Methylation of hMLH1 promoter correlates with the gene silencing with a region-specific manner in colorectal cancer

Microsatellite instability is present in over 80% of the hereditary non-polyposis colorectal carcinoma and about 15–20% of the sporadic cancer. Microsatellite instability is caused by the inactivation of the mismatch repair genes, such as primarily hMLH1, hMSH2. To study the mechanisms of the inactivation of mismatch repair genes in colorectal cancers, especially the region-specific methylation of hMLH1 promoter and its correlation with gene expression, we analysed microsatellite instability, expression and methylation of hMLH1 and loss of heterozygosity at hMLH1 locus in these samples. Microsatellite instability was present in 17 of 71 primary tumours of colorectal cancer, including 14 of 39 (36%) mucinous cancer and three of 32 (9%) non-mucinous cancer. Loss of hMLH1 and hMSH2 expression was detected in nine and three of 16 microsatellite instability tumours respectively. Methylation at CpG sites in a proximal region of hMLH1 promoter was detected in seven of nine tumours that showed no hMLH1 expression, while no methylation was present in normal mucosa and tumours which express hMLH1. However, methylation in the distal region was observed in all tissues including normal mucosa and hMLH1 expressing tumours. This observation indicates that methylation of hMLH1 promoter plays an important role in microsatellite instability with a region-specific manner in colorectal cancer. Loss of heterozygosity at hMLH1 locus was present in four of 17 cell lines and 16 of 54 tumours with normal hMLH1 status, while loss of heterozygosity was absent in all nine cell lines and nine tumours with abnormal hMLH1 status (mutation or loss of expression), showing loss of heterozygosity is not frequently involved in the inactivation of hMLH1 gene in sporadic colorectal cancer

Selection-Free Zinc-Finger Nuclease Engineering by Context-Dependent Assembly (CoDA)

Engineered zinc-finger nucleases (ZFNs) enable targeted genome modification. Here we describe Context-Dependent Assembly (CoDA), a platform for engineering ZFNs using only standard cloning techniques or custom DNA synthesis. Using CoDA ZFNs, we rapidly altered 20 genes in zebrafish, Arabidopsis, and soybean. The simplicity and efficacy of CoDA will enable broad adoption of ZFN technology and make possible large-scale projects focused on multi-gene pathways or genome-wide alterations

Metaphors We Think With: The Role of Metaphor in Reasoning

The way we talk about complex and abstract ideas is suffused with metaphor. In five experiments, we explore how these metaphors influence the way that we reason about complex issues and forage for further information about them. We find that even the subtlest instantiation of a metaphor (via a single word) can have a powerful influence over how people attempt to solve social problems like crime and how they gather information to make “well-informed” decisions. Interestingly, we find that the influence of the metaphorical framing effect is covert: people do not recognize metaphors as influential in their decisions; instead they point to more “substantive” (often numerical) information as the motivation for their problem-solving decision. Metaphors in language appear to instantiate frame-consistent knowledge structures and invite structurally consistent inferences. Far from being mere rhetorical flourishes, metaphors have profound influences on how we conceptualize and act with respect to important societal issues. We find that exposure to even a single metaphor can induce substantial differences in opinion about how to solve social problems: differences that are larger, for example, than pre-existing differences in opinion between Democrats and Republicans

Mutation Rates of TGFBR2 and ACVR2 Coding Microsatellites in Human Cells with Defective DNA Mismatch Repair

