Association between 3'UTR polymorphisms in genes ACVR2A, AGTR1 and RGS2 and preeclampsia.

Preeclampsia (PE) is a pregnancy specific disease with several risk factors such as genetic polymorphisms, environmental and social factors participating in its development. The aim of this study was to investigate whether distribution of three putative regulatory SNPs rs13430086, rs5186, rs4606 in 3'UTR of genes ACVR2A, AGTR1 and RGS2, respectively, that have been associated with hypertension and regulation of trophoblast invasion differ between women with PE and control group. The associations of rs13430086, rs5186 and rs4606 with preeclampsia were tested in two groups - the group of 50 women with PE and the control group of 42 healthy pregnant women at term. DNA was isolated from blood samples and the determination of genotypes was performed using Real-Time PCR. Power analysis for the size of the cohort was performed and the results were analyzed using Fisher exact test. The AA genotype of ACVR2A rs13430086 was significantly associated with higher risk to preeclampsia compared with TT genotype (p = 0.026, OR: 5.39, 95%CI: 1.21-31.54). Results showed no association between genotypes and preeclampsia for polymorphisms rs5186, rs4606. Further studies are important in order to better understand the role of ACVR2A in the pathogenesis of PE.

Active cigarette smoking and the risk of breast cancer at the level of N-acetyltransferase 2 (NAT2) gene polymorphisms.

The aim of our study was to assess the correlation between the tobacco exposure and NAT2 gene (rs1041983 C/T, rs1801280 T/C, rs1799930 G/A) polymorphisms in association with breast cancer development. We wanted to determine the prognostic clinical importance of these polymorphisms in association with smoking and breast cancer. For the detection of possible association between smoking, NAT2 gene polymorphisms, and the risk of breast cancer, we designed a case-controlled study with 198 patients enrolled, 98 breast cancer patients and 100 healthy controls. Ten milliliters of peripheral blood from the cubital vein was withdrawn from every patient. The HRM (high resolution melting) analysis was used for the detection of three abovementioned NAT2 gene polymorphisms. When comparing a group of women smoking more than 5 cigarettes a day with the patients smoking fewer than 5 cigarettes a day, we found out that if women were the carriers of aberrant AA genotype for rs1799930, the first group of women had higher risk of breast carcinoma than the second group. If patients were the carriers of aberrant TT genotype for rs1041983, for rs1801280CC genotype, and rs1799930AA genotype and they smoked more than 5 cigarettes a day, they had higher risk of malignant breast disease than never-smoking women. Our results confirm the hypothesis that NAT2 gene polymorphisms (rs1041983 C/T, rs1801280 T/C, and rs1799930 G/A) in association with long-period active smoking could be the possible individual risk-predicting factors for breast cancer development in the population of Slovak women.

Methylation in promoter regions of PITX2 and RASSF1A genes in association with clinicopathological features in breast cancer patients.

Breast cancer is a heterogeneous disease with very different responses to therapy and different length of survival. In many cases, however, the determination of the stage and histopathological characteristics of breast cancer is insufficient to predict prognosis and response to treatment for the vast heterogeneity of the disease. To understand the molecular signature of subtypes of breast cancer, we attempted to identify the methylation status of key tumour suppressor gene Ras association (RalGDS/AF-6) domain family member 1 isoform a (RASSF1A) and a member of the paired-like homeodomain transcription factor family which functions in left-right asymmetry development (PITX2) and to correlate results with known clinicopathological features of breast cancer. Formalin-fixed, paraffin-embedded (FFPE) tissues of breast carcinomas (n = 149) were used for DNA extraction. DNA was modified by bisulphite conversion. Detection of the methylation level of the genes mentioned above was performed by methylation-sensitive high-resolution melting assay (MS-HRM). Based on MS-HRM results for RASSF1A and PITX2, we subdivided the samples into four groups according to methylation level (≤50 % methylated, >50 % methylated, 100 % methylated and completely unmethylated alleles). All degrees of methylation status for both genes underwent analysis of dependence with known clinicopathological features, and we found significant associations. In 134 of 149 (89.9 %) primary breast carcinomas, the RASSF1A promoter was methylated. Total hypermethylation of PITX2 was observed in 60 of 135 (44.4 %) breast cancer cases. RASSF1A hypermethylation had significant association with increased age (p < 0.05), tumour grade (p < 0.0001) and stage (p < 0.0001) in the 100 % methylated group. There was significant association of PITX2 hypermethylation with tumour grade (p < 0.0001) and stage (p < 0.0001). Association between the methylation level of both investigated genes and tumour type was significant for ductal invasive carcinoma cases only (p < 0.0001). This study shows different levels of heterogeneous methylation acquired by MS-HRM assay of the promoter region of RASSF1A and PITX2 and its relationship with clinicopathological features of 149 breast cancer patients. We noticed that immunohistopathological subtypes of breast cancer contain distinct promoter methylation patterns. All these data suggest that hypermethylation of the CpG island promoters of RASSF1A and PITX2 might play an essential role in the very early stages of breast cancer pathogenesis.

