Veronika Vymetalkova

Polymorphisms in microRNA genes as predictors of clinical outcomes in colorectal cancer patients

Colorectal cancer (CRC) is one of the most frequently diagnosed malignancies worldwide. It is routinely cured by a 5-fluorouracil (5-FU)-based chemotherapy which improves outcomes in patients. We investigated the effect of single nucleotide polymorphisms (SNPs) in two microRNA (miRNA)-encoding genes that have been previously reported as important in prognosis in patients with stage III CRC and treated with 5-FU-based chemotherapy. Two SNPs (rs4919510 in miR-608 and rs213210 in miR-219-1) were genotyped in 1083 CRC patients recruited in the Czech Republic to evaluate their effect on clinical outcomes. Carriers of the variant T allele in rs213210 and receiving 5-FU chemotherapy were associated with a significantly worse survival [hazard ratio (HR) = 2.18; 95% confidence interval (CI): 1.20-3.98; adjusted P = 0.01] and an increased risk of relapse (HR = 1.94; 95% CI: 1.16-3.25; adjusted P = 0.01). After further stratification for tumor grading, stage III patients carrying the G allele of rs4919510 and undergoing adjuvant chemotherapy were at decreased risk of relapse (HR = 0.44; 95% CI: 0.20-0.94; adjusted P = 0.03). The present study confirms that variations in miRNA-encoding genes may be an important factor for modulating CRC prognosis and predicting therapy response.

Metabolic gene variants associated with chromosomal aberrations in healthy humans.

Nonspecific chromosomal aberrations (CAs) are found in about 1% of lymphocytes drawn from healthy individuals. They include chromosome‐type aberrations (CSAs), which are increased in exposure to ionizing radiation, and chromatid‐type aberrations (CTAs) which in experimental systems are formed by DNA binding carcinogens and mutagens. The frequency of CAs is associated with the risk of cancer, but the causes of CAs in general population are unknown. Here, we want to test whether variants in metabolic genes associate with CAs in healthy volunteers. Cases were considered those whose total CA (CAtot) frequency was >2% and for CSA and CTA the limit was >1%. Controls had lower frequencies of CAs. Functional polymorphisms in seven genes were selected for analysis: cytochrome P450 1B1 (CYP1B1), epoxide hydrolase 1 (EPHX1), NAD(P)H:quinone oxidoreductase 1 (NQO1), each coding for phase 1 enzymes, and glutathione S‐transferase P1 (GSTP1), glutathione S‐transferases M1 (GSTM1) and T1 (GSTT1), coding for enzymes which conjugate reactive metabolites, that is, phase 2 enzymes. The number of volunteers genotyped for each gene varied from 550 to 1,500. Only EPHX1 was individually associated with CAtot; high activity genotypes decreased CAtot. A total of six significant (P < 0.01) pair‐wise interactions were observed, most including a GST variant as one of the pair. In all genotype combinations with significant odds ratios for CAs a GST variant was involved. The present data provide evidence that variants in genes coding for metabolic enzymes, which individually have small effects, interact and are associated with CA frequencies in peripheral lymphocytes of healthy volunteers.

Double-strand break repair and colorectal cancer: gene variants within 3′ UTRs and microRNAs binding as modulators of cancer risk and clinical outcome

Genetic variations in 3′ untranslated regions of target genes may affect microRNA binding, resulting in differential protein expression. microRNAs regulate DNA repair, and single-nucleotide polymorphisms in miRNA binding sites (miRSNPs) may account for interindividual differences in the DNA repair capacity. Our hypothesis is that miRSNPs in relevant DNA repair genes may ultimately affect cancer susceptibility and impact prognosis. In the present study, we analysed the association of polymorphisms in predicted microRNA target sites of double-strand breaks (DSBs) repair genes with colorectal cancer (CRC) risk and clinical outcome. Twenty-one miRSNPs in non-homologous end-joining and homologous recombination pathways were assessed in 1111 cases and 1469 controls. The variant CC genotype of rs2155209 in MRE11A was strongly associated with decreased cancer risk when compared with the other genotypes (OR 0.54, 95% CI 0.38–0.76, p = 0.0004). A reduced expression of the reporter gene was observed for the C allele of this polymorphism by in vitro assay, suggesting a more efficient interaction with potentially binding miRNAs. In colon cancer patients, the rs2155209 CC genotype was associated with shorter survival while the TT genotype of RAD52 rs11226 with longer survival when both compared with their respective more frequent genotypes (HR 1.63, 95% CI 1.06-2.51, p = 0.03 HR 0.60, 95% CI 0.41–0.89, p = 0.01, respectively). miRSNPs in DSB repair genes involved in the maintenance of genomic stability may have a role on CRC susceptibility and clinical outcome.

