Plant-specific environmental and developmental signals regulate the mismatch repair protein MSH6 in Arabidopsis thaliana.

The DNA mismatch repair (MMR) is a postreplicative system that guarantees genomic stability by correcting mispaired and unpaired nucleotides. In eukaryotic nuclei, MMR is initiated by the binding of heterodimeric MutS homologue (MSH) complexes to the DNA error or lesion. Among these proteins, MSH2-MSH6 is the most abundant heterodimer. Even though the MMR mechanism and proteins are highly conserved throughout evolution, physiological differences between species can lead to different regulatory features. Here, we investigated how light, sugar, and/or hormones modulate Arabidopsis thaliana MSH6 expression pattern. We first characterized the promoter region of MSH6. Phylogenetic shadowing revealed three highly conserved regions. These regions were analyzed by the generation of deletion constructs of the MSH6 full-length promoter fused to the ß-glucuronidase (GUS) gene. Combined, our in silico and genetic analyses revealed that a 121-bp promoter fragment was necessary for MSH6 expression and contained potential cis-acting elements involved in light- and hormone-responsive gene expression. Accordingly, light exposure or sugar treatment of four-day old A. thaliana seedlings triggered an upregulation of MSH6 in shoot and root apical meristems. Appropriately, MSH6 was also induced by the stem cell inducer WUSCHEL. Further, the stimulatory effect of light was dependent on the presence of phyA. In addition, treatment of seedlings with auxin or cytokinin also caused an upregulation of MSH6 under darkness. Consistent with auxin signals, MSH6 expression was suppressed in the GATA23 RNAi line compared with the wild type. Our results provide evidence that endogenous factors and environmental signals controlling plant growth and development regulate the MSH6 protein in A. thaliana.

Deficiency of the Arabidopsis mismatch repair MSH6 attenuates Pseudomonas syringae invasion.

The MutS homolog 6 (MSH6) is a nuclear DNA mismatch repair (MMR) gene that encodes the MSH6 protein. MSH6 interacts with MSH2 to form the MutSα heterodimer. MutSα corrects DNA mismatches and unpaired nucleotides arising during DNA replication, deamination of 5-methylcytosine, and recombination between non-identical DNA sequences. In addition to correcting DNA biosynthetic errors, MutSα also recognizes chemically damaged DNA bases. Here, we show that inactivation of MSH6 affects the basal susceptibility of Arabidopsis thaliana to Pseudomonas syringae pv tomato DC3000. The msh6 T-DNA insertional mutant exhibited a reduced susceptibility to the bacterial invasion. This heightened basal resistance of msh6 mutants appears to be dependent on an increased stomatal closure, an accumulation of H2O2 and double-strand breaks (DSBs) and a constitutive expression of pathogenesis-related (NPR1 and PR1) and DNA damage response (RAD51D and SOG1) genes. Complementation of this mutant with the MSH6 wild type allele under the control of its own promoter resulted in reversal of the basal bacterial resistance phenotype and the stomatal closure back to wild type levels. Taken together, these results demonstrate that inactivation of MSH6 increases Arabidopsis basal susceptibility to the bacterial pathogen and suggests a link between DNA repair and stress signaling in plants.

A Previously Unrecognized Molecular Landscape of Lynch Syndrome in the Mexican Population.

Lynch syndrome (LS) is the main hereditary colorectal cancer syndrome. There have been few reports regarding the clinical and molecular characteristics of LS patients in Latin America; this is particularly true in the Mexican population, where no information is available. The present study aims to describe the clinical and molecular spectrum of variants in a cohort of patients diagnosed with LS in Mexico. We present a retrospective analysis of 412 patients with suspected LS, whose main site of cancer diagnosis was the colon (58.25%), followed by the endometrium (18.93%). Next-generation sequencing analysis, with an extensive multigene panel, showed that 27.1% (112/414) had a variant in one of the genes of the mismatch repair pathway (MMR); 30.4% (126/414) had a variant in non-MMR genes such as CHEK2, APC, MUTYH, BRCA1, and BRCA2; and 42.5% (176/414) had no genetic variants. Most of the variants were found in MLH1. Pathogenic variants (PVs) in MMR genes were identified in 65.7% (96/146) of the total PVs, and 34.24% (45/146) were in non-MMR genes. Molecular and clinical characterization of patients with LS in specific populations allowed personalized follow-up, with the option for targeted treatment with immune checkpoint inhibitors and the development of public health policies. Moreover, such characterization allows for family cascade testing and consequent prevention strategies.

Trypanosoma brucei and Trypanosoma cruzi DNA Mismatch Repair Proteins Act Differently in the Response to DNA Damage Caused by Oxidative Stress.