Gene expression abnormalities in histologically normal breast epithelium from patients with luminal type of breast cancer.

The gene expression profile of breast cancer has been described as a great breakthrough on the way to comprehend differences in cancer origin, behavior and therapy. However, gene expression profile in histologically normal epithelium (HNEpi) which could harbor genetic abnormalities predisposing breast tissue to develop malignancy was minor scope for scientists in the past. Thus, we aimed to analyze gene expressions in HNEpi and breast cancer tissue (BCTis) in order to establish its value as potential diagnostic marker for cancer development. We evaluated a panel of disease-specific genes in luminal type (A/B) of breast cancer and tumor surrounding HNEpi by qRT-PCR Array in 32 microdissected samples. There was 20.2 and 2.4% deregulation rate in genes with at least 2-fold or 5-fold over-expression between luminal (A/B) type breast carcinomas and tumor surrounding HNEpi, respectively. The high-grade luminal carcinomas showed higher number of deregulated genes compared to low-grade cases (50.6 vs. 23.8% with at least 2-fold deregulation rate). The main overexpressed genes in HNEpi were KLK5, SCGB1D2, GSN, EGFR and NGFR. The significant differences in gene expression between BCTis and HNEpi samples were revealed for BAG1, C3, CCNA2, CD44, FGF1, FOSL1, ID2, IL6R, NGFB, NGFR, PAPPA, PLAU, SERPINB5, THBS1 and TP53 gene (p < 0.05) and BCL2L2, CTSB, ITGB4, JUN, KIT, KLF5, SCGB1D2, SCGB2A1, SERPINE1 (p < 0.01), and EGFR, GABRP, GSN, MAP2K7 and THBS2 (p < 0.001), and GSN, KLK5 (p < 0.0001). The ontological gene analyses revealed high deregulations in gene group directly associated with breast cancer prognosis and origin.

mRNA expression in cervical specimens for determination of severe dysplasia or worse in HPV-16/18-positive squamous lesions.

OBJECTIVES: The objective of current study was to determine the p16 mRNA level in cervical cells by relative quantification (RQ) and to test viral E6 expression in human papillomavirus (HPV) -16 or -18-positive specimens by widely used methods. We targeted the pivotal mRNA level associated with severe dysplasia or worse. MATERIALS AND METHODS: Cervical specimens were taken from 134 women with cervical disease and 132 women with normal cytologic results. The presence of HPV was analyzed by sequencing. The results of p16 and E6 analyses were statistically processed in receiver operating characteristic curve analysis to predict severe dysplasia or worse. RESULTS: The HPV DNA was detected in 81.4% (109/134) of women with cervical disease and in 27.3% (36/132) of women with normal cytologic results. HPV-16 or -18 were present in 59.7% (80/134) of abnormal specimens. p16 and E6 mRNA expression was increasing with severity of cervical dysplasia. p16 mRNA expression was found 4.35-fold and 13.15-fold increased in high-grade squamous intraepithelial lesions and squamous cell carcinomas, respectively. E6 mRNA expression was significantly increased (p = .0038) in severe dysplasias or worse. The RQ method achieved better sensitivity (82.6%), and E6 mRNA got better specificity (80.6%) for the prediction of severe dysplasia or worse. CONCLUSIONS: An increasing level of p16 and E6 mRNA transcripts could mean the potential of cervical dysplasia progression to cancer, but further studies should be done to confirm this proposition. Nevertheless, we consider using both tests to improve the sensitivity and specificity for prediction of severe dysplasia or worse.

Antifungal Activities of Essential Oils in Vapor Phase against Botrytis cinerea and Their Potential to Control Postharvest Strawberry Gray Mold.

Essential oils (EOs) from aromatic plants seem to have the potential to control several fungal pathogens and food contaminants. Botrytis cinerea is the main strawberry fruit contaminant causing high losses during storage. Here, thirteen EOs applied in the vapor phase were evaluated for their potential to inhibit the growth of three different strains of B. cinerea isolated from strawberry fruits. Eight EOs (lemongrass, litsea, lavender, peppermint, mint, petitgrain, sage, and thyme) were able to completely inhibit the growth of B. cinerea for 7 days when applied at a concentration of 625 µL·L-1. Four EOs with the lowest minimal inhibition concentrations (thyme, peppermint, lemongrass, and litsea) have been tested on strawberry fruits intentionally inoculated by B. cinerea. All four EOs showed high inhibition at a concentration of 250 or 500 µL·L-1, but only peppermint EO was able to completely inhibit B. cinerea lesion development at a concentration of 125 µL·L-1. The sensory evaluation of strawberries treated by EOs at a concentration 125 µL·L-1 resulted in a statistically significant decrease in taste, aftertaste, aroma, and overall quality. Lemongrass and litsea EOs scored better than thyme and peppermint ones, thus forming two viable methods for B. cinerea suppression and the extension of packed strawberries' shelf life.

