FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / O cÃncer oral representa um sÃrio problema de saÃde pÃblica mundial. Entre os tumores deste sÃtio anatÃmico, os carcinomas de cÃlulas escamosas orais (CCEO) respondem por atà 94% do total. Os mecanismos moleculares envolvidos na gÃnese e desenvolvimento tumoral ainda nÃo estÃo completamente elucidados. Algumas evidÃncias tÃm sugerido a participaÃÃo viral neste processo. AlÃm disso, estes tumores ainda carecem de marcadores confiÃveis para determinar o perfil de agressividade. Neste contexto, o presente estudo teve como objetivo avaliar a expressÃo das proteÃnas p53, E-caderina, COX-2, p16, MLH1 e MYC numa sÃrie de CCEO, considerando tambÃm a marcaÃÃo citoplasmÃtica eventualmente observada para as Ãltimas trÃs proteÃnas, confrontando os resultados entre elas e com as caracterÃsticas demogrÃficas e clÃnico-patolÃgicas. AlÃm de avaliar a prevalÃncia do PapilomavÃrus Humano (HPV) e do VÃrus Epstein-Barr (EBV) na amostra e comparÃ-las com a expressÃo das referidas proteÃnas. Materiais e MÃtodos â Cem espÃcimes de CCEO, fixados em formalina e incluÃdos em blocos de parafina, foram submetidos à imunohistoquÃmica para a detecÃÃo das referidas proteÃnas e à hibridaÃÃo in situ para detecaÃà de HPV e EBV. Resultados â Foi observada associaÃÃo referente à perda de expressÃo concomitante de p16 e MLH1 (p=0,029) e na coexpressÃo de p53 e COX-2 (p=0,045). Ademais, foi verificado que a COX-2 e o MYC nuclear estavam relacionados com a marcaÃÃo citoplasmÃtica de MLH1 (p=0,060 e p=0,018; respectivamente). A anÃlise combinada dos marcadores revelou cinco grupos principais de expressÃo alterada que eram constituÃdos, em sua maioria, de tumores mais agressivos, principalmente o grupo MLH1(-)/COX-2(+)/p16(-). Os casos com marcaÃÃo citoplasmÃtica para p16, MLH1 e/ou MYC foram mais frequentes em tumores avanÃados (p=0,009) e naqueles com metÃstases em linfonodos (p=0,001). Trinta e um casos demonstraram marcaÃÃo para HPV em tecido tumoral. O EBV nÃo foi detectado em nenhum dos casos investigados, nem no tecido tumoral nem no epitÃlio nÃo neoplÃsico. O grupo HPV(+) exibiu elevada positividade para o p16 nuclear (p=0,029) e MYC cytoplasmÃtico (p=0,039), tambÃm uma maior perda de expressÃo nuclear de MLH1 (p=0,031). Houve ainda uma tendÃncia referente ao aumento da positividade de COX-2 no grupo infectado (p=0,084). ConclusÃes â As significÃncias verificadas entre p16 e MLH1 sugerem que a ausÃncia do membro do sistema de reparo de encaixe (MMR) tambÃm favoreÃa a ocorrÃncia de mutaÃÃes no gene p16, culminando na inativaÃÃo deste supressor tumoral. As associaÃÃes de COX-2 e MYC com o MLH1 de expressÃo citoplasmÃtica suscitam um mecanismo de bloqueio de entrada de MLH1 no nÃcleo. A anÃlise combinada das proteÃnas, bem como, a marcaÃÃo citoplasmÃtica de p16, MLH1 e MYC, podem representar indicadores Ãteis na avaliaÃÃo do perfil de agressividade e, provavelmente, de prognÃstico em CCEO. Acerca dos vÃrus, nossos achados sugerem que o HPV esteja envolvido em uma importante parcela de casos de CCEO e que possa promover a expressÃo de p16 nuclear, MYC citoplasmÃtico e COX-2, e suprimir a expressÃo nuclear