Das hereditäre nicht-polypöse kolorektale Karzinom (HNPCC) bildet mit 2-3 % aller kolorektalen Karzinomen die häufigste Form der erblichen Darmkrebserkrankungen. Sie wird meist durch Mutationen in den Genen MLH1 und MSH2 verursacht. Beide Gene codieren zusammen mit anderen Genen (MSH6, MLH3, PMS1 und PMS) für Proteine des DNA-Mismatch-Repair-Systems, welches wichtig für die Stabilität des Genoms ist. Um Träger dieser Mutationen zu identifizieren und frühzeitig Vorsorgeprogrammen zuzuführen, ist ein zuverlässiges Auswahlverfahren notwendig. Aus diesem Grund wird in der vorliegenden Arbeit untersucht, ob die Methode der denaturierenden Hochdruckflüssigkeits-Chromatographie (DHPLC) geeignet ist, um nach Mutationen in den Genen MLH1 und MSH2 zu suchen. Bei 26 weiblichen Patienten und 2 männlichen Probanden im Alter von 23 bis 69 Jahren, die an einem Mammakarzinom (26), einer Dysplasie der Portio (1) sowie an einem Ovarialkarzinom (1) erkrankt waren, und bei deren Familienmitglieder gehäuft Tumoren auftraten, wurden die Gene MLH1 und MSH2 mittels DHPLC gescreent. Dabei wurden alle Exons von MLH1 außer 3, 5-7, 9-10, 14-15 und 18 sowie alle Exons von MSH2 außer Exon 1-2, 4, 9 und 14 einschließlich der flankierenden Exon-/Intron-Grenzen untersucht. Bei insgesamt 41 einzelnen Patientenkurven wich das Muster des DHPLC-Chromatogramms als Hinweis auf eine Genveränderung durch mehrfache oder verbreiterte Peaks vom normalen Kurvenverlauf ab. Die sich daran anschließende DNA-Sequenzanalyse ergab bei 35 der auffälligen Kurven polymorphe Sequenz-Varianten. Keine der gefundenen Sequenzveränderungen stellte eine krankheitsauslösende Mutation für HNPCC dar. Die in dieser Arbeit erreichte Sensitivität des DHPLC-Verfahrens liegt damit bei 85,4 %. Das DHPLC-Verfahren konnte als zuverlässige und vergleichsweise kostengünstige Voruntersuchung etabliert werden.

Das hereditäre nicht-polypöse kolorektale Karzinom (HNPCC) bildet mit 2-3 % aller kolorektalen Karzinomen die häufigste Form der erblichen Darmkrebserkrankungen. Sie wird meist durch Mutationen in den Genen MLH1 und MSH2 verursacht. Beide Gene codieren zusammen mit anderen Genen (MSH6, MLH3, PMS1 und PMS) für Proteine des DNA-Mismatch-Repair-Systems, welches wichtig für die Stabilität des Genoms ist. Um Träger dieser Mutationen zu identifizieren und frühzeitig Vorsorgeprogrammen zuzuführen, ist ein zuverlässiges Auswahlverfahren notwendig. Aus diesem Grund wird in der vorliegenden Arbeit untersucht, ob die Methode der denaturierenden Hochdruckflüssigkeits-Chromatographie (DHPLC) geeignet ist, um nach Mutationen in den Genen MLH1 und MSH2 zu suchen. Bei 26 weiblichen Patienten und 2 männlichen Probanden im Alter von 23 bis 69 Jahren, die an einem Mammakarzinom (26), einer Dysplasie der Portio (1) sowie an einem Ovarialkarzinom (1) erkrankt waren, und bei deren Familienmitglieder gehäuft Tumoren auftraten, wurden die Gene MLH1 und MSH2 mittels DHPLC gescreent. Dabei wurden alle Exons von MLH1 außer 3, 5-7, 9-10, 14-15 und 18 sowie alle Exons von MSH2 außer Exon 1-2, 4, 9 und 14 einschließlich der flankierenden Exon-/Intron-Grenzen untersucht. Bei insgesamt 41 einzelnen Patientenkurven wich das Muster des DHPLC-Chromatogramms als Hinweis auf eine Genveränderung durch mehrfache oder verbreiterte Peaks vom normalen Kurvenverlauf ab. Die sich daran anschließende DNA-Sequenzanalyse ergab bei 35 der auffälligen Kurven polymorphe Sequenz-Varianten. Keine der gefundenen Sequenzveränderungen stellte eine krankheitsauslösende Mutation für HNPCC dar. Die in dieser Arbeit erreichte Sensitivität des DHPLC-Verfahrens liegt damit bei 85,4 %. Das DHPLC-Verfahren konnte als zuverlässige und vergleichsweise kostengünstige Voruntersuchung etabliert werden.:Inhaltsverzeichnis 1 Einleitung 8 1.1 Das hereditäre nicht-polypöse kolorektale Karzinom (hereditary non-polyposis colorectal