Constitutional mismatch repair deficiency (CMMRD) syndrome is a rare autosomal recessive genetic disorder caused by biallelic germline mutations in one of the mismatch repair genes. Carriers are at... Show moreConstitutional mismatch repair deficiency (CMMRD) syndrome is a rare autosomal recessive genetic disorder caused by biallelic germline mutations in one of the mismatch repair genes. Carriers are at exceptionally high risk for developing, typically in early life, hematological and brain malignancies, as well as cancers observed in Lynch syndrome. We report a homozygous MLH1 missense variant (c.1918C>A p.(Pro640Thr)) in a Tunisian patient with CMMRD syndrome and a family history of early-age colorectal cancer. The proband presented initially with colonic oligopolyposis and adenosquamous carcinoma of the caecum. He later developed several malignancies, including undifferentiated carcinoma of the parotid, grade 4 IDH-mutant astrocytoma, and ampulla of Vater adenocarcinoma. The patient was older than typical for this disease and had a remarkably prolonged survival despite developing four distinct aggressive malignancies. The current report highlights the challenges in assessing the pathogenicity of the identified variant and the remarkable phenotypic diversity in CMMRD. Show less
Pieters, W.; Hugenholtz, F.; Kos, K.; Cammeraat, M.; Moliej, T.C.; Kaldenbach, D.; ... ; Riele, H. te 2022
The gut microbiota strongly impacts the development of sporadic colorectal cancer (CRC), but it is largely unknown how the microbiota affects the pathogenesis of mismatch-repair-deficient CRC in... Show moreThe gut microbiota strongly impacts the development of sporadic colorectal cancer (CRC), but it is largely unknown how the microbiota affects the pathogenesis of mismatch-repair-deficient CRC in the context of Lynch syndrome. In a mouse model for Lynch syndrome, we found a nearly complete loss of intestinal tumor development when animals were transferred from a conventional "open" animal facility to specific-pathogen-free (SPF) conditions. Using 16S sequencing we detected large changes in microbiota composition between the two facilities. Transcriptomic analyses of tumor-free intestinal tissues showed signs of strong intestinal inflammation in conventional mice. Whole exome sequencing of tumors developing in Msh2-Lynch mice revealed a much lower mutational load in the single SPF tumor than in tumors developing in conventional mice, suggesting reduced epithelial proliferation in SPF mice. Fecal microbiota transplantations with conventional feces altered the immune landscape and gut homeostasis, illustrated by increased gut length and elevated epithelial proliferation and migration. This was associated with drastic changes in microbiota composition, in particular increased relative abundances of different mucus-degrading taxa such as Desulfovibrio and Akkermansia, and increased bacterial-epithelial contact. Strikingly, transplantation of conventional microbiota increased microsatellite instability in untransformed intestinal epithelium of Msh2-Lynch mice, indicating that the composition of the microbiota influences the rate of mutagenesis in MSH2-deficient crypts. Show less
Helderman, N.C.; Elsayed, F.A.; Wezel, T. van; Terlouw, D.; Langers, A.M.J.; Egmond, D. van; ... ; Suerink, M. 2022
& nbsp;Small intestine-neuroendocrine tumors (SI-NETs) are one of the most common tumors of the small bowel. Despite an increasing incidence, the exact mechanisms driving underlying pathology... Show more& nbsp;Small intestine-neuroendocrine tumors (SI-NETs) are one of the most common tumors of the small bowel. Despite an increasing incidence, the exact mechanisms driving underlying pathology remain to be determined. Interestingly, recent studies linked the development of (SI-)NETs to both Lynch syndrome (LS) and MUTYH variants. If confirmed, these associations would have important consequences for treatment. In this study we therefore investigated the prevalence of mismatch repair (MMR) deficiency and MUTYH variants in 64 primary resected SI-NETs. Immunohistochemistry was used to assess the expression of the MMR genes, and competitive allele-specific PCR (KASPar) targeting two hotspot MUTYH variants [p.(Tyr179Cys), p.(Gly396Asp)] was performed to determine their prevalence in SI-NETs. Strikingly, all 64 SI-NETs stained positive for MSH6 and PMS2, indicating & nbsp;MMR proficiency. In addition, no MUTYH hotspot variant was found in any of the 64 SI-NETs. As such, these results do not support an association between SI-NET development and LS or MUTYH variants. In order to gain insight into SI-NET pathogenesis and optimally manage patients, future research should therefore focus on other candidate genes. (C) 2022 Published by Elsevier Inc Show less
Rayner, E.; Tiersma, Y.; Fortuno, C.; Hees-Stuivenberg, S. van; Drost, M.; Thompson, B.; ... ; Wind, N. de 2022