de MLH1. Quanto ao EBV, nÃo foram detectados EBERs (EBV-encoded small RNAs) na amostra. / The oral cancer represents a serious world public health problem. The oral squamous cell carcinomas (OSCC) account for up to 94% of the tumors of this anatomic site. The molecular mechanisms involved in the genesis and progression are still not well elucidated. Some evidences have suggested the involvement of viruses in this process. Also, these tumors still lack of reliable markers to determine the aggressiveness profile. In this context, the aim of the present study was to evaluate the expression of the proteins p53, E-cadherin, COX-2, p16, MLH1 and MYC in a serie of OSCC, including the cytoplasmic staining eventually observed for the latter three proteins, confronting the results between them and with demographic and clinico-pathological features. Besides evaluating the prevalence of Human Papillomavirus (HPV) and Epstein-Barr virus (EBV) in the sample and compare them with the expression of the referred proteins. Materials and Methods â One hundred formalin-fixed paraffin-embedded OSCC specimens were submitted to immunohistochemistry for detection of the referred proteins, and to in situ hybridization for HPV and EBV detection. Results â OSCC was associated with a concomitant lack of expression of p16 and MLH1 (p=0.029) and coexpression of p53 and COX-2 (p=0.045). Additionally, COX-2 and nuclear MYC were found to be related to exclusively cytoplasmic staining of MLH1 (p=0.060 and p=0.018, respectively). The combination analyses of the markers revealed five main groups of altered protein expression, which were mostly of the more aggressive tumors, mainly the MLH1(-)/COX-2(+)/p16(-) group. The cases with cytoplasmic staining for p16, MLH1 and/or MYC were more frequent in advanced tumors (p=0.009) and in those with lymph node metastasis (p=0.001). Thirty-one cases showed staining for HPV in tumor tissue. The EBV was not detected in any case investigated, neither in the tumor tissue nor in the non-neoplastic epithelium. The HPV(+) group demonstrated high positivity for nuclear p16 (p=0,029) and cytoplasmic MYC (p=0,039), and an increase of the lack of MLH1 nuclear expression (p=0,031). There was also a trend related to the increase of the COX-2 positivity in the HPV(+) group (p=0,084). Conclusions â The significance between p16 and MLH1 suggests that the lack of this member of mismatch repair system also favors the occurrence of mutations in the p16 gene, culminating in inactivation of this tumor suppressor. The associations of COX-2 and MYC with cytoplasmic MLH1 suggest a blocking mechanism for the entry of MLH1 into the nucleus. The combined analyses of the proteins investigated, as well as the cytoplasmic staining of p16, MLH1 and MYC, may be useful in the evaluation of the aggressive profile and probably prognosis of OSCC. Regarding the viruses, our findings suggest that the HPV is involved in an important portion of OSCC cases and that may promote the expression of the nuclear p16, cytoplasmic MYC and COX-2, and suppress the nuclear expression of MLH1. About EBV, it was not detected the EBV-encoded small RNAs (EBERs) in the sample.