Cancer; HNPCC) 8 1.1.1 Definition, Epidemiologie und Krankheitsbild 8 1.1.2 Klinische Diagnostik 9 1.1.3 Genetische Diagnostik 11 1.1.4 Bedeutung der Diagnosestellung bei HNPCC-Patienten für die Behandlung 11 1.1.5 Molekulargenetische Grundlagen des HNPCC 13 1.2 Die hMLH1- und hMSH2-Gene 15 1.2.1 Lokalisation, Struktur und Funktion der hMHL-Gene 16 1.3 Detektion von Struktur- und Sequenzveränderungen bei HNPCC 17 1.3.1 Die Einzelstrang-Konformationspolymorphismus-Analyse (SSCP) 18 1.3.2 Die denaturierende Gradientengelelektrophorese (DGGE) 18 1.3.3 Die denaturierende Hochdruckflüssigkeits-Chromatographie (DHPLC) 19 2 Ziele dieser Arbeit 23 3 Patienten, Material und Methoden 24 3.1 Patienten 24 3.2 Materialien und Geräte 27 3.2.1 Materialien 27 3.2.2 Technische Geräte 28 3.2.3 Reagenzien 29 3.2.4 Lösungen und Puffer 30 3.2.5 Standards und Farbstoffe 31 3.2.6 Molekulardiagnostische Kits 31 3.2.7 Enzyme 32 3.3 Primer 32 3.4 Softwareprogramme, Datenbanken und Sequenzquellen 34 3.4.1 Software zur Schmelzpunktbestimmung 34 3.4.2 ABI PRISMTM Software zur Sequenzanalyse 34 3.4.3 Mutationsdatenbanken (Online) 34 3.4.4 DNA-Sequenzen 34 3.5 Methoden 35 3.5.1 DNA-Isolierung aus Vollblut 35 3.5.2 DNA-Amplifikation mittels Polymerase-Kettenreaktion (PCR) 36 3.5.3 Denaturierende Hochdruckflüssigkeits-Chromatographie (DHPLC) 38 3.5.4 DNA-Sequenzierung 41 4 Ergebnisse 46 4.1 Quantität und Qualität der isolierten DNA 46 4.1.1 DNA-Schmelzpunkt- und Fließgradienten-Bestimmung mittels WAVE®Maker-Software 47 4.1.2 Fragmentanalyse mittels DHPLC 49 4.2 DNA-Sequenzierung 54 4.2.1 Polymorphismen 55 5 Diskussion 61 5.1 Genotyp- und Phänotyp-Beziehung 61 5.1.1 Erstmanifestationsalter und Tumorspektrum 61 5.1.2 Fehlender Mutationsnachweis 62 5.2 DHPLC-Methode als Screening-Verfahren 65 5.2.1 Zeitersparnis 65 5.2.2 Kostenersparnis 67 5.2.3 Nachweisgenauigkeit 67 5.2.4 Nachweis von Sequenzvarianten 68 5.2.5 Chromatogramm-Interpretation 70 5.2.6 Anfälligkeiten für Störfaktoren 71 6 Zusammenfassung der Arbeit 73 7 Literaturverzeichnis 75 7.1 Web-Adressen 86 8 Erklärung über die eigenständige Abfassung der Arbeit 89 9 Lebenslauf 90 10 Danksagung 92

The von Hippel-Lindau (VHL) tumor suppressor pVHL is lost in 90% of clear-cell renal-cell carcinomas (ccRCCs). Jade-1 is a renal tumor suppressor that is normally stabilized by pVHL. MutS Homolog2 (MSH2) is a key initiator in DNA mismatch repair (MMR). Defects in MMR are associated with genome-wide instability and predisposition to certain types of cancer. Mass spectrometry data of immunoprecipitated Flag-tagged Jade-1 lysates showed signal for MSH2, suggesting Jade-1 may participate in MMR. Here, we confirmed an interaction between endogenous MSH2 and endogenous Jade-1 by coimmunoprecipitation. Using cell fractionation, we found that MSH2 and Jade-1 translocated to the nucleus in response to alkylating agent MNNG in kidney proximal tubule cells. We also visualized the translocation of Jade-1 by immunofluorescence. Silencing JADE1 also influenced the kinetics of MSH2 translocation. In addition, by colony forming assay, JADE1-silenced cells were resistant to mismatch damage induced by MNNG, which is a feature of cells with an MMR defect. Furthermore, reintroducing pVHL into renal cancer cells also changed the amount of translocated MSH2 and Jade-1. In contrast to wild-type mice, Jade1 heterozygous mice got spontaneous tumors, and those tumors continued to show heterozygosity for Jade1. Taken together, our results identify a mechanism for Jade-1 regulation of MMR through its nuclear translocation. pVHL may also contribute to MSH2 and Jade-1 translocation by increasing Jade-1 abundance. These findings establish an early role for Jade-1 in MMR, provide further indication that Jade-1 helps maintain genomic stability in the kidney and support that Jade-1 is a haploinsufficient renal tumor suppressor.