Microsatellite instability promotes colonic tumorigenesis through generating frameshift mutations at coding microsatellites of tumor suppressor genes, such as TGFBR2 and ACVR2. As a consequence, signaling through these TGFβ family receptors is abrogated in DNA Mismatch repair (MMR)-deficient tumors. How these mutations occur in real time and mutational rates of these human coding sequences have not previously been studied. We utilized cell lines with different MMR deficiencies (hMLH1−/−, hMSH6−/−, hMSH3−/−, and MMR-proficient) to determine mutation rates. Plasmids were constructed in which exon 3 of TGFBR2 and exon 10 of ACVR2 were cloned +1 bp out of frame, immediately after the translation initiation codon of an enhanced GFP (EGFP) gene, allowing a −1 bp frameshift mutation to drive EGFP expression. Mutation-resistant plasmids were constructed by interrupting the coding microsatellite sequences, preventing frameshift mutation. Stable cell lines were established containing portions of TGFBR2 and ACVR2, and nonfluorescent cells were sorted, cultured for 7–35 days, and harvested for flow cytometric mutation detection and DNA sequencing at specific time points. DNA sequencing revealed a −1 bp frameshift mutation (A9 in TGFBR2 and A7 in ACVR2) in the fluorescent cells. Two distinct fluorescent populations, M1 (dim, representing heteroduplexes) and M2 (bright, representing full mutants) were identified, with the M2 fraction accumulating over time. hMLH1 deficiency revealed 11 (5.91×10−4) and 15 (2.18×10−4) times higher mutation rates for the TGFBR2 and ACVR2 microsatellites compared to hMSH6 deficiency, respectively. The mutation rate of the TGFBR2 microsatellite was ∼3 times higher in both hMLH1 and hMSH6 deficiencies than the ACVR2 microsatellite. The −1 bp frameshift mutation rates of TGFBR2 and ACVR2 microsatellite sequences are dependent upon the human MMR background

Mutations in APC, CTNNB1 and K-ras genes and expression of hMLH1 in sporadic colorectal carcinomas from the Netherlands Cohort Study

BACKGROUND: The early to intermediate stages of the majority of colorectal tumours are thought to be driven by aberrations in the Wnt (APC, CTNNB1) and Ras (K-ras) pathways. A smaller proportion of cancers shows mismatch repair deficiency. The aim of this study was to analyse the co-occurrence of these genetic alterations in relation to tumour and patient characteristics. METHODS: In a group of 656 unselected sporadic colorectal cancer patients, aberrations in the APC, K-ras, CTNNB1 genes, and expression of hMLH1 were investigated. Additionally, tumours were divided in groups based on molecular features and compared with respect to patient's age at diagnosis, sex, family history of colorectal cancer, tumour sub-localisation, Dukes' stage and differentiation. RESULTS: Mutations at the phosphorylation sites (codons 31, 33, 37, and 45) in the CTNNB1 gene were observed in tumours from only 5/464 patients. Tumours with truncating APC mutations and activating K-ras mutations in codons 12 and 13 occurred at similar frequencies (37% (245/656) and 36% (235/656), respectively). Seventeen percent of tumours harboured both an APC and a K-ras mutation (109/656). Nine percent of all tumours (58/656) lacked hMLH1 expression. Patients harbouring a tumour with absent hMLH1 expression were older, more often women, more often had proximal colon tumours that showed poorer differentiation when compared to patients harbouring tumours with an APC and/or K-ras mutation. CONCLUSION: CTNNB1 mutations seem to be of minor importance in sporadic colorectal cancer. The main differences in tumour and patient characteristics are found between groups of patients based on mismatch repair deficiency

Colorectal carcinomas with microsatellite instability display a different pattern of target gene mutations according to large bowel site of origin

