Mitotic abnormalities precede microsatellite instability in lynch syndrome-associated colorectal tumourigenesis.

BACKGROUND: Lynch syndrome (LS) is one of the most common hereditary cancer syndromes worldwide. Dominantly inherited mutation in one of four DNA mismatch repair genes combined with somatic events leads to mismatch repair deficiency and microsatellite instability (MSI) in tumours. Due to a high lifetime risk of cancer, regular surveillance plays a key role in cancer prevention; yet the observation of frequent interval cancers points to insufficient cancer prevention by colonoscopy-based methods alone. This study aimed to identify precancerous functional changes in colonic mucosa that could facilitate the monitoring and prevention of cancer development in LS. METHODS: The study material comprised colon biopsy specimens (n = 71) collected during colonoscopy examinations from LS carriers (tumour-free, or diagnosed with adenoma, or diagnosed with carcinoma) and a control group, which included sporadic cases without LS or neoplasia. The majority (80%) of LS carriers had an inherited genetic MLH1 mutation. The remaining 20% included MSH2 mutation carriers (13%) and MSH6 mutation carriers (7%). The transcriptomes were first analysed with RNA-sequencing and followed up with Gorilla Ontology analysis and Reactome Knowledgebase and Ingenuity Pathway Analyses to detect functional changes that might be associated with the initiation of the neoplastic process in LS individuals. FINDINGS: With pathway and gene ontology analyses combined with measurement of mitotic perimeters from colonic mucosa and tumours, we found an increased tendency to chromosomal instability (CIN), already present in macroscopically normal LS mucosa. Our results suggest that CIN is an earlier aberration than MSI and may be the initial cancer driving aberration, whereas MSI accelerates tumour formation. Furthermore, our results suggest that MLH1 deficiency plays a significant role in the development of CIN. INTERPRETATION: The results validate our previous findings from mice and highlight early mitotic abnormalities as an important contributor and precancerous marker of colorectal tumourigenesis in LS. FUNDING: This work was supported by grants from the Jane and Aatos Erkko Foundation, the Academy of Finland (330606 and 331284), Cancer Foundation Finland sr, and the Sigrid Jusélius Foundation. Open access is funded by Helsinki University Library.

Epigenome-450K-wide methylation signatures of active cigarette smoking: The Young Finns Study.

Smoking as a major risk factor for morbidity affects numerous regulatory systems of the human body including DNA methylation. Most of the previous studies with genome-wide methylation data are based on conventional association analysis and earliest threshold-based gene set analysis that lacks sensitivity to be able to reveal all the relevant effects of smoking. The aim of the present study was to investigate the impact of active smoking on DNA methylation at three biological levels: 5'-C-phosphate-G-3' (CpG) sites, genes and functionally related genes (gene sets). Gene set analysis was done with mGSZ, a modern threshold-free method previously developed by us that utilizes all the genes in the experiment and their differential methylation scores. Application of such method in DNA methylation study is novel. Epigenome-wide methylation levels were profiled from Young Finns Study (YFS) participants' whole blood from 2011 follow-up using Illumina Infinium HumanMethylation450 BeadChips. We identified three novel smoking related CpG sites and replicated 57 of the previously identified ones. We found that smoking is associated with hypomethylation in shore (genomic regions 0-2 kilobases from CpG island). We identified smoking related methylation changes in 13 gene sets with false discovery rate (FDR) ≤ 0.05, among which is olfactory receptor activity, the flagship novel finding of the present study. Overall, we extended the current knowledge by identifying: (i) three novel smoking related CpG sites, (ii) similar effects as aging on average methylation in shore, and (iii) a novel finding that olfactory receptor activity pathway responds to tobacco smoke and toxin exposure through epigenetic mechanisms.

Mlh1 deficiency in normal mouse colon mucosa associates with chromosomally unstable colon cancer.

Colorectal cancer (CRC) genome is unstable and different types of instabilities, such as chromosomal instability (CIN) and microsatellite instability (MSI) are thought to reflect distinct cancer initiating mechanisms. Although 85% of sporadic CRC reveal CIN, 15% reveal mismatch repair (MMR) malfunction and MSI, the hallmarks of Lynch syndrome with inherited heterozygous germline mutations in MMR genes. Our study was designed to comprehensively follow genome-wide expression changes and their implications during colon tumorigenesis. We conducted a long-term feeding experiment in the mouse to address expression changes arising in histologically normal colonic mucosa as putative cancer preceding events, and the effect of inherited predisposition (Mlh1+/-) and Western-style diet (WD) on those. During the 21-month experiment, carcinomas developed mainly in WD-fed mice and were evenly distributed between genotypes. Unexpectedly, the heterozygote (B6.129-Mlh1tm1Rak) mice did not show MSI in their CRCs. Instead, both wildtype and heterozygote CRC mice showed a distinct mRNA expression profile and shortage of several chromosomal segregation gene-specific transcripts (Mlh1, Bub1, Mis18a, Tpx2, Rad9a, Pms2, Cenpe, Ncapd3, Odf2 and Dclre1b) in their colon mucosa, as well as an increased mitotic activity and abundant numbers of unbalanced/atypical mitoses in tumours. Our genome-wide expression profiling experiment demonstrates that cancer preceding changes are already seen in histologically normal colon mucosa and that decreased expressions of Mlh1 and other chromosomal segregation genes may form a field-defect in mucosa, which trigger MMR-proficient, chromosomally unstable CRC.

