Sample records for abnormal dna hypermethylation

  1. DNA methylation abnormalities in congenital heart disease.

    PubMed

    Serra-Juhé, Clara; Cuscó, Ivon; Homs, Aïda; Flores, Raquel; Torán, Núria; Pérez-Jurado, Luis A

    2015-01-01

    Congenital heart defects represent the most common malformation at birth, occurring also in ∼50% of individuals with Down syndrome. Congenital heart defects are thought to have multifactorial etiology, but the main causes are largely unknown. We have explored the global methylation profile of fetal heart DNA in comparison to blood DNA from control subjects: an absolute correlation with the type of tissue was detected. Pathway analysis revealed a significant enrichment of differential methylation at genes related to muscle contraction and cardiomyopathies in the developing heart DNA. We have also searched for abnormal methylation profiles on developing heart-tissue DNA of syndromic and non-syndromic congenital heart defects. On average, 3 regions with aberrant methylation were detected per sample and 18 regions were found differentially methylated between groups. Several epimutations were detected in candidate genes involved in growth regulation, apoptosis and folate pathway. A likely pathogenic hypermethylation of several intragenic sites at the MSX1 gene, involved in outflow tract morphogenesis, was found in a fetus with isolated heart malformation. In addition, hypermethylation of the GATA4 gene was present in fetuses with Down syndrome with or without congenital heart defects, as well as in fetuses with isolated heart malformations. Expression deregulation of the abnormally methylated genes was detected. Our data indicate that epigenetic alterations of relevant genes are present in developing heart DNA in fetuses with both isolated and syndromic heart malformations. These epimutations likely contribute to the pathogenesis of the malformation by cis-acting effects on gene expression.

  2. Developmentally linked human DNA hypermethylation is associated with down-modulation, repression, and upregulation of transcription

    PubMed Central

    Baribault, Carl; Ehrlich, Kenneth C.; Ponnaluri, V. K. Chaithanya; Pradhan, Sriharsa; Lacey, Michelle; Ehrlich, Melanie

    2018-01-01

    ABSTRACT DNA methylation can affect tissue-specific gene transcription in ways that are difficult to discern from studies focused on genome-wide analyses of differentially methylated regions (DMRs). To elucidate the variety of associations between differentiation-related DNA hypermethylation and transcription, we used available epigenomic and transcriptomic profiles from 38 human cell/tissue types to focus on such relationships in 94 genes linked to hypermethylated DMRs in myoblasts (Mb). For 19 of the genes, promoter-region hypermethylation in Mb (and often a few heterologous cell types) was associated with gene repression but, importantly, DNA hypermethylation was absent in many other repressed samples. In another 24 genes, DNA hypermethylation overlapped cryptic enhancers or super-enhancers and correlated with down-modulated, but not silenced, gene expression. However, such methylation was absent, surprisingly, in both non-expressing samples and highly expressing samples. This suggests that some genes need DMR hypermethylation to help repress cryptic enhancer chromatin only when they are actively transcribed. For another 11 genes, we found an association between intergenic hypermethylated DMRs and positive expression of the gene in Mb. DNA hypermethylation/transcription correlations similar to those of Mb were evident sometimes in diverse tissues, such as aorta and brain. Our findings have implications for the possible involvement of methylated DNA in Duchenne's muscular dystrophy, congenital heart malformations, and cancer. This epigenomic analysis suggests that DNA methylation is not simply the inevitable consequence of changes in gene expression but, instead, is often an active agent for fine-tuning transcription in association with development. PMID:29498561

  3. Transgelin gene is frequently downregulated by promoter DNA hypermethylation in breast cancer.

    PubMed

    Sayar, Nilufer; Karahan, Gurbet; Konu, Ozlen; Bozkurt, Betul; Bozdogan, Onder; Yulug, Isik G

    2015-01-01

    CpG hypermethylation in gene promoters is a frequent mechanism of tumor suppressor gene silencing in various types of cancers. It usually occurs at early steps of cancer progression and can be detected easily, giving rise to development of promising biomarkers for both detection and progression of cancer, including breast cancer. 5-aza-2'-deoxycytidine (AZA) is a DNA demethylating and anti-cancer agent resulting in induction of genes suppressed via DNA hypermethylation. Using microarray expression profiling of AZA- or DMSO-treated breast cancer and non-tumorigenic breast (NTB) cells, we identified for the first time TAGLN gene as a target of DNA hypermethylation in breast cancer. TAGLN expression was significantly and frequently downregulated via promoter DNA hypermethylation in breast cancer cells compared to NTB cells, and also in 13/21 (61.9 %) of breast tumors compared to matched normal tissues. Analyses of public microarray methylation data showed that TAGLN was also hypermethylated in 63.02 % of tumors compared to normal tissues; relapse-free survival of patients was worse with higher TAGLN methylation; and methylation levels could discriminate between tumors and healthy tissues with 83.14 % sensitivity and 100 % specificity. Additionally, qRT-PCR and immunohistochemistry experiments showed that TAGLN expression was significantly downregulated in two more independent sets of breast tumors compared to normal tissues and was lower in tumors with poor prognosis. Colony formation was increased in TAGLN silenced NTB cells, while decreased in overexpressing BC cells. TAGLN gene is frequently downregulated by DNA hypermethylation, and TAGLN promoter methylation profiles could serve as a future diagnostic biomarker, with possible clinical impact regarding the prognosis in breast cancer.

  4. Silencing of GSTP1 gene by CpG island DNA hypermethylation in HBV-associated hepatocellular carcinomas.

    PubMed

    Zhong, Sheng; Tang, Mandy W; Yeo, Winnie; Liu, Cuiling; Lo, Y M Dennis; Johnson, Philip J

    2002-04-01

    Glutathione S-transferases, enzymes that defend cells against damage mediated by oxidant and electrophilic carcinogens, may be critical determinants of cancer pathogenesis. In this report, we assess the role of epigenetic silencing of the GSTP1 gene, a gene encoding the pi-class glutathione S-transferase, in the pathogenesis of hepatitis B virus (HBV)-associated hepatocellular carcinomas (HCC). The cell lines Hep3B, HepG2, and a cohort of 43 HBV-associated HCC tissue specimens and corresponding nontumor tissues were subjected to analysis for GSTP1 epigenetic alteration and expression. GSTP1 "CpG" island DNA hypermethylation in the liver cell lines, and the tissue specimens were determined by methylation-specific PCR and correlated with expression of the gene using reverse-transcription PCR, immunoblotting, and immunohistochemistry. GSTP1 CpG island DNA hypermethylation was detected in 28 of 43 (65.1%) HCC tissues and 4 of 40 (10%) corresponding nontumor tissues. GSTP1 protein was absent in those cases showing hypermethylation of the gene. Similarly, DNA from Hep3B and HepG2 cell lines displayed complete GSTP1 hypermethylation in the CpG island, and they failed to express GSTP1 mRNA and the corresponding protein product. Treatment of the cell lines with the DNA methyltransferase inhibitor 5-aza-deoxycytidine reversed the hypermethylation, and restored GSTP1 mRNA and polypeptide expression. These data indicate that epigenetic silencing of GSTP1 gene expression by CpG island DNA hypermethylation is common in human HBV-associated HCC. In addition, somatic GSTP1 inactivation via CpG island hypermethylation may contribute to the pathogenesis of this malignancy.

  5. Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is associated with G to A mutations in K-ras in colorectal tumorigenesis.

    PubMed

    Esteller, M; Toyota, M; Sanchez-Cespedes, M; Capella, G; Peinado, M A; Watkins, D N; Issa, J P; Sidransky, D; Baylin, S B; Herman, J G

    2000-05-01

    O6-methylguanine DNA methyltransferase (MGMT) is a DNA repair protein that removes mutagenic and cytotoxic adducts from the O6 position of guanine. O6-methylguanine mispairs with thymine during replication, and if the adduct is not removed, this results in conversion from a guanine-cytosine pair to an adenine-thymine pair. In vitro assays show that MGMT expression avoids G to A mutations and MGMT transgenic mice are protected against G to A transitions at ras genes. We have recently demonstrated that the MGMT gene is silenced by promoter methylation in many human tumors, including colorectal carcinomas. To study the relevance of defective MGMT function by aberrant methylation in relation to the presence of K-ras mutations, we studied 244 colorectal tumor samples for MGMT promoter hypermethylation and K-ras mutational status. Our results show a clear association between the inactivation of MGMT by promoter hypermethylation and the appearance of G to A mutations at K-ras: 71% (36 of 51) of the tumors displaying this particular type of mutation had abnormal MGMT methylation, whereas only 32% (12 of 37) of those with other K-ras mutations not involving G to A transitions and 35% (55 of 156) of the tumors without K-ras mutations demonstrated MGMT methylation (P = 0.002). In addition, MGMT loss associated with hypermethylation was observed in the small adenomas, including those that do not yet contain K-ras mutations. Hypermethylation of other genes such as p16INK4a and p14ARF was not associated with either MGMT hypermethylation or K-ras mutation. Our data suggest that epigenetic silencing of MGMT by promoter hypermethylation may lead to a particular genetic change in human cancer, specifically G to A transitions in the K-ras oncogene.

  6. Fundamental differences in promoter CpG island DNA hypermethylation between human cancer and genetically engineered mouse models of cancer.

    PubMed

    Diede, Scott J; Yao, Zizhen; Keyes, C Chip; Tyler, Ashlee E; Dey, Joyoti; Hackett, Christopher S; Elsaesser, Katrina; Kemp, Christopher J; Neiman, Paul E; Weiss, William A; Olson, James M; Tapscott, Stephen J

    2013-12-01

    Genetic and epigenetic alterations are essential for the initiation and progression of human cancer. We previously reported that primary human medulloblastomas showed extensive cancer-specific CpG island DNA hypermethylation in critical developmental pathways. To determine whether genetically engineered mouse models (GEMMs) of medulloblastoma have comparable epigenetic changes, we assessed genome-wide DNA methylation in three mouse models of medulloblastoma. In contrast to human samples, very few loci with cancer-specific DNA hypermethylation were detected, and in almost all cases the degree of methylation was relatively modest compared with the dense hypermethylation in the human cancers. To determine if this finding was common to other GEMMs, we examined a Burkitt lymphoma and breast cancer model and did not detect promoter CpG island DNA hypermethylation, suggesting that human cancers and at least some GEMMs are fundamentally different with respect to this epigenetic modification. These findings provide an opportunity to both better understand the mechanism of aberrant DNA methylation in human cancer and construct better GEMMs to serve as preclinical platforms for therapy development.

  7. Development of Castration Resistant Prostate Cancer can be Predicted by a DNA Hypermethylation Profile.

    PubMed

    Angulo, Javier C; Andrés, Guillermo; Ashour, Nadia; Sánchez-Chapado, Manuel; López, Jose I; Ropero, Santiago

    2016-03-01

    Detection of DNA hypermethylation has emerged as a novel molecular biomarker for prostate cancer diagnosis and evaluation of prognosis. We sought to define whether a hypermethylation profile of patients with prostate cancer on androgen deprivation would predict castrate resistant prostate cancer. Genome-wide methylation analysis was performed using a methylation cancer panel in 10 normal prostates and 45 tumor samples from patients placed on androgen deprivation who were followed until castrate resistant disease developed. Castrate resistant disease was defined according to EAU (European Association of Urology) guideline criteria. Two pathologists reviewed the Gleason score, Ki-67 index and neuroendocrine differentiation. Hierarchical clustering analysis was performed and relationships with outcome were investigated by Cox regression and log rank analysis. We found 61 genes that were significantly hypermethylated in greater than 20% of tumors analyzed. Three clusters of patients were characterized by a DNA methylation profile, including 1 at risk for earlier castrate resistant disease (log rank p = 0.019) and specific mortality (log rank p = 0.002). Hypermethylation of ETV1 (HR 3.75) and ZNF215 (HR 2.89) predicted disease progression despite androgen deprivation. Hypermethylation of IRAK3 (HR 13.72), ZNF215 (HR 4.81) and SEPT9 (HR 7.64) were independent markers of prognosis. Prostate specific antigen greater than 25 ng/ml, Gleason pattern 5, Ki-67 index greater than 12% and metastasis at diagnosis also predicted a negative response to androgen deprivation. Study limitations included the retrospective design and limited number of cases. Epigenetic silencing of the mentioned genes could be novel molecular markers for the prognosis of advanced prostate cancer. It might predict castrate resistance during hormone deprivation and, thus, disease specific mortality. Gene hypermethylation is associated with disease progression in patients who receive hormone therapy. It

  8. Redox/methylation mediated abnormal DNA methylation as regulators of ambient fine particulate matter-induced neurodevelopment related impairment in human neuronal cells

    NASA Astrophysics Data System (ADS)

    Wei, Hongying; Liang, Fan; Meng, Ge; Nie, Zhiqing; Zhou, Ren; Cheng, Wei; Wu, Xiaomeng; Feng, Yan; Wang, Yan

    2016-09-01

    Fine particulate matter (PM2.5) has been implicated as a risk factor for neurodevelopmental disorders including autism in children. However, the underlying biological mechanism remains unclear. DNA methylation is suggested to be a fundamental mechanism for the neuronal responses to environmental cues. We prepared whole particle of PM2.5 (PM2.5), water-soluble extracts (Pw), organic extracts (Po) and carbon core component (Pc) and characterized their chemical constitutes. We found that PM2.5 induced significant redox imbalance, decreased the levels of intercellular methyl donor S-adenosylmethionine and caused global DNA hypomethylation. Furthermore, PM2.5 exposure triggered gene-specific promoter DNA hypo- or hypermethylation and abnormal mRNA expression of autism candidate genes. PM2.5-induced DNA hypermethylation in promoter regions of synapse related genes were associated with the decreases in their mRNA and protein expression. The inhibiting effects of antioxidative reagents, a methylation-supporting agent and a DNA methyltransferase inhibitor demonstrated the involvement of redox/methylation mechanism in PM2.5-induced abnormal DNA methylation patterns and synaptic protein expression. The biological effects above generally followed a sequence of PM2.5 ≥ Pwo > Po > Pw > Pc. Our results implicated a novel epigenetic mechanism for the neurodevelopmental toxicity of particulate air pollution, and that eliminating the chemical components could mitigate the neurotoxicity of PM2.5.

  9. Redox/methylation mediated abnormal DNA methylation as regulators of ambient fine particulate matter-induced neurodevelopment related impairment in human neuronal cells.

    PubMed

    Wei, Hongying; Liang, Fan; Meng, Ge; Nie, Zhiqing; Zhou, Ren; Cheng, Wei; Wu, Xiaomeng; Feng, Yan; Wang, Yan

    2016-09-14

    Fine particulate matter (PM2.5) has been implicated as a risk factor for neurodevelopmental disorders including autism in children. However, the underlying biological mechanism remains unclear. DNA methylation is suggested to be a fundamental mechanism for the neuronal responses to environmental cues. We prepared whole particle of PM2.5 (PM2.5), water-soluble extracts (Pw), organic extracts (Po) and carbon core component (Pc) and characterized their chemical constitutes. We found that PM2.5 induced significant redox imbalance, decreased the levels of intercellular methyl donor S-adenosylmethionine and caused global DNA hypomethylation. Furthermore, PM2.5 exposure triggered gene-specific promoter DNA hypo- or hypermethylation and abnormal mRNA expression of autism candidate genes. PM2.5-induced DNA hypermethylation in promoter regions of synapse related genes were associated with the decreases in their mRNA and protein expression. The inhibiting effects of antioxidative reagents, a methylation-supporting agent and a DNA methyltransferase inhibitor demonstrated the involvement of redox/methylation mechanism in PM2.5-induced abnormal DNA methylation patterns and synaptic protein expression. The biological effects above generally followed a sequence of PM2.5 ≥ Pwo > Po > Pw > Pc. Our results implicated a novel epigenetic mechanism for the neurodevelopmental toxicity of particulate air pollution, and that eliminating the chemical components could mitigate the neurotoxicity of PM2.5.

  10. Redox/methylation mediated abnormal DNA methylation as regulators of ambient fine particulate matter-induced neurodevelopment related impairment in human neuronal cells

    PubMed Central

    Wei, Hongying; Liang, Fan; Meng, Ge; Nie, Zhiqing; Zhou, Ren; Cheng, Wei; Wu, Xiaomeng; Feng, Yan; Wang, Yan

    2016-01-01

    Fine particulate matter (PM2.5) has been implicated as a risk factor for neurodevelopmental disorders including autism in children. However, the underlying biological mechanism remains unclear. DNA methylation is suggested to be a fundamental mechanism for the neuronal responses to environmental cues. We prepared whole particle of PM2.5 (PM2.5), water-soluble extracts (Pw), organic extracts (Po) and carbon core component (Pc) and characterized their chemical constitutes. We found that PM2.5 induced significant redox imbalance, decreased the levels of intercellular methyl donor S-adenosylmethionine and caused global DNA hypomethylation. Furthermore, PM2.5 exposure triggered gene-specific promoter DNA hypo- or hypermethylation and abnormal mRNA expression of autism candidate genes. PM2.5-induced DNA hypermethylation in promoter regions of synapse related genes were associated with the decreases in their mRNA and protein expression. The inhibiting effects of antioxidative reagents, a methylation-supporting agent and a DNA methyltransferase inhibitor demonstrated the involvement of redox/methylation mechanism in PM2.5-induced abnormal DNA methylation patterns and synaptic protein expression. The biological effects above generally followed a sequence of PM2.5 ≥ Pwo > Po > Pw > Pc. Our results implicated a novel epigenetic mechanism for the neurodevelopmental toxicity of particulate air pollution, and that eliminating the chemical components could mitigate the neurotoxicity of PM2.5. PMID:27624276

  11. Aging-dependent DNA hypermethylation and gene expression of GSTM1 involved in T cell differentiation.

    PubMed

    Yeh, Shu-Hui; Liu, Cheng-Ling; Chang, Ren-Chieh; Wu, Chih-Chiang; Lin, Chia-Hsueh; Yang, Kuender D

    2017-07-25

    This study investigated whether aging was associated with epigenetic changes of DNA hypermethylation on immune gene expression and lymphocyte differentiation. We screened CG sites of methylation in blood leukocytes from different age populations, picked up genes with age-related increase of CG methylation content more than 15%, and validated immune related genes with CG hypermethylation involved in lymphocyte differentiation in the aged population. We found that 12 genes (EXHX1、 IL-10、 TSP50、 GSTM1、SLC5A5、SPI1、F2R、LMO2、PTPN6、FGFR2、MMP9、MET) were associated with promoter or exon one DNA hypermethylation in the aged group. Two immune related genes, GSTM1 and LMO2, were chosen to validate its aging-related CG hypermethylation in different leukocytes. We are the first to validate that GSTM1_P266 and LMO2_E128 CG methylation contents in T lymphocytes but not polymorphonuclear cells (PMNs) or mononuclear cells (MNCs) were significantly increased in the aged population. The GSTM1 mRNA expression in T lymphocytes but not PMNs or MNCs was inversely associated with the GSTM1 CG hypermethylation levels in the aged population studied. Further studies showed that lower GSTM1 CG methylation content led to the higher GSTM1 mRNA expression in T cells and knockdown of GSTM1 mRNA expression decreased type 1 T helper cell (Th1) differentiation in Jurkat T cells and normal adult CD4 T cells. The GSTM1_P266 hypermethylation in the aged population associated with lower GSTM1 mRNA expression was involved in Th1 differentiation, highlighting that modulation of aging-associated GSTM1 methylation may be able to enhance T helper cell immunity in the elders.

