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Chez les mammifères, la méthylation est une marque épigénétique ciblant la cytosine principalement dans un contexte CpG pour produire une 5mC. 5mC est très sensible à une déamination spontanée ou enzymatique, conduisant à la formation d'un mésappariement G/T. La 5mCpeut également être oxydée pour former successivement la 5hmC, la 5fC et la 5caC. Ces modifications de la 5mC participent aux processus actifs de déméthylation de l’ADN. Chez les mammifères, la thymine, dans le mésappariement G/T, est clivée par TDG et MBD4. TDG est également en mesure d'exciser 5fC et 5caC. Cette thèse avait pour but de clarifier la fonction de TDG et MBD4 dans la dynamique de la 5mC. Nous avons montré que MBD4 est associée aux protéines de réparation des mésappariements. Les tests enzymatiques, in vitro, montrent que le complexe MBD4/MMR a une activité bifonctionnelle (glycosylase/lyase) spécifique pour G/T, qui est régulée par la méthylation. Pour TDG, nous avons ciblé cette enzyme dans les cellules MEF et caractérisé la distribution des cytosines modifiées. Les résultats montrent des profils de méthylation/oxydation d'ADN qui sont régulés par TDG et surviennent principalement au niveau des répétitions de CA et dans les rétroéléments spécifiques de la lignée souris. / In mammals, methylation is an epigenetic mark targeting cytosine mainly in a CpG context, producing 5mC. 5mC is highly sensitive to a spontaneous or enzymatic deamination leading to G/Tmismatch. 5mC can also be oxidized to 5- 5hmC, 5fC and 5caC. These modifications of 5mC participate in the active demethylation processes. In mammals, the thymine in G/T mismatch is cleaved by TDG and MBD4 glycosylases. TDG is able also to excise the 5fC and 5caC.This thesis was to clarify the function of TDG and MBD4 in the dynamics of 5mC. We showed that MBD4 is associated with PMS2, MLH1, MSH2 and MSH6 proteins, four proteins involved in DNA mismatch repair (MMR). The in vitro enzymatic tests show that MBD4/MMR complex has a bifunctional glycosylase/lyase activity specific for G/T and is regulated by methylation.For TDG, we targeted this enzyme in MEF cells and characterized the distribution of modified cytosines. The results show that DNA methylation/oxidation patterns are regulated by TDG and occur mainly at CA repeats and at the mouse-lineage specific retro-elements.

Genomic instability underlies diseases of unregulated cell growth that result in cancers and developmental abnormalities in humans. Similar genome destabilizing mechanisms are used to create genetic variety in crops for use in breeding and trait development. Errors that occur during DNA replication may cause mutations if they are not corrected before further cell divisions. DNA mismatch repair (MMR) corrects misinsertions and insertion/deletion DNA loop-outs that arise during DNA replication in plants, animals, prokaryotes, and some archaea, all of which incur mutations at rates 100 to 1,000-fold greater when subjected to inherited or somatic-mismatch repair deficiencies. An understanding of the effects of mismatch repair on somatic and germ-line cells in Arabidopsis thaliana is critical to the development of this plant as a model system for the study of genomic instability. Insertions and deletions of multiples of two base pairs in dinucleotide repeat sequences (microsatellites) occur more frequently in the absence of mismatch repair, and the mismatch-repair status of an individual, tissue, or cell may be inferred on the basis of microsatellite mutation frequency. Single-template PCR analysis measured microsatellite mutation frequencies in leaves and shoot-apical-meristem stem cells, and allowed me to address for the first time an important question: Do plants relax mismatch repair in vegetative tissues relative to meristematic germ-line and floral tissue? Analyses of four microsatellite loci in mismatch repair-deficient and wild type plants surprisingly suggest that there is little difference in mismatch repair activity between leaves and seeds. Mismatch-repair-deficient leaves displayed only two-fold higher microsatellite mutation frequency compared to wild type, and wild-type leaves also displayed a two-fold higher microsatellite mutation frequency compared to shoot-apical- meristems. The high frequency of microsatellite mutation in these wildtype tissues is unexpected, and it suggests that plants relax mismatch repair in differentiated tissues while maintaining genetic fidelity in a small set of stem cells in the shoot apical meristem (SAM). Genome sequencing of msh2⁻/⁻ mutation accumulation A. thaliana lines provides an estimated germ-line mutation rate of 3.9 × 10⁻⁷ in the absence of mismatch repair. Comparison of the rates of base substitution mutation per chromosome in mismatch repair-deficient plants with rates reported for wild-type plants suggests mismatch repair is more efficient on chromosome 5 than on chromosomes 1-4. Bias towards G:C → A:T mutations among transitions is maintained but increased nearly 100-fold in the absence of mismatch repair. / Graduation date: 2012

