinical significance of germline variation in these alternative exons remains unknown. In the search for novel colorectal cancer susceptibility genes, we identified a germline variant in an alternative TP53 exon 9 and we show that this variant is associated with a predisposition to multiple cancer types. Material and methods Exome sequencing was performed on 118 unexplained familial and/or early-onset colorectal cancer patients. Somatic TP53 and cancer hotspot mutations were assessed in tumour tissues of carriers. TP53 isoform expression and non-sense mediated decay were studied in cell lines from colorectal cancer patients using (allele specific) qPCR and Western blotting. Results and discussions We identified a germline heterozygous variant in an alternative TP53 exon 9 in twelve individuals from three families severely affected by colorectal cancer and multiple other cancer types. The carriers developed one or more primary tumours after 40 years of age, including colorectal cancer, breast cancer, thyroid cancer and Non-Hodgkin lymphoma. Only two out of eleven tumours harboured a somatic TP53 mutation and a single tumour showed loss of heterozygosity of the alternative exon 9 variant. The cell line carrying the TP53 variant showed (i) an allelic imbalance towards the expression of variant containing transcripts and (ii) a higher expression of the alternative TP53 exon 9 isoforms compared to wild-type cell lines. Increased protein expression of the alternative exon 9 isoforms could be confirmed in the cell line using Western blotting. The increased expression was not caused by disturbance of non-sense mediated decay mechanisms. Conclusion We have identified a variant in an alternative TP53 isoform that predisposes carriers to multiple cancers. Carriers presented at a slightly older age and with a different spectrum of tumours compared to classical Li-Fraumeni syndrome patients. Therefore we recommend the screening of the alternative exons 9 of the TP53 gene in all unexplained cancer-prone families.

Biallelic germline mutations affecting NTHL1 predispose carriers to adenomatous polyposis and colorectal cancer, but the complete phenotype is unknown. We describe 29 individuals carrying biallelic germline NTHL1 mutations from 17 families, of which 26 developed one (n = 10) or multiple (n = 16) malignancies in 14 different tissues. An unexpected high breast cancer incidence was observed in female carriers (60%). Mutational signature analysis of 14 tumors from 7 organs revealed that NTHL1 deficiency underlies the main mutational process in all but one of the tumors (93%). These results reveal NTHL1 as a multi-tumor predisposition gene with a high lifetime risk for extracolonic cancers and a typical mutational signature observed across tumor types, which can assist in the recognition of this syndrome.

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.

Lynch syndrome (LS), one of the most frequent forms of hereditary colorectal cancer (CRC), is caused by a defect in one of the mismatch repair (MMR) genes. Carriers of MMR defects have a strongly increased risk of developing CRC and endometrial cancer. Over the last few years, value-based healthcare has been introduced as an approach to the cost-effective delivery of measurable patient value over complete cycles of care. This requires all involved stakeholders to formulate and validate ‘patient value’ for Lynch syndrome, as well as to identify targets and associated costs. The aim of this study was to develop a value-based care model for Lynch syndrome that can determine patient value and associated costs, and to design a coordinated care pathway from existing guidelines. All specialists in our hospital involved in the management of LS patients evaluated the care delivered to these patients at their department and formulated outcome measures relevant to patient value. Patients were then invited to complete a questionnaire that assessed the importance of these measures on a scale of 1–10. Six high-value outcomes were identified: (1) prevention of cancer or detection of early stage cancer (2) rapid results from MMR gene mutation testing (3) rapid investigation of the colon and uterus (4) no/little pain during colonoscopy and gynaecologic examination/biopsy (5) the offer of psychological help and (6) registration with the Dutch Lynch syndrome registry. A total of 38 (59 %) out of 62 patients completed the questionnaire. The relevance of all outcomes was confirmed by the patients and mean scores varied from 7.2 to 9.9. Patients underscored the relevance of both proper patient education and the efficiency of surveillance during their care cycle. Value-based care delivery for Lynch syndrome includes the implementation of six parameters related to prevention and early detection of cancer, a short cycle time and registration to ensure continuation of care. Estimated costs are € 3320 for the first cycle of care (€ 3550 including gynaecologic surveillance) and approximately 720 per subsequent annual cycle (€ 950 including gynaecologic surveillance).Keywords: Lynch syndrome, Colorectal cancer, Endometrial cancer, Prevention, Value-based health care, Patient value, Outcome measures, Lynch syndrome registry