Orientador: Carmen Sílvia Bertuzzo / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T00:44:00Z (GMT). No. of bitstreams: 1 Rueda_LidianeCamila_D.pdf: 2777204 bytes, checksum: dc2eb94ba9de0c91e1c7a3fed2fbd3e4 (MD5) Previous issue date: 2013 / Resumo: O câncer colorretal tem importância elevada frente a sua incidência e morbidade. Dentre os casos hereditários, o câncer colorretal hereditário sem polipose (HNPCC), ou Síndrome de Lynch, é responsável por cerca de 5% do total de casos. No HNPCC, a alteração genética herdada é a inativação de um dos alelos dos genes envolvidos em reparo do DNA, sendo os principais os genes hMLH1 e hMSH2. O objetivo deste trabalho foi investigar, em indivíduos com diagnóstico clínico de HNPCC, a presença de mutações nos genes MLH1 e MSH2, associar as variáveis clínicas com o gene mutado e investigar os familiares de portadores de HNPCC aos quais tivemos acesso, com relação a mutações germinativas. A investigação das mutações foi realizada por meio de sequenciamento direto dos éxons, região promotora e regiões de junção. Foram analisados 65 indivíduos divididos em três grupos, sendo (I) 46 pacientes portadores de câncer colorretal inclusos nos Critérios de Amsterdã, (II) dois familiares portadores de câncer colorretal e (III) 17 familiares sem câncer, todos da região metropolitana de Campinas, atendidos no Hospital de Clínicas da UNICAMP. Em 21 (45,65%) dos pacientes foram encontradas mutações deletérias. As mutações deletérias nos genes MLH1 e MSH2 estavam na proporção de 34,78% (16 pacientes) e 10,86% (5 pacientes), respectivamente. As mutações não deletérias nos genes MLH1 e MSH2 estavam na proporção de 65,22% dos pacientes (30 alterações) e 50% dos pacientes (23 alterações), respectivamente. Foi possível identificar 23 mutações potencialmente deletérias entre os pacientes com HNPCC por meio de sequenciamento dos genes MLH1 e MSH2, com uma porcentagem de detecção de 50%. Parece não haver variações nas características clínicas do tumor quando a mutação germinativa ocorre no gene MLH1 ou MSH2, com exceção da relação entre presença de mutação no gene MLH1 e idade de manifestação da doença. Como ocorre no resto do mundo a doença mostrou-se extremamente heterogênea em termos moleculares, pois apenas duas mutações se repetiram em dois pacientes. A partir da análise das duas famílias foi possível mostrar a dificuldade para estabelecer a presença da mutação germinativa deletéria que poderia levar à predisposição ao HNPCC, bem como a importância da analise familial no diagnóstico molecular dessa alteração / Abstract: Colorectal cancer has high importance because of its incidence and morbidity. Among the hereditary cases, the hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome, accounts for about 5% of cases. In HNPCC, the genetic alteration inherited is the inactivation of one of the alleles of genes involved in the DNA repair, being hMSH2 and hMLH1 the main genes. The objective of this study is to investigate the presence of mutations in MLH1 and MSH2 in patients with clinical diagnosis of HNPCC, correlate clinical variables with the mutated gene, and investigate the relatives of patients with HNPCC who we had access to, in relation to germline mutations. Investigation of the mutations was performed by éxons direct sequencing, the promoter and junction regions. Sixty-five individuals, divided into three groups, were studied: (I) 46 patients with colorectal cancer included in the Amsterdam Criteria, (II) two family members of colorectal cancer patients and (III) 17 relatives without cancer, all of them treated at Hospital das Clínicas at UNICAMP and living in the Campinas metropolitan area. Deleterious mutations were found in 21 patients (45.65%). The ratio of deleterious mutations in MLH1 and MSH2 was 34.78% (16 patients) and 10.86% (5 patients) respectively. The ratio of non deleterious mutations in genes MLH1 and MSH2 was 65.22% of