The large majority of germline alterations identified in the DNA mismatch repair (MMR) gene PMS2, a low-penetrance gene for the cancer predisposition Lynch syndrome, represent variants of uncertain... Show moreThe large majority of germline alterations identified in the DNA mismatch repair (MMR) gene PMS2, a low-penetrance gene for the cancer predisposition Lynch syndrome, represent variants of uncertain significance (VUS). The inability to classify most VUS interferes with personalized healthcare. The complete in vitro MMR activity (CIMRA) assay, that only requires sequence information on the VUS, provides a functional analysis-based quantitative tool to improve the classification of VUS in MMR proteins. To derive a formula that translates CIMRA assay results into the odds of pathogenicity (OddsPath) for VUS in PMS2 we used a set of clinically classified PMS2 variants supplemented by inactivating variants that were generated by an in cellulo genetic screen, as proxies for cancer-predisposing variants. Validation of this OddsPath revealed high predictive values for benign and predisposing PMS2 VUS. We conclude that the OddsPath provides an integral metric that, following the other, higher penetrance, MMR proteins MSH2, MSH6 and MLH1 can be incorporated as strong evidence type into the upcoming criteria for MMR gene VUS classification of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP). Show less
Helderman, N.C.; Bajwa-ten Broeke, S.W.; Morreau, H.; Suerink, M.; Terlouw, D.; Werf-' t Lam, A.S. van der; ... ; Nielsen, M. 2021
Lynch syndrome (LS) is a hereditary cancer syndrome that accounts for 3% of all new colorectal cancer (CRC) cases. Patients carry a germline pathogenic variant in one of the mismatch repair (MMR)... Show moreLynch syndrome (LS) is a hereditary cancer syndrome that accounts for 3% of all new colorectal cancer (CRC) cases. Patients carry a germline pathogenic variant in one of the mismatch repair (MMR) genes (MLH1, MSH2, MSH6 or PMS2), which encode proteins involved in a post-replicative proofreading and editing mechanism. The clinical presentation of LS is highly heterogeneous, showing high variability in age at onset and penetrance of cancer, which may be partly attributable to the molecular profiles of carcinomas. This review discusses the frequency of alterations in the WNT/B-CATENIN, RAF/MEK/ERK and PI3K/PTEN/AKT pathways identified in all four LS subgroups and how these changes may relate to the 'three pathway model' of carcinogenesis, in which LS CRCs develop from MMR-proficient adenomas, MMR-deficient adenomas or directly from MMR-deficient crypts. Understanding the specific differences in carcinogenesis for each LS subgroup will aid in the further optimization of guidelines for diagnosis, surveillance and treatment. Show less
Mismatch repair (MMR) testing is recommended in the Netherlands for all patients under 70 years of age with newly diagnosed colorectal cancer (CRC) in order to identify Lynch syndrome. T1 CRC can... Show moreMismatch repair (MMR) testing is recommended in the Netherlands for all patients under 70 years of age with newly diagnosed colorectal cancer (CRC) in order to identify Lynch syndrome. T1 CRC can be removed by local excision or oncological surgical resection. We evaluated the frequency of MMR testing in pT1 lesions within the Dutch CRC screening cohort. pT1 CRC diagnosed within the Dutch population-based screening program from 2016-2018 were identified by the Dutch pathology registry (PALGA). Pathology reports were evaluated, including registration of MMR testing (by immunohistochemistry and/or microsatellite instability PCR). Frequency of MMR testing was compared between pT1 tumors that were treated by local (endoscopic or transanal) excision and oncological surgical resections. A total of 3.692 pT1 CRCs were diagnosed (median age 63 years, 61.4% males). MMR testing was performed in 83% and uptake increased over time (71% in 2016 to 92% in 2018, p<0.01). MMR testing was significantly more often performed in younger patients and in academic hospitals. When pT1 CRC was treated by oncological surgical resection (n=1.132), MMR testing was performed in 89% of cases and was known prior to oncological resection in 51% of cases. MMR testing occurred significantly less often in case of local excision (80% of n=2.560) compared to oncological surgical resection (p<0.01). MMR testing was performed in 83% of T1 CRCs and uptake increased over time. MMR testing was more frequently performed in pT1 CRC resected by oncological surgical resection compared with local excision. Show less