Germline mutations in mismatch repair genes predispose individuals to Lynch Syndrome, the most common colorectal cancer predisposition syndrome. MLH1 is a key mismatch repair gene that is mutated in Lynch syndrome and about a third of the genetic alterations identified in MLH1 are missense variants of unclassified clinical significance. We hypothesize that missense alterations in distinct domains of MLH1 likely affect its expression and function(s) to varying degrees. To address this we utilized several approaches to investigate the molecular basis of the pathogenicity of a panel of unclassified variants. Our results demonstrate that the MLH1 variants p.R265C and p.K618A significantly decrease the stability of the MLH1 protein, while the variant p.L749Q compromises heterodimerization of the MLH1-PMS2 complex. Given the limitations and complexity of in vitro assessment strategies, we conducted a proof-of-principle study to investigate whether missense variants in MLH1 lead to allelic imbalances at the transcriptional level. Our analysis using the PeakPicker software indicated that the missense variants c.350C>T, c.793C>T, c.1852_1853AA>GC, as well as the truncating variant c.1528C>T were associated with significantly unbalanced allelic expression. This illustrates a novel method of investigating the pathogenicity of unclassified genetic variants, which has the potential to be applied in the diagnostic setting. Previous genetic epidemiology studies from our laboratory have demonstrated that the MLH1 c.-93G>A promoter variant is strongly associated with the microsatellite instability phenotype in colorectal tumours. Additionally, this promoter variant was associated with an elevated risk of endometrial cancer in case-control studies. Results from our functional studies indicate that the c.-93G>A variant significantly alters the promoter activity of MLH1. The MLH1 promoter is bi-directional with the EPM2AIP1 gene located on the antisense strand. Interestingly, we observed that this variant significantly affected EPM2AIP1 transcription as well. Furthermore, our experiments suggest that c.-93G>A variant affects transcription by altering the affinity of nuclear factors that bind this region. Combined, these findings shed light on the possible mechanisms by which missense variants affect MLH1 expression and function, which in conjunction with results from other functional assays will help increase the accuracy and efficiency of genetic testing of inherited cancers.

Germline mutations in mismatch repair genes predispose individuals to Lynch Syndrome, the most common colorectal cancer predisposition syndrome. MLH1 is a key mismatch repair gene that is mutated in Lynch syndrome and about a third of the genetic alterations identified in MLH1 are missense variants of unclassified clinical significance. We hypothesize that missense alterations in distinct domains of MLH1 likely affect its expression and function(s) to varying degrees. To address this we utilized several approaches to investigate the molecular basis of the pathogenicity of a panel of unclassified variants. Our results demonstrate that the MLH1 variants p.R265C and p.K618A significantly decrease the stability of the MLH1 protein, while the variant p.L749Q compromises heterodimerization of the MLH1-PMS2 complex. Given the limitations and complexity of in vitro assessment strategies, we conducted a proof-of-principle study to investigate whether missense variants in MLH1 lead to allelic imbalances at the transcriptional level. Our analysis using the PeakPicker software indicated that the missense variants c.350C>T, c.793C>T, c.1852_1853AA>GC, as well as the truncating variant c.1528C>T were associated with significantly unbalanced allelic expression. This illustrates a novel method of investigating the pathogenicity of unclassified genetic variants, which has the potential to be applied in the diagnostic setting. Previous genetic epidemiology studies from our laboratory have demonstrated that the MLH1 c.-93G>A promoter variant is strongly associated with the microsatellite instability phenotype in colorectal tumours. Additionally, this promoter variant was associated with an elevated risk of endometrial cancer in case-control studies. Results from our functional studies indicate that the c.-93G>A variant significantly alters the promoter activity of MLH1. The MLH1 promoter is bi-directional with the EPM2AIP1 gene located on the antisense strand. Interestingly, we observed that this variant significantly affected EPM2AIP1 transcription as well. Furthermore, our experiments suggest that c.-93G>A variant affects transcription by altering the affinity of nuclear factors that bind this region. Combined, these findings shed light on the possible mechanisms by which missense variants affect MLH1 expression and function, which in conjunction with results from other functional assays will help increase the accuracy and efficiency of genetic testing of inherited cancers.

