Fetal hydrops as detected by prenatal ultrasound usually carries a poor prognosis depending on the underlying aetiology. We describe the prenatal and postnatal clinical course of two unrelated... Show moreFetal hydrops as detected by prenatal ultrasound usually carries a poor prognosis depending on the underlying aetiology. We describe the prenatal and postnatal clinical course of two unrelated female probands in whom de novo heterozygous missense variants in the planar cell polarity gene CELSR1 were detected using exome sequencing. Using several in vitro assays, we show that the CELSR1 p.(Cys1318Tyr) variant disrupted the subcellular localisation, affected cell-cell junction, impaired planar cell polarity signalling and lowered proliferation rate. These observations suggest that deleterious rare CELSR1 variants could be a possible cause of fetal hydrops. Show less
Terlouw, D.; Boot, A.; Ducarmon, Q.R.; Nooij, S.; Suerink, M.; Leerdam, M.E. van; ... ; Morreau, H. 2024
BackgroundColibactin, a genotoxin produced by polyketide synthase harboring (pks+) bacteria, induces double-strand breaks and chromosome aberrations. Consequently, enrichment of pks+Escherichia... Show moreBackgroundColibactin, a genotoxin produced by polyketide synthase harboring (pks+) bacteria, induces double-strand breaks and chromosome aberrations. Consequently, enrichment of pks+Escherichia coli in colorectal cancer and polyposis suggests a possible carcinogenic effect in the large intestine. Additionally, specific colibactin-associated mutational signatures; SBS88 and ID18 in the Catalogue of Somatic Mutations in Cancer database, are detected in colorectal carcinomas. Previous research showed that a recurrent APC splice variant perfectly fits SBS88.MethodsIn this study, we explore the presence of colibactin-associated signatures and fecal pks in an unexplained polyposis cohort. Somatic targeted Next-Generation Sequencing (NGS) was performed for 379 patients. Additionally, for a subset of 29 patients, metagenomics was performed on feces and mutational signature analyses using Whole-Genome Sequencing (WGS) on Formalin-Fixed Paraffin Embedded (FFPE) colorectal tissue blocks.ResultsNGS showed somatic APC variants fitting SBS88 or ID18 in at least one colorectal adenoma or carcinoma in 29% of patients. Fecal metagenomic analyses revealed enriched presence of pks genes in patients with somatic variants fitting colibactin-associated signatures compared to patients without variants fitting colibactin-associated signatures. Also, mutational signature analyses showed enrichment of SBS88 and ID18 in patients with variants fitting these signatures in NGS compared to patients without.ConclusionsThese findings further support colibactins ability to mutagenize colorectal mucosa and contribute to the development of colorectal adenomas and carcinomas explaining a relevant part of patients with unexplained polyposis. Show less
Werf-'t, A.S. van der; Terlouw, D.; Tops, C.M.; Kan, M.S. van; Hest, L.P. van; Gille, H.J.P.; ... ; Nielsen, M. 2023
Diagnosis of Lynch syndrome (LS) caused by a pathogenic germline MSH6 variant may be complicated by discordant immunohistochemistry (IHC) and/or by a microsatellite stable (MSS) phenotype. This... Show moreDiagnosis of Lynch syndrome (LS) caused by a pathogenic germline MSH6 variant may be complicated by discordant immunohistochemistry (IHC) and/or by a microsatellite stable (MSS) phenotype. This study aimed to identify the various causes of the discordant phenotypes of colorectal cancer (CRC) and endometrial cancer (EC) in MSH6-associated LS. Data were collected from Dutch family cancer clinics. Carriers of a (likely) pathogenic MSH6 variant diagnosed with CRC or EC were categorized based on an microsatellite instability (MSI)/IHC test outcome that might fail to result in a diagnosis of LS (eg, retained staining of all 4 mismatch repair proteins, with or without an MSS phenotype, and other staining patterns). When tumor tissue was available, MSI and/or IHC were repeated. Next-generation sequencing (NGS) was performed in cases with discordant staining patterns. Data were obtained