Introduction contains a general overview of the research topics discussed in this thesis.Chapter 1 addresses a fundamental question in DNA mismatch repair, which is how ATP binding and hydrolysis... Show moreIntroduction contains a general overview of the research topics discussed in this thesis.Chapter 1 addresses a fundamental question in DNA mismatch repair, which is how ATP binding and hydrolysis drive the conformational changes in MutS thatare needed for the mismatch repair cascade.Chapter 2 focuses on the final stages of the DNA mismatch repair pathway, which are the resection and subsequent resynthesis of the mismatch containing strand.Chapter 3 presents an example of how DNA polymerases can be targeted for the development of novel antibiotics against Mycobacterium tuberculosis (Mtb).Chapter 4 describes a new instrument named the Puffalot, developed for the preparation of cryo-EM grids, which aims to improve the reliability of cryo-EM sample preparation.Discussion provides a summary of the scientific findings described in this thesis in light of the published literature as well as an overview of the future directions and perspectives. 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
Background Hodgkin's lymphoma (HL) survivors treated with abdominal radiotherapy and/or procarbazine have an increased risk of developing colorectal neoplasia.Aims We evaluated the... Show moreBackground Hodgkin's lymphoma (HL) survivors treated with abdominal radiotherapy and/or procarbazine have an increased risk of developing colorectal neoplasia.Aims We evaluated the clinicopathological characteristics and risk factors for developing (advanced) neoplasia (AN) in HL survivors.Methods In all, 101 HL survivors (median age 51 years, median age of HL diagnosis 25 years) underwent colonoscopy and 350 neoplasia and 44 AN (classified as advanced adenomas/serrated lesions or colorectal cancer), mostly right-sided, were detected, as published previously. An average-risk asymptomatic cohort who underwent screening colonoscopy were controls (median age 60 years). Clinicopathological characteristics of AN were evaluated in both groups. Mismatch repair (MMR) status was assessed using immunohistochemistry (MLH1/MSH2/MSH6/PMS2). Logistic regression analysis was performed to evaluate the risk factors for AN in HL survivors, including age at HL diagnosis and interval between HL and colonoscopy.Results In 101 colonoscopies in HL survivors, AN was primarily classified based on polyp size >= 10 mm, whereas (high-grade)dysplasia was more often seen in AN in controls. An interval between HL diagnosis and colonoscopy >26 years was associated with more AN compared with an interval of <26 years, with an odds ratio for AN of 3.8 (95% confidence interval 1.4-9.1) (p < 0.01). All 39 AN that were assessed were MMR proficient.Conclusions Colorectal neoplasia in HL survivors differ from average-risk controls; classification AN was primarily based on polyp size (>= 10 mm) in HL survivors. Longer follow-up between HL diagnosis and colonoscopy was associated with a higher prevalence of AN in HL survivors. 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
Canonical DNA mismatch repair (MMR) excises base-base mismatches to increase the fidelity of DNA replication. Thus, loss of MMR leads to increased spontaneous mutagenesis. MMR genes also are... Show moreCanonical DNA mismatch repair (MMR) excises base-base mismatches to increase the fidelity of DNA replication. Thus, loss of MMR leads to increased spontaneous mutagenesis. MMR genes also are involved in the suppression of mutagenic, and the induction of protective, responses to various types of DNA damage. In this review we describe these non-canonical roles of MMR at different lesion types. Loss of non-canonical MMR gene functions may have important ramifications for the prevention, development and treatment of colorectal cancer associated with inherited MMR gene defects in Lynch syndrome. This graphical review pays tribute to Samuel H. Wilson. Sam not only made seminal contributions to understanding base excision repair, particularly with respect to structure-function relationships in DNA polymerase beta but also, as Editor of DNA Repair, has maintained a high standard of the journal. Show less
Houlleberghs, H.; Dekker, M.; Lusseveld, J.; Pieters, W.; Ravesteyn, T. van; Verhoef, S.; ... ; Riele, H. te 2020
BackgroundInactivating mutations in the MLH1 DNA mismatch repair (MMR) gene underlie 42% of Lynch syndrome (LS) cases. LS is a cancer predisposition causing early onset colorectal and endometrial... Show moreBackgroundInactivating mutations in the MLH1 DNA mismatch repair (MMR) gene underlie 42% of Lynch syndrome (LS) cases. LS is a cancer predisposition causing early onset colorectal and endometrial cancer. Nonsense and frameshift alterations unambiguously cause LS. The phenotype of missense mutations that only alter a single amino acid is often unclear. These variants of uncertain significance (VUS) hinder LS diagnosis and family screening and therefore functional tests are urgently needed. We developed a functional test for MLH1 VUS termed 'oligonucleotide-directed mutation screening' (ODMS).MethodsThe MLH1 variant was introduced by oligonucleotide-directed gene modification in mouse embryonic stem cells that were subsequently exposed to the guanine analogue 6-thioguanine to determine whether the variant abrogated MMR.ResutsIn a proof-of-principle analysis, we demonstrate that ODMS can distinguish pathogenic and non-pathogenic MLH1 variants with a sensitivity of >95% and a specificity of >91%. We subsequently applied the screen to 51 MLH1 VUS and identified 31 pathogenic variants.ConclusionODMS is a reliable tool to identify pathogenic MLH1 variants. Implementation in clinical diagnostics will improve clinical care of patients with suspected LS and their relatives. 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
Lynch syndrome (LS) is caused by germline mutations in DNA mismatch repair (MMR) genes and is the most prevalent hereditary colorectal cancer syndrome. A significant proportion of variants... Show moreLynch syndrome (LS) is caused by germline mutations in DNA mismatch repair (MMR) genes and is the most prevalent hereditary colorectal cancer syndrome. A significant proportion of variants identified in MMR and other common cancer susceptibility genes are missense or noncoding changes whose consequences for pathogenicity cannot be easily interpreted. Such variants are designated as __variants of uncertain significance__ (VUS). Management of LS can be significantly improved by identifying individuals who carry a pathogenic variant and thus benefit from screening, preventive, and therapeutic measures. Also, identifying family members that do not carry the variant is important so they can be released from the intensive surveillance. Determining which genetic variants are pathogenic and which are neutral is a major challenge in clinical genetics. The profound mechanistic knowledge on the genetics and biochemistry of MMR enables the development and use of targeted assays to evaluate the pathogenicity of variants found in suspected patients with LS. I describe different approaches for the functional analysis of MMR gene VUS. Show less