<p>Abstract</p> <p>Background</p> <p>Only a few studies have addressed the molecular pathways specifically involved in carcinogenesis of the distal colon and rectum. We aimed to identify potential differences among genetic alterations in distal colon and rectal carcinomas as compared to cancers arising elsewhere in the large bowel.</p> <p>Methods</p> <p>Constitutional and tumor DNA from a test series of 37 patients with rectal and 25 patients with sigmoid carcinomas, previously analyzed for microsatellite instability (MSI), was studied for <it>BAX</it>, <it>IGF2R</it>, <it>TGFBR2</it>, <it>MSH3</it>, and <it>MSH6 </it>microsatellite sequence alterations, <it>BRAF </it>and <it>KRAS </it>mutations, and <it>MLH1 </it>promoter methylation. The findings were then compared with those of an independent validation series consisting of 36 MSI-H carcinomas with origin from each of the large bowel regions. Immunohistochemical and germline mutation analyses of the mismatch repair system were performed when appropriate.</p> <p>Results</p> <p>In the test series, <it>IGFR2 </it>and <it>BAX </it>mutations were present in one and two out of the six distal MSI-H carcinomas, respectively, and no mutations were detected in <it>TGFBR2</it>, <it>MSH3</it>, and <it>MSH6</it>. We confirmed these findings in the validation series, with <it>TGFBR2 </it>and <it>MSH3 </it>microsatellite mutations occurring less frequently in MSI-H rectal and sigmoid carcinomas than in MSI-H colon carcinomas elsewhere (<it>P </it>= 0.00005 and <it>P </it>= 0.0000005, respectively, when considering all MSI-carcinomas of both series). No <it>MLH1 </it>promoter methylation was observed in the MSI-H rectal and sigmoid carcinomas of both series, as compared to 53% found in MSI-H carcinomas from other locations (<it>P </it>= 0.004). <it>KRAS </it>and <it>BRAF </it>mutational frequencies were 19% and 43% in proximal carcinomas and 25% and 17% in rectal/sigmoid carcinomas, respectively.</p> <p>Conclusion</p> <p>The mechanism and the pattern of genetic changes driving MSI-H carcinogenesis in distal colon and rectum appears to differ from that occurring elsewhere in the colon and further investigation is warranted both in patients with sporadic or hereditary disease.</p

Modulations of the Chicken Cecal Microbiome and Metagenome in Response to Anticoccidial and Growth Promoter Treatment

With increasing pressures to reduce or eliminate the use of antimicrobials for growth promotion purposes in production animals, there is a growing need to better understand the effects elicited by these agents in order to identify alternative approaches that might be used to maintain animal health. Antibiotic usage at subtherapeutic levels is postulated to confer a number of modulations in the microbes within the gut that ultimately result in growth promotion and reduced occurrence of disease. This study examined the effects of the coccidiostat monensin and the growth promoters virginiamycin and tylosin on the broiler chicken cecal microbiome and metagenome. Using a longitudinal design, cecal contents of commercial chickens were extracted and examined using 16S rRNA and total DNA shotgun metagenomic pyrosequencing. A number of genus-level enrichments and depletions were observed in response to monensin alone, or monensin in combination with virginiamycin or tylosin. Of note, monensin effects included depletions of Roseburia, Lactobacillus and Enterococcus, and enrichments in Coprococcus and Anaerofilum. The most notable effect observed in the monensin/virginiamycin and monensin/tylosin treatments, but not in the monensin-alone treatments, was enrichments in Escherichia coli. Analysis of the metagenomic dataset identified enrichments in transport system genes, type I fimbrial genes, and type IV conjugative secretion system genes. No significant differences were observed with regard to antimicrobial resistance gene counts. Overall, this study provides a more comprehensive glimpse of the chicken cecum microbial community, the modulations of this community in response to growth promoters, and targets for future efforts to mimic these effects using alternative approaches

Molecular Genetic Analysis of 103 Sporadic Colorectal Tumours in Czech Patients

The Czech Republic has one of the highest incidences of colorectal cancer (CRC) in Europe. To evaluate whether sporadic CRCs in Czech patients have specific mutational profiles we analysed somatic genetic changes in known CRC genes (APC, KRAS, TP53, CTNNB1, MUTYH and BRAF, loss of heterozygosity (LOH) at the APC locus, microsatellite instability (MSI), and methylation of the MLH1 promoter) in 103 tumours from 102 individuals. The most frequently mutated gene was APC (68.9% of tumours), followed by KRAS (31.1%), TP53 (27.2%), BRAF (8.7%) and CTNNB1 (1.9%). Heterozygous germline MUTYH mutations in 2 patients were unlikely to contribute to the development of their CRCs. LOH at the APC locus was found in 34.3% of tumours, MSI in 24.3% and MLH1 methylation in 12.7%. Seven tumours (6.9%) were without any changes in the genes tested. The analysis yielded several findings possibly specific for the Czech cohort. Somatic APC mutations did not cluster in the mutation cluster region (MCR). Tumours with MSI but no MLH1 methylation showed earlier onset and more severe mutational profiles compared to MSI tumours with MLH1 methylation. TP53 mutations were predominantly located outside the hot spots, and transitions were underrepresented. Our analysis supports the observation that germline MUTYH mutations are rare in Czech individuals with sporadic CRCs. Our findings suggest the influence of specific ethnic genetic factors and/or lifestyle and dietary habits typical for the Czech population on the development of these cancers