Purpose Patients with Lynch syndrome are at high risk for colon and endometrial cancer, but also at an elevated risk for other less common cancers. The purpose of this retrospective cohort study was to provide risk estimates for these less common cancers in proven carriers of pathogenic mutations in the mismatch repair (MMR) genes MLH1, MSH2, and MSH6. Patients and Methods Data were pooled from the German and Dutch national Lynch syndrome registries. Seven different cancer types were analyzed: stomach, small bowel, urinary bladder, other urothelial, breast, ovarian, and prostate cancer. Age-, sex- and MMR gene–specific cumulative risks (CRs) were calculated using the Kaplan-Meier method. Sex-specific incidence rates were compared with general population incidence rates by calculating standardized incidence ratios (SIRs). Multivariate Cox regression analysis was used to estimate the impact of sex and mutated gene on cancer risk. Results The cohort comprised 2,118 MMR gene mutation carriers (MLH1, n = 806; MSH2, n = 1,004; MSH6, n = 308). All cancers were significantly more frequent than in the general population. The highest risks were found for male small bowel cancer (SIR, 251; 95% CI, 177 to 346; CR at 70 years, 12.0; 95% CI, 5.7 to 18.2). Breast cancer showed an SIR of 1.9 (95% CI, 1.4 to 2.4) and a CR of 14.4 (95% CI, 9.5 to 19.3). MSH2 mutation carriers had a considerably higher risk of developing urothelial cancer than MLH1 or MSH6 carriers. Conclusion The sex- and gene-specific differences of less common cancer risks should be taken into account in cancer surveillance and prevention programs for patients with Lynch syndrome.

Contains fulltext : 167193.pdf (Publisher’s version ) (Open Access) Familial adenomatous polyposis (FAP) is a dominantly inherited syndrome caused by germline mutations in the APC gene and characterized by the development of multiple colorectal adenomas and a high risk of developing colorectal cancer (CRC). The severity of polyposis is correlated with the site of the APC mutation. However, there is also phenotypic variability within families with the same underlying APC mutation, suggesting that additional factors influence the severity of polyposis. Genome-wide association studies identified several single nucleotide polymorphisms (SNPs) that are associated with CRC. We assessed whether these SNPs are associated with polyp multiplicity in proven APC mutation carriers. Sixteen CRC-associated SNPs were analysed in a cohort of 419 APC germline mutation carriers from 182 families. Clinical data were retrieved from the Dutch Polyposis Registry. Allele frequencies of the SNPs were compared for patients with /=100 adenomas, using generalized estimating equations with the APC genotype as a covariate. We found a trend of association of two of the tested SNPs with the >/=100 adenoma phenotype: the C alleles of rs16892766 at 8q23.3 (OR 1.71, 95 % CI 1.05-2.76, p = 0.03, dominant model) and rs3802842 at 11q23.1 (OR 1.51, 95 % CI 1.03-2.22, p = 0.04, dominant model). We identified two risk variants that are associated with a more severe phenotype in APC mutation carriers. These risk variants may partly explain the phenotypic variability in families with the same APC gene defect. Further studies with a larger sample size are recommended to evaluate and confirm the phenotypic effect of these SNPs in FAP.