MSH2, associated with MSH3 or MSH6, is a central component of the eukaryotic DNA Mismatch Repair (MMR) pathway responsible for the recognition and correction of base mismatches that occur during DNA replication and recombination. Previous studies have shown that MSH2 plays an additional DNA repair role in response to oxidative damage in Trypanosoma cruzi and Trypanosoma brucei. By performing co-immunoprecipitation followed by mass spectrometry with parasites expressing tagged proteins, we confirmed that the parasites' MSH2 forms complexes with MSH3 and MSH6. To investigate the involvement of these two other MMR components in the oxidative stress response, we generated knockout mutants of MSH6 and MSH3 in T. brucei bloodstream forms and MSH6 mutants in T. cruzi epimastigotes. Differently from the phenotype observed with T. cruzi MSH2 knockout epimastigotes, loss of one or two alleles of T. cruzi msh6 resulted in increased susceptibility to H2O2 exposure, besides impaired MMR. In contrast, T. brucei msh6 or msh3 null mutants displayed increased tolerance to MNNG treatment, indicating that MMR is affected, but no difference in the response to H2O2 treatment when compared to wild type cells. Taken together, our results suggest that, while T. cruzi MSH6 and MSH2 are involved with the oxidative stress response in addition to their role as components of the MMR, the DNA repair pathway that deals with oxidative stress damage operates differently in T. brucei.

The mismatch repair protein MSH6 regulates somatic recombination in Arabidopsis thaliana.

The mismatch repair (MMR) pathway promotes genome stability by controlling the fidelity of replication and recombination. The first step of the pathway involves recognition of the mismatch by heterodimers composed of MutS homologs (MSH). Although MSH6 has been well characterized in yeasts and humans, the role of the plant protein has not been extensively studied. We first analyzed gene expression in Arabidopsis thaliana. The use of transgenic plants expressing the ß-glucuronidase (GUS) reporter gene under the control of approximately 1-kb region upstream of the start codon of the AtMSH6 gene demonstrated that MSH6 is preferentially expressed in undifferentiated cells with an intense cell division rate. We then examined protein function in meiotic and somatic recombination. Suppression of AtMSH6 did not affect the rate of meiotic recombination, but increased the frequency of recombination between two homeologous repeats of a marker gene by 3-fold relative to wild-type plants. Expression of the AtMSH6 gene under the control of its own promoter in msh6 homozygous mutant plants rescued the altered somatic recombination phenotype. We conclude that MSH6 shows a functional conservation across different biological kingdoms and a functional specificity in plants.

Alterações germinativas em pacientes com câncer de reto em idade jovem / Germline alterations in patients with rectal cancer at a young age

O câncer colorretal (CCR) é o terceiro tipo de câncer mais comum no mundo e a segunda causa de morte por câncer. Dados atuais apontam que o câncer de reto (CaRe) contribui para a maior incidência de CCR observada em pacientes jovens e é responsável por um aumento destes tumores em cerca de 75% nos últimos 40 anos. As variantes germinativas são reportadas em 14 a 16% dos indivíduos jovens com CCR, independente de história familiar de câncer. Ainda assim, a causa do desenvolvimento de CCR na maioria dos casos de indivíduos jovens ou famílias com múltiplos indivíduos afetados permanece desconhecida. Neste estudo, foram investigadas variantes germinativas em 76 pacientes com CaRe incluindo 29 classificados pelos critérios de Amsterdam I/II para a Síndrome de Lynch (Grupo 1) e 47 pacientes com idade igual ou inferior 40 anos (Grupo 2). O objetivo principal foi identificar variantes em 93 genes relacionados ao câncer que possam contribuir para o risco de desenvolvimento da doença usando sequenciamento de alto desempenho. Quinze variantes foram selecionadas e avaliadas em membros de oito famílias. Foram identificadas 153 variantes envolvendo 65 genes. Pacientes com câncer de reto apresentaram alta frequência de variantes germinativas em ATM (19%), APC (10%) e BRCA2 (9%). Variantes patogênicas ou likely-patogênicas foram identificadas em genes de alta (MSH2, MSH6, MLH1, MUTYH e BRCA2), moderada (ATM) e baixa (MTHFR e NOTCH1) penetrância, em 18% (14/76) dos pacientes, concordante com dados da literatura. Variantes características da Síndrome de Lynch foram identificadas em seis pacientes (6/76) e variantes associadas à polipose no gene MUTYH, em dois pacientes (2/76). Variantes patogênicas ou likely-patogênicas foram identificadas em 17% (8/47) dos pacientes jovens, especialmente em MUTYH (3/47), MTHFR (2/47), ATM, MSH6 e MLH1 (1/47 cada). Dentre estes, dois de 14 pacientes não reportaram câncer na família e seis tiveram pelo menos um caso de câncer na família, mas não preencheram os critérios clínicos para síndrome de predisposição hereditária ao câncer. Este estudo revelou 25 variantes novas (não identificadas em bancos de dados públicos ou reportadas em literatura). Em adição, foram observadas variantes germinativas em diferentes genes das vias MMR e de recombinação homóloga, incluindo ATM, BRCA2 e POLD1. Foram identificadas 15 variantes candidatas e ou associadas ao fenótipo nos genes ATM, APC, MSH2, MTHFR, CDKN2A, MUTYH e POLD1. Essas variantes foram avaliadas em 20 indivíduos de 8 famílias, sendo confirmadas nos probandos e identificadas em mais de um membro das famílias investigadas. A identificação de genes associados à predisposição ao CaRe tem potencial importância para o delineamento de estratégias mais eficientes de diagnóstico e aconselhamento genético em famílias com um alto risco de desenvolver este tumor (AU)