Heterozygous carriers of a pathogenic variant in the mismatch repair genes have an increased risk to develop colorectal cancer and various other types of cancer during adulthood. This cancer... Show moreHeterozygous carriers of a pathogenic variant in the mismatch repair genes have an increased risk to develop colorectal cancer and various other types of cancer during adulthood. This cancer predisposition syndrome is called Lynch syndrome. Children who carry a mutation on both copies of a mismatch repair gene develop malignancies during childhood or adolescence. This syndrome is called constitutional mismatch repair deficiency (CMMRD). The aim of this thesis is 1) to provide insights that may help in the identification of patients with Lynch syndrome and CMMRD, and 2) to further elucidate the phenotype and potential modifying factors that result from carrying a germline pathogenic variant in one of the mismatch repair genes. Both aims are important to further facilitate adequate detection and surveillance. Show less
Simple SummaryFemale Lynch syndrome (LS) carriers have an increased risk to develop endometrial and ovarian cancer. In the Netherlands, carriers are therefore advised annual gynecological... Show moreSimple SummaryFemale Lynch syndrome (LS) carriers have an increased risk to develop endometrial and ovarian cancer. In the Netherlands, carriers are therefore advised annual gynecological surveillance and eventually, risk-reducing surgery. Global gynecological LS surveillance guidelines are scarce and based on limited evidence. These are, however, warranted to offer accurate surveillance. To provide more insight into surveillance outcomes, this study assessed outcomes of gynecological surveillance and risk-reducing surgery in 164 LS carriers diagnosed in our center, with a median follow-up of 5.6 years per carrier. Although most surveillance visits happened within an advised timeframe, we observed large variability in how gynecological surveillance visits were performed. This finding stresses the need for development of clear and evidence-based guidelines. Endometrial cancers identified at surveillance were all found in early stage, mostly symptomatic, questioning surveillance benefit. Large, prospective studies should assess to what extent current LS surveillance programs contribute to early detection of gynecological tumors.Lynch syndrome (LS) is caused by pathogenic germline variants in DNA mismatch repair (MMR) genes, predisposing female carriers for endometrial cancer (EC) and ovarian cancer (OC). Since gynecological LS surveillance guidelines are based on little evidence, we assessed its outcomes. Data regarding gynecological tumors, surveillance, and (risk-reducing) surgery were collected from female LS carriers diagnosed in our center since 1993. Of 505 female carriers, 104 had a gynecological malignancy prior to genetic LS diagnosis. Of 264 carriers eligible for gynecological management, 164 carriers gave informed consent and had available surveillance data: 38 MLH1, 25 MSH2, 82 MSH6, and 19 PMS2 carriers (median follow-up 5.6 years). Surveillance intervals were within advised time in >80%. Transvaginal ultrasound, endometrial sampling, and CA125 measurements were performed in 76.8%, 35.9%, and 40.6%, respectively. Four symptomatic ECs, one symptomatic OC, and one asymptomatic EC were diagnosed. Endometrial hyperplasia was found in eight carriers, of whom three were symptomatic. Risk-reducing surgery was performed in 73 (45.5%) carriers (median age 51 years), revealing two asymptomatic ECs. All ECs were diagnosed in FIGO I. Gynecological management in LS carriers varied largely, stressing the need for uniform, evidence-based guidelines. Most ECs presented early and symptomatically, questioning the surveillance benefit in its current form. Show less
People with Lynch syndrome (LS), who carry a pathogenic mutation in a DNA mismatch repair gene, have increased risks of colorectal cancer (CRC) and endometrial cancer (EC). A high reported... Show morePeople with Lynch syndrome (LS), who carry a pathogenic mutation in a DNA mismatch repair gene, have increased risks of colorectal cancer (CRC) and endometrial cancer (EC). A high reported variability in cancer risk suggests the existence of factors that modify cancer risk for persons with LS. We aimed to investigate the associations between height and CRC and EC risk for persons with LS using data from 2 large studies. Information on 1,115 men and 1,553 women with LS from the Colon Cancer Family Registry (1998-2007) and the GEOLynch Cohort Study (2006-2017) was harmonized. We used weighted Cox proportional hazards regression models with age on the time axis to estimate adjusted hazard ratios and 95% confidence intervals for each 5-cm increment in self-reported height. CRC was diagnosed in 947 persons during 65,369 person-years of observation, and 171 women were diagnosed with EC during 39,227 person-years. Height was not associated with CRC for either men (per 5-cm increment, hazard ratio (HR) = 1.00, 95% confidence interval (CI): 0.91, 1.11) or women (per 5-cm increment, HR = 1.01, 95% CI: 0.92, 1.11), nor was height associated with EC (per 5-cm increment, HR = 1.08, 95% CI: 0.94, 1.24). Hence, we observed no evidence for an association of height with either CRC or EC among persons with LS. Show less