from 360 families with 1763 (obligate) carriers. MSH6 variant carriers with CRC or EC (n = 590) were included, consisting of 418 CRCs and 232 ECs. Discordant staining was reported in 77 cases (36% of MSI/IHC results). Twelve patients gave informed consent for further analysis of tumor material. Upon revision, 2 out of 3 MSI/IHC cases were found to be concordant with the MSH6 variant, and NGS showed that 4 discordant IHC results were sporadic rather than LS-associated tumors. In 1 case, somatic events explained the discordant phenotype. The use of reflex IHC mismatch repair testing, the current standard in most Western countries, may lead to the misdiagnosis of germline MSH6 variant carriers. The pathologist should point out that further diagnostics for inheritable colon cancer, including LS, should be considered in case of a strong positive family history. Germline DNA analysis of the mismatch repair genes, preferably as part of a larger gene panel, should therefore be considered in potential LS patients. Show less
Background & AimsConstitutional mismatch repair deficiency (CMMRD) is a rare recessive childhood cancer predisposition syndrome caused by germline mismatch repair variants. Constitutional microsate...Show moreBackground & AimsConstitutional mismatch repair deficiency (CMMRD) is a rare recessive childhood cancer predisposition syndrome caused by germline mismatch repair variants. Constitutional microsatellite instability (cMSI) is a CMMRD diagnostic hallmark and may associate with cancer risk. We quantified cMSI in a large CMMRD patient cohort to explore genotype–phenotype correlations using novel MSI markers selected for instability in blood.MethodsThree CMMRD, 1 Lynch syndrome, and 2 control blood samples were genome sequenced to >120× depth. A pilot cohort of 8 CMMRD and 38 control blood samples and a blinded cohort of 56 CMMRD, 8 suspected CMMRD, 40 Lynch syndrome, and 43 control blood samples were amplicon sequenced to 5000× depth. Sample cMSI score was calculated using a published method comparing microsatellite reference allele frequencies with 80 controls.ResultsThirty-two mononucleotide repeats were selected from blood genome and pilot amplicon sequencing data. cMSI scoring using these MSI markers achieved 100% sensitivity (95% CI, 93.6%–100.0%) and specificity (95% CI 97.9%–100.0%), was reproducible, and was superior to an established tumor MSI marker panel. Lower cMSI scores were found in patients with CMMRD with MSH6 deficiency and patients with at least 1 mismatch repair missense variant, and patients with biallelic truncating/copy number variants had higher scores. cMSI score did not correlate with age at first tumor.ConclusionsWe present an inexpensive and scalable cMSI assay that enhances CMMRD detection relative to existing methods. cMSI score is associated with mismatch repair genotype but not phenotype, suggesting it is not a useful predictor of cancer risk. Show less
Terlouw, D.; Hes, F.J.; Suerink, M.; Boot, A.; Langers, A.M.J.; Tops, C.M.; ... ; Nielsen, 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
Objective: Human genomics established that pathogenic variation in diverse genes can underlie a single disorder. For example, hereditary spastic paraplegia is associated with >80 genes, with... Show moreObjective: Human genomics established that pathogenic variation in diverse genes can underlie a single disorder. For example, hereditary spastic paraplegia is associated with >80 genes, with frequently only few affected individuals described for each gene. Herein, we characterize a large cohort of individuals with biallelic variation in ENTPD1, a gene previously linked to spastic paraplegia 64 (Mendelian Inheritance in Man # 615683). Methods: Individuals with biallelic ENTPD1 variants were recruited worldwide. Deep phenotyping and molecular characterization were performed. Results: A total of 27 individuals from 17 unrelated families were studied; additional phenotypic information was collected from published cases. Twelve novel pathogenic ENTPD1 variants are described (NM 001776.6): c.398_399delinsAA; p.(Gly133Glu), c.540del; p.