  12. Hypermethylations of RASAL1 and KLOTHO is associated with renal dysfunction in a Chinese population environmentally exposed to cadmium

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhang, Chen; Liang, Yihuai; Key Laboratory of Public Health Safety, Ministry of Education, 130 DongAn Road, Shanghai 200032

    2013-08-15

    Exposure to cadmium (Cd) can affect both DNA methylation and renal function, but there are few examples of the association between epigenetic markers and Cd-induced kidney damage. It has been suggested that hypermethylation of the genes RASAL1 and KLOTHO is associated with renal fibrogenesis. To investigate whether hypermethylation of RASAL1 and KLOTHO in peripheral blood DNA can be associated with Cd exposure and/or Cd-induced renal dysfunction, the degrees of methylation of RASAL1 and KLOTHO in peripheral blood DNA from 81 residents in Cd-polluted and non-polluted areas were measured using bisulfate-PCR-pyrosequencing. Changes in blood cadmium (BCd), urinary cadmium (UCd), and kidneymore » parameters were measured, and the glomerular filtration rate (eGFR) was estimated. The levels of BCd and UCd correlated positively with the levels of DNA methylation in RASAL1 and in KLOTHO. The more heavily exposed residents (BCd, 4.23–13.22 μg/L; UCd, 8.65–32.90 μg/g creatinine) exhibited obvious renal dysfunction. Notably, when Cd concentration in blood and urine was adjusted, the increased methylation level in RASAL1 was inversely correlated with eGFR (P < 0.01) but the relationship between hypermethylation of KLOTHO and eGFR was not statistically significant. The methylation of RASAL1 increased along with the increased abnormal prevalence of eGFR. Our findings suggest that Cd exposure can induce the hypermethylation of RASAL1 and KLOTHO. Hypermethylation of RASAL1 may be an indicator of the progress for chronic kidney disease. - Highlights: • A long term heavily Cd exposure induced renal dysfunction. • Cd exposure correlated positively with DNA methylation in RASAL1 and KLOTHO. • Hypermethylation of RASAL1 correlated with adjusted renal function indicators.« less

  13. Hypermethylated ERG as a cell-free fetal DNA biomarker for non-invasive prenatal testing of Down syndrome.

    PubMed

    Chen, Xi; Xiong, Likuan; Zeng, Ting; Xiao, Kelin; Huang, Yanping; Guo, Hui; Ren, Jinghui

    2015-04-15

    Previous reports have shown that the ERG gene is hypermethylated in the placenta and hypomethylated in maternal blood cells. In this study, we explore the feasibility of hypermethylated ERG as a cell-free fetal (cff) DNA biomarker for non-invasive prenatal testing (NIPT) of Down syndrome. We randomly selected 90 healthy pregnant women, including 30 cases at each trimester of pregnancy. In addition, 15 pregnant women were recruited as the case group whose fetuses had been confirmed to have trisomy 21 by amniotic fluid analysis at 18th to 26th week gestation. Using HpaII, MspІ to digest cell-free maternal plasma DNA, we performed SYBR Green PCR to detect methylated sites of ERG sequences, and analyzed the concentrations of cff DNA in maternal plasma in different gestational trimesters and the case group. The ERG median concentrations of the maternal plasma after Hpa II digestion (LG copies/ml) in first, second and third-trimesters were 5.38, 6.10, and 7.04, respectively (Kruskal-Wallis, P<0.01); and that in the trisomy 21 case group was 6.85, which was higher than the second-trimester (Mann-Whitney, P<0.01). The study demonstrated that ERG gene is hypermethylated in cff DNA but hypomethylated in maternal DNA; and the median concentration of ERG gene in the trisomy 21 case group is higher than that in the gestational trimester matched normal group. ERG gene, as a fetal DNA biomarker, may be useful for NIPT of Down syndrome. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Hypermethylation of testis derived transcript gene promoter significantly correlates with worse outcomes in glioblastoma patients.

    PubMed

    Wang, Li-jia; Bai, Yu; Bao, Zhao-shi; Chen, Yan; Yan, Zhuo-hong; Zhang, Wei; Zhang, Quan-geng

    2013-01-01

    Glioblastoma is the most common and lethal cancer of the central nervous system. Global genomic hypomethylation and some CpG island hypermethylation are common hallmarks of these malignancies, but the effects of these methylation abnormalities on glioblastomas are still largely unclear. Methylation of the O6-methylguanine-DNA methyltransferase promoter is currently an only confirmed molecular predictor of better outcome in temozolomide treatment. To better understand the relationship between CpG island methylation status and patient outcome, this study launched DNA methylation profiles for thirty-three primary glioblastomas (pGBMs) and nine secondary glioblastomas (sGBMs) with the expectation to identify valuable prognostic and therapeutic targets. We evaluated the methylation status of testis derived transcript (TES) gene promoter by microarray analysis of glioblastomas and the prognostic value for TES methylation in the clinical outcome of pGBM patients. Significance analysis of microarrays was used for genes significantly differently methylated between 33 pGBM and nine sGBM. Survival curves were calculated according to the Kaplan-Meier method, and differences between curves were assessed using the log-rank test. Then, we treated glioblastoma cell lines (U87 and U251) with 5-aza-2-deoxycytidines (5-aza-dC) and detected cell biological behaviors. Microarray data analysis identified TES promoter was hypermethylated in pGBMs compared with sGBMs (P < 0.05). Survival curves from the Kaplan-Meier method analysis revealed that the patients with TES hypermethylation had a short overall survival (P < 0.05). This abnormality is also confirmed in glioblastoma cell lines (U87 and U251). Treating these cells with 5-aza-dC released TES protein expression resulted in significant inhibition of cell growth (P = 0.013). Hypermethylation of TES gene promoter highly correlated with worse outcome in pGBM patients. TES might represent a valuable prognostic marker for glioblastoma.

  15. [Promoter hypermethylation status of the mismatch repair gene hMLH1 in patients with sporadic renal cell carcinoma].

    PubMed

    Salinas-Sánchez, Antonio S; Rubio-del-Campo, Antonio; Sánchez-Sánchez, Francisco; Giménez-Bachs, José M; Donate-Moreno, María J; García-Olmo, Dolores C; Escribano-Martínez, Julio

    2006-04-01

    Epigenetic inactivation is a gene function abnormality that produces no changes in the DNA sequence, with the most frequent epigenetic alteration being hypermethylation of CpG islands in the promoter regions of the genes. Based on recent indications of a potential relationship between mismatch repair genes and renal cell carcinoma (RCC), we were interested in investigating the existence of promoter hypermethylation of the hMLH1 gene in tumor DNA samples from patients with sporadic RCC. Sixty-five tumor tissue specimens were collected consecutively. The DNA was first obtained and purified, then digested with the restriction enzymes Hpa II and Msp I, followed by polimerase chain reaction amplification of 3 promoter regions of the hMLH1 gene, agarose gel electrophoresis, and densitometric analysis of the images of the amplified bands. Mean patient age was 63.7 years. The most frequent cell type was clear cell carcinoma (67.7%). 73.9% of tumors were diagnosed in stages below pT2, 9.3% had gland involvement and 20%, distant metastasis. No somatic hypermethylation was detected in the promoter region of the hMLH1 gene in any of the patients studied. Our data indicate that promoter hypermethylation of the hMLH1 gene is not implicated in the pathogenesis of sporadic RCC, and therefore the existence of another type of mutation, microsatellite instability and/or loss of heterozygosity should be examined to determine the possible role of this gene in sporadic RCC.

  16. IMBALANCE OF DNA METHYLATION, BOTH HYPERMETHYLATION AND HYPOMETHYLATION, OCCUR AFTER EXPOSURE OF HUMAN CELLS TO NANOMOLAR CONCENTRATIONS OF ARSENITE IN CULTURE.

    EPA Science Inventory

    Imbalance of DNA methylation, BOTH hypermethylation and hypomethylation, occur after exposure of human cells to nanomolar concentrations of arsenite in culture.

    We and others have hypothesized that a mechanism of arsenic carcinogenesis could involve alteration of DNA methy...

  17. Clinical Implications of Promoter Hypermethylation in RASSF1A and MGMT in Retinoblastoma1

    PubMed Central

    Choy, Kwong Wai; Lee, Tom C; Cheung, Kin Fai; Fan, Dorothy S P; Lo, Kwok Wai; Beaverson, Katherine L; Abramson, David H; Lam, Dennis S C; Yu, Christopher B O; Pang, Chi Pui

    2005-01-01

    Abstract We investigated the epigenetic silencing and genetic changes of the RAS-associated domain family 1A (RASSF1A) gene and the O6-methylguanine-DNA methyltransferase (MGMT) gene in retinoblastoma. We extracted DNA from microdissected tumor and normal retina tissues of the same patient in 68 retinoblastoma cases. Promoter methylation in RASSF1A and MGMT was analyzed by methylation-specific PCR, RASSF1A sequence alterations in all coding exons by direct DNA sequencing, and RASSF1A expression by RT-PCR. Cell cycle staging was analyzed by flow cytometry. We detected RASSF1A promoter hypermethylation in 82% of retinoblastoma, in tumor tissues only but not in adjacent normal retinal tissue cells. There was no expression of RASSF1A transcripts in all hypermethylated samples, but RASSF1A transcripts were restored after 5-aza-2′-deoxycytidine treatment with no changes in cell cycle or apoptosis. No mutation in the RASSF1A sequence was found. MGMT hypermethylation was present in 15% of theretinoblastoma samples, and the absence of MGMT hypermethylation was associated (P = .002) with retinoblastoma at advanced Reese-Ellsworth tumor stage. Our results revealed a high RASSF1A hypermethylation frequency in retinoblastoma. The correlation of MGMT inactivation by promoter hypermethylation with lower-stage diseases indicated that MGMT hypermethylation provides useful prognostic information. Epigenetic mechanism plays an important role in the progression of retinoblastoma. PMID:15799820

  18. Diagnostic Performance of DNA Hypermethylation Markers in Peripheral Blood for the Detection of Colorectal Cancer: A Meta-Analysis and Systematic Review

    PubMed Central

    Li, Bingsheng; Gan, Aihua; Chen, Xiaolong; Wang, Xinying; He, Weifeng; Zhang, Xiaohui; Huang, Renxiang; Zhou, Shuzhu; Song, Xiaoxiao; Xu, Angao

    2016-01-01

    DNA hypermethylation in blood is becoming an attractive candidate marker for colorectal cancer (CRC) detection. To assess the diagnostic accuracy of blood hypermethylation markers for CRC in different clinical settings, we conducted a meta-analysis of published reports. Of 485 publications obtained in the initial literature search, 39 studies were included in the meta-analysis. Hypermethylation markers in peripheral blood showed a high degree of accuracy for the detection of CRC. The summary sensitivity was 0.62 [95% confidence interval (CI), 0.56–0.67] and specificity was 0.91 (95% CI, 0.89–0.93). Subgroup analysis showed significantly greater sensitivity for the methylated Septin 9 gene (SEPT9) subgroup (0.75; 95% CI, 0.67–0.81) than for the non-methylated SEPT9 subgroup (0.58; 95% CI, 0.52–0.64). Sensitivity and specificity were not affected significantly by target gene number, CRC staging, study region, or methylation analysis method. These findings show that hypermethylation markers in blood are highly sensitive and specific for CRC detection, with methylated SEPT9 being particularly robust. The diagnostic performance of hypermethylation markers, which have varied across different studies, can be improved by marker optimization. Future research should examine variation in diagnostic accuracy according to non-neoplastic factors. PMID:27158984

  19. Alpha-Lipoic Acid Downregulates IL-1β and IL-6 by DNA Hypermethylation in SK-N-BE Neuroblastoma Cells.

    PubMed

    Dinicola, Simona; Proietti, Sara; Cucina, Alessandra; Bizzarri, Mariano; Fuso, Andrea

    2017-09-26

    Alpha-lipoic acid (ALA) is a pleiotropic molecule with antioxidant and anti-inflammatory properties, of which the effects are exerted through the modulation of NF-kB. This nuclear factor, in fact, modulates different inflammatory cytokines, including IL-1b and IL-6, in different tissues and cell types. We recently showed that IL-1b and IL-6 DNA methylation is modulated in the brain of Alzheimer's disease patients, and that IL-1b expression is associated to DNA methylation in the brain of patients with tuberous sclerosis complex. These results prompted us to ask whether ALA-induced repression of IL-1b and IL-6 was dependent on DNA methylation. Therefore, we profiled DNA methylation in the 5'-flanking region of the two aforementioned genes in SK-N-BE human neuroblastoma cells cultured in presence of ALA 0.5 mM. Our experimental data pointed out that the two promoters are hypermethylated in cells supplemented with ALA, both at CpG and non-CpG sites. Moreover, the observed hypermethylation is associated with decreased mRNA expression and decreased cytokine release. These results reinforce previous findings indicating that IL-1b and IL-6 undergo DNA methylation-dependent modulation in neural models and pave the road to study the epigenetic mechanisms triggered by ALA.

  20. Downregulation of TES by hypermethylation in glioblastoma reduces cell apoptosis and predicts poor clinical outcome.

    PubMed

    Bai, Yu; Zhang, Quan-Geng; Wang, Xin-Hua

    2014-12-11

    Gliomas are the most common human brain tumors. Glioblastoma, also known as glioblastoma multiform (GBM), is the most aggressive, malignant, and lethal glioma. The investigation of prognostic and diagnostic molecular biomarkers in glioma patients to provide direction on clinical practice is urgent. Recent studies demonstrated that abnormal DNA methylation states play a key role in the pathogenesis of this kind of tumor. In this study, we want to identify a novel biomarker related to glioma initiation and find the role of the glioma-related gene. We performed a methylation-specific microarray on the promoter region to identify methylation gene(s) that may affect outcome of GBM patients. Normal and GBM tissues were collected from Tiantan Hospital. Genomic DNA was extracted from these tissues and analyzed with a DNA promoter methylation microarray. Testis derived transcript (TES) protein expression was analyzed by immunohistochemistry in paraffin-embedded patient tissues. Western blotting was used to detect TES protein expression in the GBM cell line U251 with or without 5-aza-dC treatment. Cell apoptosis was evaluated by flow cytometry analysis using Annexin V/PI staining. We found that the TES promoter was hypermethylated in GBM compared to normal brain tissues under DNA promoter methylation microarray analysis. The GBM patients with TES hypermethylation had a short overall survival (P <0.05, log-rank test). Among GBM samples, reduced TES protein level was detected in 33 (89.2%) of 37 tumor tissues by immunohistochemical staining. Down regulation of TES was also correlated with worse patient outcome (P <0.05, log-rank test). Treatment on the GBM cell line U251 with 5-aza-dC can greatly increase TES expression, confirming the hypermethylation of TES promoter in GBM. Up-regulation of TES prompts U251 apoptosis significantly. This study demonstrated that both TES promoter hypermethylation and down-regulated protein expression significantly correlated with worse

  1. Regulatory network analysis of LINC00472, a long noncoding RNA downregulated by DNA hypermethylation in colorectal cancer.

    PubMed

    Chen, L; Zhang, W; Li, D Y; Wang, X; Tao, Y; Zhang, Y; Dong, C; Zhao, J; Zhang, L; Zhang, X; Guo, J; Zhang, X; Liao, Q

    2018-06-01

    Colorectal cancer (CRC), one of the common malignant cancers in the world, is caused by accumulated alterations of genetic and epigenetic factors over a long period of time. Along with that protein-coding genes being identified as oncogenes or tumor suppressors in CRC, a number of lncRNAs have also been found to be associated with CRC. Considering the important regulatory role of lncRNAs, the first goal of this study was to identify CRC-associated lncRNAs from a public database. One such lncRNA, LINC00472, was verified to be downregulated in CRC cell lines and cancer tissues compared with adjacent tissues. In addition, the down-regulation of LINC00472 seemed to be caused by DNA hypermethylation at its promoter region. Furthermore, the expression of LINC00472 and DNA methylation of promoter were significantly correlated with clinicopathological features. And DNA hypermethylation of LINC00472 may serve as a better diagnostic biomarker than its expression for CRC. Finally, we predicted the functions of LINC00472 and constructed a regulatory network and found LINC00472 may be involved in cell cycle and cell proliferation processes. Our results may provide a clue to further research into the function and regulatory mechanism of LINC00472 in CRC. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  2. Systemic Age-Associated DNA Hypermethylation of ELOVL2 Gene: In Vivo and In Vitro Evidences of a Cell Replication Process.

    PubMed

    Bacalini, Maria Giulia; Deelen, Joris; Pirazzini, Chiara; De Cecco, Marco; Giuliani, Cristina; Lanzarini, Catia; Ravaioli, Francesco; Marasco, Elena; van Heemst, Diana; Suchiman, H Eka D; Slieker, Roderick; Giampieri, Enrico; Recchioni, Rina; Mercheselli, Fiorella; Salvioli, Stefano; Vitale, Giovanni; Olivieri, Fabiola; Spijkerman, Annemieke M W; Dollé, Martijn E T; Sedivy, John M; Castellani, Gastone; Franceschi, Claudio; Slagboom, Pieternella E; Garagnani, Paolo

    2017-08-01

    Epigenetic remodeling is one of the major features of the aging process. We recently demonstrated that DNA methylation of ELOVL2 and FHL2 CpG islands is highly correlated with age in whole blood. Here we investigated several aspects of age-associated hypermethylation of ELOVL2 and FHL2. We showed that ELOVL2 methylation is significantly different in primary dermal fibroblast cultures from donors of different ages. Using epigenomic data from public resources, we demonstrated that most of the tissues show ELOVL2 and FHL2 hypermethylation with age. Interestingly, ELOVL2 hypermethylation was not found in tissues with very low replication rate. We demonstrated that ELOVL2 hypermethylation is associated with in vitro cell replication rather than with senescence. We confirmed intra-individual hypermethylation of ELOVL2 and FHL2 in longitudinally assessed participants from the Doetinchem Cohort Study. Finally we showed that, although the methylation of the two loci is not associated with longevity/mortality in the Leiden Longevity Study, ELOVL2 methylation is associated with cytomegalovirus status in nonagenarians, which could be informative of a higher number of replication events in a fraction of whole-blood cells. Collectively, these results indicate that ELOVL2 methylation is a marker of cell divisions occurring during human aging. © The Author 2016. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  3. Genome-wide methylation analysis identifies a core set of hypermethylated genes in CIMP-H colorectal cancer.

    PubMed

    McInnes, Tyler; Zou, Donghui; Rao, Dasari S; Munro, Francesca M; Phillips, Vicky L; McCall, John L; Black, Michael A; Reeve, Anthony E; Guilford, Parry J

    2017-03-28

    Aberrant DNA methylation profiles are a characteristic of all known cancer types, epitomized by the CpG island methylator phenotype (CIMP) in colorectal cancer (CRC). Hypermethylation has been observed at CpG islands throughout the genome, but it is unclear which factors determine whether an individual island becomes methylated in cancer. DNA methylation in CRC was analysed using the Illumina HumanMethylation450K array. Differentially methylated loci were identified using Significance Analysis of Microarrays (SAM) and the Wilcoxon Signed Rank (WSR) test. Unsupervised hierarchical clustering was used to identify methylation subtypes in CRC. In this study we characterized the DNA methylation profiles of 94 CRC tissues and their matched normal counterparts. Consistent with previous studies, unsupervized hierarchical clustering of genome-wide methylation data identified three subtypes within the tumour samples, designated CIMP-H, CIMP-L and CIMP-N, that showed high, low and very low methylation levels, respectively. Differential methylation between normal and tumour samples was analysed at the individual CpG level, and at the gene level. The distribution of hypermethylation in CIMP-N tumours showed high inter-tumour variability and appeared to be highly stochastic in nature, whereas CIMP-H tumours exhibited consistent hypermethylation at a subset of genes, in addition to a highly variable background of hypermethylated genes. EYA4, TFPI2 and TLX1 were hypermethylated in more than 90% of all tumours examined. One-hundred thirty-two genes were hypermethylated in 100% of CIMP-H tumours studied and these were highly enriched for functions relating to skeletal system development (Bonferroni adjusted p value =2.88E-15), segment specification (adjusted p value =9.62E-11), embryonic development (adjusted p value =1.52E-04), mesoderm development (adjusted p value =1.14E-20), and ectoderm development (adjusted p value =7.94E-16). Our genome-wide characterization of DNA

  4. Promoter CpG island hypermethylation of the DNA repair enzyme MGMT predicts clinical response to dacarbazine in a phase II study for metastatic colorectal cancer.

    PubMed

    Amatu, Alessio; Sartore-Bianchi, Andrea; Moutinho, Catia; Belotti, Alessandro; Bencardino, Katia; Chirico, Giuseppe; Cassingena, Andrea; Rusconi, Francesca; Esposito, Anna; Nichelatti, Michele; Esteller, Manel; Siena, Salvatore

    2013-04-15

    O(6)-methylguanine-DNA-methyltransferase (MGMT) is a DNA repair protein removing mutagenic and cytotoxic adducts from O(6)-guanine in DNA. Approximately 40% of colorectal cancers (CRC) display MGMT deficiency due to the promoter hypermethylation leading to silencing of the gene. Alkylating agents, such as dacarbazine, exert their antitumor activity by DNA methylation at the O(6)-guanine site, inducing base pair mismatch; therefore, activity of dacarbazine could be enhanced in CRCs lacking MGMT. We conducted a phase II study with dacarbazine in CRCs who had failed standard therapies (oxaliplatin, irinotecan, fluoropyrimidines, and cetuximab or panitumumab if KRAS wild-type). All patients had tumor tissue assessed for MGMT as promoter hypermethylation in double-blind for treatment outcome. Patients received dacarbazine 250 mg/m(2) intravenously every day for four consecutive days, every 21 days, until progressive disease or intolerable toxicity. We used a Simon two-stage design to determine whether the overall response rate would be 10% or more. Secondary endpoints included association of response, progression-free survival, and disease control rate with MGMT status. Sixty-eight patients were enrolled from May 2011 to March 2012. Patients received a median of three cycles of dacarbazine (range 1-12). Grades 3 and 4 toxicities included: fatigue (41%), nausea/vomiting (29%), constipation (25%), platelet count decrease (19%), and anemia (18%). Overall, two patients (3%) achieved partial response and eight patients (12%) had stable disease. Disease control rate (partial response + stable disease) was significantly associated with MGMT promoter hypermethylation in the corresponding tumors. Objective clinical responses to dacarbazine in patients with metastatic CRC are confined to those tumors harboring epigenetic inactivation of the DNA repair enzyme MGMT.