Type 1 diabetes is an autoimmune disease leading to insulin deficiency. Autoantibodies to beta cell proteins are already present in the asymptomatic phase of type 1 diabetes. Recent findings have suggested a number of additional minor autoantigens in patients with type 1 diabetes. We have established luciferase immunoprecipitation systems (LIPS) for anti-MTIF3, anti-PPIL2, anti-NUP50 and anti-MLH1 and analyzed samples from 500 patients with type 1 diabetes at onset of clinical disease and 200 healthy individuals who had a family history of type 1 diabetes but no evidence of beta cell autoantibodies. We show significantly higher frequencies of anti-MTIF3, anti-PPIL2 and anti-MLH1 in recent onset type 1 diabetes patients in comparison to controls. In addition, antibodies to NUP50 were associated with HLA-DRB1*03 and antibodies to MLH1 were associated with HLA-DRB1*04 genotypes.:1. Introduction 2. Material and methods 2.1 Participants 2.2. Cloning and expression of antigens 2.3. Luciferase ImmunoPrecipitation (LIPS) assays 2.4. Antinuclear antibodies (ANA) 2.5. Statistics 3. Results 4. Discussion 5. Conclusion Declaration of interest Funding Acknowledgements Appendix A Suplementary data References

The base excision repair protein, MBD4 (also known as MED1) is known to be transcriptionally coupled to a mismatch repair protein MLH1. To date the significance of this coupling has not been elucidated and the significance of MBD4 within the mismatch repair system and apoptotic pathway is still being understood. Recently a novel alternatively spliced form of MBD4 has been identified and termed sMBD4. To date the significance of sMBD4 is unknown. MBD4 and sMBD4 share a common glycosylase domain and this is the domain through which MBD4 is reported to interact with MLH1. It was the aim of this study to determine if sMBD4 was also a binding partner of MLH1 to help elucidate a potential role of sMBD4 and to further characterise the binding domain between MLH1 and MBD4. Recombinant proteins were utilised in binding assays however, a specific protein – protein interaction could not be determined. Regular aspirin intake is associated with a reduction in the incidence of colorectal cancer. Aspirin has been shown to be cytotoxic to colorectal cancer cells in vitro. The molecular basis for this cytotoxicity is controversial, with a number of competing hypotheses in circulation. One suggestion is that the protective effect is related to the induction of DNA mismatch repair (MMR) proteins in DNA MMR proficient cells. As MBD4 has previously been suggested to be coupled to MLH1 expression by a post‐translational mechanism the cytotoxicy of aspirin in relation to MBD4 expression was examined. This study reports that aspirin does not up‐regulate MBD4 gene transcription in vitro in the DNA mismatch repair proficient/p53 mutant colorectal cancer cell line SW480. However, MBD4 gene transcription was up‐regulated upon treatment with the aspirin precursor, salicylic acid. The suggested involvement of the DNA repair proteins in the mechanism of action of aspirin promoted the investigation into the expression of DNA damage signalling pathways genes upon aspirin exposure. This study utilised a commercially available PCR array to analyse the expression of 84 DNA damage signalling genes in the SW480 colorectal cancer cell line upon aspirin treatment. It is reported that treatment of the SW480 cell line with aspirin caused changes in mRNA expression of several key genes involved in DNA damage signalling including a significant down‐regulation in expression of the genes encoding ATR, BRCA1 and MAPK12 and increases in the expression of XRCC3 and GADD45α genes. Regulation of these genes could potentially have profound effects on colorectal cancer cells and may play a role in the observed chemo‐protective effect of aspirin in vivo.Further to this, protein expression was analysed to determine if correlation could be established with the changes in mRNA expression observed. Although a correlation was not seen between transcript and protein levels of ATR, BRCA1 and GADD45α, an increase in XRCC3 protein expression upon aspirin treatment in SW480 cells was observed by immunoblotting, immunofluorescence and immunohistochemical analysis. This study indicates that alterations in gene expression seen in microarray studies need to be verified at the protein level. Furthermore, this study reports the novel discovery of XRCC3 gene and protein expression being susceptible to exposure to the non‐steroidal anti‐inflammatory drug, aspirin.

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.