Mutations in SDHD and SDHAF2 (both located on chromosome 11) give rise to hereditary paraganglioma almost exclusively after paternal transmission of the mutation, and tumours often show loss of the entire maternal copy of chromosome 11. The 'Hensen' model postulates that a tumour modifier gene located on chromosome 11p15, a region known to harbour a cluster of imprinted genes, is essential to tumour formation. We observed decreased protein expression of the 11p15 candidate genes CDKN1C, SLC22A18 and ZNF215 evaluated in 60 SDHD-mutated tumours compared to normal carotid body tissue and non-SDH mutant tumours.We then created stable knockdown in vitro models, reasoning that the simultaneous knockdown of SDHD and a maternally expressed 11p15 modifier gene would enhance paraganglioma-related cellular characteristics compared to SDHD knockdown alone. Knockdown of SDHD in SNB19 and SHSY5Y cells resulted in the accumulation of succinate, the stabilization of HIF1 protein and a reduction in cell proliferation.Compared to single knockdown of SDHD, knockdown of SDHD together with SLC22A18 or with CDKN1C led to small but significant increases in cell proliferation and resistance to apoptosis, and to a gene expression profile closely related to the known transcriptional profile of SDH-deficient tumours. Of the 60 SDHD tumours investigated, four tumours showing retention of chromosome 11 showed SLC22A18 and CDKN1C expression levels comparable to levels in tumours showing loss of chromosome 11, suggesting loss of protein expression despite chromosomal retention.Our data strongly suggest that SLC22A18 and/or CDKN1C are tumour modifier genes involved in the tumourigenesis of SDHD-linked paraganglioma.

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.

Recent genome-wide association studies have identified several loci that confer an increased risk of colorectal cancer (CRC). We studied the role of the 8q24.21 (rs6983267), 18q21.1 (rs12953717), 15q13.3 (rs4779584), 11q23.1 (rs3802842), 8q23.3 (rs16892766), and 10p14 (rs10795668) risk variants in a series of 995 Dutch CRC cases and 1340 controls. The CRC cases were selected on basis of having a family history of CRC and/or early-onset disease. The detailed clinical and molecular data available on the cases allowed us to examine the relationship between risk variants and clinicopathologic characteristics. We replicated the association with an increased risk of CRC cancer for all loci, except 10p14. The association with the variant on chromosome 15q13.3 was confirmed for the first time. The risks associated with variants in our series were higher (not significant) than those previously reported, consistent with our series reflecting genetic enrichment. Moreover, we show that familial CRC cases possess an increased number of risk alleles compared with solitary CRC cases (early-onset; mean age at diagnosis of 48.5 years). We also identified a significant increase in the number of risk alleles in families with early-onset disease (≤50 years) compared with late-onset families (>50 years). In solitary CRC patients, enrichment for risk alleles was not observed, suggesting that other causes of increased CRC risk play a role in these cases. Overall, our results suggest that clustering of low-risk variants may explain part of the excess risk in CRC families. (Cancer Epidemiol Biomarkers Prev 2009;18(11):3062–7)

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 advance online publication 25 June 2015Genetics in Medicine (2015); doi:10.1038/gim.2015.83.

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.

AIM: Patients with germline phosphatase and tensin homologue (PTEN) mutations develop hamartomatous lesions in several organs and are at increased risk of various malignancies. We assessed the lifetime risk of benign and malignant gastrointestinal lesions in patients with a proven PTEN mutation. METHOD: Data on gender, mutation, dates of birth, last contact, and diagnosis, location and type of gastrointestinal lesions were collected from nine countries. The lifetime risk of gastrointestinal lesions was calculated by Kaplan-Meier methods. RESULTS: A total of 156 patients (67 men, 43%) from 101 families with a PTEN mutation were included. Patients were born between 1928 and 2008. Benign gastrointestinal polyps were reported in 49 (31%) patients at a mean age of 38 years (range 18-62 years) and were most often hamartomas. Twenty-two (44%) patients had upper as well as lower gastrointestinal lesions, 14 (29%) had only colonic lesions and 13 (27%) had gastrointestinal lesions at unknown sites. The cumulative risk of developing benign gastrointestinal polyps was 70% at age 60. Four patients (two men) developed colorectal carcinoma at 53, 57, 59 and 62 years, respectively. The cumulative risk of developing colorectal carcinoma was 18% at age 60. Except for one carcinoid in the small intestine, no upper gastrointestinal cancers were observed. CONCLUSION: Benign gastrointestinal lesions are common in PTEN mutation carriers, and a three- to four-fold increased lifetime risk of colorectal cancer compared with the general population may exist. Colorectal screening of patients with germline PTEN mutations is recommended, starting at age 40 years.