Lynch syndrome is the most frequent hereditary colorectal cancer (CRC) syndrome, affecting approximately 1 in 300 in the Western population. It is caused by pathogenic variants in the mismatch... Show moreLynch syndrome is the most frequent hereditary colorectal cancer (CRC) syndrome, affecting approximately 1 in 300 in the Western population. It is caused by pathogenic variants in the mismatch repair (MMR) genes including MLH1, MSH2 (EPCAM), MSH6 and PMS2, and is associated with high risks of CRC, endometrial cancer and other cancers. In view of these risks, carriers of such variants are encouraged to participate in colonoscopic surveillance programs that are known to substantially improve their prognosis. In the last decade several important studies have been published that provide detailed cancer risk estimates and prognoses based on large numbers of patients. These studies also provided new insights regarding the pathways of carcinogenesis in CRC, which appear to differ depending on the specific MMR gene defect. In this report, we will discuss the implications of these new findings for the development of new surveillance protocols. Show less
Sina, M.; Ghorbanoghli, Z.; Abedrabbo, A.; Al-Mulla, F.; Sghaier, R. ben; Buisine, M.P.; ... ; Middle East Netw 2020
Background Lynch syndrome (LS), the most common inherited form of colorectal cancer (CRC), is responsible for 3% of all cases of CRC. LS is caused by a mismatch repair gene defect and is... Show moreBackground Lynch syndrome (LS), the most common inherited form of colorectal cancer (CRC), is responsible for 3% of all cases of CRC. LS is caused by a mismatch repair gene defect and is characterized by a high risk for CRC, endometrial cancer and several other cancers. Identification of LS is of utmost importance because colonoscopic surveillance substantially improves a patient's prognosis. Recently, a network of physicians in Middle Eastern and North African (ME/NA) countries was established to improve the identification and management of LS families. The aim of the present survey was to evaluate current healthcare for families with LS in this region. Methods A questionnaire was developed that addressed the following issues: availability of clinical management guidelines for LS; attention paid to family history of cancer; availability of genetic services for identification and diagnosis of LS; and assessment of knowledge of LS surveillance. Members of the network and authors of recent papers on LS from ME/NA and neighbouring countries were invited to participate in the survey and complete the online questionnaire. Results A total of 55 individuals were invited and 19 respondents from twelve countries including Algeria, Azerbaijan, Cyprus, Egypt, Iran, Jordan, Kuwait, Lebanon, Morocco, Palestine, Tunisia, and Turkey completed the questionnaire. The results showed that family history of CRC is considered in less than half of the surveyed countries. Guidelines for the management of LS are available in three out of twelve countries. The identification and selection of families for genetic testing were based on clinical criteria (Amsterdam criteria II or Revised Bethesda criteria) in most countries, and only one country performed universal screening. In most of the surveyed countries genetic services were available in few hospitals or only in a research setting. However, surveillance of LS families was offered in the majority of countries and most frequently consisted of regular colonoscopy. Conclusion The identification and management of LS in ME/NA countries are suboptimal and as a result most LS families in the region remain undetected. Future efforts should focus on increasing awareness of LS amongst both the general population and doctors, and on the improvement of the infrastructure in these countries. Show less
Schubert, S.A.; Ruano, D.; Tiersma, Y.; Drost, M.; Wind, N. de; Nielsen, M.; ... ; Wezel, T. van 2020
We describe a family severely affected by colorectal cancer (CRC) where whole-exome sequencing identified the coinheritance of the germline variants encoding MSH6 p.Thr1100Met and MUTYH p.Tyr179Cys... Show moreWe describe a family severely affected by colorectal cancer (CRC) where whole-exome sequencing identified the coinheritance of the germline variants encoding MSH6 p.Thr1100Met and MUTYH p.Tyr179Cys in, at least, three CRC patients diagnosed before 60 years of age. Digenic inheritance of monoallelic MSH6 variants of uncertain significance and MUTYH variants has been suggested to predispose to Lynch syndrome-associated cancers; however, cosegregation with disease in the familial setting has not yet been established. The identification of individuals carrying multiple potential cancer risk variants is expected to rise with the increased application of whole-genome sequencing and large multigene panel testing in clinical genetic counseling of familial cancer patients. Here we demonstrate the coinheritance of monoallelic variants in MSH6 and MUTYH consistent with cosegregation with CRC, further supporting a role for digenic inheritance in cancer predisposition. Show less