  5. Validation study of genes with hypermethylated promoter regions associated with prostate cancer recurrence

    PubMed Central

    Stott-Miller, Marni; Zhao, Shanshan; Wright, Jonathan L.; Kolb, Suzanne; Bibikova, Marina; Klotzle, Brandy; Ostrander, Elaine A.; Fan, Jian-Bing; Feng, Ziding; Stanford, Janet L.

    2014-01-01

    Background One challenge in prostate cancer (PCa) is distinguishing indolent from aggressive disease at diagnosis. DNA promoter hypermethylation is a frequent epigenetic event in PCa, but few studies of DNA methylation in relation to features of more aggressive tumors or PCa recurrence have been completed. Methods We used the Infinium® HumanMethylation450 BeadChip to assess DNA methylation in tumor tissue from 407 patients with clinically localized PCa who underwent radical prostatectomy. Recurrence status was determined by follow-up patient surveys, medical record review, and linkage with the SEER registry. The methylation status of 14 genes for which promoter hypermethylation was previously correlated with advanced disease or biochemical recurrence was evaluated. Average methylation level for promoter region CpGs in patients who recurred compared to those with no evidence of recurrence was analyzed. For two genes with differential methylation, time to recurrence was examined. Results During an average follow-up of 11.7 years, 104 (26%) patients recurred. Significant promoter hypermethylation in at least 50% of CpG sites in two genes, ABHD9 and HOXD3, was found in tumors from patients who recurred compared to those without recurrence. Evidence was strongest for HOXD3 (lowest P = 9.46x10−6), with higher average methylation across promoter region CpGs associated with reduced recurrence-free survival (P = 2×10−4). DNA methylation profiles did not differ by recurrence status for the other genes. Conclusions These results validate the association between promoter hypermethylation of ADHB9 and HOXD3 and PCa recurrence. Impact Tumor DNA methylation profiling may help distinguish PCa patients at higher risk for disease recurrence. PMID:24718283

  6. Hyper-Methylated Loci Persisting from Sessile Serrated Polyps to Serrated Cancers.

    PubMed

    Andrew, Angeline S; Baron, John A; Butterly, Lynn F; Suriawinata, Arief A; Tsongalis, Gregory J; Robinson, Christina M; Amos, Christopher I

    2017-03-02

    Although serrated polyps were historically considered to pose little risk, it is now understood that progression down the serrated pathway could account for as many as 15%-35% of colorectal cancers. The sessile serrated adenoma/polyp (SSA/P) is the most prevalent pre-invasive serrated lesion. Our objective was to identify the CpG loci that are persistently hyper-methylated during serrated carcinogenesis, from the early SSA/P lesion through the later cancer phases of neoplasia development. We queried the loci hyper-methylated in serrated cancers within our rightsided SSA/Ps from the New Hampshire Colonoscopy Registry, using the Illumina Infinium Human Methylation 450 k panel to comprehensively assess the DNA methylation status. We identified CpG loci and regions consistently hyper-methylated throughout the serrated carcinogenesis spectrum, in both our SSA/P specimens and in serrated cancers. Hyper-methylated CpG loci included the known the tumor suppressor gene RET (p = 5.72 x 10-10), as well as loci in differentially methylated regions for GSG1L, MIR4493, NTNG1, MCIDAS, ZNF568, and RERG. The hyper-methylated loci that we identified help characterize the biology of SSA/P development, and could be useful as therapeutic targets, or for future identification of patients who may benefit from shorter surveillance intervals.

  7. Acquired hypermethylation of the P16INK4A promoter in abdominal paraganglioma: relation to adverse tumor phenotype and predisposing mutation

    PubMed Central

    Kiss, Nimrod B; Muth, Andreas; Andreasson, Adam; Juhlin, C Christofer; Geli, Janos; Bäckdahl, Martin; Höög, Anders; Wängberg, Bo; Nilsson, Ola; Ahlman, Håkan; Larsson, Catharina

    2013-01-01

    Recurrent alterations in promoter methylation of tumor suppressor genes (TSGs) and LINE1 (L1RE1) repeat elements were previously reported in pheochromocytoma and abdominal paraganglioma. This study was undertaken to explore CpG methylation abnormalities in an extended tumor panel and assess possible relationships between metastatic disease and mutation status. CpG methylation was quantified by bisulfite pyrosequencing for selected TSG promoters and LINE1 repeats. Methylation indices above normal reference were observed for DCR2 (TNFRSF10D), CDH1, P16 (CDKN2A), RARB, and RASSF1A. Z-scores for overall TSG, and individual TSG methylation levels, but not LINE1, were significantly correlated with metastatic disease, paraganglioma, disease predisposition, or outcome. Most strikingly, P16 hypermethylation was strongly associated with SDHB mutation as opposed to RET/MEN2, VHL/VHL, or NF1-related disease. Parallel analyses of constitutional, tumor, and metastasis DNA implicate an order of events where constitutional SDHB mutations are followed by TSG hypermethylation and 1p loss in primary tumors, later transferred to metastatic tissue. In the combined material, P16 hypermethylation was prevalent in SDHB-mutated samples and was associated with short disease-related survival. The findings verify the previously reported importance of P16 and other TSG hypermethylation in an independent tumor series. Furthermore, a constitutional SDHB mutation is proposed to predispose for an epigenetic tumor phenotype occurring before the emanation of clinically recognized malignancy. PMID:23154831

  8. Epigenetic profiling of cutaneous T-cell lymphoma: promoter hypermethylation of multiple tumor suppressor genes including BCL7a, PTPRG, and p73.

    PubMed

    van Doorn, Remco; Zoutman, Willem H; Dijkman, Remco; de Menezes, Renee X; Commandeur, Suzan; Mulder, Aat A; van der Velden, Pieter A; Vermeer, Maarten H; Willemze, Rein; Yan, Pearlly S; Huang, Tim H; Tensen, Cornelis P

    2005-06-10

    To analyze the occurrence of promoter hypermethylation in primary cutaneous T-cell lymphoma (CTCL) on a genome-wide scale, focusing on epigenetic alterations with pathogenetic significance. DNA isolated from biopsy specimens of 28 patients with CTCL, including aggressive CTCL entities (transformed mycosis fungoides and CD30-negative large T-cell lymphoma) and an indolent entity (CD30-positive large T-cell lymphoma), were investigated. For genome-wide DNA methylation screening, differential methylation hybridization using CpG island microarrays was applied, which allows simultaneous detection of the methylation status of 8640 CpG islands. Bisulfite sequence analysis was applied for confirmation and detection of hypermethylation of eight selected tumor suppressor genes. The DNA methylation patterns of CTCLs emerging from differential methylation hybridization analysis included 35 CpG islands hypermethylated in at least four of the 28 studied CTCL samples when compared with benign T-cell samples. Hypermethylation of the putative tumor suppressor genes BCL7a (in 48% of CTCL samples), PTPRG (27%), and thrombospondin 4 (52%) was confirmed and demonstrated to be associated with transcriptional downregulation. BCL7a was hypermethylated at a higher frequency in aggressive (64%) than in indolent (14%) CTCL entities. In addition, the promoters of the selected tumor suppressor genes p73 (48%), p16 (33%), CHFR (19%), p15 (10%), and TMS1 (10%) were hypermethylated in CTCL. Malignant T cells of patients with CTCL display widespread promoter hypermethylation associated with inactivation of several tumor suppressor genes involved in DNA repair, cell cycle, and apoptosis signaling pathways. In view of this, CTCL may be amenable to treatment with demethylating agents.

  9. Promoter hypermethylation of the DNA repair gene O(6)-methylguanine-DNA methyltransferase is associated with the presence of G:C to A:T transition mutations in p53 in human colorectal tumorigenesis.

    PubMed

    Esteller, M; Risques, R A; Toyota, M; Capella, G; Moreno, V; Peinado, M A; Baylin, S B; Herman, J G

    2001-06-15

    Defects in DNA repair may be responsible for the genesis of mutations in key genes in cancer cells. The tumor suppressor gene p53 is commonly mutated in human cancer by missense point mutations, most of them G:C to A:T transitions. A recognized cause for this type of change is spontaneous deamination of the methylcytosine. However, the persistence of a premutagenic O(6)-methylguanine can also be invoked. This last lesion is removed in the normal cell by the DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT). In many tumor types, epigenetic silencing of MGMT by promoter hypermethylation has been demonstrated and linked to the appearance of G to A mutations in the K-ras oncogene in colorectal tumors. To study the relevance of defective MGMT function by aberrant methylation in relation to the presence of p53 mutations, we studied 314 colorectal tumors for MGMT promoter hypermethylation and p53 mutational spectrum. Inactivation of MGMT by aberrant methylation was associated with the appearance of G:C to A:T transition mutations at p53 (Fischer's exact test, two-tailed; P = 0.01). Overall, MGMT methylated tumors displayed p53 transition mutations in 43 of 126 (34%) cases, whereas MGMT unmethylated tumors only showed G:C to A:T changes in 37 of 188 (19%) tumors. A more striking association was found in G:C to A:T transitions in non-CpG dinucleotides; 71% (12 of 17) of the total non-CpG transition mutations in p53 were observed in MGMT aberrantly methylated tumors (Fischer's exact test, two-tailed; P = 0.008). Our data suggest that epigenetic silencing of MGMT by promoter hypermethylation may lead to G:C to A:T transition mutations in p53.

  10. Genome hypermethylation in Pinus silvestris of Chernobyl--a mechanism for radiation adaptation?

    PubMed

    Kovalchuk, Olga; Burke, Paula; Arkhipov, Andrey; Kuchma, Nikolaj; James, S Jill; Kovalchuk, Igor; Pogribny, Igor

    2003-08-28

    Adaptation is a complex process by which populations of organisms respond to long-term environmental stresses by permanent genetic change. Here we present data from the natural "open-field" radiation adaptation experiment after the Chernobyl accident and provide the first evidence of the involvement of epigenetic changes in adaptation of a eukaryote-Scots pine (Pinus silvestris), to chronic radiation exposure. We have evaluated global genome methylation of control and radiation-exposed pine trees using a method based on cleavage by a methylation-sensitive HpaII restriction endonuclease that leaves a 5' guanine overhang and subsequent single nucleotide extension with labeled [3H] dCTP. We have found that genomic DNA of exposed pine trees was considerably hypermethylated. Moreover, hypermethylation appeared to be dependent upon the radiation dose absorbed by the trees. Such hypermethylation may be viewed as a defense strategy of plants that prevents genome instability and reshuffling of the hereditary material, allowing survival in an extreme environment. Further studies are clearly needed to analyze in detail the involvement of DNA methylation and other epigenetic mechanisms in the complex process of radiation stress and adaptive response.

  11. Preeclampsia is associated with hypermethylation of IGF-1 promoter mediated by DNMT1.

    PubMed

    Ma, Min; Zhou, Qiong-Jie; Xiong, Yu; Li, Bin; Li, Xiao-Tian

    2018-01-01

    Previous studies have demonstrated a dynamic epigenetic regulation of genes expression in placenta trophoblasts and a dynamic imbalance of DNA methylation and hydroxymethylation. Reduced IGF-1 has been observed in preeclampsia. This study was to investigate the interactive roles between IGF-1 and the global DNA methylation/hydroxymethylation, and the status of DNA methylation/hydroxymethylation and associated enzymes such as DNMTs and TETs in peeeclamptic placentas and hypoxic trophoblasts. It was found that IGF-1 was decreased in preeclamptic placentas and hypoxic trophoblasts when compared to the control group using immunohistochemisty, western blot, qRT-PCR and ELISA. Pyrophosphate sequencing showed IGF-1 promoter was significantly hypermethylated in preeclamptic placentas, which was responsible for reduced IGF-1 expression. Preeclamptic placentas and hypoxic trophoblasts were hypermethylated and hypohydroxymethylated accompanied by remarkably higher 5mC, DNMT1 and DNMT3b, and lower DNMT3a, 5hmC, TET1, TET2 and TET3 detected by immunohistochemisty, western blot, qRT-PCR and ELISA. Pearson's correlation confirmed a statistically significant negative correlation between IGF-1 and DNMT1. Furthermore, both treatment with 5-Aza-dc and DNMT1-siRNA significantly increased the expression of IGF-1 in HTR8 cells, indicating the potential mechanism of DNMT1-mediated DNA methylation in IGF-1 regulation. However, IGF-1 didn't change DNA methylation or hydroxymethylation. These findings suggest that preeclampsia is associated with hypermethylation of IGF-1 promoter mediated by DNMT1 and provide new insights into the diagnosis and treatment of preeclampsia.

  12. Preeclampsia is associated with hypermethylation of IGF-1 promoter mediated by DNMT1

    PubMed Central

    Ma, Min; Zhou, Qiong-Jie; Xiong, Yu; Li, Bin; Li, Xiao-Tian

    2018-01-01

    Previous studies have demonstrated a dynamic epigenetic regulation of genes expression in placenta trophoblasts and a dynamic imbalance of DNA methylation and hydroxymethylation. Reduced IGF-1 has been observed in preeclampsia. This study was to investigate the interactive roles between IGF-1 and the global DNA methylation/hydroxymethylation, and the status of DNA methylation/hydroxymethylation and associated enzymes such as DNMTs and TETs in peeeclamptic placentas and hypoxic trophoblasts. It was found that IGF-1 was decreased in preeclamptic placentas and hypoxic trophoblasts when compared to the control group using immunohistochemisty, western blot, qRT-PCR and ELISA. Pyrophosphate sequencing showed IGF-1 promoter was significantly hypermethylated in preeclamptic placentas, which was responsible for reduced IGF-1 expression. Preeclamptic placentas and hypoxic trophoblasts were hypermethylated and hypohydroxymethylated accompanied by remarkably higher 5mC, DNMT1 and DNMT3b, and lower DNMT3a, 5hmC, TET1, TET2 and TET3 detected by immunohistochemisty, western blot, qRT-PCR and ELISA. Pearson’s correlation confirmed a statistically significant negative correlation between IGF-1 and DNMT1. Furthermore, both treatment with 5-Aza-dc and DNMT1-siRNA significantly increased the expression of IGF-1 in HTR8 cells, indicating the potential mechanism of DNMT1-mediated DNA methylation in IGF-1 regulation. However, IGF-1 didn’t change DNA methylation or hydroxymethylation. These findings suggest that preeclampsia is associated with hypermethylation of IGF-1 promoter mediated by DNMT1 and provide new insights into the diagnosis and treatment of preeclampsia. PMID:29422991

  13. NR3C1 hypermethylation in depressed and bullied adolescents.

    PubMed

    Efstathopoulos, Paschalis; Andersson, Filip; Melas, Philippe A; Yang, Liu L; Villaescusa, J Carlos; Rȕegg, Joëlle; Ekström, Tomas J; Forsell, Yvonne; Galanti, Maria Rosaria; Lavebratt, Catharina

    2018-06-19

    The disruption of key epigenetic processes during critical periods of brain development can increase an individual's vulnerability to psychopathology later in life. For instance, DNA methylation in the glucocorticoid receptor gene (NR3C1) in adulthood is known to be associated with early-life adversities and has been suggested to mediate the development of stress-related disorders. However, the association between NR3C1 methylation and the emergence of internalizing symptoms in childhood and adolescence has not been studied extensively. In the present report, we used saliva DNA from a cohort of Swedish adolescents (13-14 years old; N = 1149) to measure NR3C1 methylation in the exon 1F region. Internalizing psychopathological symptoms were assessed using the Center for Epidemiologic Studies Depression Scale for Children (CES-DC). We found that NR3C1 hypermethylation was cross-sectionally associated with high score for internalizing symptoms in the whole group as well as among the female participants. In addition, an analysis of social environmental stressors revealed that reports of bullied or lacking friends were significantly associated with NR3C1 hypermethylation. This cross-sectional association of NR3C1 exon 1F hypermethylation with internalizing psychopathology in adolescents, as well as with bullying and lack of friends are novel results in this field. Longitudinal studies are needed to address whether NR3C1 methylation mediates the link between social stressors and psychopathology in adolescence.

  14. Low temperature-induced DNA hypermethylation attenuates expression of RhAG, an AGAMOUS homolog, and increases petal number in rose (Rosa hybrida).

    PubMed

    Ma, Nan; Chen, Wen; Fan, Tiangang; Tian, Yaran; Zhang, Shuai; Zeng, Daxing; Li, Yonghong

    2015-10-05

    Flower development is central to angiosperm reproduction and is regulated by a broad range of endogenous and exogenous stimuli. It has been well documented that ambient temperature plays a key role in controlling flowering time; however, the mechanisms by which temperature regulates floral organ differentiation remain largely unknown. In this study, we show that low temperature treatment significantly increases petal number in rose (Rosa hybrida) through the promotion of stamen petaloidy. Quantitative RT-PCR analysis revealed that the expression pattern of RhAG, a rose homolog of the Arabidopsis thaliana AGAMOUS C-function gene, is associated with low temperature regulated flower development. Silencing of RhAG mimicked the impact of low temperature treatments on petal development by significantly increasing petal number through an increased production of petaloid stamens. In situ hybridization studies further revealed that low temperature restricts its spatial expression area. Analysis of DNA methylation level showed that low temperature treatment enhances the methylation level of the RhAG promoter, and a specific promoter region that was hypermethylated at CHH loci under low temperature conditions, was identified by bisulfite sequencing. This suggests that epigenetic DNA methylation contributes to the ambient temperature modulation of RhAG expression. Our results provide highlights in the role of RhAG gene in petal number determination and add a new layer of complexity in the regulation of floral organ development. We propose that RhAG plays an essential role in rose flower patterning by regulating petal development, and that low temperatures increase petal number, at least in part, by suppressing RhAG expression via enhancing DNA CHH hypermethylation of the RhAG promoter.

  15. Genome-scale analysis of aberrant DNA methylation in colorectal cancer

    PubMed Central

    Hinoue, Toshinori; Weisenberger, Daniel J.; Lange, Christopher P.E.; Shen, Hui; Byun, Hyang-Min; Van Den Berg, David; Malik, Simeen; Pan, Fei; Noushmehr, Houtan; van Dijk, Cornelis M.; Tollenaar, Rob A.E.M.; Laird, Peter W.

    2012-01-01

    Colorectal cancer (CRC) is a heterogeneous disease in which unique subtypes are characterized by distinct genetic and epigenetic alterations. Here we performed comprehensive genome-scale DNA methylation profiling of 125 colorectal tumors and 29 adjacent normal tissues. We identified four DNA methylation–based subgroups of CRC using model-based cluster analyses. Each subtype shows characteristic genetic and clinical features, indicating that they represent biologically distinct subgroups. A CIMP-high (CIMP-H) subgroup, which exhibits an exceptionally high frequency of cancer-specific DNA hypermethylation, is strongly associated with MLH1 DNA hypermethylation and the BRAFV600E mutation. A CIMP-low (CIMP-L) subgroup is enriched for KRAS mutations and characterized by DNA hypermethylation of a subset of CIMP-H-associated markers rather than a unique group of CpG islands. Non-CIMP tumors are separated into two distinct clusters. One non-CIMP subgroup is distinguished by a significantly higher frequency of TP53 mutations and frequent occurrence in the distal colon, while the tumors that belong to the fourth group exhibit a low frequency of both cancer-specific DNA hypermethylation and gene mutations and are significantly enriched for rectal tumors. Furthermore, we identified 112 genes that were down-regulated more than twofold in CIMP-H tumors together with promoter DNA hypermethylation. These represent ∼7% of genes that acquired promoter DNA methylation in CIMP-H tumors. Intriguingly, 48/112 genes were also transcriptionally down-regulated in non-CIMP subgroups, but this was not attributable to promoter DNA hypermethylation. Together, we identified four distinct DNA methylation subgroups of CRC and provided novel insight regarding the role of CIMP-specific DNA hypermethylation in gene silencing. PMID:21659424

  16. Clinical accuracy of abnormal cell-free fetal DNA results for the sex chromosomes.

    PubMed

    Scibetta, Emily W; Gaw, Stephanie L; Rao, Rashmi R; Silverman, Neil S; Han, Christina S; Platt, Lawrence D

    2017-12-01

    To investigate factors associated with abnormal cell-free DNA (cfDNA) results for sex chromosomes (SCs). This is a retrospective cohort study of abnormal cfDNA results for SC at a referral practice from March 2013 to July 2015. Cell-free DNA results were abnormal if they were positive for SC aneuploidy (SCA), inconclusive, or discordant with ultrasound (US) findings. Primary outcome was concordance with karyotype or postnatal evaluation. Of 50 abnormal cfDNA results for SC, 31 patients (62%) were positive for SCA, 13 (26%) were inconclusive, and 6 (12%) were sex discordant on US. Of SCA results, 19 (61%) were reported as 45,X and 12 (39%) were SC trisomy. Abnormal karyotypes were confirmed in 8/23 (35%) of SC aneuploidy and 1/5 (20%) of inconclusive results. Abnormal SC cfDNA results were associated with in vitro fertilization (P = .001) and twins (P < .001). Sex discordance between cfDNA and US was associated with twin gestation (P < .001). In our cohort, abnormal SC cfDNA results were associated with in vitro fertilization and twins. Our results indicate cfDNA for sex prediction in twins of limited utility. Positive predictive value and sensitivity for SC determination were lower than previously reported. © 2017 John Wiley & Sons, Ltd.