(Thr181Leufs*18), c.640del; p.(Gly216Glufs*75), c.185 T > G; p.(Leu62*), c.1531 T > C; p.(*511Glnext*100), c.967C > T; p.(Gln323*), c.414-2_414-1del, and c.146 A > G; p.(Tyr49Cys) including 4 recurrent variants c.1109 T > A; p.(Leu370*), c.574-6_574-3del, c.770_771del; p.(Gly257Glufs*18), and c.1041del; p.(Ile348Phefs*19). Shared disease traits include childhood onset, progressive spastic paraplegia, intellectual disability (ID), dysarthria, and white matter abnormalities. In vitro assays demonstrate that ENTPD1 expression and function are impaired and that c.574-6_574-3del causes exon skipping. Global metabolomics demonstrate ENTPD1 deficiency leads to impaired nucleotide, lipid, and energy metabolism. Interpretation: The ENTPD1 locus trait consists of childhood disease onset, ID, progressive spastic paraparesis, dysarthria, dysmorphisms, and white matter abnormalities, with some individuals showing neurocognitive regression. Investigation of an allelic series of ENTPD1 (1) expands previously described features of ENTPD1-related neurological disease, (2) highlights the importance of genotype-driven deep phenotyping, (3) documents the need for global collaborative efforts to characterize rare autosomal recessive disease traits, and (4) provides insights into disease trait neurobiology. ANN NEUROL 2022 Show less
Suerink, M.; Kilinc, G.; Terlouw, D.; Hristova, H.; Sensuk, L.; Egmond, D. van; ... ; Nielsen, M. 2021
Aims Previous estimates of the prevalence of mismatch repair (MMR) deficiency and Lynch syndrome in small bowel cancer have varied widely. The aim of this study was to establish the prevalence of... Show moreAims Previous estimates of the prevalence of mismatch repair (MMR) deficiency and Lynch syndrome in small bowel cancer have varied widely. The aim of this study was to establish the prevalence of MMR deficiency and Lynch syndrome in a large group of small bowel adenocarcinomas. Methods To this end, a total of 400 small bowel adenocarcinomas (332 resections, 68 biopsies) were collected through the Dutch nationwide registry of histopathology and cytopathology (Pathologisch-Anatomisch Landelijk Geautomatiseerd Archief (PALGA)). No preselection criteria, such as family history, were applied, thus avoiding (ascertainment) bias. MMR deficiency status was determined by immunohistochemical staining of MMR proteins, supplemented by MLH1 promoter hypermethylation analysis and next generation sequencing of the MMR genes. Results MMR deficiency was observed in 22.3% of resected and 4.4% of biopsied small bowel carcinomas. Prevalence of Lynch syndrome was 6.2% in resections and 0.0% in biopsy samples. Patients with Lynch syndrome-associated small bowel cancer were significantly younger at the time of diagnosis than patients with MMR-proficient and sporadic MMR-deficient cancers (mean age of 54.6 years vs 66.6 years and 68.8 years, respectively, p<0.000). Conclusions The prevalence of MMR deficiency and Lynch syndrome in resected small bowel adenocarcinomas is at least comparable to prevalence in colorectal cancers, a finding relevant both for treatment (immunotherapy) and family management. We recommend that all small bowel adenocarcinomas should be screened for MMR deficiency. Show less
Bajwa-ten Broeke, S.W.; Ballhausen, A.; Ahadova, A.; Suerink, M.; Bohaumilitzky, L.; Seidler, F.; ... ; Kloor, M. 2021
Lynch syndrome (LS) is caused by a pathogenic heterozygous germline variant in one of the DNA mismatch repair (MMR) genes: MLH1, MSH2, MSH6 or PMS2. LS-associated colorectal carcinomas (CRCs) are... Show moreLynch syndrome (LS) is caused by a pathogenic heterozygous germline variant in one of the DNA mismatch repair (MMR) genes: MLH1, MSH2, MSH6 or PMS2. LS-associated colorectal carcinomas (CRCs) are characterized by MMR deficiency and by accumulation of multiple insertions/deletions at coding microsatellites (cMS). MMR deficiency-induced variants at defined cMS loci have a driver function and promote tumorigenesis. Notably, PMS2 variant carriers face only a slightly increased risk of developing CRC. Here, we investigate whether this lower penetrance is also reflected by