Single Nucleotide Polymorphisms within Interferon Signaling Pathway Genes Are Associated with Colorectal Cancer Susceptibility and Survival

Interferon (IFN) signaling has been suggested to play an important role in colorectal carcinogenesis. Our study aimed to examine potentially functional genetic variants in interferon regulatory factor 3 (IRF3), IRF5, IRF7, type I and type II IFN and their receptor genes with respect to colorectal cancer (CRC) risk and clinical outcome. Altogether 74 single nucleotide polymorphisms (SNPs) were covered by the 34 SNPs genotyped in a hospital-based case-control study of 1327 CRC cases and 758 healthy controls from the Czech Republic. We also analyzed these SNPs in relation to overall survival and event-free survival in a subgroup of 483 patients. Seven SNPs in IFNA1, IFNA13, IFNA21, IFNK, IFNAR1 and IFNGR1 were associated with CRC risk. After multiple testing correction, the associations with the SNPs rs2856968 (IFNAR1) and rs2234711 (IFNGR1) remained formally significant (Pu200a=u200a0.0015 and P<0.0001, respectively). Multivariable survival analyses showed that the SNP rs6475526 (IFNA7/IFNA14) was associated with overall survival of the patients (Pu200a=u200a0.041 and event-free survival among patients without distant metastasis at the time of diagnosis, Pu200a=u200a0.034). The hazard ratios (HRs) for rs6475526 remained statistically significant even after adjustment for age, gender, grade and stage (Pu200a=u200a0.029 and Pu200a=u200a0.036, respectively), suggesting that rs6475526 is an independent prognostic marker for CRC. Our data suggest that genetic variation in the IFN signaling pathway genes may play a role in the etiology and survival of CRC and further studies are warranted.

Variations in mismatch repair genes and colorectal cancer risk and clinical outcome

DNA mismatch repair (MMR) deficiency is one of the best understood forms of genetic instability in colorectal cancer (CRC). CRC is routinely cured by 5-fluorouracil (5-FU)-based chemotherapy, with a prognostic effect and resistance to such therapy conferred by MMR status. In this study, we aimed to analyse the effect of genetic variants in classical coding regions or in less-explored predicted microRNA (miRNA)-binding sites in the 3 untranslated region (3UTR) of MMR genes on the risk of CRC, prognosis and the efficacy of 5-FU therapy. Four single nucleotide polymorphisms (SNPs) in MMR genes were initially tested for susceptibility to CRC in a case-control study (1095 cases and 1469 healthy controls). Subsequently, the same SNPs were analysed for their role in survival on a subset of patients with complete follow-up. Two SNPs in MLH3 and MSH6 were associated with clinical outcome. Among cases with colon and sigmoideum cancer, carriers of the CC genotype of rs108621 in the 3UTR of MLH3 showed a significantly increased survival compared to those with the CT + TT genotype (log-rank test, P = 0.05). Moreover, this polymorphism was also associated with an increased risk of relapse or metastasis in patients with heterozygous genotype (log-rank test, P = 0.03). Patients carrying the CC genotype for MSH6 rs1800935 (D180D) and not undergoing 5-FU-based chemotherapy showed a decreased number of recurrences (log-rank test, P = 0.03). No association with CRC risk was observed. We provide the first evidence that variations in potential miRNA target-binding sites in the 3UTR of MMR genes may contribute to modulate CRC prognosis and predictivity of therapy.

Interactions of DNA repair gene variants modulate chromosomal aberrations in healthy subjects

Human cancers are often associated with numerical and structural chromosomal instability. Structural chromosomal aberrations (CAs) in peripheral blood lymphocytes (PBL) arise as consequences of direct DNA damage or due to replication on a damaged DNA template. In both cases, DNA repair is critical and inter-individual differences in its capacity are probably due to corresponding genetic variations. We investigated functional variants in DNA repair genes (base and nucleotide excision repair, double-strand break repair) in relation to CAs, chromatid-type aberrations (CTAs) and chromosome-type aberrations (CSAs) in healthy individuals. Chromosomal damage was determined by conventional cytogenetic analysis. The genotyping was performed by both restriction fragment length polymorphism and TaqMan allelic discrimination assays. Multivariate logistic regression was applied for testing individual factors on CAs, CTAs and CSAs. Pair-wise genotype interactions of 11 genes were constructed for all possible pairs of single-nucleotide polymorphisms. Analysed individually, we observed significantly lower CTA frequencies in association with XPD Lys751Gln homozygous variant genotype [odds ratio (OR) 0.64, 95% confidence interval (CI) 0.48-0.85, P = 0.004; n = 1777]. A significant association of heterozygous variant genotype in RAD54L with increased CSA frequency (OR 1.96, 95% CI 1.01-4.02, P = 0.03) was determined in 282 subjects with available genotype. By addressing gene-gene interactions, we discovered 14 interactions significantly modulating CAs, 9 CTAs and 12 CSAs frequencies. Highly significant interactions included always pairs from two different pathways. Although individual variants in genes encoding DNA repair proteins modulate CAs only modestly, several gene-gene interactions in DNA repair genes evinced either enhanced or decreased CA frequencies suggesting that CAs accumulation requires complex interplay between different DNA repair pathways.