O câncer é a principal causa de morte nos paises economicamente desenvolvidos e a segunda em paises em desenvolvimento, resultado, em parte, da grande falta de especificidade dos tratamentos atualmente disponíveis. Por outro lado, uma aplicação clínica muito específica, denominada letalidade sintética, foi recentemente proposta. Nesta abordagem terapêutica os inibidores de poli(ADP-ribose) polimerases (PARP), também conhecidos como PARPis, mostraram-se capazes de induzir a morte celular seletiva em células tumorais com defeitos em BRCA1 e BRCA2 (ambas envolvidas no reparo de quebras duplas - DSBR). Assim, a excitante possibilidade de eliminar as células cancerígenas de maneira seletiva fez com que os PARPis passassem de interessantes ferramentas moleculares às mais promissoras drogas anticâncer da atualidade. Contudo, os mecanismos básicos envolvidos na citotoxicidade dos PARPis continuam pouco conhecidos e suas aplicações restritas a um pequeno grupo de cânceres. Por este motivo, neste trabalho, a citotoxicidade do Olaparib (um inibidor de PARP) foi investigada em um painel de linhagens de câncer colorretal (CRC). Os resultados demonstraram que o Olaparib é uma droga de ação lenta, cuja citotoxicidade pode ser modulada por defeitos genéticos em MLH1 (envolvido no reparo de bases mal-emparelhadas) e no supressor tumoral PTEN. Por outro lado, observou-se que o fenótipo MSI (Instabilidade de microssatélites) e os defeitos genéticos em p53 não influenciaram a citotoxicidade do Olaparib. Além disso, linhagens com resistência adquirida a Oxaliplatina (Oxp) e a 5-Fluorouracil (5- Fu) não apresentaram efeito refratário ao Olaparib, enquanto que linhagens com resistência adquirida a SN-38 (metabólito ativo do Irinotecano) apresentaram um forte efeito refratário. Finalmente, as associações de Oxp ou 5-Fu com Olaparib foram capazes de sensibilizar células com resistência relativa e adquirida. Juntos, estes resultados sugerem uma série de novas possibilidades para o emprego de inibidores de PARP no tratamento de CRC. / Cancer is the main cause of death in developed countries and the second in lessdeveloped countries, that results in part from the low specific treatments available. However, a very specific therapeutic approach, called synthetic lethality, was recently proposed. The best documented synthetic lethal interaction was reported between poly(ADP-ribose) polymerases inhibitors (PARPis) and defects in BRCA1 and BRCA2 (both involved in double-strand break repair - DSBR), which may induce selective cancer cells death. Therefore, the exciting possibility to selectively kill cancer cells has been moving PARPis from interesting molecular tools to the forefront of cancer therapy research. However, the basic mechanisms involved in PARPis cytotoxicity are still poorly studied and its clinical applications are restricted to a small number of malignances. Herein, the Olaparib (PARPi) cytotoxicity was investigated in a colorectal cancer (CRC) cell line panel. The results demonstrated that Olaparib is a slow action drug, which may have its effects increased in cells with MLH1 (involved in mismatch repair) and PTEN (tumor supressor) defects. On the other hand, neither the MSI (microsatellite instability) phenotype nor the p53 defects were observed to influence on Olaparib cytotoxicity. Further, neither Oxp nor 5-Fu resistant cell lines presented cross-resistance to Olaparib, whereas a pronounced cross-resistance was observed for SN-38 (Irinotecan metabolite) resistant cell line. Finally, Olaparib associations with Oxaliplatin or 5-Fluorouracil were shown to sensitize cells with both relative and acquired resistances. Together, these results suggest a series of new possible uses for PARP inhibitors in CRC treatment.