patients (30 alterations) and 50% of patients (23 alterations) respectively. Among patients with HNPCC, 23 potentially deleterious mutations were identified, via sequences of MLH1 and MSH2 with a 50% detection rate. It doesn't seem to appear variations in the clinical characteristics of the tumor when a germline mutation occurs in MLH1 or MSH2, with the exception of the relationship between the presence of mutation in the MLH1 gene and age of disease onset. As it occurs throughout the world, the disease present a his molecular extremely heterogeneoty, where only two mutations were repeated in two patients. The analysis of the two families demonstrated not only the difficulty to establish the presence of deleterious germline mutation that could lead to a predisposition to of HNPCC, but also the importance of familial analysis in molecular diagnostics of this alteration / Doutorado / Clinica Medica / Doutora em Ciências

Submitted by Leonardo Cavalcante (leo.ocavalcante@gmail.com) on 2018-04-24T17:10:12Z No. of bitstreams: 1 Arquivototal.pdf: 1560244 bytes, checksum: 121c5ccc4c7de435dbb28f4f19393b40 (MD5) / Made available in DSpace on 2018-04-24T17:10:13Z (GMT). No. of bitstreams: 1 Arquivototal.pdf: 1560244 bytes, checksum: 121c5ccc4c7de435dbb28f4f19393b40 (MD5) Previous issue date: 2017-05-03 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Basal cell carcinoma (BCC) is considered a tumor involving genetic, epigenetic and environmental factors. UVB radiation is considered to be one of the main physical agents involved in the carcinogenesis of the epidermis promoting DNA damage. In response to DNA damage the DNA repair mechanisms are activated, among them, the mismatch repair mechanism (MMR). The MMR system is an extremely important to maintain the fidelity of replication, however single nucleotide polymorphisms (SNPs) in genes involved in MMR should be considered an important factor for CBC carcinogenesis. The present study carried out the genotyping of SNPs rs560246973 (T> C), rs2303425 (-118 T> C) in the MSH2 gene and rs565410865 (G> T) in the MLH1 gene, in 100 samples of paraffin-embedded tissue from patients diagnosed with basal cell carcinoma. The results were obtained by means of the Dideoxy Single Genotype Allele Specific PCR-DSASP genotyping method. SNPs rs565410865 MLH1 and rs560246973 (C> T) MSH2 showed a statistically significant association with the susceptibility and risk of developing BCC. The results suggest that SNPs rs565410865 MLH1 and rs560246973 (C> T) MSH2 are potential molecular markers associated with susceptibility to the development of BCC in the analyzed samples. . / O carcinoma basocelular (CBC) é considerado um tumor que envolve fatores genéticos, epigenéticos e ambientais. Sendo a radiação UVB considerada um dos principais agentes físicos envolvido na carcinogênese da epiderme promovendo danos ao DNA. Em resposta aos danos no DNA os mecanismos de reparo do DNA são ativados, entre eles, o Mecanismo de Reparo de Mal Pareamento (MMR). O sistema MMR é uma via extremamente importante para manter a fidelidade da replicação, no entanto polimorfismos de nucleotídeo único (SNPs) em genes envolvidos no MMR deve ser considerado fator importante para carcinogênese do CBC. O presente estudo realizou a genotipagem dos SNPs rs560246973 (T>C), rs2303425 (-118 T>C) no gene MSH2 e rs565410865 (G>T) no gene MLH1, em 100 amostras de tecido parafinado de pacientes diagnosticados com carcinoma basocelular. Os resultados foram obtidos por meio do método de genotipagem Didesoxi Único Alelo Específico PCR- DSASP. Os SNPs rs565410865 MLH1 e rs560246973 (C>T) MSH2 apresentaram associação estatisticamente significativa à susceptibilidade e o risco de desenvolver CBC. Os resultados sugerem que SNPs rs565410865 MLH1 e rs560246973 (C>T) MSH2 são potenciais marcadores moleculares associados à susceptibilidade ao desenvolvimento do CBC nas amostras analisadas.