Cyclin D1 splice site variant triggers chromosomal aberrations in healthy humans.

Chromosomal aberrations (CAs) were suggested to be the cause of cancer by Boveri already a 100 years ago, but the detection of the Philadelphia chromosome about 50 years later provided the first direct evidence on the relationship (reviewed by Mitelman). CAs include recurrent aberrations, including specific translocations, deletions and amplifications of chromosomal regions or whole chromosomes. The detection of recurrent CAs was greatly facilitated when fluorescence in situ hybridization techniques were developed for interphase chromosomes. Non-specific CAs include missing, fragmented or fused chromosomal segments, divided into chromosome-type aberrations (CSAs) and chromatidtype aberrations (CTAs). These have been conventionally analyzed by microscopic scoring of metaphase nuclei from cultured lymphocytes. Modern sequencing techniques have revealed numerous gene fusions and rearrangements, in the extreme, chromothripsis, a catastrophic parsing of illegitimate chromosomal segments together. Non-specific CAs may arise as a result of direct DNA damage by, for example, ionizing radiation or replication on a damaged DNA template; the former lesions would be detected as CSAs, whereas the latter may be CSAs or CTAs. Non-specific CAs remain in lymphocytes for their lifetime. They have been used in monitoring of radiation exposure and exposure to genotoxic compounds and, together with sister chromatid exchanges and micronuclei, CAs have offered the only available method for human biomonitoring for genotoxic exposures. There are convincing data on the association of CA frequency with subsequent risk of many cancers. Furthermore, patients with many types of cancer show increased frequencies of CAs at diagnosis. CAs were assumed to be somatic events but our recent study on multiple myeloma showed that the recurrent t(11;14) (q13;q32) translocation was strongly associated with the cyclin D1 (CCND1) G870A genotype at a splice site. However, no effect was found for any other translocations or other CAs in myeloma. Here, we wanted to test the possible dependence of non-specific CAs on the CCND1 genotype by assaying for the G870A genotype in a large cohort of persons for whom CAs were measured. This polymorphism has been implicated as a risk factor for a number of cancers. The subjects (N1⁄4 731) were identified from occupational settings, including persons with defined exposures (553), and hospital staff without exposures (178) from Slovakia. Occupational exposures included small organic compounds (330), cytostatics (81), anesthetics (74) and metals (68). The median age was 40 years (range 19 to 71), whereas the mean was 40.6 years (s.d. 10.3); characteristics of the population are shown in Table 1. The sampling of peripheral blood was carried out according to the Helsinki Declaration and the study design was approved by the local Ethical Committee of the Jessenius Medical Faculty. The participants provided an informed consent. Cytogenetic analysis was performed on cultured lymphocytes, as previously described, by microscopically analyzing (two microscopists) in a double-blind fashion coded slides of 100 mitoses per person for the frequency of total CAs, CTAs and CSAs. Inter-arm and intra-arm interchanges of CTAs and CSAs (including dicentrics and centric rings) were also scored. In statistical analysis, non-parametric Mann–Whitney U-test was used for testing of differences between the groups. The CAs were categorized into a high-frequency group (42%) and low-frequency group (p2%); this arbitrary cutoff point was based on our previous experience. For CTA and CSA, the cutoff was 1%. Odds ratios (ORs) from multivariable linear regression analysis were employed to consider simultaneous effects of particular occupational exposures, age, gender and smoking habits on the frequencies of CAs, CTAs and CSAs. Genotyping of G870A (rs603965, also known as rs9344) was carried out by the PCR-restriction-fragment length polymorphism method (primer F 50-GTGAAGTTCATTTCCAATCCGC-30, primer R 50-GGGACATCACCCTCACTTAC-30). The amplified fragment was digested with the MspI restriction endonuclease and the digested PCR products were resolved on 3% agarose gels containing ethidium bromide for visualization under UV light. The quality control was performed by random re-genotyping of about 10% of sample. Occupational exposure information was lacking for six individuals. Genotyping of one idividual was not successful, thus 730 persons were successfully genotyped. The results for total CAs showed significant effects of occupational exposure (OR 1.68) and CCND1 AA genotype (OR 1.85, Table 1). In the separate analysis of CTAs and CSAs, the only significant effect of OR 1.99 (P1⁄4 0.003) was on CSAs (Table 2). Our recent study on multiple myeloma showed that the t(11;14) translocation was associated with the CCND1 GG genotype with an OR 1.95 (P1⁄4 2.07 10 ) (Weinhold et al.). The present results showed that AA genotype was associated with overall lymphocyte CAs and, specifically, CSAs in healthy individuals. The opposite genotype effects are likely to be explained by the cell type in question. In the earlier study, we found no genotype effect in mantle cell lymphoma, for which the t(11;14) translocation is a hallmark, most likely because in mantle cell lymphoma the translocations are formed during VDJ recombination when antigen receptor genes are rearranged, earlier than in multiple myeloma. Myeloma is thought to arise from post-germinal center memory B-cells in which immunoglobulin H (IgH) translocations take place during germinal center maturation when somatic hypermutation and class switch recombination take place. Both VDJ and class switch recombination involve complex doublestranded DNA repair events. VDJ recombination is initiated by recombination-activating genes, whereas the germinal center events are catalyzed by activation-induced cytidine deaminase, followed by classical non-homologous DNA end-joining and alternative end-joining repair activities. It is thus likely that the