Brouwer, J.G.M.; Snellen, M.; Bisseling, T.M.; Koornstra, J.J.; Vasen, H.F.A.; Kampman, E.; Duijnhoven, F.J.B. van 2020
A cancer diagnosis is suggested to be associated with changes in dietary and lifestyle habits. Whether this applies to persons with familial cancer, such as Lynch syndrome (LS) is unknown. We... Show moreA cancer diagnosis is suggested to be associated with changes in dietary and lifestyle habits. Whether this applies to persons with familial cancer, such as Lynch syndrome (LS) is unknown. We investigated whether a colorectal neoplasm (CRN) diagnosis in persons with LS is associated with changes in dietary and lifestyle habits over time. We used data of confirmed LS mutation carriers from the GEOLynch study, a prospective cohort study. Information on dietary intake and lifestyle habits was collected with a validated semi-quantitative food frequency questionnaire and a general questionnaire administered at baseline (2006-2008) and follow-up (2012-2017). Participants' medical records were used to identify CRN diagnoses. Changes in dietary and lifestyle habits in the CRN and the no-CRN group were compared using multivariable linear regression models for continuous variables and cross-tables with percentage change at follow-up compared with baseline for categorical variables. Of the 324 included participants, 146 developed a CRN (CRN group) between baseline and follow-up, while 178 did not (no-CRN group). Smoking cessation was more often reported in the CRN than in the no-CRN group (41.4% vs. 35.0%). There were no differences in changes of energy intake, alcohol, red meat, processed meat, dairy, fruit, vegetables and dietary fiber consumption, BMI, physical activity and NSAID use. Apart from a potentially higher likelihood of smoking cessation, we found little evidence that a CRN diagnosis is associated with changes in lifestyle habits in persons with LS. Show less
Thompson, B.A.; Walters, R.; Parsons, M.T.; Dumenil, T.; Drost, M.; Tiersma, Y.; ... ; InSiGHT Variant Interpretation Com 2020
Functional assays that assess mRNA splicing can be used in interpretation of the clinical significance of sequence variants, including the Lynch syndrome-associated mismatch repair (MMR) genes. The... Show moreFunctional assays that assess mRNA splicing can be used in interpretation of the clinical significance of sequence variants, including the Lynch syndrome-associated mismatch repair (MMR) genes. The purpose of this study was to investigate the contribution of splicing assay data to the classification of MMR gene sequence variants. We assayed mRNA splicing for 24 sequence variants inMLH1,MSH2, andMSH6, including 12 missense variants that were also assessed using a cell-freein vitroMMR activity (CIMRA) assay. Multifactorial likelihood analysis was conducted for each variant, combining CIMRA outputs and clinical data where available. We collated these results with existing public data to provide a dataset of splicing assay results for a total of 671 MMR gene sequence variants (328 missense/in-frame indel), and published and unpublished repair activity measurements for 154 of these variants. There were 241 variants for which a splicing aberration was detected: 92 complete impact, 33 incomplete impact, and 116 where it was not possible to determine complete versus incomplete splicing impact. Splicing results mostly aided in the interpretation of intronic (72%) and silent (92%) variants and were the least useful for missense substitutions/in-frame indels (10%). MMR protein functional activity assays were more useful in the analysis of these exonic variants but by design they were not able to detect clinically important splicing aberrations identified by parallel mRNA assays. The development of high throughput assays that can quantitatively assess impact on mRNA transcript expression and protein function in parallel will streamline classification of MMR gene sequence variants. Show less
Drost, M.; Tiersma, Y.; Glubb, D.; Kathe, S.; Hees, S. van; Calleja, F.; ... ; Wind, N. de 2020