  17. Stochastic anomaly of methylome but persistent SRY hypermethylation in disorder of sex development in canine somatic cell nuclear transfer

    PubMed Central

    Jeong, Young-Hee; Lu, Hanlin; Park, Chi-Hun; Li, Meiyan; Luo, Huijuan; Kim, Joung Joo; Liu, Siyang; Ko, Kyeong Hee; Huang, Shujia; Hwang, In Sung; Kang, Mi Na; Gong, Desheng; Park, Kang Bae; Choi, Eun Ji; Park, Jung Hyun; Jeong, Yeon Woo; Moon, Changjong; Hyun, Sang-Hwan; Kim, Nam Hyung; Jeung, Eui-Bae; Yang, Huanming; Hwang, Woo Suk; Gao, Fei

    2016-01-01

    Somatic cell nuclear transfer (SCNT) provides an excellent model for studying epigenomic reprogramming during mammalian development. We mapped the whole genome and whole methylome for potential anomalies of mutations or epimutations in SCNT-generated dogs with XY chromosomal sex but complete gonadal dysgenesis, which is classified as 78, XY disorder of sex development (DSD). Whole genome sequencing revealed no potential genomic variations that could explain the pathogenesis of DSD. However, extensive but stochastic anomalies of genome-wide DNA methylation were discovered in these SCNT DSD dogs. Persistent abnormal hypermethylation of the SRY gene was observed together with its down-regulated mRNA and protein expression. Failure of SRY expression due to hypermethylation was further correlated with silencing of a serial of testis determining genes, including SOX9, SF1, SOX8, AMH and DMRT1 in an early embryonic development stage at E34 in the XYDSD gonad, and high activation of the female specific genes, including FOXL2, RSPO1, CYP19A1, WNT4, ERα and ERβ, after one postnatal year in the ovotestis. Our results demonstrate that incomplete demethylation on the SRY gene is the driving cause of XYDSD in these XY DSD dogs, indicating a central role of epigenetic regulation in sex determination. PMID:27501986

  18. Promoter Hypermethylation of the ATM Gene as a Novel Biomarker for Breast Cancer

    PubMed

    Begam, Nasrin; Jamil, Kaiser; Raju, Suryanarayana G

    2017-11-26

    Background: Breast cancer may be induced by activation of protooncogenes to oncogenes and in many cases inactivation of tumor suppressor genes. Ataxia telangiectasia mutated (ATM) is an important tumor suppressor gene which plays central roles in the maintenance of genomic integrity by activating cell cycle checkpoints and promoting repair of double-strand breaks of DNA. In breast cancer, decrease ATM expression correlates with a poor outcome; however, the molecular mechanisms underlying downregulation are still unclear. Promoter hypermethylation may contribute in downregulation. Hence the present investigation was designed to evaluate promoter methylation and expression of the ATM gene in breast cancer cases, and to determine links with clinical and demographic manifestations, in a South Indian population. Methods: Tumor biopsy samples were collected from 50 pathologically confirmed sporadic breast cancer cases. DNA was isolated from tumor and adjacent non-tumorous regions, and sodium bisulfite conversion and methylation-specific PCR were performed using MS-PCR primers for the ATM promoter region. In addition, ATM mRNA expression was also analyzed for all samples using real-time PCR. Results: Fifty eight percent (58%) of cancer tissue samples showed promoter hypermethylation for the ATM gene, in contrast to only 4.44% of normal tissues (p= 0.0001). Furthermore, ATM promoter methylation was positively associated with age (p = 0.01), tumor size (p=0.045) and advanced stage of disease i.e. stages III and IV (p =0.019). An association between promoter hypermethylation and lower expression of ATM mRNA was also found (p=0.035). Conclusion: We report for the first time that promoter hypermethylation of ATM gene may be useful as a potential new biomarker for breast cancer, especially in the relatively young patients. Creative Commons Attribution License

  19. High-level dietary cadmium exposure is associated with global DNA hypermethylation in the gastropod hepatopancreas

    PubMed Central

    Popescu, Cristina; Draghici, George A.; Andrica, Florina-Maria; Privistirescu, Ionela A.; Gergen, Iosif I.; Stöger, Reinhard

    2017-01-01

    5-methylcytosine (5mC) is a key epigenetic mark which influences gene expression and phenotype. In vertebrates, this epigenetic mark is sensitive to Cd exposure, but there is no information linking such an event with changes in global 5mC levels in terrestrial gastropods despite their importance as excellentecotoxicological bioindicators of metal contamination. Therefore, we first evaluated total 5mC content in DNA of the hepatopancreas of adult Cantareus aspersus with the aim to determine whether this epigenetic mark is responsive to Cd exposure. The experiment was conducted under laboratory conditions and involved a continuous exposure, multiple dose- and time-point (14, 28, and 56 days) study design. Hepatopancreas cadmium levels were measured using Flame Atomic Absorption Spectrometry and the percentage of 5-mC in samples using an ELISA-based colorimetric assay. Snail death rates were also assessed. Our results, for the first time, reveal the presence of 5mC in C. aspersus and provide evidence for Cd-induced changes in global 5mC levels in DNA of gastropods and mollusks. Although less sensitive than tissue accumulation, DNA methylation levels responded in a dose- and time-dependent manner to dietary cadmium, with exposure dose having a much stronger effect than exposure duration. An obvious trend of increasing 5mC levels was observed starting at 28 days of exposure to the second highest dose and this trend persisted at the two highest treatments for close to one month, when the experiment was terminated after 56 days. Moreover, a strong association was identified between Cd concentrations in the hepatopancreas and DNA methylation levels in this organ. These data indicate an overall trend towards DNA hypermethylation with elevated Cd exposure. No consistent lethal effect was observed, irrespective of time point and Cd-dosage. Overall, our findings suggest that the total 5mC content in DNA of the hepatopancreas of land snails is responsive to sublethal Cd

  20. Immunofluorescent staining reveals hypermethylation of microchromosomes in the central bearded dragon, Pogona vitticeps.

    PubMed

    Domaschenz, Renae; Livernois, Alexandra M; Rao, Sudha; Ezaz, Tariq; Deakin, Janine E

    2015-01-01

    Studies of model organisms have demonstrated that DNA cytosine methylation and histone modifications are key regulators of gene expression in biological processes. Comparatively little is known about the presence and distribution of epigenetic marks in non-model amniotes such as non-avian reptiles whose genomes are typically packaged into chromosomes of distinct size classes. Studies of chicken karyotypes have associated the gene-richness and high GC content of microchromosomes with a distinct epigenetic landscape. To determine whether this is likely to be a common feature of amniote microchromosomes, we have analysed the distribution of epigenetic marks using immunofluorescence on metaphase chromosomes of the central bearded dragon (Pogona vitticeps). This study is the first to study the distribution of epigenetic marks on non-avian reptile chromosomes. We observed an enrichment of DNA cytosine methylation, active modifications H3K4me2 and H3K4me3, as well as the repressive mark H3K27me3 in telomeric regions on macro and microchromosomes. Microchromosomes were hypermethylated compared to macrochromosomes, as they are in chicken. However, differences between macro- and microchromosomes for histone modifications associated with actively transcribed or repressed DNA were either less distinct or not detectable. Hypermethylation of microchromosomes compared to macrochromosomes is a shared feature between P. vitticeps and avian species. The lack of the clear distinction between macro- and microchromosome staining patterns for active and repressive histone modifications makes it difficult to determine at this stage whether microchrosome hypermethylation is correlated with greater gene density as it is in aves, or associated with the greater GC content of P. vitticeps microchromosomes compared to macrochromosomes.

  1. Combination of methylated-DNA precipitation and methylation-sensitive restriction enzymes (COMPARE-MS) for the rapid, sensitive and quantitative detection of DNA methylation.

    PubMed

    Yegnasubramanian, Srinivasan; Lin, Xiaohui; Haffner, Michael C; DeMarzo, Angelo M; Nelson, William G

    2006-02-09

    Hypermethylation of CpG island (CGI) sequences is a nearly universal somatic genome alteration in cancer. Rapid and sensitive detection of DNA hypermethylation would aid in cancer diagnosis and risk stratification. We present a novel technique, called COMPARE-MS, that can rapidly and quantitatively detect CGI hypermethylation with high sensitivity and specificity in hundreds of samples simultaneously. To quantitate CGI hypermethylation, COMPARE-MS uses real-time PCR of DNA that was first digested by methylation-sensitive restriction enzymes and then precipitated by methyl-binding domain polypeptides immobilized on a magnetic solid matrix. We show that COMPARE-MS could detect five genome equivalents of methylated CGIs in a 1000- to 10,000-fold excess of unmethylated DNA. COMPARE-MS was used to rapidly quantitate hypermethylation at multiple CGIs in >155 prostate tissues, including benign and malignant prostate specimens, and prostate cell lines. This analysis showed that GSTP1, MDR1 and PTGS2 CGI hypermethylation as determined by COMPARE-MS could differentiate between malignant and benign prostate with sensitivities >95% and specificities approaching 100%. This novel technology could significantly improve our ability to detect CGI hypermethylation.

  2. Genetic Determinants for Promoter Hypermethylation in the Lungs of Smokers: A Candidate Gene-Based Study

    PubMed Central

    Leng, Shuguang; Stidley, Christine A.; Liu, Yushi; Edlund, Christopher K.; Willink, Randall P.; Han, Younghun; Landi, Maria Teresa; Thun, Michael; Picchi, Maria A.; Bruse, Shannon E.; Crowell, Richard E.; Van Den Berg, David; Caporaso, Neil E.; Amos, Christopher I.; Siegfried, Jill M.; Tesfaigzi, Yohannes; Gilliland, Frank D.; Belinsky, Steven A.

    2011-01-01

    The detection of tumor suppressor gene promoter methylation in sputum-derived exfoliated cells predicts early lung cancer. Here we identified genetic determinants for this epigenetic process and examined their biological effects on gene regulation. A two-stage approach involving discovery and replication was employed to assess the association between promoter hypermethylation of a 12-gene panel and common variation in 40 genes involved in carcinogen metabolism, regulation of methylation, and DNA damage response in members of the Lovelace Smokers Cohort (n=1434). Molecular validation of three identified variants was conducted using primary bronchial epithelial cells. Association of study-wide significance (P<8.2×10−5) was identified for rs1641511, rs3730859, and rs1883264 in TP53, LIG1, and BIK, respectively. These SNPs were significantly associated with altered expression of the corresponding genes in primary bronchial epithelial cells. In addition, rs3730859 in LIG1 was also moderately associated with increased risk for lung cancer among Caucasian smokers. Together, our findings suggest that genetic variation in DNA replication and apoptosis pathways impacts the propensity for gene promoter hypermethylation in the aerodigestive tract of smokers. The incorporation of genetic biomarkers for gene promoter hypermethylation with clinical and somatic markers may improve risk assessment models for lung cancer. PMID:22139380

  3. Abnormal Expressions of DNA Glycosylase Genes NEIL1, NEIL2, and NEIL3 Are Associated with Somatic Mutation Loads in Human Cancer.

    PubMed

    Shinmura, Kazuya; Kato, Hisami; Kawanishi, Yuichi; Igarashi, Hisaki; Goto, Masanori; Tao, Hong; Inoue, Yusuke; Nakamura, Satoki; Misawa, Kiyoshi; Mineta, Hiroyuki; Sugimura, Haruhiko

    2016-01-01

    The effects of abnormalities in the DNA glycosylases NEIL1, NEIL2, and NEIL3 on human cancer have not been fully elucidated. In this paper, we found that the median somatic total mutation loads and the median somatic single nucleotide mutation loads exhibited significant inverse correlations with the median NEIL1 and NEIL2 expression levels and a significant positive correlation with the median NEIL3 expression level using data for 13 cancer types from the Cancer Genome Atlas (TCGA) database. A subset of the cancer types exhibited reduced NEIL1 and NEIL2 expressions and elevated NEIL3 expression, and such abnormal expressions of NEIL1, NEIL2, and NEIL3 were also significantly associated with the mutation loads in cancer. As a mechanism underlying the reduced expression of NEIL1 in cancer, the epigenetic silencing of NEIL1 through promoter hypermethylation was found. Finally, we investigated the reason why an elevated NEIL3 expression level was associated with an increased number of somatic mutations in cancer and found that NEIL3 expression was positively correlated with the expression of APOBEC3B, a potent inducer of mutations, in diverse cancers. These results suggested that the abnormal expressions of NEIL1, NEIL2, and NEIL3 are involved in cancer through their association with the somatic mutation load.

  4. Helicobacter pylori CagA induces tumor suppressor gene hypermethylation by upregulating DNMT1 via AKT-NFκB pathway in gastric cancer development.

    PubMed

    Zhang, Bao-gui; Hu, Lei; Zang, Ming-de; Wang, He-xiao; Zhao, Wei; Li, Jian-fang; Su, Li-ping; Shao, Zhifeng; Zhao, Xiaodong; Zhu, Zheng-gang; Yan, Min; Liu, Bingya

    2016-03-01

    Methylation of CpG islands in tumor suppressor gene prompter is one of the most characteristic abnormalities in Helicobacter pylori (HP)-associated gastric carcinoma (GC). Here, we investigated the pathogenic and molecular mechanisms underlying hypermethylation of tumor suppressor genes in HP induced GC development. We found that tumor suppressor genes hypermethylation, represented by MGMT, positively correlated with CagA in clinical specimens, gastric tissues from HP infected C57 mice and GC cell lines transfected by CagA or treated by HP infection. CagA enhanced PDK1 and AKT interaction and increased AKT phosphorylation. The P-AKT subsequent activated NFκB, which then bound to DNMT1 promoter and increased its expression. Finally, the upregulated DNMT1 promoted tumor suppressor genes hypermethylation with MGMT as a representative. In conclusion, CagA increased tumor suppressor genes hypermethylation via stimulating DNMT1 expression through the AKT-NFκB pathway.

  5. Helicobacter pylori CagA induces tumor suppressor gene hypermethylation by upregulating DNMT1 via AKT-NFκB pathway in gastric cancer development

    PubMed Central

    Wang, He-xiao; Zhao, Wei; Li, Jian-fang; Su, Li-ping; Shao, Zhifeng; Zhao, Xiaodong; Zhu, Zheng-gang; Yan, Min; Liu, Bingya

    2016-01-01

    Methylation of CpG islands in tumor suppressor gene prompter is one of the most characteristic abnormalities in Helicobacter pylori (HP)-associated gastric carcinoma (GC). Here, we investigated the pathogenic and molecular mechanisms underlying hypermethylation of tumor suppressor genes in HP induced GC development. We found that tumor suppressor genes hypermethylation, represented by MGMT, positively correlated with CagA in clinical specimens, gastric tissues from HP infected C57 mice and GC cell lines transfected by CagA or treated by HP infection. CagA enhanced PDK1 and AKT interaction and increased AKT phosphorylation. The P-AKT subsequent activated NFκB, which then bound to DNMT1 promoter and increased its expression. Finally, the upregulated DNMT1 promoted tumor suppressor genes hypermethylation with MGMT as a representative. In conclusion, CagA increased tumor suppressor genes hypermethylation via stimulating DNMT1 expression through the AKT-NFκB pathway. PMID:26848521

  6. Gene-body hypermethylation of ATM in peripheral blood DNA of bilateral breast cancer patients

    PubMed Central

    Flanagan, James M.; Munoz-Alegre, Marta; Henderson, Stephen; Tang, Thomas; Sun, Ping; Johnson, Nichola; Fletcher, Olivia; dos Santos Silva, Isabel; Peto, Julian; Boshoff, Chris; Narod, Steven; Petronis, Arturas

    2009-01-01

    Bilaterality of breast cancer is an indicator of constitutional cancer susceptibility; however, the molecular causes underlying this predisposition in the majority of cases is not known. We hypothesize that epigenetic misregulation of cancer-related genes could partially account for this predisposition. We have performed methylation microarray analysis of peripheral blood DNA from 14 women with bilateral breast cancer compared with 14 unaffected matched controls throughout 17 candidate breast cancer susceptibility genes including BRCA1, BRCA2, CHEK2, ATM, ESR1, SFN, CDKN2A, TP53, GSTP1, CDH1, CDH13, HIC1, PGR, SFRP1, MLH1, RARB and HSD17B4. We show that the majority of methylation variability is associated with intragenic repetitive elements. Detailed validation of the tiled region around ATM was performed by bisulphite modification and pyrosequencing of the same samples and in a second set of peripheral blood DNA from 190 bilateral breast cancer patients compared with 190 controls. We show significant hypermethylation of one intragenic repetitive element in breast cancer cases compared with controls (P = 0.0017), with the highest quartile of methylation associated with a 3-fold increased risk of breast cancer (OR 3.20, 95% CI 1.78–5.86, P = 0.000083). Increased methylation of this locus is associated with lower steady-state ATM mRNA level and correlates with age of cancer patients but not controls, suggesting a combined age–phenotype-related association. This research demonstrates the potential for gene-body epigenetic misregulation of ATM and other cancer-related genes in peripheral blood DNA that may be useful as a novel marker to estimate breast cancer risk. Accession numbers: The microarray data and associated .BED and .WIG files can be accessed through Gene Expression Omnibus accession number: GSE14603. PMID:19153073

  7. [Relationship of abnormal sperm DNA methylation with early spontaneous abortion].

    PubMed

    Pan, Lian-Jun; Ma, Jie-Hua; Zhang, Feng-Lei; Zhao, Dan; Pan, Feng; Zhang, Xing-Yuan

    2016-10-01

    To investigate the relationship between the abnormal sperm DNA methylation level and early spontaneous abortion. We randomly selected 98 males who met the inclusion criteria and whose wives suffered from unexplained abortion or embryo abortion, and included another 46 normal healthy men present for pre-pregnancy check-up as controls. We examined the semen quality and sperm morphology, obtained the sperm DNA fragmentation index (DFI) by modified sperm chromatin dispersion, and measured the sperm DNA methylation level using the methylated DNA quantification kit and the colorimetric method. Compared with the normal controls, the men in the unexplained abortion group showed a significantly lower rate of big-halo sperm ([45.50 ± 26.27] vs [36.49 ± 23.06]%, P = 0.038), a higher rate of abnormal-head sperm ([77.08± 12.21] vs [81.09± 10.89]%, P = 0.049), and a lower level of sperm DNA methylation ([0.47 ± 0.33] vs [0.36 ± 0.26] ng/μl, P = 0.035). The sperm DNA methylation level was positively correlated with the percentage of big-halo sperm (OR=0.546, P<0.01). Multivariate regression analysis manifested that sperm head abnormality was an independent risk factor of early spontaneous abortion or embryo abortion (OR=1.032, P = 0.049), while the high methylation level was protective factor against early spontaneous abortion or embryo abortion (OR=0.244, P = 0.03). The abnormal level of sperm DNA methylation may be one of the important reasons for early spontaneous abortion or embryo abortion.

  8. Differential role of gene hypermethylation in adenocarcinomas, squamous cell carcinomas and cervical intraepithelial lesions of the uterine cervix.

    PubMed

    Blanco-Luquin, Idoia; Guarch, Rosa; Ojer, Amaya; Pérez-Janices, Noemí; Martín-Sánchez, Esperanza; Maria-Ruiz, Sergio; Monreal-Santesteban, Iñaki; Blanco-Fernandez, Laura; Pernaut-Leza, Eduardo; Escors, David; Guerrero-Setas, David

    2015-09-01

    Cervical cancer is the third most common cancer in women worldwide. The hypermethylation of P16, TSLC-1 and TSP-1 genes was analyzed in squamous cell carcinomas (SCC), cervical intraepithelial lesions (CIN) and adenocarcinomas (ADC) of the uterine cervix (total 181 lesions). Additionally human papillomavirus (HPV) type, EPB41L3, RASSF1 and RASSF2 hypermethylation were tested in ADC and the results were compared with those obtained previously by our group in SCC. P16, TSLC-1 and TSP-1 hypermethylation was more frequent in SCCs than in CINs. These percentages and the corresponding ones for EPB41L3, RASSF1 and RASSF2 genes were also higher in SCCs than in ADCs, except for P16. The presence of HPV in ADCs was lower than reported previously in SCC and CIN. Patients with RASSF1A hypermethylation showed significantly longer disease-free survival (P = 0.015) and overall survival periods (P = 0.009) in ADC patients. To our knowledge, this is the first description of the EPB41L3 and RASSF2 hypermethylation in ADCs. These results suggest that the involvement of DNA hypermethylation in cervical cancer varies depending on the histological type, which might contribute to explaining the different prognosis of patients with these types of tumors. © 2015 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd.