O câncer é a principal causa de morte nos paises economicamente desenvolvidos e a segunda em paises em desenvolvimento, resultado, em parte, da grande falta de especificidade dos tratamentos atualmente disponíveis. Por outro lado, uma aplicação clínica muito específica, denominada letalidade sintética, foi recentemente proposta. Nesta abordagem terapêutica os inibidores de poli(ADP-ribose) polimerases (PARP), também conhecidos como PARPis, mostraram-se capazes de induzir a morte celular seletiva em células tumorais com defeitos em BRCA1 e BRCA2 (ambas envolvidas no reparo de quebras duplas - DSBR). Assim, a excitante possibilidade de eliminar as células cancerígenas de maneira seletiva fez com que os PARPis passassem de interessantes ferramentas moleculares às mais promissoras drogas anticâncer da atualidade. Contudo, os mecanismos básicos envolvidos na citotoxicidade dos PARPis continuam pouco conhecidos e suas aplicações restritas a um pequeno grupo de cânceres. Por este motivo, neste trabalho, a citotoxicidade do Olaparib (um inibidor de PARP) foi investigada em um painel de linhagens de câncer colorretal (CRC). Os resultados demonstraram que o Olaparib é uma droga de ação lenta, cuja citotoxicidade pode ser modulada por defeitos genéticos em MLH1 (envolvido no reparo de bases mal-emparelhadas) e no supressor tumoral PTEN. Por outro lado, observou-se que o fenótipo MSI (Instabilidade de microssatélites) e os defeitos genéticos em p53 não influenciaram a citotoxicidade do Olaparib. Além disso, linhagens com resistência adquirida a Oxaliplatina (Oxp) e a 5-Fluorouracil (5- Fu) não apresentaram efeito refratário ao Olaparib, enquanto que linhagens com resistência adquirida a SN-38 (metabólito ativo do Irinotecano) apresentaram um forte efeito refratário. Finalmente, as associações de Oxp ou 5-Fu com Olaparib foram capazes de sensibilizar células com resistência relativa e adquirida. Juntos, estes resultados sugerem uma série de novas possibilidades para o emprego de inibidores de PARP no tratamento de CRC. / Cancer is the main cause of death in developed countries and the second in lessdeveloped countries, that results in part from the low specific treatments available. However, a very specific therapeutic approach, called synthetic lethality, was recently proposed. The best documented synthetic lethal interaction was reported between poly(ADP-ribose) polymerases inhibitors (PARPis) and defects in BRCA1 and BRCA2 (both involved in double-strand break repair - DSBR), which may induce selective cancer cells death. Therefore, the exciting possibility to selectively kill cancer cells has been moving PARPis from interesting molecular tools to the forefront of cancer therapy research. However, the basic mechanisms involved in PARPis cytotoxicity are still poorly studied and its clinical applications are restricted to a small number of malignances. Herein, the Olaparib (PARPi) cytotoxicity was investigated in a colorectal cancer (CRC) cell line panel. The results demonstrated that Olaparib is a slow action drug, which may have its effects increased in cells with MLH1 (involved in mismatch repair) and PTEN (tumor supressor) defects. On the other hand, neither the MSI (microsatellite instability) phenotype nor the p53 defects were observed to influence on Olaparib cytotoxicity. Further, neither Oxp nor 5-Fu resistant cell lines presented cross-resistance to Olaparib, whereas a pronounced cross-resistance was observed for SN-38 (Irinotecan metabolite) resistant cell line. Finally, Olaparib associations with Oxaliplatin or 5-Fluorouracil were shown to sensitize cells with both relative and acquired resistances. Together, these results suggest a series of new possible uses for PARP inhibitors in CRC treatment.

O câncer é a principal causa de morte nos paises economicamente desenvolvidos e a segunda em paises em desenvolvimento, resultado, em parte, da grande falta de especificidade dos tratamentos atualmente disponíveis. Por outro lado, uma aplicação clínica muito específica, denominada letalidade sintética, foi recentemente proposta. Nesta abordagem terapêutica os inibidores de poli(ADP-ribose) polimerases (PARP), também conhecidos como PARPis, mostraram-se capazes de induzir a morte celular seletiva em células tumorais com defeitos em BRCA1 e BRCA2 (ambas envolvidas no reparo de quebras duplas - DSBR). Assim, a excitante possibilidade de eliminar as células cancerígenas de maneira seletiva fez com que os PARPis passassem de interessantes ferramentas moleculares às mais promissoras drogas