DNA mismatch repair ensures faithful transmission of genetic material from parents to progeny, which is required for the survival of the organism. The studies on E. coli MMR proteins have formed the basis for the study of the MMR system in eukaryotic organisms, because the functions of MMR proteins believed to be been conserved. In organisms that harbor MutH protein, it is known that MutH acts as a monomer which nicks the unmethylated daughter strand and is activated in a MutS-MutL- dependent manner. The cleavage specificity of MutH is very stringent. Till recently, it was not clear as to how MutH distinguishes hemimethylated DNA from fully or unmethylated DNA. The co-crystal structures of MutH-DNA complexes revealed that Y212, R184 and P185 were in close proximity to the methyl-adenine. Clustal-W sequence alignment of MutH with Sau3AI showed that Sau3AI has PCT residues instead of L183, R184, and P185. A triple mutant MutH-L183P-R184C-P185T was found to cleave both unmethylated and methylated DNA. The nicking endonuclease activity of the LRP→ PCT triple mutant was enhanced in the presence of Haemophilus influenzae MutL. The mutL gene of Neisseria gonorrhoeae was cloned and the gene product purified. It was shown that the homodimeric Neisseria gonorrhoeae MutL (NgoL) protein displays an endonuclease activity that incises covalently closed circular DNA in the presence of manganese or magnesium or calcium ions unlike human MutLα which shows endonuclease activity only in the presence of manganese. Further more the C-terminal domain of Neisseria gonorrhoeae MutL (NgoL-CTD) consisting of amino acids 460 to 658 also exhibits Mn2+ dependent endonuclease activity. Sedimentation velocity, sedimentation equilibrium and dynamic light scattering experiments show NgoL-CTD to be a dimer. By in vitro comparison of wild-type and a mutant NgoL-CTD protein, it was shown that the latter protein exhibits highly reduced endonuclease activity. Surface plasmon resonance spectroscopy was used to determine the kinetics of DNA binding by NgoL. The DNA binding was carried out in absence of metal ions. Interaction studies with NgoL with ssDNA in SPR spectroscopy revealed a KD value of 4.7 × 10–8 M. While the human MutLα endonuclease activity was shown to be stimulated by ATP, ATP inhibits NgoL endonuclease activity. By in vitro comparison of wild-type and a mutant NgoL-CTD protein, it was shown that the latter protein exhibits highly reduced endonuclease activity. NgoL ATPase activity was enhanced in the presence of DNA. The fact that NgoL ATPase activity is stimulated ~ 2.5-fold by dsDNA and ~ 2-fold by ssDNA is a further evidence for the interaction between NgoL and DNA. The results presented above show that NgoL harbors a nicking endonuclease activity which is present in the C-terminal domain. NgoL and NgoL-CTD are dimers in solution and DMHA(X)2E(X)4E motif present in the CTD is required for the nicking endonuclease activity. These results suggest that DNA mismatch repair mechanism in N. gonorrhoeae is different from that in E. coli. In the absence of MutH homolog, N. gonorrhoeae is able to repair the DNA by virtue of MutL nicking endonuclease activity.

The evaluation of microsatellite instability in cancer patients might be of clinical importance as a prognostic and predictor factor. The aim of this study − evaluate the frequency and status of microsatellite instability and DNA mismatch repair protein expression in endometrial cancer and to relate the obtained results to clinicopathologic characteristics as well as patient survival. This study enrolled 109 patients.The objectives of the study: to determine the frequency and status of microsatellite instability by using two marker panels (earlier created BAT-25, BAT-26, NR-21, NR-24, MONO-27 and a new BAT-52, BAT-55, BAT-56, BAT-57, BAT-59 panels) recommended by Promega Corporation (USA); to compare the frequency and status of microsatellite instability with tumor clinicopathologic characteristics; to investigate the expression of DNA mismatch repair proteins (MLH1, PMS2, MSH2, and MSH6) in tumors with high-frequency microsatellite instability; to evaluate the impact of microsatellite instability on the survival of patients. The results showed that high-frequency microsatellite instability was detected two times more frequently with the new panel of markers in comparison while using earlier created panel. A statistically significant difference regarding tumor grade and myometrial invasion was found between the groups with high-frequency microsatellite instability and stable phenotype. There was