In order to build any structure, a good supply of materials, accurate workers and quality control are needed. This is even the case when constructing DNA, the so-called “Code of Life.” For a species to continue to exist, this DNA code must be copied with incredibly high accuracy when each and every cell replicates. In fact, just one mistake in the 12 million bases that comprise the genome of budding yeast, Saccharomyces cerevisiae, can be fatal. DNA is composed of a double strand helix made up of just four different bases repeated millions of times. The building blocks of DNA are the deoxyribonucleotides (dNTPs); dCTP, dTTP, dATP and dGTP. Their production and balance are carefully controlled within each cell, largely by the key enzyme Ribonucleotide Reductase (RNR). Here, we studied how the enzymes that copy DNA, the replicative polymerases α, δ and ε, cope with the effects of an altered dNTP pool balance. An introduced mutation in the allosteric specificity site of RNR in a strain of S. cerevisiae, rnr1-Y285A, leads to elevated dCTP and dTTP levels and has been shown to have a 14-fold increase in mutation rate compared to wild type. To ascertain the full effects of the dNTP pool imbalance upon the replicative polymerases, we disabled one of the major quality control systems in a cell that corrects replication errors, the post-replicative Mismatch Repair system. Using both the CAN1 reporter assay and whole genome sequencing, we found that, despite inherent differences between the polymerases, their replication fidelity was affected very similarly by this dNTP pool imbalance. Hence, the high dCTP and dTTP forced Pol ε and Pol α/δ to make the same mistakes. In addition, the mismatch repair machinery was found to correct replication errors driven by this dNTP pool imbalance with highly variable efficiencies. Another mechanism to protect cells from DNA damage during replication is a checkpoint that can be activated to delay the cell cycle and activate repair mechanisms. In yeast, Mec1 and Rad53 (human ATR and Chk1/Chk2) are two key S-phase checkpoint proteins. They are essential as they are also required for normal DNA replication and dNTP pool regulation. However the reason why they are essential is not well understood. We investigated this by mutating RAD53 and analyzing dNTP pools and gene interactions. We show that Rad53 is essential in S-phase due to its role in regulating basal dNTP levels by action in the Dun1 pathway that regulates RNR and Rad53’s compensatory kinase function if dNTP levels are perturbed. In conclusion we present further evidence of the importance of dNTP pools in the maintenance of genome integrity and shed more light on the complex regulation of dNTP levels.

Msh2 and Mlh1 proteins from Tetrahymena thermophla are homologues of MutS and MutL from Escherichia coli respectively. MutS and MutL are DNA mismatch repair proteins. In eukaryotes, MutS homologues recognize the replication errors and MutL homologues interact with MutS homologues and other proteins to make the repair occur. Biolistic transformation has been done to make the msh2 and mlh1 single knockouts in the macronuclei of different strains and the knockouts were verified complete. Two strains of WT crossing KO or KO crossing KO, with different mating types, were induced to conjugate. The processes were studied by microscopy using DAPI staining. For the msh2 knockouts, there were no crescent micronuclei formed throughout the conjugation of two knockout cells, and the pairing level was reduced severely. However, a knockout cell and a wild-type cell could conjugate normally at a high level pairing efficiency. Msh2 protein seems to be important to cell pairing and indispensible for the formation of the crescent micronuclei during cell conjugation. For the mlh1 knockouts, the pairing level of a knockout and a wild-type was reduced by half and the pairing level of two knockouts was reduced more than 80%; however, the paired cells in both could complete the conjugation with delay. Pms2 protein may have redundant roles in the MutL heterodimer (Mlh1-Pms2). In addition, chemical mutagens treated knockout was crossed with non-treated wild-type and the conjugation was compared with treated wild-types. Most of the treated knockout cells could not pair after starvation and mixing with non-treated wild-type cells, which means most of the cells could not enter meiotic phase. It is probable that G2/M checkpoint arrested the meiotic cell cycle and the intra-S phase was inactivated. Thus, Msh2 protein may have a role in the meiotic intra-S phase checkpoint system.