Purpose Variants in the DNA mismatch repair (MMR) gene MSH6, identified in individuals suspected of Lynch syndrome, are difficult to classify owing to the low cancer penetrance of defects in that... Show morePurpose Variants in the DNA mismatch repair (MMR) gene MSH6, identified in individuals suspected of Lynch syndrome, are difficult to classify owing to the low cancer penetrance of defects in that gene. This not only obfuscates personalized health care but also the development of a rapid and reliable classification procedure that does not require clinical data. Methods The complete in vitro MMR activity (CIMRA) assay was calibrated against clinically classified MSH6 variants and, employing Bayes' rule, integrated with computational predictions of pathogenicity. To enable the validation of this two-component classification procedure we have employed a genetic screen to generate a large set of inactivating Msh6 variants, as proxies for pathogenic variants. Results The genetic screen-derived variants established that the two-component classification procedure displays high sensitivities and specificities. Moreover, these inactivating variants enabled the direct reclassification of human variants of uncertain significance (VUS) as (likely) pathogenic. Conclusion The two-component classification procedure and the genetic screens provide complementary approaches to rapidly and cost-effectively classify the large majority of human MSH6 variants. The approach followed here provides a template for the classification of variants in other disease-predisposing genes, facilitating the translation of personalized genomics into personalized health care. Show less
Ryan, N.; Wall, J.; Crosbie, E.J.; Arends, M.; Bosse, T.; Arif, S.; ... ; Singh, N. 2019
Purpose: To enhance classification of variants of uncertain significance (VUS) in the DNA mismatch repair (MMR) genes in the cancer predisposition Lynch syndrome, we developed the cellfree in vitro... Show morePurpose: To enhance classification of variants of uncertain significance (VUS) in the DNA mismatch repair (MMR) genes in the cancer predisposition Lynch syndrome, we developed the cellfree in vitro MMR activity (CIMRA) assay. Here, we calibrate and validate the assay, enabling its integration with in silico and clinical data.Methods: Two sets of previously classified MLH1 and MSH2 variants were selected from a curated MMR gene database, and their biochemical activity determined by the CIMRA assay. The assay was calibrated by regression analysis followed by symmetric cross-validation and Bayesian integration with in silico predictions of pathogenicity. CIMRA assay reproducibility was assessed in four laboratories.Results: Concordance between the training runs met our prespecified validation criterion. The CIMRA assay alone correctly classified 65% of variants, with only 3% discordant classification. Bayesian integration with in silico predictions of pathogenicity increased the proportion of correctly classified variants to 87%, without changing the discordance rate. Interlaboratory results were highly reproducible.Conclusion: The CIMRA assay accurately predicts pathogenic and benign MMR gene variants. Quantitative combination of assay results with in silico analysis correctly classified the majority of variants. Using this calibration, CIMRA assay results can be integrated into the diagnostic algorithm for MMR gene variants. Show less
Sghaier, R. ben; Jansen, A.M.L.; Bdioui, A.; Wezel, T. van; Ksiaa, M.; Elgolli, L.; ... ; Morreau, H. 2019
A high colorectal cancer (CRC) incidence is observed in Tunisia, with a relatively high proportion of patients developing CRC before the age of 40. While this suggests a genetic susceptibility,... Show moreA high colorectal cancer (CRC) incidence is observed in Tunisia, with a relatively high proportion of patients developing CRC before the age of 40. While this suggests a genetic susceptibility, only a few Tunisian Lynch Syndrome families have been described. In this study we aimed to identify the underlying genetic cause in 32 patients with early onset CRC and/or a positive family history. Of twenty-four patients' tumor or biopsies could be analyzed with immunohistochemical staining to detect loss of expression of one of the MMR proteins. Ten tumors showed loss of expression, of which one tumor was from a patient where a germline pathogenic MSH2 variant was detected previously with Sanger sequencing. Next generation sequencing of the MMR, POLE and POLD1 genes was performed in leukocyte and tumor DNA of the remaining nine patients, as well as in two patients with MMR-proficient tumors, but with severe family history. In six of 11 patients a germline variant was detected in MLH1 (n=5) or MSH2 (n=1). Two of six patients were from the same family and both were found to carry a novel in-frame MLH1 deletion, predicted to affect MLH1 function. All MLH1 variant carriers had loss of heterozygosity with retention of the variant in the tumors, while a somatic pathogenic variant was detected in the patient with the germline MSH2 variant. Show less