Two forms of genomic instability can be distinguished in colorectal cancer (CRC) tumourigenesis. One is characterised by pronounced chromosomal instability (CIN), while the other relates to alterations produced at the nucleotide level that preferentially target microsatellite sequences. Tumours developing under the latter form of genomic instability possess a microsatellite instability-high (MSI-H) phenotype due to inactivation of the DNA mismatch repair system. The most recently described CRC syndrome, MUTYH-associated polyposis (MAP), shares characteristics with both MSI-H and CIN cancers. MAP carcinomas develop from the impairment of the base excision repair system, where MUTYH is involved, but also present a peculiar form of CIN. Several clinicopathological characteristics of MSI-H and MAP CRCs overlap such as tumour location, clinical prognosis and histological features. We propose that MSI-H and MAP CRCs are particularly prone to interact with their tumour microenvironment. A great deal of this interaction is probably stimulated by the immunogenic character of those tumours, known to possess a high mutagenic potential. The accumulation of mutations in coding regions of the genome of MSI-H and MAP carcinomas is likely to translate into a surplus of neo-antigens that trigger an anti-tumour immune response. The immune system constitutes thus an important vector of selective pressure that favours the outgrowth of tumour clones with immune-evasive phenotypes. In this review, we summarise the evidence for the influence of the tumour microenvironment in MSI-H and MAP tumourigenesis. Furthermore, we discuss how particular features of MSI-H and MAP CRCs can be exploited for the development of therapeutic strategies for affected patients.

Objective: We studied the reciprocal effect of pregnancy and von Hippel-Lindau (VHL) disease by analyzing the influence of pregnancy on VHL disease-related lesions and VHL disease on pregnancy outcome. Methods: Medical charts and imaging reports from the VHL disease expertise centers in the Netherlands were used to retrospectively assess lesion progression score before and after pregnancy and to obtain data on pregnancy outcome and VHL disease-related lesions. The Friedman test was used for analysis (p

Germline variants in subunit D of the succinate dehydrogenase gene (SDHD variants) are associated with an increased risk of developing paragangliomas. The aim of this study was to compare mortality rates and survival in a Dutch cohort of SDHD variant carriers with those in the general population. The study was conducted at the Leiden University Medical Center, a tertiary referral center for patients with paragangliomas. Included subjects all tested positive for SDHD variants before 1 July 2012 and visited the departments of Otorhinolaryngology or Endocrinology at least once or had a diagnosed paraganglioma and a SDHD variant-positive family history. Clinical data were retrieved from medical records, information on mortality was obtained from the Municipal Personal Records Database, and mortality rates for the Dutch population were obtained from the Dutch Central Bureau of Statistics, stratified by sex, age and date. SDHD variant carriers were followed from the date of first SDHD variant-related contact until death, emigration or 12 December 2012 and the standardized mortality ratio (SMR) was calculated. Two-hundred and seventy-five SDHD variant carriers were included in the study, of which 80% carried the c.274G>T, p.(Asp92Tyr) variant, had a mean duration of follow-up of 7.6 years, yielding 2242 person-years of observation for analysis. There were 18 deaths in the SDHD variant carrier group; two were paraganglioma related. The SMR for the whole cohort was 1.07 (95% confidence interval 0.67-1.73). In conclusion, mortality in SDHD variant carriers is not substantially increased. Additional studies are required to confirm these findings.

Objective: Li Fraumeni syndrome (LFS) and Von Hippel-Lindau disease (VHL) are two rare hereditary tumor syndromes, characterized by a high risk of developing multiple tumors at various sites and ages for which preventive and treatment options are limited. For partners, it may be difficult to deal with the on-going threat of tumors in both their spouse and children. Therefore, this study aims to evaluate the prevalence of and factors associated with psychological distress among partners of individuals with or at high risk of LFS or VHL. Methods: As part of a nationwide, cross-sectional study, partners of individuals diagnosed with or at high risk of LFS or VHL were invited to complete a self-report questionnaire assessing distress, worries, and health-related quality of life. Results: Fifty-five (58%) of those high-risk individuals with a partner consented to having their partner approached for the study. In total, 50 partners (91%) completed the questionnaire, of whom 28% reported clinically relevant levels of syndrome-related distress. Levels of distress and worries of the partners and their high-risk spouse were significantly correlated. Younger age and a lack of social support were also associated significantly with heightened levels of distress and worries. The majority of partners (76%) believed that professional psychosocial support should be routinely offered to them. Conclusions: Approximately one-quarter of the partners exhibit clinically relevant levels of distress that warrant psychological support. The distress levels of the ‘patient’ could potentially be used to identify partners at risk of developing clinically relevant levels of distress. Copyright © 2011 John Wiley & Sons, Ltd.