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

Background. Lynch syndrome is the most common genetic predisposition for hereditary cancer. Carriers of pathogenic changes in mismatch repair (MMR) genes have an increased risk of developing colorectal (CRC), endometrial, ovarian, urinary tract, prostate, and other cancers, depending on which gene is malfunctioning. In Lynch syndrome, differences in cancer incidence (penetrance) according to the gene involved have led to the stratification of cancer surveillance. By contrast, any differences in penetrance determined by the type of pathogenic variant remain unknown. Objective. To determine cumulative incidences of cancer in carriers of truncating and missense or aberrant splicing pathogenic variants of the MLH1 and MSH2 genes. Methods. Carriers of pathogenic variants of MLH1 (path_MLH1) and MSH2 (path_MSH2) genes filed in the Prospective Lynch Syndrome Database (PLSD) were categorized as truncating or missense/aberrant splicing according to the InSiGHT criteria for pathogenicity. Results. Among 5199 carriers, 1045 had missense or aberrant splicing variants, and 3930 had truncating variants. Prospective observation years for the two groups were 8205 and 34,141 years, respectively, after which there were no significant differences in incidences for cancer overall or for colorectal cancer or endometrial cancers separately. Conclusion. Truncating and missense or aberrant splicing pathogenic variants were associated with similar average cumulative incidences of cancer in carriers of path MLH1 and path_MSH2.

Lynch syndrome (Hereditary Nonpolyposis Colorectal Cancer, HNPCC) is the most common hereditary syndrome predisposing to colorectal cancer, accounting for 1-3% of all colorectal cancer. This multi-organ cancer predisposition syndrome is caused by mutations in the mismatch repair (MMR) genes, especially MLH1 and MSH2, and to lesser extents MSH6 and PMS2, which lead to widespread genetic instability and thus microsatellite instability (MSI). Hereditary cancer often manifests in two or more tumours in a single individual; 35-40% of Lynch syndrome patients have synchronous or metachronous tumours of the two major Lynch syndrome-related cancers: colorectal and endometrial. The main purposes of the work underlying this thesis were to identify persons at risk of Lynch syndrome or other types of hereditary colorectal cancer, to estimate the cancer risks associated with these predispositions and to identify the underlying genetic causes. A population-based cohort of 78 persons with double primary colorectal or colorectal and endometrial cancer was identified. Cancer risks in their 649 first-degree relatives were estimated in relation to tumour MSI status (positive or negative) and age at diagnosis (before or after 50 years of age) in the probands. The overall standardised incidence ratio was 1.69 (95% CI; 1.39-2.03). The highest risks for Lynch syndrome-associated cancers: (colorectal, endometrial, ovarian and gastric) were found in families with young MSI-positive probands, likely representing Lynch syndrome families. Importantly, no overall risk was found in families with old probands, irrespective of MSI status. Blood samples were available from 24 MSI-positive patients for mutation screening of MLH1, MSH2 and MSH6. Sequence variants or rearrangements predicted to affect protein function were found in 16 patients. Six novel variants were found: two large rearrangements, two truncating and two missense mutations. The missense mutations were found to segregate in the families. Studies of allele frequencies, MSI and loss of immunostaning in tumours from family members further supports the hypothesis that these missense changes play a role in Lynch syndrome, as do the non-conservative nature and evolutionary conservation of the amino acid exchanges. Five families had mutations in MLH1, five in MSH2, and six in MSH6. The unexpectedly large impact of MSH6 was in genealogical studies shown to be due to a founder effect. Cumulative risk studies showed that the MSH6 families, despite their late age of onset, have a high lifetime risk for all Lynch syndrome-related cancers, significantly higher in women (89% by age 80 years) than in men (69%). The gender differences are in part due to high endometrial (70%) and ovarian cancer risk (33%) in addition to the high colorectal cancer risk (60%). These findings are of great importance for counselling and surveillance of families with MSH6 mutations. Finally, in a large family with MSI-negative hereditary colorectal cancer for which the MMR genes and APC had been excluded as possible causes, a genome-wide linkage analysis was performed, resulting in a suggested linkage to chromosome 7. Conclusions: Relatives of probands with MSI-positive, double primary colorectal and endometrial cancer diagnosed before the age of 50 years have significantly increased risks of Lynch syndrome-related cancers. MSH6 mutations, which have unusually high impact in this study population due to a founder effect, confer high cumulative risks of cancer despite the generally late age of onset.