  9. DNA hypermethylation as a predictor of extramural vascular invasion (EMVI) in rectal cancer.

    PubMed

    Kokelaar, Rory F; Jones, Huw G; Williamson, Jeremy; Williams, Namor; Griffiths, A Paul; Beynon, John; Jenkins, Gareth J; Harris, Dean A

    2018-03-04

    DNA hypermethylation in gene promoter regions (CpG islands) is emerging as an important pathway in colorectal cancer tumourigenesis. Whilst genetic mutations have been associated with extramural vascular invasion (EMVI) in rectal cancer, no such association has yet been made with epigenetic factors. 100 consecutive neoadjuvant-naïve patients undergoing curative surgery for rectal were classified according to the presence or absence of EMVI on histopathological examination. DNA was extracted from tumours and subjected to bisulfite conversion and methylation-specific PCR to determine CIMP status (high, intermediate, or low; according to a validated panel of 8 genes). CIMP status was correlated with EMVI status, histopathological, clinical, and demographic variables, in addition to overall (OS) and disease free (DFS) survival. 51 patients were characterised as CIMP-low, 48 CIMP-intermediate, and one patient CIMP-high. EMVI-positivity was associated with CIMP-intermediate epigenotype (p < 0.001). Patients with EMVI-positive tumours were found to have significantly more advanced disease by pT, pN, and pAJCC categorisation (p = 0.002, p < 0.001, and = p < 0.001, respectively). EMVI-positivity was significantly associated with the requirement for adjuvant chemotherapy (p < 0.001), and worse DFS but not OS (p = 0.012 and p = 0.052). Given the association between CIMP-intermediate epigenotype and EMVI-positivity, and the subsequent disadvantage in pathological stage, requirement for adjuvant therapy and worse survival, tumour epigenotyping could potentially play an important role in personalising patients' cancer care. Further work is required to understand the mechanisms that underlie the observed effect, with the hope that they may provide novel opportunities for intervention and inform treatment decisions in rectal cancer.

  10. Smoking is associated with hypermethylation of the APC 1A promoter in colorectal cancer: the ColoCare Study.

    PubMed

    Barrow, Timothy M; Klett, Hagen; Toth, Reka; Böhm, Jürgen; Gigic, Biljana; Habermann, Nina; Scherer, Dominique; Schrotz-King, Petra; Skender, Stephanie; Abbenhardt-Martin, Clare; Zielske, Lin; Schneider, Martin; Ulrich, Alexis; Schirmacher, Peter; Herpel, Esther; Brenner, Hermann; Busch, Hauke; Boerries, Melanie; Ulrich, Cornelia M; Michels, Karin B

    2017-11-01

    Smoking tobacco is a known risk factor for the development of colorectal cancer and for mortality associated with the disease. Smoking has been reported to be associated with changes in DNA methylation in blood and in lung tumour tissues, although there has been scant investigation of how epigenetic factors may be implicated in the increased risk of developing colorectal cancer. To identify epigenetic changes associated with smoking behaviours, we performed epigenome-wide analysis of DNA methylation in colorectal tumours from 36 never-smokers, 47 former smokers, and 13 active smokers, and in adjacent mucosa from 49 never-smokers, 64 former smokers, and 18 active smokers. Our analyses identified 15 CpG sites within the APC 1A promoter that were significantly hypermethylated and 14 CpG loci within the NFATC1 gene body that were significantly hypomethylated (pLIS < 1 × 10 -5 ) in the tumours of active smokers. The APC 1A promoter was hypermethylated in 7 of 36 tumours from never-smokers (19%), 12 of 47 tumours from former smokers (26%), and 8 of 13 tumours from active smokers (62%). Promoter hypermethylation was positively associated with duration of smoking (Spearman rank correlation, ρ = 0.26, p = 0.03) and was confined to tumours, with hypermethylation never being observed in adjacent mucosa. Further analysis of adjacent mucosa revealed significant hypomethylation of four loci associated with the TNXB gene in tissue from active smokers. Our findings provide exploratory evidence for hypermethylation of the key tumour suppressor gene APC being implicated in smoking-associated colorectal carcinogenesis. Further work is required to establish the validity of our observations in independent cohorts. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  11. DNA hypermethylation profiles in squamous cell carcinoma of the vulva.

    PubMed

    Stephen, Josena K; Chen, Kang Mei; Raitanen, Misa; Grénman, Seija; Worsham, Maria J

    2009-01-01

    Gene silencing through promoter hypermethylation is a growing concept in the development of human cancers. In this study, we examined the contribution of aberrant methylation of promoter regions in methylation-prone tumor suppressors to the pathogenesis of vulvar cancer. Thirteen cell lines from 12 patients with squamous cell carcinoma of the vulva were evaluated for aberrant methylation status and gene copy number alterations, concomitantly, using the methylation-specific multiplex ligation-dependent probe amplification assay. Of the 22 tumor suppressor genes examined, aberrant methylation was observed for 9 genes: tumor protein p73 (TP73), fragile histidine triad (FHIT), von Hippel-Lindau (VHL), adenomatosis polyposis coli (APC), estrogen receptor 1 (ESR1), cyclin-dependent kinase inhibitor 2B (CDKN2B), death-associated protein kinase 1 (DAPK1), glutathione S-transferase pi (GSTP1), and immunoglobin superfamily, member 4 (IGSF4). The most frequently methylated genes included TP73 in 9 of 13 cell lines, and IGSF4, DAPK1, and FHIT in 3 of 13 cell lines. Methylation-specific polymerase chain reaction was performed for TP73 and FHIT to confirm aberrant methylation by methylation-specific multiplex ligation-dependent probe amplification. In the context of gene copy number and methylation status, both copies of the TP73 gene were hypermethylated. Loss or decreased mRNA expression of TP73 and IGSF4 by reverse transcription polymerase chain reaction confirmed aberrant methylation. Frequent genetic alterations of loss and gain of gene copy number included gain of GSTP1 and multiple endocrine neoplasia type 1 (MEN1), and loss of malignant fibrous histiocytoma amplified sequence 1 (MFHAS1) and IGSF4 in over 50% of the squamous cell carcinoma of the vulva cell lines. These findings underscore the contribution of both genetic and epigenetic events to the underlying pathogenesis of squamous cell carcinoma of the vulva.

  12. Identification and functional validation of HPV-mediated hypermethylation in head and neck squamous cell carcinoma

    PubMed Central

    2013-01-01

    Background Human papillomavirus-positive (HPV+) head and neck squamous cell carcinoma (HNSCC) represents a distinct clinical and epidemiological condition compared with HPV-negative (HPV-) HNSCC. To test the possible involvement of epigenetic modulation by HPV in HNSCC, we conducted a genome-wide DNA-methylation analysis. Methods Using laser-capture microdissection of 42 formalin-fixed paraffin wax-embedded (FFPE) HNSCCs, we generated DNA-methylation profiles of 18 HPV+ and 14 HPV- samples, using Infinium 450 k BeadArray technology. Methylation data were validated in two sets of independent HPV+/HPV- HNSCC samples (fresh-frozen samples and cell lines) using two independent methods (Infinium 450 k and whole-genome methylated DNA immunoprecipitation sequencing (MeDIP-seq)). For the functional analysis, an HPV- HNSCC cell line was transduced with lentiviral constructs containing the two HPV oncogenes (E6 and E7), and effects on methylation were assayed using the Infinium 450 k technology. Results and discussion Unsupervised clustering over the methylation variable positions (MVPs) with greatest variation showed that samples segregated in accordance with HPV status, but also that HPV+ tumors are heterogeneous. MVPs were significantly enriched at transcriptional start sites, leading to the identification of a candidate CpG island methylator phenotype in a sub-group of the HPV+ tumors. Supervised analysis identified a strong preponderance (87%) of MVPs towards hypermethylation in HPV+ HNSCC. Meta-analysis of our HNSCC and publicly available methylation data in cervical and lung cancers confirmed the observed DNA-methylation signature to be HPV-specific and tissue-independent. Grouping of MVPs into functionally more significant differentially methylated regions identified 43 hypermethylated promoter DMRs, including for three cadherins of the Polycomb group target genes. Integration with independent expression data showed strong negative correlation, especially for the

  13. RUNX3 promoter hypermethylation is frequent in leukaemia cell lines and associated with acute myeloid leukaemia inv(16) subtype.

    PubMed

    Estécio, Marcos R H; Maddipoti, Sirisha; Bueso-Ramos, Carlos; DiNardo, Courtney D; Yang, Hui; Wei, Yue; Kondo, Kimie; Fang, Zhihong; Stevenson, William; Chang, Kun-Sang; Pierce, Sherry A; Bohannan, Zachary; Borthakur, Gautam; Kantarjian, Hagop; Garcia-Manero, Guillermo

    2015-05-01

    Correlative and functional studies support the involvement of the RUNX gene family in haematological malignancies. To elucidate the role of epigenetics in RUNX inactivation, we evaluated promoter DNA methylation of RUNX1, 2, and 3 in 23 leukaemia cell lines and samples from acute myeloid leukaemia (AML), acute lymphocytic leukaemia (ALL) and myelodysplatic syndromes (MDS) patients. RUNX1 and RUNX2 gene promoters were mostly unmethylated in cell lines and clinical samples. Hypermethylation of RUNX3 was frequent among cell lines (74%) and highly variable among patient samples, with clear association to cytogenetic status. High frequency of RUNX3 hypermethylation (85% of the 20 studied cases) was found in AML patients with inv(16)(p13.1q22) compared to other AML subtypes (31% of the other 49 cases). RUNX3 hypermethylation was also frequent in ALL (100% of the six cases) but low in MDS (21%). In support of a functional role, hypermethylation of RUNX3 was correlated with low levels of protein, and treatment of cell lines with the DNA demethylating agent, decitabine, resulted in mRNA re-expression. Furthermore, relapse-free survival of non-inv(16)(p13.1q22) AML patients without RUNX3 methylation was significantly better (P = 0·016) than that of methylated cases. These results suggest that RUNX3 silencing is an important event in inv(16)(p13.1q22) leukaemias. © 2015 John Wiley & Sons Ltd.

  14. Loss of maintenance DNA methylation results in abnormal DNA origin firing during DNA replication

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Haruta, Mayumi; Shimada, Midori, E-mail: midorism@med.nagoya-cu.ac.jp; Nishiyama, Atsuya

    The mammalian maintenance methyltransferase DNMT1 [DNA (cytosine-5-)-methyltransferase 1] mediates the inheritance of the DNA methylation pattern during replication. Previous studies have shown that depletion of DNMT1 causes a severe growth defect and apoptosis in differentiated cells. However, the detailed mechanisms behind this phenomenon remain poorly understood. Here we show that conditional ablation of Dnmt1 in murine embryonic fibroblasts (MEFs) resulted in an aberrant DNA replication program showing an accumulation of late-S phase replication and causing severely defective growth. Furthermore, we found that the catalytic activity and replication focus targeting sequence of DNMT1 are required for a proper DNA replication program.more » Taken together, our findings suggest that the maintenance of DNA methylation by DNMT1 plays a critical role in proper regulation of DNA replication in mammalian cells. - Highlights: • DNMT1 depletion results in an abnormal DNA replication program. • Aberrant DNA replication is independent of the DNA damage checkpoint in DNMT1cKO. • DNMT1 catalytic activity and RFT domain are required for proper DNA replication. • DNMT1 catalytic activity and RFT domain are required for cell proliferation.« less

  15. A DNA 3′-phosphatase functions in active DNA demethylation in Arabidopsis

    PubMed Central

    Martínez-Macías, María Isabel; Qian, Weiqiang; Miki, Daisuke; Pontes, Olga; Liu, Yunhua; Tang, Kai; Liu, Renyi; Morales-Ruiz, Teresa; Ariza, Rafael R.; Roldán-Arjona, Teresa; Zhu, Jian-Kang

    2012-01-01

    SUMMARY DNA methylation is an important epigenetic mark established by the combined actions of methylation and demethylation reactions. Plants use a base excision repair pathway for active DNA demethylation. After 5-methylcytosine removal, the Arabidopsis DNA glycosylase/lyase ROS1 incises the DNA backbone and part of the product has a single-nucleotide gap flanked by 3′- and 5′-phosphate termini. Here we show that the DNA phosphatase ZDP removes the blocking 3′-phosphate, allowing subsequent DNA polymerization and ligation steps needed to complete the repair reactions. ZDP and ROS1 interact in vitro and co-localize in vivo in nucleoplasmic foci. Extracts from zdp mutant plants are unable to complete DNA demethylation in vitro, and the mutations cause DNA hypermethylation and transcriptional silencing of a reporter gene. Genome-wide methylation analysis in zdp mutant plants identified hundreds of hypermethylated endogenous loci. Our results show that ZDP functions downstream of ROS1 in one branch of the active DNA demethylation pathway. PMID:22325353

  16. Promoter hypermethylation in Indian primary oral squamous cell carcinoma

    PubMed Central

    Kaur, Jatinder; Demokan, Semra; Tripathi, Satyendra Chandra; Macha, Muzafar Ahmad; Begum, Shahnaz; Califano, Joseph A.; Ralhan, Ranju

    2010-01-01

    We evaluated promoter hypermethylation of a panel of tumor suppressor genes as a means to detect epigenetic alterations in oral squamous cell carcinomas (OSCC) of Indian-origin and compare with North-American head and neck squamous cell carcinomas (HNSCC). Quantitative-methylation-specific PCR was used to investigate the promoter methylation status of DCC, EDNRB, p16INK4a and KIF1A in 92 OSCC, and compared to 48 paired normal tissues and 30 saliva and sera samples from healthy control subjects. Aberrant methylation of at-least one of these genes was detected in 74/92 (80.4%) OSCC; 72.8% at EDNRB, 71.7% at KIF1A, 47.8% at p16INK4a and 58.7% at DCC; and in 5 of 48 (10.4%) normal oral tissues. None of the saliva and sera samples from controls exhibited DNA methylation in these four target genes. Thirty-two of 72 node positive cases harbored p16INK4a and DCC hypermethylation (p = 0.005). Thus, promoter hypermethylation in genes analyzed herein is a common event in Indian OSCC and may represent promising markers for the molecular staging of OSCC patients. We found higher frequency of p16INK4a methylation (47.8%) in this Indian cohort in comparison with a North-American cohort (37.5%). In conclusion, aberrant methylation of EDNRB, KIF1A, DCC and p16INK4a genes is a common event in Indian OSCC, suggesting that epigenetic alterations of these genes warrant validation in larger studies for their potential use as biomarkers. PMID:20473870

  17. Cell-free DNA screening in clinical practice: abnormal autosomal aneuploidy and microdeletion results.

    PubMed

    Valderramos, Stephanie G; Rao, Rashmi R; Scibetta, Emily W; Silverman, Neil S; Han, Christina S; Platt, Lawrence D

    2016-11-01

    Since its commercial release in 2011 cell-free DNA screening has been rapidly adopted as a routine prenatal genetic test. However, little is known about its performance in actual clinical practice. We sought to investigate factors associated with the accuracy of abnormal autosomal cell-free DNA results. We conducted a retrospective cohort study of 121 patients with abnormal cell-free DNA results from a referral maternal-fetal medicine practice from March 2013 through July 2015. Patients were included if cell-free DNA results for trisomy 21, trisomy 18, trisomy 13, or microdeletions (if reported by the laboratory) were positive or nonreportable. The primary outcome was confirmed aneuploidy or microarray abnormality on either prenatal or postnatal karyotype or microarray. Secondary outcomes were identifiable associations with in vitro fertilization, twins, ultrasound findings, testing platform, and testing laboratory. Kruskal-Wallis or Fisher exact tests were used as appropriate. A total of 121 patients had abnormal cell-free DNA results for trisomy 21, trisomy 18, trisomy 13, and/or microdeletions. In all, 105 patients had abnormal cell-free DNA results for trisomy 21, trisomy 18, and trisomy 13. Of these, 92 (87.6%) were positive and 13 (12.4%) were nonreportable. The results of the 92 positive cell-free DNA were for trisomy 21 (48, 52.2%), trisomy 18 (22, 23.9%), trisomy 13 (17, 18.5%), triploidy (2, 2.2%), and positive for >1 parameter (3, 3.3%). Overall, the positive predictive value of cell-free DNA was 73.5% (61/83; 95% confidence interval, 63-82%) for all trisomies (by chromosome: trisomy 21, 83.0% [39/47; 95% confidence interval, 69-92%], trisomy 18, 65.0% [13/20; 95% confidence interval, 41-84%], and trisomy 13, 43.8% [7/16; 95% confidence interval, 21-70%]). Abnormal cell-free DNA results were associated with positive serum screening (by group: trisomy 21 [17/48, 70.8%]; trisomy 18 [7/22, 77.8%]; trisomy 13 [3/17, 37.5%]; nonreportable [2/13, 16.7%]; P

  18. DNA methylation aberrancies as a guide for surveillance and treatment of human cancers

    PubMed Central

    Liang, Gangning; Weisenberger, Daniel J.

    2017-01-01

    ABSTRACT DNA methylation aberrancies are hallmarks of human cancers and are characterized by global DNA hypomethylation of repetitive elements and non-CpG rich regions concomitant with locus-specific DNA hypermethylation. DNA methylation changes may result in altered gene expression profiles, most notably the silencing of tumor suppressors, microRNAs, endogenous retorviruses and tumor antigens due to promoter DNA hypermethylation, as well as oncogene upregulation due to gene-body DNA hypermethylation. Here, we review DNA methylation aberrancies in human cancers, their use in cancer surveillance and the interplay between DNA methylation and histone modifications in gene regulation. We also summarize DNA methylation inhibitors and their therapeutic effects in cancer treatment. In this context, we describe the integration of DNA methylation inhibitors with conventional chemotherapies, DNA repair inhibitors and immune-based therapies, to bring the epigenome closer to its normal state and increase sensitivity to other therapeutic agents to improve patient outcome and survival. PMID:28358281

  19. Genome-Wide DNA Methylation Indicates Silencing of Tumor Suppressor Genes in Uterine Leiomyoma

    PubMed Central

    Navarro, Antonia; Yin, Ping; Monsivais, Diana; Lin, Simon M.; Du, Pan; Wei, Jian-Jun; Bulun, Serdar E.

    2012-01-01

    Background Uterine leiomyomas, or fibroids, represent the most common benign tumor of the female reproductive tract. Fibroids become symptomatic in 30% of all women and up to 70% of African American women of reproductive age. Epigenetic dysregulation of individual genes has been demonstrated in leiomyoma cells; however, the in vivo genome-wide distribution of such epigenetic abnormalities remains unknown. Principal Findings We characterized and compared genome-wide DNA methylation and mRNA expression profiles in uterine leiomyoma and matched adjacent normal myometrial tissues from 18 African American women. We found 55 genes with differential promoter methylation and concominant differences in mRNA expression in uterine leiomyoma versus normal myometrium. Eighty percent of the identified genes showed an inverse relationship between DNA methylation status and mRNA expression in uterine leiomyoma tissues, and the majority of genes (62%) displayed hypermethylation associated with gene silencing. We selected three genes, the known tumor suppressors KLF11, DLEC1, and KRT19 and verified promoter hypermethylation, mRNA repression and protein expression using bisulfite sequencing, real-time PCR and western blot. Incubation of primary leiomyoma smooth muscle cells with a DNA methyltransferase inhibitor restored KLF11, DLEC1 and KRT19 mRNA levels. Conclusions These results suggest a possible functional role of promoter DNA methylation-mediated gene silencing in the pathogenesis of uterine leiomyoma in African American women. PMID:22428009

  20. Abnormal DNA methylation may contribute to the progression of osteosarcoma.

    PubMed

    Chen, Xiao-Gang; Ma, Liang; Xu, Jia-Xin

    2018-01-01

    The identification of optimal methylation biomarkers to achieve maximum diagnostic ability remains a challenge. The present study aimed to elucidate the potential molecular mechanisms underlying osteosarcoma (OS) using DNA methylation analysis. Based on the GSE36002 dataset obtained from the Gene Expression Omnibus database, differentially methylated genes were extracted between patients with OS and controls using t‑tests. Subsequently, hierarchical clustering was performed to segregate the samples into two distinct clusters, OS and normal. Gene Ontology (GO) and pathway enrichment analyses for differentially methylated genes were performed using the Database for Annotation, Visualization and Integrated Discovery tool. A protein‑protein interaction (PPI) network was established, followed by hub gene identification. Using the cut‑off threshold of ≥0.2 average β‑value difference, 3,725 unique CpGs (2,862 genes) were identified to be differentially methylated between the OS and normal groups. Among these 2,862 genes, 510 genes were differentially hypermethylated and 2,352 were differentially hypomethylated. The differentially hypermethylated genes were primarily involved in 20 GO terms, and the top 3 terms were associated with potassium ion transport. For differentially hypomethylated genes, GO functions principally included passive transmembrane transporter activity, channel activity and metal ion transmembrane transporter activity. In addition, a total of 10 significant pathways were enriched by differentially hypomethylated genes; notably, neuroactive ligand‑receptor interaction was the most significant pathway. Based on a connectivity degree >90, 7 hub genes were selected from the PPI network, including neuromedin U (NMU; degree=103) and NMU receptor 1 (NMUR1; degree=103). Functional terms (potassium ion transport, transmembrane transporter activity, and neuroactive ligand‑receptor interaction) and hub genes (NMU and NMUR1) may serve as potential

  1. Mitochondrial DNA copy number is regulated in a tissue specific manner by DNA methylation of the nuclear-encoded DNA polymerase gamma A

    PubMed Central

    Kelly, Richard D. W.; Mahmud, Arsalan; McKenzie, Matthew; Trounce, Ian A.; St John, Justin C.