Blackcurrant (Ribes nigrum L.) and Kamchatka Honeysuckle (Lonicera caerulea var. Kamtschatica) Extract Effects on Technological Properties, Sensory Quality, and Lipid Oxidation of Raw-Cooked Meat Product (Frankfurters).

Oxidation is one of the most prevalent factors responsible for meat product deterioration. Due to their potential health risks, commonly used synthetic antioxidants are beginning to be frowned upon by customers. The industry is searching for a natural replacement. In our study, we incorporated blackcurrant (Ribes nigrum L.) and Kamchatka honeysuckle (Lonicera caerulea var. Kamtschatica) extracts into raw-cooked meat products (frankfurters) as natural antioxidants. We observed that both extracts at concentrations of 3 mL·kg-1 were able to significantly (α = 0.05) postpone lipid oxidation in our samples, with results comparable to vitamin C (0.5 mg·kg-1) addition. Moreover, we did not observe negative effects of the extracts on the product's color, pH, or textural properties. Negative results were reported in the sensory evaluation of honeysuckle addition samples. This could have been caused by the natural strong and bitter taste of honeysuckle, which was transferred to the extracts and, subsequently, into the meat product.

Association of Polymorphisms in CYBA, SOD1, and CAT Genes with Type 1 Diabetes and Diabetic Peripheral Neuropathy in Children and Adolescents.

AIMS: The aim of our study was to investigate possible associations between three SNPs: rs4673 in the CYBA gene; rs1041740 in the SOD1 gene; and rs1001179 in the CAT gene, and type 1 diabetes (T1D) or diabetic peripheral neuropathy (DPN) in T1D patients. MATERIALS AND METHODS: Allelic variants of the selected SNPs were determined by allelic discrimination assays in 114 T1D patients enrolled in the study group and in 90 healthy individuals from a control group. Associations between each of the three SNPs were tested in subgroups of T1D patients divided according to the presence of DPN. RESULTS: The TT genotype of rs4673 in the CYBA gene was associated with DPN in T1D patients (OR 4.997, 95% CI 1.403-19.083, p = 0.016). Weak significance was observed for a protective effect of the TT genotype of rs1041740 in the SOD1 gene relative to T1D development (OR 0.318, 95% CI 0.092-0.959, p = 0.056). There was no significant association between the CAT gene SNP rs1001179 and T1D or DPN. CONCLUSION: We showed a strong association of the CYBA polymorphism rs4673 with DPN in Slovak children and adolescents with T1D. Further studies are necessary to assess the relationship between rs1041740 and T1D or DPN.

Methylation pattern of CDH1 promoter and its association with CDH1 gene expression in cytological cervical specimens.

Cervical cancer is the fourth leading cause of cancer mortality in females worldwide. Infection with high-risk human papillomavirus (HPV) is essential but insufficient to cause cervical cancer, and the clearance of HPV infection is mediated by the immune system. The deficit of molecules responsible for adhesion may play a role in the development of cervical cancer. E-cadherin is encoded by the cadherin 1 (CDH1) gene, and is involved in cell adhesion by forming adherens junctions. The aim of present study was to investigate the methylation pattern of the CDH1 promoter and to identify the association between CDH1 promoter hypermethylation, CDH1 gene expression and HPV infection in cervical specimens obtained from 93 patients with low-grade squamous intraepithelial lesions (SILs), high-grade SILs or squamous cell carcinomas, and from 47 patients with normal cervical cytology (HPV-negative). The methylation pattern of the CDH1 promoter was investigated by methylation-specific polymerase chain reaction and quantitative pyrosequencing. CDH1 gene expression was measured by relative quantification. CDH1 methylation was significantly higher in both types of lesions and in cervical cancer than in normal samples, and CDH1 gene expression was significantly reduced during SIL progression (P=0.0162). However, the influence of HPV infection or HPV E6 expression on the methylation pattern of the CDH1 gene or its gene expression levels could not be confirmed. The present results support that the methylation of the CDH1 gene is age-related in patients with cervical lesions (P=0.01085), and therefore, older patients could be more susceptible to cancer than younger patients. The important methylation of the CDH1 promoter occurred near the transcription factor binding sites on nucleotides -13 and +103, which are close to the translational start codon. These results suggest that methylation at these sites may be an important event in the transcriptional regulation of E-cadherin, and in patients harboring these methylated cytosines, this event may facilitate HPV-driven carcinogenesis.