Summary 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 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 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. Funding Sacha Swarttouw-Hijmans Foundation, Dutch Cancer Society, Deutsche Krebshilfe (German Cancer Aid), Hong Kong Cancer Fund, Hungarian Research Grant OTKA, Norwegian EEA Financial Mechanism (Hungarian National Institute of Oncology), and US National Cancer Institute.

Background: Carney complex (CNC) is a familial multiple neoplasia syndrome frequently associated with primary pigmented nodular adrenocortical disease (PPNAD), a bilateral form of micronodular adrenal hyperplasia that leads to Cushing's syndrome (CS). Germline PRKAR1A mutations cause CNC and only rarely isolated PPNAD. Patients and Methods: PRKAR1A mutation analysis in two large families with CS and no other CNC manifestations demonstrated a M1V germline mutation; a total of 21 asymptomatic individuals were screened, and mutation carriers were evaluated for CNC. The mutation was expressed in vitro and functionally tested for its effects on protein kinase A function. Results: Presymptomatic testing identified five first-degree relatives who were M1V carriers and who were all diagnosed with subclinical, mild CS at ages ranging from 20-56 yr. There were no other signs of CNC. In a cell-free system, we detected a shorter compared with the wild-type type 1 alpha regulatory subunit of protein kinase A (PRKAR1A) protein (43 kDa). This was not identified in cell lines from the patients or in transfection experiments in HEK293 cells that showed no detectable PRKAR1A protein from the M1V-bearing constructs. In these cells, the mutant mRNA was expressed in a 1: 1 ratio. Conclusion: In two large families, the M1V PRKAR1A mutation resulted in a PPNAD-only phenotype with significant variability both in terms of age of onset and clinical severity. Expression studies showed a unique effect of this sequence change. This study has implications for genetic counseling of carriers of this PRKAR1A mutation and patients with CNC and PPNAD and for the study of PRKAR1A-related tumorigenesis. (J Clin Endocrinol Metab 95: 338-342, 2010)

Item does not contain fulltext Familial colorectal cancer (CRC) accounts for 10-15% of all CRCs. In about 5% of all cases, CRC is associated with a highly penetrant dominant inherited syndrome. The most common inherited form of non-polyposis CRC is the Lynch syndrome which is responsible for about 2-4% of all cases. Surveillance of individuals at high risk for CRC prevents the development of advanced CRC. About 1 million individuals in Western Europe are at risk for Lynch syndrome. We performed a survey to evaluate the strategies currently used to identify individuals at high risk for CRC in 14 Western European countries. Questionnaires were distributed amongst members of a European collaborative group of experts that aims to improve the prognosis of families with hereditary CRC. The survey showed that in all countries obtaining a family history followed by referral to clinical genetics centres of suspected cases was the main strategy to identify familial and hereditary CRC. In five out of seven countries with a (regional or national) CRC population screening program, attention was paid in the program to the detection of familial CRC. In only one country were special campaigns organized to increase the awareness of familial CRC among the general population. In almost all countries, the family history is assessed when a patient visits a general practitioner or hospital. However, the quality of family history taking was felt to be rather poor. Microsatellite instability testing (MSI) or immunohistochemical analysis (IHC) of CRC are usually recommended as tools to select high-risk patients for genetic testing and are performed in most countries in patients suspected of Lynch syndrome. In one country, IHC was recommended in all new cases of CRC. In most countries there are no specific programs on cancer genetics in the teaching curriculum for medical doctors. In conclusion, the outcome of this survey and the discussions within an European expert group may be used to improve the strategies to identify individuals at high risk of CRC. More attention should be given to increasing the awareness of the general population of hereditary CRC. Immunohistochemical analysis or MSI-analysis of all CRCs may be an effective tool for identifying all Lynch syndrome families. The cost-effectiveness of this approach should be further evaluated. All countries with a CRC population screening program should obtain a full family history as part of patient assessment. 01 juni 2010