Colorectal cancer (CRC) is the third most common cancer worldwide and the second leading cause of cancer-related death. Recent studies suggest that rectal cancer (ReCa) contributes to the higher CRC incidence observed in young patients, and it is responsible for 75% of the increased incidence in colorectal tumors over the last 40 years. Germline variants have been reported in 14-16% of patients with early-onset CRC, regardless of family history of cancer. Nonetheless, the causes of CRC onset in most young patients or families with multiple affected cases remain unknown. We investigated germline variants in 76 ReCa patients, including 29 cases classified by the Amsterdam I/II criteria for Lynch Syndrome (Group 1) and 47 early-onset ReCa patients (≤40 years old, Group 2). The main objective was to identify variants in 93 cancer-related genes that can contribute to increased risk of the disease development using next-generation sequencing. Fifteen candidate variants were evaluated in eight selected families. Next-generation sequencing revealed 153 variants involving 65 genes. ReCa patients showed high frequency of germline variants in ATM (19%), APC (10%) and BRCA2 (9%). Pathogenic or likely-pathogenic variants were observed in high (MSH2, MSH6, MLH1, MUTYH, and BRCA2), moderate (ATM) and low (MTHFR and NOTCH1) penetrance genes, in 18% (14/76) of the patients, consistent with the literature data. Variants associated with Lynch Syndrome were identified in six patients (6/76), and variants involved in MUTYH-associated polyposis in two patients (2/76). Pathogenic or likely-pathogenic variants were identified in 17% (8/47) of early-onset patients, especially in the MUTYH (3/47), MTHFR (2/47), ATM, MSH6, and MLH1 (1/47 each) genes. Among these, two of fourteen patients had no family history of cancer and six reported at least one case of cancer in the family, but none of them met clinical criteria for the hereditary cancer syndrome. This study revealed 25 new variants (not reported in public databases or previous studies). In addition, germline variants were observed in several genes involved in MMR and homologous recombination (HR) pathways, including ATM, BRCA2 and POLD1. Fifteen candidates and associated to the phenotype variants were identified in the ATM, APC, MSH2, MTHFR, CDKN2A, MUTYH, and POLD1 genes. These variants were evaluated in 20 individuals (8 families), being confirmed in the index cases and identified in more than one relative of the evaluated families. The identification of genes associated with ReCa predisposition is crucial for outlining more efficient diagnostic strategies and for improving genetic counseling for families with high risk to develop this tumor type (AU)

Presence of microsatellite instability in esophageal squamous cell carcinoma associated with chagasic megaesophagus.

AIM: The molecular pathogenesis of esophageal squamous cell carcinoma (ESCC) has been increasingly studied, but there is no report on the role of MSI in ESCC development associated with chagasic megaesophagus (CM).Results/methodology: In four ESCC/CM (4/19) we found microsatellite instability (MSI) alterations (21.1%), being three MSI-L (15.8%) and one MSI-H (5.3%). Four out of 35 ESCC cases showed MSI-L (11.4%) and only one out of 26 CM cases presented MSI-L (3.9%). The MSI-H was observed in an ESCC/CM patient that presents lack of MSH6 immunostaining corroborating deficiency in MMR pathway. Interestingly, the MSI-H ESCC/CM case also presented a deletion the HSP110 poly(T)17 gene. DISCUSSION/CONCLUSION: Taking together, we concluded that MSI is a rare event in esophageal squamous cell carcinoma, but can be associated with CM.