no association between the survival of patients and microsatellite instability. / Mikrosatelitų nestabilumo tyrimai svarbūs vėžio ligos prognozei vertinti bei parenkant individualų gydymą. Darbo tikslas − ištirti mikrosatelitų nestabilumo dažnį, pobūdį ir klaidingai suporuotų nukleotidų DNR pažaidų taisymo sistemos baltymų raišką sergant gimdos kūno vėžiu ir gautus rezultatus susieti su pacienčių klinikinėmis-patologinėmis charakteristikomis bei jų išgyvenamumu. Į stebėsenos perspektyvinį tyrimą buvo įtrauktos 109 pacientės. Darbo uždaviniai: nustatyti mikrosatelitų nestabilumo dažnį ir pobūdį tarp sergančiųjų gimdos kūno vėžiu, panaudojus Promega Corporation (JAV) sukurtus du žymenų rinkinius (anksčiau sukurtą BAT-25, BAT-26, NR-21, NR-24, MONO-27 ir naująjį BAT-52, BAT-55, BAT-56, BAT-57, BAT-59); palyginti mikrosatelitų nestabilumo dažnį ir pobūdį su pacienčių klinikinėmis-patologinėmis charakteristikomis; ištirti DNR pažaidų taisymo sistemos baltymų (MLH1, PMS2, MSH2, MSH6) raišką gimdos kūno navikuose, kuriuose nustatytas didelio dažnio mikrosatelitų nestabilumas; įvertinti mikrosatelitų nestabilumo įtaką pacienčių išgyvenamumui. Tyrimų rezultatai parodė, kad mikrosatelitų didelio dažnio nestabilumas dvigubai dažniau nustatytas naudojant naująjį žymenų rinkinį, palyginti su anksčiau sukurtu žymenų rinkiniu. Statistiškai reikšminga sąsaja konstatuota tarp mikrosatelitų didelio dažnio nestabilumo arba jo nebuvimo ir naviko diferenciacijos laipsnio bei invazijos į miometriumą gylio. Sergančiųjų išgyvenamumas nebuvo susijęs su mikrosatelitų nestabilumu. ... [toliau žr. visą tekstą]

Mikrosatelitų nestabilumo tyrimai svarbūs vėžio ligos prognozei vertinti bei parenkant individualų gydymą. Darbo tikslas − ištirti mikrosatelitų nestabilumo dažnį, pobūdį ir klaidingai suporuotų nukleotidų DNR pažaidų taisymo sistemos baltymų raišką sergant gimdos kūno vėžiu ir gautus rezultatus susieti su pacienčių klinikinėmis-patologinėmis charakteristikomis bei jų išgyvenamumu. Į stebėsenos perspektyvinį tyrimą buvo įtrauktos 109 pacientės. Darbo uždaviniai: nustatyti mikrosatelitų nestabilumo dažnį ir pobūdį tarp sergančiųjų gimdos kūno vėžiu, panaudojus Promega Corporation (JAV) sukurtus du žymenų rinkinius (anksčiau sukurtą BAT-25, BAT-26, NR-21, NR-24, MONO-27 ir naująjį BAT-52, BAT-55, BAT-56, BAT-57, BAT-59); palyginti mikrosatelitų nestabilumo dažnį ir pobūdį su pacienčių klinikinėmis-patologinėmis charakteristikomis; ištirti DNR pažaidų taisymo sistemos baltymų (MLH1, PMS2, MSH2, MSH6) raišką gimdos kūno navikuose, kuriuose nustatytas didelio dažnio mikrosatelitų nestabilumas; įvertinti mikrosatelitų nestabilumo įtaką pacienčių išgyvenamumui. Tyrimų rezultatai parodė, kad mikrosatelitų didelio dažnio nestabilumas dvigubai dažniau nustatytas naudojant naująjį žymenų rinkinį, palyginti su anksčiau sukurtu žymenų rinkiniu. Statistiškai reikšminga sąsaja konstatuota tarp mikrosatelitų didelio dažnio nestabilumo arba jo nebuvimo ir naviko diferenciacijos laipsnio bei invazijos į miometriumą gylio. Sergančiųjų išgyvenamumas nebuvo susijęs su mikrosatelitų nestabilumu. / The evaluation of microsatellite instability in cancer patients might be of clinical importance as a prognostic and predictor factor. The aim of this study − evaluate the frequency and status of microsatellite instability and DNA mismatch repair protein expression in endometrial cancer and to relate the obtained results to clinicopathologic characteristics as well as patient survival. This study enrolled 109 patients.The objectives of the study: to determine the frequency and status of microsatellite instability by using two marker panels (earlier created BAT-25, BAT-26, NR-21, NR-24, MONO-27 and a new BAT-52, BAT-55, BAT-56, BAT-57, BAT-59 panels) recommended by Promega Corporation (USA); to compare the frequency and status of microsatellite instability with tumor clinicopathologic characteristics; to investigate the expression of DNA mismatch repair proteins (MLH1, PMS2, MSH2, and MSH6) in tumors with high-frequency microsatellite instability; to evaluate the impact of microsatellite instability on the survival of patients. The results showed that high-frequency microsatellite instability was detected two times more frequently with the new panel of markers in comparison while using earlier created panel. A statistically significant difference regarding tumor grade and myometrial invasion was found between the groups with high-frequency microsatellite instability and stable phenotype. There was no association between the survival of patients and microsatellite instability.