Polymorphisms in Non-coding RNA Genes and Their Targets Sites as Risk Factors of Sporadic Colorectal Cancer

Colorectal cancer (CRC) is a complex disease that develops as a consequence of both genetic and environmental risk factors in interplay with epigenetic mechanisms, such as microRNAs (miRNAs). CRC cases are predominantly sporadic in which the disease develops with no apparent hereditary syndrome. The last decade has seen the progress of genome-wide association studies (GWAS) that allowed the discovery of several genetic regions and variants associated with weak effects on sporadic CRC. Collectively these variants may enable a more accurate prediction of an individuals risk to the disease and its prognosis. However, the number of variants contributing to CRC is still not fully explored.SNPs in genes encoding the miRNA sequence or in 3UTR regions of the corresponding binding sites may affect miRNA transcription, miRNA processing, and/or the fidelity of the miRNA-mRNA interaction. These variants could plausibly impact miRNA expression and target mRNA translation into proteins critical for cellular integrity, differentiation, and proliferation.In the present chapter, we describe the different aspects of variations related to miRNAs and other non-coding RNAs (ncRNAs) and evidence from studies investigating these candidate genetic alterations in support to their role in CRC development and progression.

DNA and chromosomal damage in medical workers exposed to anaesthetic gases assessed by the lymphocyte cytokinesis-block micronucleus (CBMN) assay. A critical review

The lymphocyte cytokinesis-block micronucleus (CBMN) assay has been applied in hundreds of in vivo biomonitoring studies of humans exposed either environmentally or occupationally to genotoxic chemicals. However, there is an emerging need to re-evaluate the use of MN and other biomarkers within the lymphocyte CBMN cytome assay as quantitative indicators of exposure to main classes of chemical genotoxins. The main aim of the present report is to systematically review published studies investigating the use of the lymphocyte CBMN assay to determine DNA damage in subjects exposed to anaesthetic gases. We also compared performance of the CBMN assay with other DNA damage assays employed and identified strengths and weaknesses of the published studies. We have retrieved 11 studies, published between 1996 and 2013, reporting MN associated with occupational exposures (operating room personnel). The individual job categories were often described (anaesthesiologists, technicians, radiologists) among cases, as well as duration of exposure. All studies reported the compounds present at the workplace and, in some instances, the exposure levels were measured. Controls were usually recruited among personnel at the hospital not exposed to anaesthetics or they were healthy unexposed subjects from general population. The number of investigated subjects, due to the character of the occupation, was relatively smaller than those investigated in other occupational monitoring settings. Overall, the majority of the studies were age- and gender- matched (or investigated only males or females) while less attention was given to lifestyle confounders. Appropriate measurement of exposure, available in approximately half of the studies only, was compromised by the lack of the personal dosimetry-based determinations. In all studies, higher MN frequencies were observed in exposed individuals. The meta-analysis of mean MN frequency of combined studies confirmed this tendency (log mean ratio=0.56 [0.34-0.77]; P=3.51×10-7). Similar differences between the exposed and controls were also observed for other biomarkers.

Epigenome-wide analysis of DNA methylation reveals a rectal cancer-specific epigenomic signature

AIMnThe aim of the present study is to address a genome-wide search for novel methylation biomarkers in the rectal cancer (RC), as only scarce information on methylation profile is available.nnnMATERIALS & METHODSnWe analyzed methylation status in 25 pairs of RC and adjacent healthy mucosa using the Illumina Human Methylation 450 BeadChip.nnnRESULTSnWe found significantly aberrant methylation in 33 genes. After validation of our results by pyrosequencing, we found a good agreement with our findings. The BPIL3 and HBBP1 genes resulted hypomethylated in RC, whereas TIFPI2, ADHFE1, FLI1 and TLX1 were hypermethylated. An external validation by TCGA datasets confirmed the results.nnnCONCLUSIONnOur study, with external validation, has demonstrated the feasibility of using specific methylated DNA signatures for developing biomarkers in RC.