anticâncer da atualidade. Contudo, os mecanismos básicos envolvidos na citotoxicidade dos PARPis continuam pouco conhecidos e suas aplicações restritas a um pequeno grupo de cânceres. Por este motivo, neste trabalho, a citotoxicidade do Olaparib (um inibidor de PARP) foi investigada em um painel de linhagens de câncer colorretal (CRC). Os resultados demonstraram que o Olaparib é uma droga de ação lenta, cuja citotoxicidade pode ser modulada por defeitos genéticos em MLH1 (envolvido no reparo de bases mal-emparelhadas) e no supressor tumoral PTEN. Por outro lado, observou-se que o fenótipo MSI (Instabilidade de microssatélites) e os defeitos genéticos em p53 não influenciaram a citotoxicidade do Olaparib. Além disso, linhagens com resistência adquirida a Oxaliplatina (Oxp) e a 5-Fluorouracil (5- Fu) não apresentaram efeito refratário ao Olaparib, enquanto que linhagens com resistência adquirida a SN-38 (metabólito ativo do Irinotecano) apresentaram um forte efeito refratário. Finalmente, as associações de Oxp ou 5-Fu com Olaparib foram capazes de sensibilizar células com resistência relativa e adquirida. Juntos, estes resultados sugerem uma série de novas possibilidades para o emprego de inibidores de PARP no tratamento de CRC. / Cancer is the main cause of death in developed countries and the second in lessdeveloped countries, that results in part from the low specific treatments available. However, a very specific therapeutic approach, called synthetic lethality, was recently proposed. The best documented synthetic lethal interaction was reported between poly(ADP-ribose) polymerases inhibitors (PARPis) and defects in BRCA1 and BRCA2 (both involved in double-strand break repair - DSBR), which may induce selective cancer cells death. Therefore, the exciting possibility to selectively kill cancer cells has been moving PARPis from interesting molecular tools to the forefront of cancer therapy research. However, the basic mechanisms involved in PARPis cytotoxicity are still poorly studied and its clinical applications are restricted to a small number of malignances. Herein, the Olaparib (PARPi) cytotoxicity was investigated in a colorectal cancer (CRC) cell line panel. The results demonstrated that Olaparib is a slow action drug, which may have its effects increased in cells with MLH1 (involved in mismatch repair) and PTEN (tumor supressor) defects. On the other hand, neither the MSI (microsatellite instability) phenotype nor the p53 defects were observed to influence on Olaparib cytotoxicity. Further, neither Oxp nor 5-Fu resistant cell lines presented cross-resistance to Olaparib, whereas a pronounced cross-resistance was observed for SN-38 (Irinotecan metabolite) resistant cell line. Finally, Olaparib associations with Oxaliplatin or 5-Fluorouracil were shown to sensitize cells with both relative and acquired resistances. Together, these results suggest a series of new possible uses for PARP inhibitors in CRC treatment.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第18110号 / 理博第3988号 / 新制||理||1575(附属図書館) / 30968 / 京都大学大学院理学研究科生物科学専攻 / (主査)准教授 秋山 秋梅, 教授 沼田 英治, 教授 疋田 努 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

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.

Colorectal carcinoma (CRC) is one of the most prevalent malignancies in the Czech Republic. In general, there are two molecular pathways leading to CRC: one is characterized by chromosomal instability, the other by the deficiency in DNA mismatch repair (MMR) genes. MutL homologue 1 (MLH1) gene, a member of the MMR gene-family, represents a key component of the MMR system, responsible for recognition of nucleotide mismatches occurring during DNA replication, and for the recruitment of repair proteins to correct the replication errors. According to literature, somatic mutations in MMR genes, and MLH1 in particular, hallmark sporadic, MMR deficient, CRC cases. We aimed at analyzing somatic events in MLH1 gene and the determination of microsatellite instability (MSI) status in 99 DNA samples from 96 patients with sporadic CRC. Mutations were screened by high resolution melting (HRM) curve analysis. Positive cases in each run were subsequently verified by automated sequencing. Mainly gene variants were found in MLH1 gene: We discovered two new variants, one in exon 2 at position c. 204 C>G, p. Ile68Met (98 C/C, 1C/G) and the other in exon 11 at position c. 973 C>T, p. Arg325Trp (98 C/C, 1 C/T). Only the latter variant c. 973 C>T was identified as somatic mutation. All other variants found in MLH1 gene...