    2012-01-01

    DNA methylation is an essential mechanism controlling gene expression during differentiation and development. We investigated the epigenetic regulation of the nuclear-encoded, mitochondrial DNA (mtDNA) polymerase γ catalytic subunit (PolgA) by examining the methylation status of a CpG island within exon 2 of PolgA. Bisulphite sequencing identified low methylation levels (<10%) within exon 2 of mouse oocytes, blastocysts and embryonic stem cells (ESCs), while somatic tissues contained significantly higher levels (>40%). In contrast, induced pluripotent stem (iPS) cells and somatic nuclear transfer ESCs were hypermethylated (>20%), indicating abnormal epigenetic reprogramming. Real time PCR analysis of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) immunoprecipitated DNA suggests active DNA methylation and demethylation within exon 2 of PolgA. Moreover, neural differentiation of ESCs promoted de novo methylation and demethylation at the exon 2 locus. Regression analysis demonstrates that cell-specific PolgA expression levels were negatively correlated with DNA methylation within exon 2 and mtDNA copy number. Finally, using chromatin immunoprecipitation (ChIP) against RNA polymerase II (RNApII) phosphorylated on serine 2, we show increased DNA methylation levels are associated with reduced RNApII transcriptional elongation. This is the first study linking nuclear DNA epigenetic regulation with mtDNA regulation during differentiation and cell specialization. PMID:22941637

  2. Promoter Hypermethylation of Tumour Suppressor Genes as Potential Biomarkers in Colorectal Cancer

    PubMed Central

    Ng, Jennifer Mun-Kar; Yu, Jun

    2015-01-01

    Colorectal cancer (CRC) is a common malignancy and the fourth leading cause of cancer deaths worldwide. It results from the accumulation of multiple genetic and epigenetic changes leading to the transformation of colon epithelial cells into invasive adenocarcinomas. In CRC, epigenetic changes, in particular promoter CpG island methylation, occur more frequently than genetic mutations. Hypermethylation contributes to carcinogenesis by inducing transcriptional silencing or downregulation of tumour suppressor genes and currently, over 600 candidate hypermethylated genes have been identified. Over the past decade, a deeper understanding of epigenetics coupled with technological advances have hinted at the potential of translating benchtop research into biomarkers for clinical use. DNA methylation represents one of the largest bodies of literature in epigenetics, and hence has the highest potential for minimally invasive biomarker development. Most progress has been made in the development of diagnostic markers and there are currently two, one stool-based and one blood-based, biomarkers that are commercially available for diagnostics. Prognostic and predictive methylation markers are still at their infantile stages. PMID:25622259

  3. Promoter hypermethylation and downregulation of the FAS gene may be involved in colorectal carcinogenesis.

    PubMed

    Manoochehri, Mehdi; Borhani, Nasim; Karbasi, Ashraf; Koochaki, Ameneh; Kazemi, Bahram

    2016-07-01

    Aberrant DNA methylation has been investigated in carcinogenesis and as biomarker for the early detection of colorectal cancer (CRC). The present study aimed to define the methylation status in the regulatory elements of two proapoptotic genes, Fas cell surface death receptor (FAS) and BCL2-associated X protein (BAX). DNA methylation analysis was performed in tumor and adjacent normal tissue using Hpa II/ Msp I restriction digestion and methylation-specific polymerase chain reaction (PCR). The results observed downregulation of the FAS and BAX genes in the CRC tissues compared with the adjacent normal samples. Furthermore, demethylation using 5-aza-2'-deoxycytidine treatment followed by reverse-transcription quantitative PCR were performed on the HT-29 cell line to measure BAX and FAS mRNA expression following demethylation. The 5-aza-2'-deoxycytidine treatment resulted in significant FAS gene upregulation in the HT-29 cell line, but no significant difference in BAX expression. Furthermore, analysis of CpG islands in the FAS gene promoter revealed that the FAS promoter was significantly hypermethylated in 53.3% of tumor tissues compared with adjacent normal samples. Taken together, the results indicate that decreased expression of the FAS gene due to hypermethylation of its promoter may lead to apoptotic resistance, and acts as an important step during colorectal carcinogenesis.

  4. Genome-wide DNA methylation sequencing reveals miR-663a is a novel epimutation candidate in CIMP-high endometrial cancer

    PubMed Central

    Yanokura, Megumi; Banno, Kouji; Adachi, Masataka; Aoki, Daisuke; Abe, Kuniya

    2017-01-01

    Aberrant DNA methylation is widely observed in many cancers. Concurrent DNA methylation of multiple genes occurs in endometrial cancer and is referred to as the CpG island methylator phenotype (CIMP). However, the features and causes of CIMP-positive endometrial cancer are not well understood. To investigate DNA methylation features characteristic to CIMP-positive endometrial cancer, we first classified samples from 25 patients with endometrial cancer based on the methylation status of three genes, i.e. MLH1, CDH1 (E-cadherin) and APC: CIMP-high (CIMP-H, 2/25, 8.0%), CIMP-low (CIMP-L, 7/25, 28.0%) and CIMP-negative (CIMP(-), 16/25, 64.0%). We then selected two samples each from CIMP-H and CIMP(-) classes, and analyzed DNA methylation status of both normal (peripheral blood cells: PBCs) and cancer tissues by genome-wide, targeted bisulfite sequencing. Genomes of the CIMP-H cancer tissues were significantly hypermethylated compared to those of the CIMP(-). Surprisingly, in normal tissues of the CIMP-H patients, promoter region of the miR-663a locus is hypermethylated relative to CIMP(-) samples. Consistent with this finding, miR-663a expression was lower in the CIMP-H PBCs than in the CIMP(-) PBCs. The same region of the miR663a locus is found to be highly methylated in cancer tissues of both CIMP-H and CIMP(-) cases. This is the first report showing that aberrant DNA methylation of the miR-663a promoter can occur in normal tissue of the cancer patients, suggesting a possible link between this epigenetic abnormality and endometrial cancer. This raises the possibility that the hypermethylation of the miR-663a promoter represents an epimutation associated with the CIMP-H endometrial cancers. Based on these findings, relationship of the aberrant DNA methylation and CIMP-H phenotype is discussed. PMID:28440489

  5. Genome-wide DNA methylation sequencing reveals miR-663a is a novel epimutation candidate in CIMP-high endometrial cancer.

    PubMed

    Yanokura, Megumi; Banno, Kouji; Adachi, Masataka; Aoki, Daisuke; Abe, Kuniya

    2017-06-01

    Aberrant DNA methylation is widely observed in many cancers. Concurrent DNA methylation of multiple genes occurs in endometrial cancer and is referred to as the CpG island methylator phenotype (CIMP). However, the features and causes of CIMP-positive endometrial cancer are not well understood. To investigate DNA methylation features characteristic to CIMP-positive endometrial cancer, we first classified samples from 25 patients with endometrial cancer based on the methylation status of three genes, i.e. MLH1, CDH1 (E-cadherin) and APC: CIMP-high (CIMP-H, 2/25, 8.0%), CIMP-low (CIMP-L, 7/25, 28.0%) and CIMP-negative (CIMP(-), 16/25, 64.0%). We then selected two samples each from CIMP-H and CIMP(-) classes, and analyzed DNA methylation status of both normal (peripheral blood cells: PBCs) and cancer tissues by genome-wide, targeted bisulfite sequencing. Genomes of the CIMP-H cancer tissues were significantly hypermethylated compared to those of the CIMP(-). Surprisingly, in normal tissues of the CIMP-H patients, promoter region of the miR-663a locus is hypermethylated relative to CIMP(-) samples. Consistent with this finding, miR-663a expression was lower in the CIMP-H PBCs than in the CIMP(-) PBCs. The same region of the miR663a locus is found to be highly methylated in cancer tissues of both CIMP-H and CIMP(-) cases. This is the first report showing that aberrant DNA methylation of the miR-663a promoter can occur in normal tissue of the cancer patients, suggesting a possible link between this epigenetic abnormality and endometrial cancer. This raises the possibility that the hypermethylation of the miR-663a promoter represents an epimutation associated with the CIMP-H endometrial cancers. Based on these findings, relationship of the aberrant DNA methylation and CIMP-H phenotype is discussed.

  6. Aberrant DNA methylation at genes associated with a stem cell-like phenotype in cholangiocarcinoma tumours

    PubMed Central

    Dai, Wei; Siddiq, Afshan; Walley, Andrew J; Limpaiboon, Temduang; Brown, Robert

    2013-01-01

    Genetic abnormalities of cholangiocarcinoma have been widely studied; however, epigenomic changes related to cholangiocarcinogenesis have been less well characterised. We have profiled the DNA methylomes of 28 primary cholangiocarcinoma and six matched adjacent normal tissues using Infinium’s HumanMethylation27 BeadChips with the aim of identifying gene sets aberrantly epigenetically regulated in this tumour type. Using a linear model for microarray data we identified 1610 differentially methylated autosomal CpG sites with 809 CpG sites (representing 603 genes) being hypermethylated and 801 CpG sites (representing 712 genes) being hypomethylated in cholangiocarcinoma versus adjacent normal tissues (false discovery rate ≤ 0.05). Gene ontology and gene set enrichment analyses identified gene sets significantly associated with hypermethylation at linked CpG sites in cholangiocarcinoma including homeobox genes and target genes of PRC2, EED, SUZ12 and histone H3 trimethylation at lysine 27. We confirmed frequent hypermethylation at the homeobox genes HOXA9 and HOXD9 by bisulfite pyrosequencing in a larger cohort of cholangiocarcinoma (n = 102). Our findings indicate a key role for hypermethylation of multiple CpG sites at genes associated with a stem cell-like phenotype as a common molecular aberration in cholangiocarcinoma. These data have implications for cholangiocarcinogenesis, as well as possible novel treatment options using histone methyltransferase inhibitors. PMID:24089088

  7. Analysis of the association between CIMP and BRAF in colorectal cancer by DNA methylation profiling.

    PubMed

    Hinoue, Toshinori; Weisenberger, Daniel J; Pan, Fei; Campan, Mihaela; Kim, Myungjin; Young, Joanne; Whitehall, Vicki L; Leggett, Barbara A; Laird, Peter W

    2009-12-21

    A CpG island methylator phenotype (CIMP) is displayed by a distinct subset of colorectal cancers with a high frequency of DNA hypermethylation in a specific group of CpG islands. Recent studies have shown that an activating mutation of BRAF (BRAF(V600E)) is tightly associated with CIMP, raising the question of whether BRAF(V600E) plays a causal role in the development of CIMP or whether CIMP provides a favorable environment for the acquisition of BRAF(V600E). We employed Illumina GoldenGate DNA methylation technology, which interrogates 1,505 CpG sites in 807 different genes, to further study this association. We first examined whether expression of BRAF(V600E) causes DNA hypermethylation by stably expressing BRAF(V600E) in the CIMP-negative, BRAF wild-type COLO 320DM colorectal cancer cell line. We determined 100 CIMP-associated CpG sites and examined changes in DNA methylation in eight stably transfected clones over multiple passages. We found that BRAF(V600E) is not sufficient to induce CIMP in our system. Secondly, considering the alternative possibility, we identified genes whose DNA hypermethylation was closely linked to BRAF(V600E) and CIMP in 235 primary colorectal tumors. Interestingly, genes that showed the most significant link include those that mediate various signaling pathways implicated in colorectal tumorigenesis, such as BMP3 and BMP6 (BMP signaling), EPHA3, KIT, and FLT1 (receptor tyrosine kinases) and SMO (Hedgehog signaling). Furthermore, we identified CIMP-dependent DNA hypermethylation of IGFBP7, which has been shown to mediate BRAF(V600E)-induced cellular senescence and apoptosis. Promoter DNA hypermethylation of IGFBP7 was associated with silencing of the gene. CIMP-specific inactivation of BRAF(V600E)-induced senescence and apoptosis pathways by IGFBP7 DNA hypermethylation might create a favorable context for the acquisition of BRAF(V600E) in CIMP+ colorectal cancer. Our data will be useful for future investigations toward understanding

  8. p16 promoter hypermethylation: A useful serum marker for early detection of gastric cancer

    PubMed Central

    Abbaszadegan, Mohammad Reza; Moaven, Omeed; Sima, Hamid Reza; Ghafarzadegan, Kamran; A'rabi, Azadeh; Forghani, Mohammad Naser; Raziee, Hamid Reza; Mashhadinejad, Ali; Jafarzadeh, Mostafa; Esmaili-Shandiz, Ehsan; Dadkhah, Ezzat

    2008-01-01

    AIM: To determine p16 promoter hypermethylation in gastric tumoral tissue and serum samples, its impact on p16-protein expression, and correlation with clinical and histological features. METHODS: Samples were obtained from 52 histologically confirmed cases of gastric adenocarcinoma. Gastric tissue and serum of 50 age- and sex-matched individuals with normal gastroscopy and biopsy were obtained as control samples. Methylation-specific polymerase chain reaction (MSP) was used to evaluate methylation status of p16 promoter. p16-protein expression was analyzed by immunohistochemical staining on paraffin-embedded sections. RESULTS: Methylation was detected in 44.2% (23/52) of tumoral tissues. 60.9% of them were also methylated in serum, i.e., 26.9% of all patients (14/52). Methylation was not detected in tissue and sera of control samples. p16-protein expression was decreased in 61.5% of cases (32/52), and was significantly associated with promoter hypermethylation (P < 0.001). Methylation was significantly more frequent in higher pathological grades (P < 0.05). Methylation was not associated with other clinicopathological features and environmental factors including H pylori infection and smoking. CONCLUSION: p16 promoter hypermethylation is an important event in gastric carcinogenesis. It is the principle mechanism of p16 gene silencing. It is related to malignant tumor behavior. Detection of DNA methylation in serum may be a biomarker for early detection of gastric cancer. PMID:18395906

  9. Promoter hypermethylation and downregulation of the FAS gene may be involved in colorectal carcinogenesis

    PubMed Central

    MANOOCHEHRI, MEHDI; BORHANI, NASIM; KARBASI, ASHRAF; KOOCHAKI, AMENEH; KAZEMI, BAHRAM

    2016-01-01

    Aberrant DNA methylation has been investigated in carcinogenesis and as biomarker for the early detection of colorectal cancer (CRC). The present study aimed to define the methylation status in the regulatory elements of two proapoptotic genes, Fas cell surface death receptor (FAS) and BCL2-associated X protein (BAX). DNA methylation analysis was performed in tumor and adjacent normal tissue using HpaII/MspI restriction digestion and methylation-specific polymerase chain reaction (PCR). The results observed downregulation of the FAS and BAX genes in the CRC tissues compared with the adjacent normal samples. Furthermore, demethylation using 5-aza-2′-deoxycytidine treatment followed by reverse-transcription quantitative PCR were performed on the HT-29 cell line to measure BAX and FAS mRNA expression following demethylation. The 5-aza-2′-deoxycytidine treatment resulted in significant FAS gene upregulation in the HT-29 cell line, but no significant difference in BAX expression. Furthermore, analysis of CpG islands in the FAS gene promoter revealed that the FAS promoter was significantly hypermethylated in 53.3% of tumor tissues compared with adjacent normal samples. Taken together, the results indicate that decreased expression of the FAS gene due to hypermethylation of its promoter may lead to apoptotic resistance, and acts as an important step during colorectal carcinogenesis. PMID:27347139

  10. Hypermethylated-capped selenoprotein mRNAs in mammals

    PubMed Central

    Wurth, Laurence; Gribling-Burrer, Anne-Sophie; Verheggen, Céline; Leichter, Michael; Takeuchi, Akiko; Baudrey, Stéphanie; Martin, Franck; Krol, Alain; Bertrand, Edouard; Allmang, Christine

    2014-01-01

    Mammalian mRNAs are generated by complex and coordinated biogenesis pathways and acquire 5′-end m7G caps that play fundamental roles in processing and translation. Here we show that several selenoprotein mRNAs are not recognized efficiently by translation initiation factor eIF4E because they bear a hypermethylated cap. This cap modification is acquired via a 5′-end maturation pathway similar to that of the small nucle(ol)ar RNAs (sn- and snoRNAs). Our findings also establish that the trimethylguanosine synthase 1 (Tgs1) interacts with selenoprotein mRNAs for cap hypermethylation and that assembly chaperones and core proteins devoted to sn- and snoRNP maturation contribute to recruiting Tgs1 to selenoprotein mRNPs. We further demonstrate that the hypermethylated-capped selenoprotein mRNAs localize to the cytoplasm, are associated with polysomes and thus translated. Moreover, we found that the activity of Tgs1, but not of eIF4E, is required for the synthesis of the GPx1 selenoprotein in vivo. PMID:25013170

  11. Linking abnormal mitosis to the acquisition of DNA damage

    PubMed Central

    Pellman, David

    2012-01-01

    Cellular defects that impair the fidelity of mitosis promote chromosome missegregation and aneuploidy. Increasing evidence reveals that errors in mitosis can also promote the direct and indirect acquisition of DNA damage and chromosome breaks. Consequently, deregulated cell division can devastate the integrity of the normal genome and unleash a variety of oncogenic stimuli that may promote transformation. Recent work has shed light on the mechanisms that link abnormal mitosis with the development of DNA damage, how cells respond to such affronts, and the potential impact on tumorigenesis. PMID:23229895

  12. Cigarette smoking and p16INK4α gene promoter hypermethylation in non-small cell lung carcinoma patients: a meta-analysis.

    PubMed

    Zhang, Bo; Zhu, Wei; Yang, Ping; Liu, Tao; Jiang, Mei; He, Zhi-Ni; Zhang, Shi-Xin; Chen, Wei-Qing; Chen, Wen

    2011-01-01

    Aberrant methylation of promoter DNA and transcriptional repression of specific tumor suppressor genes play an important role in carcinogenesis. Recently, many studies have investigated the association between cigarette smoking and p16(INK4α) gene hypermethylation in lung cancer, but could not reach a unanimous conclusion. Nineteen cross-sectional studies on the association between cigarette smoking and p16(INK4α) methylation in surgically resected tumor tissues from non-small cell lung carcinoma (NSCLC) patients were identified in PubMed database until June 2011. For each study, a 2×2 cross-table was extracted. In total, 2,037 smoker and 765 nonsmoker patients were pooled with a fixed-effects model weighting for the inverse of the variance. Overall, the frequency of p16(INK4α) hypermethylation was higher in NSCLC patients with smoking habits than that in non-smoking patients (OR = 2.25, 95% CI = 1.81-2.80). The positive association between cigarette smoking and p16(INK4α) hypermethylation was similar in adenocarcinoma and squamous-cell carcinoma. In the stratified analyses, the association was stronger in Asian patients and in the studies with larger sample sizes. Cigarette smoking is positively correlated to p16(INK4α) gene hypermethylation in NSCLC patients.

  13. Hypermethylation of miR-203 in endometrial carcinomas.

    PubMed

    Huang, Yi-Wen; Kuo, Chieh-Ti; Chen, Jo-Hsin; Goodfellow, Paul J; Huang, Tim H-M; Rader, Janet S; Uyar, Denise S

    2014-05-01

    Aberrant expression of SOX4 in endometrial cancer has been identified and partially was contributed to hypermethylation of miR-129-2. Other miRNAs are suspected to influence SOX 4 as well. The current study seeks to identify other hypermethylated miRNAs that regulate SOX4 in endometrial carcinomas. Methylation levels of miRNA promoter regions were measured by combined bisulfite restriction analysis (COBRA) and pyrosequencing assays. Gene expression was determined by RT-qPCR. Methylation level of a miRNA locus was corrected with clinicopathologic factors for 252 gynecological specimens. In silico analysis identified 13 miRNA loci bound on the 3'-UTR of SOX4. Using COBRA assays, increased methylation of miR-203, miR-219-2, miR-596, and miR-618 was detected in endometrial cancer cells relative to those seen in a normal cell line and in normal endometrium. Transfection of a miR-203 mimic decreased SOX4 gene expression. Hypermethylation of miR-203 was detected in 52% of type I endometrioid endometrial carcinomas (n=131) but was not seen in any of 10 uninvolved normal endometria (P<0.001). Methylation status of miR-203 was significantly associated with microsatellite instability and MLH1 methylation in endometrial tumors (P<0.001). Furthermore, hypermethylation of miR-203 was found in endometrioid and clear endometrial subtype tumors, but not in cervical squamous cell and ovarian carcinomas. Hypermethylation of miR-203 is a frequent event in endometrial carcinomas and is strongly associated with microsatellite instability and MLH1 methylation status. Thus, miR-203 methylation level might represent a marker for patients with endometrioid and clear endometrial sub-cancers. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. TESTIN was commonly hypermethylated and involved in the epithelial-mesenchymal transition of endometrial cancer.