Gene set analysis: limitations in popular existing methods and proposed improvements.

MOTIVATION: Gene set analysis is the analysis of a set of genes that collectively contribute to a biological process. Most popular gene set analysis methods are based on empirical P-value that requires large number of permutations. Despite numerous gene set analysis methods developed in the past decade, the most popular methods still suffer from serious limitations. RESULTS: We present a gene set analysis method (mGSZ) based on Gene Set Z-scoring function (GSZ) and asymptotic P-values. Asymptotic P-value calculation requires fewer permutations, and thus speeds up the gene set analysis process. We compare the GSZ-scoring function with seven popular gene set scoring functions and show that GSZ stands out as the best scoring function. In addition, we show improved performance of the GSA method when the max-mean statistics is replaced by the GSZ scoring function. We demonstrate the importance of both gene and sample permutations by showing the consequences in the absence of one or the other. A comparison of asymptotic and empirical methods of P-value estimation demonstrates a clear advantage of asymptotic P-value over empirical P-value. We show that mGSZ outperforms the state-of-the-art methods based on two different evaluations. We compared mGSZ results with permutation and rotation tests and show that rotation does not improve our asymptotic P-values. We also propose well-known asymptotic distribution models for three of the compared methods. AVAILABILITY AND IMPLEMENTATION: mGSZ is available as R package from cran.r-project.org.

Mismatch repair analysis of inherited MSH2 and/or MSH6 variation pairs found in cancer patients.

Mismatch repair (MMR) malfunction causes the accumulation of mismatches in the genome leading to genomic instability and cancer. The inactivation of an MMR gene (MSH2, MSH6, MLH1, or PMS2) with an inherited mutation causes Lynch syndrome (LS), a dominant susceptibility to cancer. MMR gene variants of uncertain significance (VUS) may be pathogenic mutations, which cause LS, may result in moderately increased cancer risks, or may be harmless polymorphisms. Our study suggests that an inherited MMR VUS individually assessed as proficient may, however, in a pair with another MMR VUS found in the same colorectal cancer (CRC) patient have a concomitant contribution to the MMR deficiency. Here, eight pairs of MMR gene variants found in cancer patients were functionally analyzed in an in vitro MMR assay. Although the other pairs do not suggest a compound deficiency, the MSH2 VUS pair c.380A>G/c.982G>C (p.Asn127Ser/p.Ala328Pro), which nearly halves the repair capability of the wild-type MSH2 protein, is presumed to increase the cancer risk considerably. Moreover, two MSH6 variants, c.1304T>C (p.Leu435Pro) and c.1754T>C (p.Leu585Pro), were shown to be MMR deficient. The role of one of the most frequently reported MMR gene VUS, MSH2 c.380A>G (p.Asn127Ser), is especially interesting because its concomitant defect with another variant could finally explain its recurrent occurrence in CRC patients.

Adhesive polypeptides of Staphylococcus aureus identified using a novel secretion library technique in Escherichia coli.

BACKGROUND: Bacterial adhesive proteins, called adhesins, are frequently the decisive factor in initiation of a bacterial infection. Characterization of such molecules is crucial for the understanding of bacterial pathogenesis, design of vaccines and development of antibacterial drugs. Because adhesins are frequently difficult to express, their characterization has often been hampered. Alternative expression methods developed for the analysis of adhesins, e.g. surface display techniques, suffer from various drawbacks and reports on high-level extracellular secretion of heterologous proteins in Gram-negative bacteria are scarce. These expression techniques are currently a field of active research. The purpose of the current study was to construct a convenient, new technique for identification of unknown bacterial adhesive polypeptides directly from the growth medium of the Escherichia coli host and to identify novel proteinaceous adhesins of the model organism Staphylococcus aureus. RESULTS: Randomly fragmented chromosomal DNA of S. aureus was cloned into a unique restriction site of our expression vector, which facilitates secretion of foreign FLAG-tagged polypeptides into the growth medium of E. coli ΔfliCΔfliD, to generate a library of 1663 clones expressing FLAG-tagged polypeptides. Sequence and bioinformatics analyses showed that in our example, the library covered approximately 32% of the S. aureus proteome. Polypeptides from the growth medium of the library clones were screened for binding to a selection of S. aureus target molecules and adhesive fragments of known staphylococcal adhesins (e.g coagulase and fibronectin-binding protein A) as well as polypeptides of novel function (e.g. a universal stress protein and phosphoribosylamino-imidazole carboxylase ATPase subunit) were detected. The results were further validated using purified His-tagged recombinant proteins of the corresponding fragments in enzyme-linked immunoassay and surface plasmon resonance analysis. CONCLUSIONS: A new technique for identification of unknown bacterial adhesive polypeptides was constructed. Application of the method on S. aureus allowed us to identify three known adhesins and in addition, five new polypeptides binding to human plasma and extracellular matrix proteins. The method, here used on S. aureus, is convenient due to the use of soluble proteins from the growth medium and can in principle be applied to any bacterial species of interest.