A major cause of paraganglioma and pheochromocytoma is germline mutation of the tumor suppressor genes SDHB, SDHC, and SDHD, encoding subunits of succinate dehydrogenase (SDH). While many SDH missense/nonsense mutations have been identified, few large deletions have been described. We performed multiplex ligation-dependent probe amplification deletion analysis in 126 point mutation-negative patients, and here we describe four novel deletions of SDHD and SDHC. Long-range PCR was used for the fine mapping of deletions. One patient had a 10 kb AluSg–AluSx-mediated deletion including SDHD exons 1 and 2, the entire TIMM8B gene, and deletion of exons of C11orf57. A second patient had a deletion of SDHD exons 1 and 2 and exon 1 of the TIMM8B gene. A third patient showed a deletion of exon 2 of SDHD, together with a 235 bp MIRb–Tensin gene insertion. In a fourth patient, a deletion of exons 5 and 6 of the SDHC gene was found, only the second SDHC deletion currently known. The deletions of the TIMM8B and C11orf57 genes are the first to be described, but do not appear to result in an additional phenotype in these patients. Four of the eight breakpoints occurred in Alu sequences and all three SDHD deletions showed an intron 2 breakpoint. This study underlines the fact that clinically relevant deletions may encompass neighboring genes, with the potential to modify phenotype. Gene deletions of SDHD and SDHC represent a substantial proportion of all mutations, and must be considered in paraganglioma patients shown to be negative for mutations by sequencing.

We report on multiple genomic aberrations in a patient with mental retardation. In addition, he had hypogonadism, elevated thyroid hormone levels, hearing loss, delayed speech development and mild dysmorphic features. First, we identified a mosaic karyotype, 45,X/46,X,psu dic(Y). The pseudo-dicentric Y chromosome has three short arm segments. Second, we found a germline mutation (Pro453Thr) of the thyroid hormone receptor beta (THRB) which is associated with resistance to thyroid hormone. Third, he was found to be a carrier of a heterozygous ATP7B mutation (c.2575 + 5G > C), the Wilson disease gene. Even though an array-CGH (with a density of similar to 1 Mb) did not reveal any further genomic gains or losses, we cannot exclude that all contributing factors have been identified. However, this case report shows that with increasing technological possibilities we can find more than one cause for developmental problems in a single patient. The identification of multiple causes in a single patient may complicate explaining the disorder and genetic counseling. (C) 2009 Wiley-Liss, Inc.

Mismatch repair deficiency in tumors can result from germ line mutations in one of the mismatch repair (MMR) genes (MLH1, MSH2, MSH6 and PMS2), or from sporadic promoter hypermethylation of MLH1. The role of unclassified variants (UVs) in MMR genes is subject to debate. To establish the extend of chromosomal instability and copy neutral loss of heterozygosity (cnLOH), we analyzed 41 archival microsatellite unstable carcinomas, mainly colon cancer, from 23 patients with pathogenic MMR mutations, from eight patients with UVs in one of the MMR genes and 10 cases with MLH1 promoter hypermethylation. We assessed genome wide copy number abnormalities and cnLOH using SNP arrays. SNP arrays overcome the problems of detecting LOH due to instability of polymorphic microsatellite markers. All carcinomas showed relatively few chromosomal aberrations. Also cnLOH was infrequent and in Lynch syndrome carcinomas usually confined to the locus harbouring pathogenic mutations in MLH1, MSH2 or PMS2 In the carcinomas from the MMR-UV carriers such cnLOH was less common and in the carcinomas with MLH1 promoter hypermethylation no cnLOH at MLH1 occurred. MSI-H carcinomas of most MMR-UV carriers present on average with more aberrations compared to the carcinomas from pathogenic MMR mutation carriers, suggesting that another possible pathogenic MMR mutation had not been missed. The approach we describe here shows to be an excellent way to study genome-wide cnLOH in archival mismatch repair deficient tumors.