    PubMed

    Dong, Ruofan; Pu, Hong; Wang, Yuan; Yu, Jinjin; Lian, Kuixian; Mao, Caiping

    2015-05-01

    We previously reported frequent loss of TESTIN in human endometrial carcinoma, which significantly suppressed tumor proliferation and invasion. Herein, we further explored the mechanisms underlying TESTIN loss and its roles in the epithelial-mesenchymal transition (EMT, a key step for tumor spreading). Methylation-specific PCR was performed to investigate the promoter status of TESTIN in a panel of endometrial cancer and normal endometrium tissues. The expression of TESTIN mRNA was determined by real-time PCR. Up- and down-regulation of TESTIN were achieved by transient transfection with pcDNA3.1-TESTIN and shRNA-TESTIN plasmids, respectively. The EMT alterations were observed under the optical microscope and EMT-related markers were detected by real-time PCR and western blot. Compared to the control (3.6%), TESTIN was hypermethylated in 43.7% endometrial cancer tissues (p < 0.001). Moreover, TESTIN hypermethylation was significantly correlated with advanced tumor stage, deep myometrial invasion and lymphatic node metastasis. In vitro, the demethylating agent dramatically restored the expression of TESTIN. In addition, up-regulation of TESTIN significantly suppressed the EMT procedure; whereas down-regulation of TESTIN enhanced EMT. In conclusion, we demonstrated that loss of TESTIN was mainly caused by hypermethylation, which might be a potent prognostic marker. Furthermore, we proved that TESTIN significantly suppressed the EMT procedure, proposing restoration of TESTIN to be a novel therapeutic strategy for endometrial carcinoma. © 2015 APMIS. Published by John Wiley & Sons Ltd.

  15. Cardiac abnormalities in patients with mitochondrial DNA mutation 3243A>G.

    PubMed

    Majamaa-Voltti, Kirsi; Peuhkurinen, Keijo; Kortelainen, Marja-Leena; Hassinen, Ilmo E; Majamaa, Kari

    2002-08-01

    Tissues that depend on aerobic energy metabolism suffer most in diseases caused by mutations in mitochondrial DNA (mtDNA). Cardiac abnormalities have been described in many cases, but their frequency and clinical spectrum among patients with mtDNA mutations is unknown. Thirty-nine patients with the 3243A>G mtDNA mutation were examined, methods used included clinical evaluation, electrocardiogram, Holter recording and echocardiography. Autopsy reports on 17 deceased subjects were also reviewed. The degree of 3243A>G mutation heteroplasmy was determined using an Apa I restriction fragment analysis. Better hearing level (BEHL0.5-4 kHz) was used as a measure of the clinical severity of disease. Left ventricular hypertrophy (LVH) was diagnosed in 19 patients (56%) by echocardiography and in six controls (15%) giving an odds ratio of 7.5 (95% confidence interval; 1.74-67). The dimensions of the left ventricle suggested a concentric hypertrophy. Left ventricular systolic or diastolic dysfunction was observed in 11 patients. Holter recording revealed frequent ventricular extrasystoles (>10/h) in five patients. Patients with LVH differed significantly from those without LVH in BEHL0.5-4 kHz, whereas the contribution of age or the degree of the mutant heteroplasmy in skeletal muscle to the risk of LVH was less remarkable. Structural and functional abnormalities of the heart were common in patients with 3243A>G. The risk of LVH was related to the clinical severity of the phenotype, and to a lesser degree to age, suggesting that patients presenting with any symptoms from the mutation should also be evaluated for cardiac abnormalities.

  16. Trichloroethylene-Induced DNA Methylation Changes in Male F344 Rat Liver.

    PubMed

    Jiang, Yan; Chen, Jiahong; Yue, Cong; Zhang, Hang; Chen, Tao

    2016-10-17

    Trichloroethylene (TCE), a common environmental contaminant, causes hepatocellular carcinoma in mice but not in rats. To understand the mechanisms of the species-specific hepatocarcinogenecity of TCE, we examined the methylation status of DNA in the liver of rats exposed to TCE at 0 or 1000 mg/kg b.w. for 5 days using MeDIP-chip, bisulfite sequencing, COBRA, and LC-MS/MS. The related mRNA expression levels were measured by qPCR. Although no global DNA methylation change was detected, 806 genes were hypermethylated and 186 genes were hypomethylated. The genes with hypermethylated DNA were enriched in endocytosis, MAPK, and cAMP signaling pathways. We further confirmed the hypermethylation of Uhrf2 DNA and the hypomethylation of Hadhb DNA, which were negatively correlated with their mRNA expression levels. The transcriptional levels of Jun, Ihh, and Tet2 were significantly downregulated, whereas Cdkn1a was overexpressed. No mRNA expression change was found for Mki67, Myc, Uhrf1, and Dnmt1. In conclusion, TCE-induced DNA methylation changes in rats appear to suppress instead of promote hepatocarcinogenesis, which might play a role in the species-specific hepatocarcinogenecity of TCE.

  17. Cardiac abnormalities in patients with mitochondrial DNA mutation 3243A>G

    PubMed Central

    Majamaa-Voltti, Kirsi; Peuhkurinen, Keijo; Kortelainen, Marja-Leena; Hassinen, Ilmo E; Majamaa, Kari

    2002-01-01

    Background Tissues that depend on aerobic energy metabolism suffer most in diseases caused by mutations in mitochondrial DNA (mtDNA). Cardiac abnormalities have been described in many cases, but their frequency and clinical spectrum among patients with mtDNA mutations is unknown. Methods Thirty-nine patients with the 3243A>G mtDNA mutation were examined, methods used included clinical evaluation, electrocardiogram, Holter recording and echocardiography. Autopsy reports on 17 deceased subjects were also reviewed. The degree of 3243A>G mutation heteroplasmy was determined using an Apa I restriction fragment analysis. Better hearing level (BEHL0.5–4 kHz) was used as a measure of the clinical severity of disease. Results Left ventricular hypertrophy (LVH) was diagnosed in 19 patients (56%) by echocardiography and in six controls (15%) giving an odds ratio of 7.5 (95% confidence interval; 1.74–67). The dimensions of the left ventricle suggested a concentric hypertrophy. Left ventricular systolic or diastolic dysfunction was observed in 11 patients. Holter recording revealed frequent ventricular extrasystoles (>10/h) in five patients. Patients with LVH differed significantly from those without LVH in BEHL0.5–4 kHz, whereas the contribution of age or the degree of the mutant heteroplasmy in skeletal muscle to the risk of LVH was less remarkable. Conclusions Structural and functional abnormalities of the heart were common in patients with 3243A>G. The risk of LVH was related to the clinical severity of the phenotype, and to a lesser degree to age, suggesting that patients presenting with any symptoms from the mutation should also be evaluated for cardiac abnormalities. PMID:12150714

  18. MLH1-deficient Colorectal Carcinoma With Wild-type BRAF and MLH1 Promoter Hypermethylation Harbor KRAS Mutations and Arise From Conventional Adenomas.

    PubMed

    Farchoukh, Lama; Kuan, Shih-Fan; Dudley, Beth; Brand, Randall; Nikiforova, Marina; Pai, Reetesh K

    2016-10-01

    Between 10% and 15% of colorectal carcinomas demonstrate sporadic DNA mismatch-repair protein deficiency as a result of MLH1 promoter methylation and are thought to arise from sessile serrated adenomas, termed the serrated neoplasia pathway. Although the presence of the BRAF V600E mutation is indicative of a sporadic cancer, up to 30% to 50% of colorectal carcinomas with MLH1 promoter hypermethylation will lack a BRAF mutation. We report the clinicopathologic and molecular features of MLH1-deficient colorectal carcinoma with wild-type BRAF and MLH1 promoter hypermethylation (referred to as MLH1-hypermethylated BRAF wild-type colorectal carcinoma, n=36) in comparison with MLH1-deficient BRAF-mutated colorectal carcinoma (n=113) and Lynch syndrome-associated colorectal carcinoma (n=36). KRAS mutations were identified in 31% of MLH1-hypermethylated BRAF wild-type colorectal carcinomas compared with 0% of MLH1-deficient BRAF-mutated colorectal carcinomas and 37% of Lynch syndrome-associated colorectal carcinomas. When a precursor polyp was identified, MLH1-hypermethylated BRAF wild-type colorectal carcinomas arose from precursor polyps resembling conventional tubular/tubulovillous adenomas in contrast to MLH1-deficient BRAF-mutated colorectal carcinomas, which arose from precursor sessile serrated adenomas (P<0.001). Both MLH1-hypermethylated BRAF wild-type colorectal carcinoma and MLH1-deficient BRAF-mutated colorectal carcinoma had a predilection for the right colon compared with Lynch syndrome-associated colorectal carcinoma (86% vs. 92% vs. 49%, P<0.001). There was no significant difference in mucinous differentiation, tumor-infiltrating lymphocytes, Crohn-like reaction, and medullary differentiation between the 3 tumor groups. Using Kaplan-Meier survival functions, there was no significant difference in disease-specific survival between the 3 patient groups (P>0.05). In conclusion, our results indicate that MLH1-hypermethylated BRAF wild-type colorectal carcinomas

  19. Comprehensive methylome analysis of ovarian tumors reveals hedgehog signaling pathway regulators as prognostic DNA methylation biomarkers.

    PubMed

    Huang, Rui-Lan; Gu, Fei; Kirma, Nameer B; Ruan, Jianhua; Chen, Chun-Liang; Wang, Hui-Chen; Liao, Yu-Ping; Chang, Cheng-Chang; Yu, Mu-Hsien; Pilrose, Jay M; Thompson, Ian M; Huang, Hsuan-Cheng; Huang, Tim Hui-Ming; Lai, Hung-Cheng; Nephew, Kenneth P

    2013-06-01

    Women with advanced stage ovarian cancer (OC) have a five-year survival rate of less than 25%. OC progression is associated with accumulation of epigenetic alterations and aberrant DNA methylation in gene promoters acts as an inactivating "hit" during OC initiation and progression. Abnormal DNA methylation in OC has been used to predict disease outcome and therapy response. To globally examine DNA methylation in OC, we used next-generation sequencing technology, MethylCap-sequencing, to screen 75 malignant and 26 normal or benign ovarian tissues. Differential DNA methylation regions (DMRs) were identified, and the Kaplan-Meier method and Cox proportional hazard model were used to correlate methylation with clinical endpoints. Functional role of specific genes identified by MethylCap-sequencing was examined in in vitro assays. We identified 577 DMRs that distinguished (p < 0.001) malignant from non-malignant ovarian tissues; of these, 63 DMRs correlated (p < 0.001) with poor progression free survival (PFS). Concordant hypermethylation and corresponding gene silencing of sonic hedgehog pathway members ZIC1 and ZIC4 in OC tumors was confirmed in a panel of OC cell lines, and ZIC1 and ZIC4 repression correlated with increased proliferation, migration and invasion. ZIC1 promoter hypermethylation correlated (p < 0.01) with poor PFS. In summary, we identified functional DNA methylation biomarkers significantly associated with clinical outcome in OC and suggest our comprehensive methylome analysis has significant translational potential for guiding the design of future clinical investigations targeting the OC epigenome. Methylation of ZIC1, a putative tumor suppressor, may be a novel determinant of OC outcome.

  20. Hypermethylated APC in serous carcinoma based on a meta-analysis of ovarian cancer.

    PubMed

    Shen, Chunyan; Sheng, Qifang; Zhang, Xiaojie; Fu, Yuling; Zhu, Kemiao

    2016-09-26

    The reduced expression of the Adenomatous polyposis coli (APC) gene, a tumor suppressor gene, through promoter hypermethylation has been reported to play a key role in the carcinogenesis. However, the correlation between APC promoter hypermethylation and ovarian cancer (OC) remains to be clarified. A comprehensive literature search was carried out in related research databases. The overall odds ratio (OR) and corresponding 95 % confidence interval (CI) were used to evaluate the effects of APC promoter hypermethylation on OC and clinicopathological characteristics. Ultimately, 12 eligible studies were used in our study, including 806 OC samples, 429 normal controls, 109 benign lesions and 75 LMP samples. The pooled OR showed that APC promoter hypermethylation was significantly higher in OC than in normal and benign controls (OR = 6.18 and OR = 3.26, respectively). No significant correlation was observed between OC and low malignant potential (LMP) tumors (P = 0.436). In the comparison of OC and normal controls, subgroup analysis based on race showed that the overall OR of APC promoter hypermethylation was significant and similar in Asians and Caucasians (OR = 8.34 and OR = 5.39, respectively). A subgroup analysis based on sample type found that the pooled OR was significantly higher in blood than in tissue (OR = 18.71 and OR = 5.74, respectively). A significant association was not observed between APC promoter hypermethylation and tumor grade or tumor stage. The pooled OR indicated that APC promoter hypermethylation was significantly lower in serous carcinoma than in non-serous carcinoma (OR = 0.56, P = 0.02). No obvious publication bias was detected by Egger's test (all P > 0.05). APC promoter hypermethylation may be linked to the increased risk of OC. It was associated with histological type, but not with tumor grade or tumor stage. Moreover, hypermethylated APC may be a noninvasive biomarker using blood samples. Future

  1. DNA demethylation in the Arabidopsis genome

    PubMed Central

    Penterman, Jon; Zilberman, Daniel; Huh, Jin Hoe; Ballinger, Tracy; Henikoff, Steven; Fischer, Robert L.

    2007-01-01

    Cytosine DNA methylation is considered to be a stable epigenetic mark, but active demethylation has been observed in both plants and animals. In Arabidopsis thaliana, DNA glycosylases of the DEMETER (DME) family remove methylcytosines from DNA. Demethylation by DME is necessary for genomic imprinting, and demethylation by a related protein, REPRESSOR OF SILENCING1, prevents gene silencing in a transgenic background. However, the extent and function of demethylation by DEMETER-LIKE (DML) proteins in WT plants is not known. Using genome-tiling microarrays, we mapped DNA methylation in mutant and WT plants and identified 179 loci actively demethylated by DML enzymes. Mutations in DML genes lead to locus-specific DNA hypermethylation. Reintroducing WT DML genes restores most loci to the normal pattern of methylation, although at some loci, hypermethylated epialleles persist. Of loci demethylated by DML enzymes, >80% are near or overlap genes. Genic demethylation by DML enzymes primarily occurs at the 5′ and 3′ ends, a pattern opposite to the overall distribution of WT DNA methylation. Our results show that demethylation by DML DNA glycosylases edits the patterns of DNA methylation within the Arabidopsis genome to protect genes from potentially deleterious methylation. PMID:17409185

  2. HOXA11 hypermethylation is associated with progression of non-small cell lung cancer

    PubMed Central

    Hwang, Jung-Ah; Lee, Bo Bin; Kim, Yujin; Park, Seong-Eun; Heo, Kyun; Hong, Seung-Hyun; Kim, Young-Ho; Han, Joungho; Shim, Young Mog; Lee, Yeon-Su; Kim, Duk-Hwan

    2013-01-01

    This study was aimed at understanding the functional significance of HOXA11 hypermethylation in non-small cell lung cancer (NSCLC). HOXA11 hypermethylation was characterized in six lung cancer cell lines, and its clinical significance was analyzed using formalin-fixed paraffin-embedded tissues from 317 NSCLC patients, and Ki-67 expression was analyzed using immunohistochemistry. The promoter region of HOXA11 was highly methylated in six lung cancer cell lines, but not in normal bronchial epithelial cells. The loss of expression was restored by treatment of the cells with a demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-dC). Transient transfection of HOXA11 into H23 lung cancer cells resulted in the inhibition of cell migration and proliferation. HOXA11 hypermethylation was found in 218 (69%) of 317 primary NSCLCs. HOXA11 hypermethylation was found at a higher prevalence in squamous cell carcinoma than in adenocarcinoma (74% vs. 63%, respectively). HOXA11 hypermethylation was associated with Ki-67 proliferation index (P = 0.03) and pT stage (P = 0.002), but not with patient survival. Patients with pT2 and pT3 stages were 1.85 times (95% confidence interval [CI] = 1.04-3.29; P = 0.04) and 5.47 times (95% CI = 1.18-25.50; P = 0.01), respectively, more likely to show HOXA11 hypermethylation than those with pT1 stage, after adjusting for age, sex, and histology. In conclusion, the present study suggests that HOXA11 hypermethylation may contribute to the progression of NSCLC by promoting cell proliferation or migration. PMID:24259349

  3. DNA methylation and differentiation: HOX genes in muscle cells

    PubMed Central

    2013-01-01

    Background Tight regulation of homeobox genes is essential for vertebrate development. In a study of genome-wide differential methylation, we recently found that homeobox genes, including those in the HOX gene clusters, were highly overrepresented among the genes with hypermethylation in the skeletal muscle lineage. Methylation was analyzed by reduced representation bisulfite sequencing (RRBS) of postnatal myoblasts, myotubes and adult skeletal muscle tissue and 30 types of non-muscle-cell cultures or tissues. Results In this study, we found that myogenic hypermethylation was present in specific subregions of all four HOX gene clusters and was associated with various chromatin epigenetic features. Although the 3′ half of the HOXD cluster was silenced and enriched in polycomb repression-associated H3 lysine 27 trimethylation in most examined cell types, including myoblasts and myotubes, myogenic samples were unusual in also displaying much DNA methylation in this region. In contrast, both HOXA and HOXC clusters displayed myogenic hypermethylation bordering a central region containing many genes preferentially expressed in myogenic progenitor cells and consisting largely of chromatin with modifications typical of promoters and enhancers in these cells. A particularly interesting example of myogenic hypermethylation was HOTAIR, a HOXC noncoding RNA gene, which can silence HOXD genes in trans via recruitment of polycomb proteins. In myogenic progenitor cells, the preferential expression of HOTAIR was associated with hypermethylation immediately downstream of the gene. Other HOX gene regions also displayed myogenic DNA hypermethylation despite being moderately expressed in myogenic cells. Analysis of representative myogenic hypermethylated sites for 5-hydroxymethylcytosine revealed little or none of this base, except for an intragenic site in HOXB5 which was specifically enriched in this base in skeletal muscle tissue, whereas myoblasts had predominantly 5

  4. Raman Spectroscopy of DNA Packaging in Individual Human Sperm Cells distinguishes Normal from Abnormal Cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Huser, T; Orme, C; Hollars, C

    Healthy human males produce sperm cells of which about 25-40% have abnormal head shapes. Increases in the percentage of sperm exhibiting aberrant sperm head morphologies have been correlated with male infertility, and biochemical studies of pooled sperm have suggested that sperm with abnormal shape may contain DNA that has not been properly repackaged by protamine during spermatid development. We have used micro-Raman spectroscopy to obtain Raman spectra from individual human sperm cells and examined how differences in the Raman spectra of sperm chromatin correlate with cell shape. We show that Raman spectra of individual sperm cells contain vibrational marker modesmore » that can be used to assess the efficiency of DNA-packaging for each cell. Raman spectra obtained from sperm cells with normal shape provide evidence that DNA in these sperm is very efficiently packaged. We find, however, that the relative protein content per cell and DNA packaging efficiencies are distributed over a relatively wide range for sperm cells with both normal and abnormal shape. These findings indicate that single cell Raman spectroscopy should be a valuable tool in assessing the quality of sperm cells for in-vitro fertilization.« less

  5. Analysis of DNA Methylation Status in Bodily Fluids for Early Detection of Cancer

    PubMed Central

    Yokoi, Keigo; Yamashita, Keishi; Watanabe, Masahiko

    2017-01-01

    Epigenetic alterations by promoter DNA hypermethylation and gene silencing in cancer have been reported over the past few decades. DNA hypermethylation has great potential to serve as a screening marker, a prognostic marker, and a therapeutic surveillance marker in cancer clinics. Some bodily fluids, such as stool or urine, were obtainable without any invasion to the body. Thus, such bodily fluids were suitable samples for high throughput cancer surveillance. Analyzing the methylation status of bodily fluids around the cancer tissue may, additionally, lead to the early detection of cancer, because several genes in cancer tissues are reported to be cancer-specifically hypermethylated. Recently, several studies that analyzed the methylation status of DNA in bodily fluids were conducted, and some of the results have potential for future development and further clinical use. In fact, a stool DNA test was approved by the U.S. Food and Drug Administration (FDA) for the screening of colorectal cancer. Another promising methylation marker has been identified in various bodily fluids for several cancers. We reviewed studies that analyzed DNA methylation in bodily fluids as a less-invasive cancer screening. PMID:28358330

  6. Unique DNA methylome profiles in CpG island methylator phenotype colon cancers

    PubMed Central

    Xu, Yaomin; Hu, Bo; Choi, Ae-Jin; Gopalan, Banu; Lee, Byron H.; Kalady, Matthew F.; Church, James M.; Ting, Angela H.