Gene expression profiling of histologically normal breast tissue in females with human epidermal growth factor receptor 2­positive breast cancer.

Gene expression profile­based taxonomy of breast cancer (BC) has been described as a significant breakthrough in comprehending the differences in the origin and behavior of cancer to allow individually tailored therapeutic approaches. In line with this, we hypothesized that the gene expression profile of histologically normal epithelium (HNEpi) could harbor certain genetic abnormalities predisposing breast tissue cells to develop human epidermal growth factor receptor 2 (HER2)­positive BC. Thus, the aim of the present study was to assess gene expression in normal and BC tissue (BCTis) from patients with BC in order to establish its value as a potential diagnostic marker for cancer development. An array study evaluating a panel of 84 pathway­ and disease­specific genes in HER2­positive BC and tumor­adjacent HNEpi was performed using quantitative polymerase chain reaction in 12 patients using microdissected samples from frozen tissue. Common prognostic and predictive parameters of BC were assessed by immunohistochemistry and in situ hybridization. In the BCTis and HNEpi samples of 12 HER2­positive subjects with BC, the expression of 2,016 genes was assessed. A total of 39.3% of genes were deregulated at a minimal two­fold deregulation rate and 10.7% at a five­fold deregulation rate in samples of HNEpi or BCTis. Significant differences in gene expression between BCTis and HNEpi samples were revealed for BCL2L2, CD44, CTSD, EGFR, ERBB2, ITGA6, NGFB, RPL27, SCBG2A1 and SCGB1D2 genes (P<0.05), as well as GSN, KIT, KLK5, SERPINB5 and STC2 genes (P<0.01). Insignificant differences (P<0.07) were observed for CCNA1, CLU, DLC1, GABRP and IL6 genes. The ontological gene analyses revealed that the majority of the deregulated genes in the HNEpi samples were part of the functional gene group directly associated with BC origin and prognosis. Functional analysis showed that the most frequent gene deregulations occurred in genes associated with apoptosis and cell cycle regulation in BCTis samples, and with angiogenesis, regulation of the cell cycle and transcriptional activity in HNEpi samples. The molecular profiling of HNEpi breast tissue revealed gene expression abnormalities that may represent potential markers of increased risk for HER2­positive malignant transformation of breast tissue, and may be able to be employed as predictors of prognosis.

Promoter hypermethylation of the tumor-suppressor genes RASSF1A, GSTP1 and CDH1 in endometrial cancer.

Endometrial cancer is a common gynecological malignancy with a good prognosis in early stages of the disease. The CpG island in the promoter region of tumor-suppressor genes are frequently methylated in various types of human cancers. In the present study, we examined the methylation status of the GSTP1, CDH1 and RASSF1A genes in endometrioid endometrial cancer (EEC), endometrial complex hyperplasia (EHP) and healthy endometrium with the aim to identify correlations between promoter hypermethylation, disease risk and clinicopathological parameters. A nested two-stage methylation-specific PCR (MSP) was performed to analyze the promoter CpG methylation status of GSTP1, CDH1 and RASSF1A genes in the population studied. A total of 92 subjects were initially included in the study of which 41 EEC, 19 EHP and 20 controls were processed for final analyses. A significant difference was found between the study groups and the presence of promoter CpG hypermethylation status in the GSTP1 (p<0.05) and RASSF1A (p<0.0001) genes. RASSF1A, GSTP1 and CDH1 gene promoter methylation was present in 85.4, 68.3 and 31.4% of EEC samples when compared to that in the controls with 30.0, 35.0 and 20.0%, respectively. CpG methylation of all three investigated tumor-suppressor genes was found in 12.2% of EEC patients, in 4.2% of EHP patients and in 3.7% of the controls, respectively. Positive findings for the promoter methylation of two investigated genes were found in 48.7% of EEC patients, 26.0% of EHP patients and in 18.5% of the controls. With regard to histopathological variables and CpG methylation, we found significant correlations between the RASSF1A and GSTP1 genes and higher tumor grade, deeper myometrial invasion and positive metastatic involvement of pelvic lymph nodes. No associations were noted between promoter hypermethylation of the CDH1 gene and biological features of the endometrial cancer cases. The results indicate that aberrant CpG methylation of the promoter region in the GSTP1 and RASSF1A tumor-suppressor genes is an important event in carcinogenesis of endometrial cancer and may have an impact on tumor aggressiveness. Finally, the present study suggests that epigenetic alterations may be of diagnostic value for the better clinical management of premalignant endometrial lesions.