Aims: The purpose of the research was to investigate the benefits of a hereditary colorectal cancer registry in the management of patients and families with Lynch syndrome. In study one, a systematic review was performed to quantify the impact of registration and screening on colorectal cancer (CRC) incidence and mortality, with comparison between familial adenomatous polyposis (FAP) and Lynch syndrome (LS). In study two, a regional Lynch syndrome registry was utilised to evaluate the uptake of predictive testing and colorectal screening among first-degree relatives (FDRs) and investigate novel methods for engaging at-risk relatives, including an enhanced role for the general practitioner (GP). In study three, the registry was used to investigate proposed associations between Lynch syndrome and prostate and bladder cancer. In study four, mismatch repair-deficient (dMMR) CRCs from Lynch syndrome patients and randomised-controlled trials (RCTs) were used to evaluate a novel prognostic biomarker, beta-2 microglobulin (B2M). Methods: An electronic database search was conducted to identify studies describing CRC incidence and/or mortality in FAP or LS, with comparison of either: 1) screened and unscreened patients or 2) patients ‘before and after’ establishment of the registry. Using the Manchester regional Lynch syndrome registry database, the uptake of predictive testing and colorectal screening among FDRs was assessed with Kaplan-Meier analysis. Novel strategies for improving engagement were explored via a patient advisory group discussion and a regional primary care questionnaire. Cases of prostate and bladder cancer in male mutation carriers and their male FDRs were identified, and cumulative and relative risks were calculated, using expected rates from cancer registry data. DNA from 350 dMMR CRC specimens from Lynch syndrome patients and RCTs were tested for B2M mutations using Sanger sequencing, and correlated with clinical outcome. Results: 43 studies were included in the systematic review (33 FAP; 10 Lynch). Registry-based screening was associated with a significant reduction in CRC incidence and in Lynch syndrome, CRC-related mortality was negligible in those undergoing surveillance. 242 Lynch syndrome families were recorded on the Manchester Lynch syndrome registry. 329 of 591 (55.7%) eligible FDRs had undergone predictive testing. Uptake was significantly lower in males and younger age groups (<25 yrs). Compliance with colorectal screening was excellent following a mutation positive predictive test but poor in untested individuals (97.3% vs 35.0%). Eight prostate cancers were identified in 821 male LS mutation carriers and male FDRs. MSH2 mutation carriers had a ten-fold increased risk of prostate cancer (RR 10.41; 95%CI 2.80, 26.65) but no association with bladder cancer was identified. 69/286 (24.1%) of dMMR CRCs contained significant B2M mutations. B2M mutations were associated with complete absence of recurrence (0/39) during follow-up in the QUASAR trial (stage II), compared with 14/77 (18.2%) in wild-type B2M (p=0.005). Conclusion: Studies consistently report that registration and screening result in a reduction of CRC incidence and mortality in FAP and LS (Level 2a evidence, Grade B recommendation). Funding and managerial support for registries should be made available. Uptake of predictive testing and colorectal screening in Lynch syndrome could be substantially improved, particularly among males and younger age groups, but this requires advances in communication with at-risk relatives. It is unlikely that GPs will actively participate without considerable support from genetics services. A trial of PSA screening in MSH2 mutation carriers from 50 years would be appropriate. B2M mutation status has potential clinical utility as a prognostic biomarker in stage II dMMR CRC.

Epigenetic changes represent chemical modifications of the DNA molecule and histone proteins by which gene expression is altered. Among them, DNA methylation is a known mechanism of silencing of tumor-suppressor and DNA repair genes, with an important role in carcinogenesis. Many studies have been done in order to identify the methylation signatures of these genes in different types of cancer. In our study, we investigated the methylation status of promoter regions of eight mismatch repair genes (MLH1, MSH2, MSH3, MLH3, PMS1, PMS2, MSH6 and EXO1) in 45 sporadic colorectal cancer cases and 12 head and neck cancer patients. Two out of eight genes, MLH1 and MLH3, exhibited promoter methylation. The results from both groups of patients were concordant. We summarize that the methylation profiles of MLH1 and MLH3 promoters could be potential candidates for epigenetic biomarkers in colorectal cancer, and eventually in head and neck cancer. Further investigations, which would confirm this theory, should be carried out.