Body mass index in early adulthood and colorectal cancer risk for carriers and non-carriers of germline mutations in DNA mismatch repair genes

BACKGROUND: Carriers of germline mutations in DNA mismatch repair (MMR) genes have a high risk of colorectal cancer (CRC), but the modifiers of this risk are not well established. We estimated an association between body mass index (BMI) in early adulthood and subsequent risk of CRC for carriers and, as a comparison, estimated the association for non-carriers. METHODS: A weighted Cox regression was used to analyse height and weight at 20 years reported by 1324 carriers of MMR gene mutations (500 MLH1, 648 MSH2, 117 MSH6 and 59 PMS2) and 1219 non-carriers from the Colon Cancer Family Registry. RESULTS: During 122,304 person-years of observation, we observed diagnoses of CRC for 659 carriers (50%) and 36 non-carriers (3%). For carriers, the risk of CRC increased by 30% for each 5 kg m(-2) increment in BMI in early adulthood (hazard ratio, HR: 1.30; 95% confidence interval, CI: 1.08-1.58; P=0.01), and increased by 64% for non-carriers (HR: 1.64; 95% CI: 1.02-2.64; P=0.04) after adjusting for sex, country, cigarette smoking and alcohol drinking (and the MMR gene that was mutated in carriers). The difference in HRs for carriers and non-carriers was not statistically significant (P=0.50). For MLH1 and PMS2 (MutLα heterodimer) mutation carriers combined, the corresponding increase was 36% (HR: 1.36; 95% CI: 1.05-1.76; P=0.02). For MSH2 and MSH6 (MutSα heterodimer) mutation carriers combined, the HR was 1.26 (95% CI: 0.96-1.65; P=0.09). There was no significant difference between the HRs for MutLα and MutSα heterodimer carriers (P=0.56). CONCLUSION: Body mass index in early adulthood is positively associated with risk of CRC for MMR gene mutation carriers and non-carriers

Body mass index in early adulthood and colorectal cancer risk for carriers and non-carriers of germline mutations in DNA mismatch repair genes

BACKGROUND: Carriers of germline mutations in DNA mismatch repair (MMR) genes have a high risk of colorectal cancer (CRC), but the modifiers of this risk are not well established. We estimated an association between body mass index (BMI) in early adulthood and subsequent risk of CRC for carriers and, as a comparison, estimated the association for non-carriers. METHODS: A weighted Cox regression was used to analyse height and weight at 20 years reported by 1324 carriers of MMR gene mutations (500 MLH1, 648 MSH2, 117 MSH6 and 59 PMS2) and 1219 non-carriers from the Colon Cancer Family Registry. RESULTS: During 122,304 person-years of observation, we observed diagnoses of CRC for 659 carriers (50%) and 36 non-carriers (3%). For carriers, the risk of CRC increased by 30% for each 5 kg m(-2) increment in BMI in early adulthood (hazard ratio, HR: 1.30; 95% confidence interval, CI: 1.08-1.58; P=0.01), and increased by 64% for non-carriers (HR: 1.64; 95% CI: 1.02-2.64; P=0.04) after adjusting for sex, country, cigarette smoking and alcohol drinking (and the MMR gene that was mutated in carriers). The difference in HRs for carriers and non-carriers was not statistically significant (P=0.50). For MLH1 and PMS2 (MutLα heterodimer) mutation carriers combined, the corresponding increase was 36% (HR: 1.36; 95% CI: 1.05-1.76; P=0.02). For MSH2 and MSH6 (MutSα heterodimer) mutation carriers combined, the HR was 1.26 (95% CI: 0.96-1.65; P=0.09). There was no significant difference between the HRs for MutLα and MutSα heterodimer carriers (P=0.56). CONCLUSION: Body mass index in early adulthood is positively associated with risk of CRC for MMR gene mutation carriers and non-carriers