differences in molecular features and cMS variant patterns. Tumor DNA was extracted from formalin-fixed paraffin-embedded (FFPE) tissue cores or sections (n = 90). Tumors originated from genetically proven germline pathogenic MMR variant carriers (including 14 PMS2-deficient tumors). The mutational spectrum was analyzed using fluorescently labeled primers specific for 18 cMS previously described as mutational targets in MMR-deficient tumors. Immune cell infiltration was analyzed by immunohistochemical detection of T-cells on FFPE tissue sections. The cMS spectrum of PMS2-deficient CRCs did not show any sig-nificant differences from MLH1/MSH2-deficient CRCs. PMS2-deficient tumors, however, displayed lower CD3-positive T-cell infiltration compared to other MMR-deficient cancers (28.00 vs. 55.00 per 0.1 mm(2), p = 0.0025). Our study demonstrates that the spectrum of potentially immunogenic cMS variants in CRCs from PMS2 gene variant carriers is similar to that observed in CRCs from other MMR gene variant carriers. Lower immune cell infiltration observed in PMS2-deficient CRCs could be the result of alternative mechanisms of immune evasion or immune cell exclusion, similar to those seen in MMR-proficient tumors. 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
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
Terlouw, D.; Suerink, M.; Boot, A.; Wezel, T. van; Nielsen, M.; Morreau, H. 2020
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... Show moreThis 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 <70. Patients with variants outside these criteria had FAP-related extracolonic manifestations, colorectal cancer aged <40, somatic KRAS c.34G > 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 <70 is advised. Almost all FAP and MAP patients not meeting these criteria showed other characteristics that can be used as an indication to prompt genetic testing. Show less
Suerink, M.; Rodriguez-Girondo, M.; Klift, H.M. van der; Colas, C.; Brugieres, L.; Lavoine, N.; ... ; Nielsen, M. 2019
Background: PMS2-associated Lynch syndrome is characterized by a relatively low colorectal cancer penetrance compared with other Lynch syndromes. However, age at colorectal cancer diagnosis varies... Show moreBackground: PMS2-associated Lynch syndrome is characterized by a relatively low colorectal cancer penetrance compared with other Lynch syndromes. However, age at colorectal cancer diagnosis varies widely, and a strong genetic anticipation effect has been suggested for PMS2 families. In this study, we examined proposed genetic anticipation in a sample of 152 European PMS2 families.Methods: The 152 families (637 family members) that were eligible for analysis were mainly clinically ascertained via clinical genetics centers. We used weighted Cox-type random effects model, adjusted by birth cohort and sex, to estimate the generational effect on the age of onset of colorectal cancer. Probands and young birth cohorts were excluded from the analyses. Weights represented mutation probabilities based on kinship coefficients, thus avoiding testing bias.Results: Family data across three generations, including 123 colorectal cancers, were analyzed. When compared with the first generation, the crude HR for anticipation was 2.242 [95% confidence interval (CI), 1.162-4.328] for the second generation and 2.644 (95% CI, 1.082-6.464) for the third generation. However, after correction for birth cohort and sex, the effect vanished [HR = 1.302 (95% CI, 0.648-2.619) and HR = 1.074 (95% CI, 0.406-2.842) for second and third generations, respectively].Conclusions: Our study did not confirm previous reports of genetic anticipation in PMS2-associated Lynch syndrome. Birth-cohort effect seems the most likely explanation for observed younger colorectal cancer diagnosis in subsequent generations, particularly because there is currently no commonly accepted biological mechanism that could explain genetic anticipation in Lynch syndrome.Impact: This new model for studying genetic anticipation provides a standard for rigorous analysis of families with dominantly inherited cancer predisposition. Show less