    2012-01-01

    A subset of colorectal cancers was postulated to have the CpG island methylator phenotype (CIMP), a higher propensity for CpG island DNA methylation. The validity of CIMP, its molecular basis, and its prognostic value remain highly controversial. Using MBD-isolated genome sequencing, we mapped and compared genome-wide DNA methylation profiles of normal, non-CIMP, and CIMP colon specimens. Multidimensional scaling analysis revealed that each specimen could be clearly classified as normal, non-CIMP, and CIMP, thus signifying that these three groups have distinctly different global methylation patterns. We discovered 3780 sites in various genomic contexts that were hypermethylated in both non-CIMP and CIMP colon cancers when compared with normal colon. An additional 2026 sites were found to be hypermethylated in CIMP tumors only; and importantly, 80% of these sites were located in CpG islands. These data demonstrate on a genome-wide level that the additional hypermethylation seen in CIMP tumors occurs almost exclusively at CpG islands and support definitively that these tumors were appropriately named. When these sites were examined more closely, we found that 25% were adjacent to sites that were also hypermethylated in non-CIMP tumors. Thus, CIMP is also characterized by more extensive methylation of sites that are already prone to be hypermethylated in colon cancer. These observations indicate that CIMP tumors have specific defects in controlling both DNA methylation seeding and spreading and serve as an important first step in delineating molecular mechanisms that control these processes. PMID:21990380

  7. DNA methylation profile distinguishes clear cell sarcoma of the kidney from other pediatric renal tumors.

    PubMed

    Ueno, Hitomi; Okita, Hajime; Akimoto, Shingo; Kobayashi, Kenichiro; Nakabayashi, Kazuhiko; Hata, Kenichiro; Fujimoto, Junichiro; Hata, Jun-Ichi; Fukuzawa, Masahiro; Kiyokawa, Nobutaka

    2013-01-01

    A number of specific, distinct neoplastic entities occur in the pediatric kidney, including Wilms' tumor, clear cell sarcoma of the kidney (CCSK), congenital mesoblastic nephroma (CMN), rhabdoid tumor of the kidney (RTK), and the Ewing's sarcoma family of tumors (ESFT). By employing DNA methylation profiling using Illumina Infinium HumanMethylation27, we analyzed the epigenetic characteristics of the sarcomas including CCSK, RTK, and ESFT in comparison with those of the non-neoplastic kidney (NK), and these tumors exhibited distinct DNA methylation profiles in a tumor-type-specific manner. CCSK is the most frequently hypermethylated, but least frequently hypomethylated, at CpG sites among these sarcomas, and exhibited 490 hypermethylated and 46 hypomethylated CpG sites in compared with NK. We further validated the results by MassARRAY, and revealed that a combination of four genes was sufficient for the DNA methylation profile-based differentiation of these tumors by clustering analysis. Furthermore, THBS1 CpG sites were found to be specifically hypermethylated in CCSK and, thus, the DNA methylation status of these THBS1 sites alone was sufficient for the distinction of CCSK from other pediatric renal tumors, including Wilms' tumor and CMN. Moreover, combined bisulfite restriction analysis could be applied for the detection of hypermethylation of a THBS1 CpG site. Besides the biological significance in the pathogenesis, the DNA methylation profile should be useful for the differential diagnosis of pediatric renal tumors.

  8. Hypermethylation of brain natriuretic peptide gene is associated with the risk of rheumatic heart disease

    PubMed Central

    Li, Ni; Zheng, Dawei; Sun, Lebo; Shi, Huoshun; Zhu, Xiuying; Xu, Guodong; Wang, Qinning; Zhu, Caimin

    2016-01-01

    To investigate the contribution of brain natriuretic peptide (BNP) promoter DNA methylation to the risk of rheumatic heart disease (RHD) and the influence of warfarin anticoagulant therapy on BNP methylation levels for RHD patients after surgery. BNP methylation levels were determined by bisulfite pyrosequencing from plasma samples of RHD patients compared with healthy controls. Several factors influencing the RHD patients were included like age, smoking and cholesterol levels. A fragment of five CG sites (CpG1–5) in the promoter region of BNP gene was measured. BNP gene hypermethylation was found in CpG4 and CpG5 in RHD patients compared with non-RHD controls. A significant difference was also observed between RHD patients with long-term administration of warfarin and RHD patients who had recently undergone an operation. Moreover, single CpG4 and CpG5 analysis revealed a significant increase in methylation levels in men. BNP gene body hypermethylation is associated with the risk of RHD, and also influenced by the warfarin anticoagulant therapy of RHD patients after surgery, which could represent novel and promising targets for therapeutic development. PMID:27920275

  9. Early diagnostic potential of APC hypermethylation in esophageal cancer.

    PubMed

    Wang, Bujiang; Song, Haojun; Jiang, Haizhong; Fu, Yangbo; Ding, Xiaoyun; Zhou, Chongchang

    2018-01-01

    The hypermethylation of APC gene is observed in various cancers, including esophageal cancer (EC). However, the association between APC methylation and the initiation and progression of EC is poorly understood. The current study systematically reviewed studies on abnormal methylation of APC in EC and quantitatively synthesized 18 studies by meta-analysis involving 1008 ECs, 570 Barrett's esophagus (BE), and 782 controls. Our results showed higher methylation of APC in EC (OR = 23.33, P < 0.001) and BE (OR = 9.34, P < 0.001) than in normal controls. Whereas APC methylation in EC was similar to that in BE ( P = 0.052), it was not associated with tumor stage ( P = 0.204). Additionally, APC methylation was not significantly associated with overall survival (OS) and relapse-free survival (RFS) in patients with EC. The performance of APC methylation for the detection of EC and BE achieved areas under the receiver operating characteristic curves of 0.94 and 0.88, respectively. Our results imply that APC methylation detection is a potential diagnostic biomarker for EC and BE.

  10. Hypermethylation of the Human Glutathione S-Transferase-π Gene (GSTP1) CpG Island Is Present in a Subset of Proliferative Inflammatory Atrophy Lesions but Not in Normal or Hyperplastic Epithelium of the Prostate

    PubMed Central

    Nakayama, Masashi; Bennett, Christina J.; Hicks, Jessica L.; Epstein, Jonathan I.; Platz, Elizabeth A.; Nelson, William G.; De Marzo, Angelo M.

    2003-01-01

    Somatic inactivation of the glutathione S-transferase-π gene (GSTP1) via CpG island hypermethylation occurs early during prostate carcinogenesis, present in ∼70% of high-grade prostatic intraepithelial neoplasia (high-grade PIN) lesions and more than 90% of adenocarcinomas. Recently, there has been a resurgence of the concept that foci of prostatic atrophy (referred to as proliferative inflammatory atrophy or PIA) may be precursor lesions for the development of prostate cancer and/or high-grade PIN. Many of the cells within PIA lesions contain elevated levels of GSTP1, glutathione S-transferase-α (GSTA1), and cyclooxygenase-II proteins, suggesting a stress response. Because not all PIA cells are positive for GSTP1 protein, we hypothesized that some of the cells within these regions acquire GSTP1 CpG island hypermethylation, increasing the chance of progression to high-grade PIN and/or adenocarcinoma. Separate regions (n =199) from 27 formalin-fixed paraffin-embedded prostates were microdissected by laser-capture microdissection (Arcturus PixCell II). These regions included normal epithelium (n = 48), hyperplasticepithelium from benign prostatic hyperplasia nodules (n = 22), PIA (n = 64), high-grade PIN (n = 32), and adenocarcinoma (n = 33). Genomic DNA was isolated and assessed for GSTP1 CpG island hypermethylation by methylation-specific polymerase chain reaction. GSTP1 CpG island hypermethylation was not detected in normal epithelium (0 of 48) or in hyperplastic epithelium (0 of 22), but was found in 4 of 64 (6.3%) PIA lesions. The difference in the frequency of GSTP1 CpG island hypermethylation between normal or hyperplastic epithelium and PIA was statistically significant (P = 0.049). Similar to studies using nonmicrodissected cases, hypermethylation was found in 22 of 32 (68.8%) high-grade PIN lesions and in 30 of 33 (90.9%) adenocarcinoma lesions. Unlike normal or hyperplastic epithelium, GSTP1 CpG island hypermethylation can be detected in some PIA

  11. Current controversies in prenatal diagnosis 2: Cell-free DNA prenatal screening should be used to identify all chromosome abnormalities.

    PubMed

    Chitty, Lyn S; Hudgins, Louanne; Norton, Mary E

    2018-02-01

    Noninvasive prenatal testing (NIPT) using cell-free DNA (cfDNA) from maternal serum has been clinically available since 2011. This technology has revolutionized our ability to screen for the common aneuploidies trisomy 21 (Down syndrome), trisomy 18, and trisomy 13. More recently, clinical laboratories have offered screening for other chromosome abnormalities including sex chromosome abnormalities and copy number variants (CNV) without little published data on the sensitivity, specificity, and positive predictive value. In this debate, the pros and cons of performing prenatal screening via cfDNA for all chromosome abnormalities is discussed. At the time of the debate in 2017, the general consensus was that the literature does not yet support using this technology to screen for all chromosome abnormalities and that education is key for both providers and the patients so that the decision-making process is as informed as possible. © 2018 John Wiley & Sons, Ltd.

  12. H3K4me1 marks DNA regions hypomethylated during aging in human stem and differentiated cells

    PubMed Central

    Fernández, Agustín F.; Bayón, Gustavo F.; Urdinguio, Rocío G.; Toraño, Estela G.; García, María G.; Carella, Antonella; Petrus-Reurer, Sandra; Ferrero, Cecilia; Martinez-Camblor, Pablo; Cubillo, Isabel; García-Castro, Javier; Delgado-Calle, Jesús; Pérez-Campo, Flor M.; Riancho, José A.; Bueno, Clara; Menéndez, Pablo; Mentink, Anouk; Mareschi, Katia; Claire, Fabian; Fagnani, Corrado; Medda, Emanuela; Toccaceli, Virgilia; Brescianini, Sonia; Moran, Sebastián; Esteller, Manel; Stolzing, Alexandra; de Boer, Jan; Nisticò, Lorenza; Stazi, Maria A.

    2015-01-01

    In differentiated cells, aging is associated with hypermethylation of DNA regions enriched in repressive histone post-translational modifications. However, the chromatin marks associated with changes in DNA methylation in adult stem cells during lifetime are still largely unknown. Here, DNA methylation profiling of mesenchymal stem cells (MSCs) obtained from individuals aged 2 to 92 yr identified 18,735 hypermethylated and 45,407 hypomethylated CpG sites associated with aging. As in differentiated cells, hypermethylated sequences were enriched in chromatin repressive marks. Most importantly, hypomethylated CpG sites were strongly enriched in the active chromatin mark H3K4me1 in stem and differentiated cells, suggesting this is a cell type–independent chromatin signature of DNA hypomethylation during aging. Analysis of scedasticity showed that interindividual variability of DNA methylation increased during aging in MSCs and differentiated cells, providing a new avenue for the identification of DNA methylation changes over time. DNA methylation profiling of genetically identical individuals showed that both the tendency of DNA methylation changes and scedasticity depended on nongenetic as well as genetic factors. Our results indicate that the dynamics of DNA methylation during aging depend on a complex mixture of factors that include the DNA sequence, cell type, and chromatin context involved and that, depending on the locus, the changes can be modulated by genetic and/or external factors. PMID:25271306

  13. A heterozygous IDH1R132H/WT mutation induces genome-wide alterations in DNA methylation.

    PubMed

    Duncan, Christopher G; Barwick, Benjamin G; Jin, Genglin; Rago, Carlo; Kapoor-Vazirani, Priya; Powell, Doris R; Chi, Jen-Tsan; Bigner, Darell D; Vertino, Paula M; Yan, Hai

    2012-12-01

    Monoallelic point mutations of the NADP(+)-dependent isocitrate dehydrogenases IDH1 and IDH2 occur frequently in gliomas, acute myeloid leukemias, and chondromas, and display robust association with specific DNA hypermethylation signatures. Here we show that heterozygous expression of the IDH1(R132H) allele is sufficient to induce the genome-wide alterations in DNA methylation characteristic of these tumors. Using a gene-targeting approach, we knocked-in a single copy of the most frequently observed IDH1 mutation, R132H, into a human cancer cell line and profiled changes in DNA methylation at over 27,000 CpG dinucleotides relative to wild-type parental cells. We find that IDH1(R132H/WT) mutation induces widespread alterations in DNA methylation, including hypermethylation of 2010 and hypomethylation of 842 CpG loci. We demonstrate that many of these alterations are consistent with those observed in IDH1-mutant and G-CIMP+ primary gliomas and can segregate IDH wild-type and mutated tumors as well as those exhibiting the G-CIMP phenotype in unsupervised analysis of two primary glioma cohorts. Further, we show that the direction of IDH1(R132H/WT)-mediated DNA methylation change is largely dependent upon preexisting DNA methylation levels, resulting in depletion of moderately methylated loci. Additionally, whereas the levels of multiple histone H3 and H4 methylation modifications were globally increased, consistent with broad inhibition of histone demethylation, hypermethylation at H3K9 in particular accompanied locus-specific DNA hypermethylation at several genes down-regulated in IDH1(R132H/WT) knock-in cells. These data provide insight on epigenetic alterations induced by IDH1 mutations and support a causal role for IDH1(R132H/WT) mutants in driving epigenetic instability in human cancer cells.

  14. Promoter hypermethylation of mismatch repair gene hMLH1 predicts the clinical response of malignant astrocytomas to nitrosourea.

    PubMed

    Fukushima, Takao; Katayama, Yoichi; Watanabe, Takao; Yoshino, Atsuo; Ogino, Akiyoshi; Ohta, Takashi; Komine, Chiaki

    2005-02-15

    In certain types of human cancers, transcriptional inactivation of hMLH1 by promoter hypermethylation plays a causal role in the loss of mismatch repair functions that modulate cytotoxic pathways in response to DNA-damaging agents. The aim of the present study was to investigate the role of promoter methylation of the hMLH1 gene in malignant astrocytomas. We examined the hMLH1 promoter methylation in a homogeneous cohort of patients with 41 malignant astrocytomas treated by 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-2(2-chloroethyl)-3-nitrosourea chemotherapy in combination with radiation and interferon therapy, and assessed the correlation of such methylation with clinical outcome. hMLH1 promoter methylation was found in 6 (15%) of the 41 newly diagnosed malignant astrocytomas. Hypermethylation of the hMLH1 promoter corresponded closely with a loss of immunohistochemical staining for hMLH1 protein (P = 0.0013). Patients with hMLH1-methylated tumors displayed a greater chance of responding to adjuvant therapy as compared with those with hMLH1-unmethylated tumors (P = 0.0150). The presence of hMLH1 hypermethylation was significantly associated with a longer progression-free survival on both univariate analysis (P = 0.0340) and multivariate analysis (P = 0.0161). The present study identified hMLH1 methylation status as a predictor of the clinical response of malignant astrocytomas to chloroethylnitrosourea-based adjuvant therapy. The findings obtained suggest that determination of the methylation status of hMLH1 could provide a potential basis for designing rational chemotherapeutic strategies, as well as for predicting prognosis.

  15. Progesterone impairs antigen-non-specific immune protection by CD8 T memory cells via interferon-γ gene hypermethylation.

    PubMed

    Yao, Yushi; Li, Hui; Ding, Jie; Xia, Yixin; Wang, Lei

    2017-11-01

    Pregnant women and animals have increased susceptibility to a variety of intracellular pathogens including Listeria monocytogenes (LM), which has been associated with significantly increased level of sex hormones such as progesterone. CD8 T memory(Tm) cell-mediated antigen-non-specific IFN-γ responses are critically required in the host defense against LM. However, whether and how increased progesterone during pregnancy modulates CD8 Tm cell-mediated antigen-non-specific IFN-γ production and immune protection against LM remain poorly understood. Here we show in pregnant women that increased serum progesterone levels are associated with DNA hypermethylation of IFN-γ gene promoter region and decreased IFN-γ production in CD8 Tm cells upon antigen-non-specific stimulation ex vivo. Moreover, IFN-γ gene hypermethylation and significantly reduced IFN-γ production post LM infection in antigen-non-specific CD8 Tm cells are also observed in pregnant mice or progesterone treated non-pregnant female mice, which is a reversible phenotype following demethylation treatment. Importantly, antigen-non-specific CD8 Tm cells from progesterone treated mice have impaired anti-LM protection when adoptive transferred in either pregnant wild type mice or IFN-γ-deficient mice, and demethylation treatment rescues the adoptive protection of such CD8 Tm cells. These data demonstrate that increased progesterone impairs immune protective functions of antigen-non-specific CD8 Tm cells via inducing IFN-γ gene hypermethylation. Our findings thus provide insights into a new mechanism through which increased female sex hormone regulate CD8 Tm cell functions during pregnancy.

  16. Progesterone impairs antigen-non-specific immune protection by CD8 T memory cells via interferon-γ gene hypermethylation

    PubMed Central

    Yao, Yushi; Li, Hui; Ding, Jie; Xia, Yixin

    2017-01-01

    Pregnant women and animals have increased susceptibility to a variety of intracellular pathogens including Listeria monocytogenes (LM), which has been associated with significantly increased level of sex hormones such as progesterone. CD8 T memory(Tm) cell-mediated antigen-non-specific IFN-γ responses are critically required in the host defense against LM. However, whether and how increased progesterone during pregnancy modulates CD8 Tm cell-mediated antigen-non-specific IFN-γ production and immune protection against LM remain poorly understood. Here we show in pregnant women that increased serum progesterone levels are associated with DNA hypermethylation of IFN-γ gene promoter region and decreased IFN-γ production in CD8 Tm cells upon antigen-non-specific stimulation ex vivo. Moreover, IFN-γ gene hypermethylation and significantly reduced IFN-γ production post LM infection in antigen-non-specific CD8 Tm cells are also observed in pregnant mice or progesterone treated non-pregnant female mice, which is a reversible phenotype following demethylation treatment. Importantly, antigen-non-specific CD8 Tm cells from progesterone treated mice have impaired anti-LM protection when adoptive transferred in either pregnant wild type mice or IFN-γ-deficient mice, and demethylation treatment rescues the adoptive protection of such CD8 Tm cells. These data demonstrate that increased progesterone impairs immune protective functions of antigen-non-specific CD8 Tm cells via inducing IFN-γ gene hypermethylation. Our findings thus provide insights into a new mechanism through which increased female sex hormone regulate CD8 Tm cell functions during pregnancy. PMID:29155896

  17. APC hypermethylation for early diagnosis of colorectal cancer: a meta-analysis and literature review.

    PubMed

    Liang, Tie-Jun; Wang, Hong-Xu; Zheng, Yan-Yan; Cao, Ying-Qing; Wu, Xiaoyu; Zhou, Xin; Dong, Shu-Xiao

    2017-07-11

    Adenomatous polyposis coli (APC) promoter hypermethylation has been frequently observed in colorectal cancer (CRC). The association between APC promoter methylation and clinicopathological significance in CRC is under investigation. We performed a meta-analysis to quantitatively evaluate the significance of APC methylation in CRC. The study included a total of 24 articles and 2025 CRC patients. The frequency of APC promoter hypermethylation was significantly higher in colorectal adenoma than in normal colorectal tissue, OR was 5.76, 95% CI, 2.45-13.56; p<0.0001, I2=0%. APC promoter more frequently hypermethylated in CRC stage I compared to normal colorectal tissue, OR was 13.42, 95% CI, 3.66-49.20; p<0.0001, I2=31%. The risk of incidence of CRC was significantly correlated to APC promoter hypermethylation, pooled OR was 9.80, 95%CI, 6.07-15.81; p<0.00001, I2=43%. APC methylation was not associated with grade, stage of CRC as well as tumor location, patients' gender, and smoking behavior. The results indicate that APC promoter hypermethylation is an early event in carcinogenesis of CRC, could be a valuable diagnostic marker for early-stage CRC. APC methylation is not significantly associated with overall survival in patients with CRC. APC is a potential drug target for development of personalized treatment.

  18. DNA Methylation-a Potential Source of Mitochondria DNA Base Mismatch in the Development of Diabetic Retinopathy.

    PubMed

    Mishra, Manish; Kowluru, Renu A

    2018-04-21

    In the development of diabetic retinopathy, retinal mitochondria are dysfunctional, and mitochondrial DNA (mtDNA) is damaged with increased base mismatches and hypermethylated cytosines. DNA methylation is also a potential source of mutation, and in diabetes, the noncoding region, the displacement loop (D-loop), experiences more methylation and base mismatches than other regions of the mtDNA. Our aim was to investigate a possible crosstalk between mtDNA methylation and base mismatches in the development of diabetic retinopathy. The effect of inhibition of Dnmts (by 5-aza-2'-deoxycytidine or Dnmt1-siRNA) on glucose-induced mtDNA base mismatches was investigated in human retinal endothelial cells by surveyor endonuclease digestion and validated by Sanger sequencing. The role of deamination factors on increased base mismatches was determined in the cells genetically modulated for mitochondrial superoxide dismutase (Sod2) or cytidine-deaminase (APOBEC3A). The results were confirmed in an in vivo model using retinal microvasculature from diabetic mice overexpressing Sod2. Inhibition of DNA methylation, or regulation of cytosine deamination, significantly inhibited an increase in base mismatches at the D-loop and prevented mitochondrial dysfunction. Overexpression of Sod2 in mice also prevented diabetes-induced D-loop hypermethylation and increase in base mismatches. The crosstalk between DNA methylation and base mismatches continued even after termination of hyperglycemia, suggesting its role in the metabolic memory phenomenon associated with the progression of diabetic retinopathy. Inhibition of DNA methylation limits the availability of methylated cytosine for deamination, suggesting a crosstalk between DNA methylation and base mismatches. Thus, regulation of DNA methylation, or its deamination, should impede the development of diabetic retinopathy by preventing formation of base mismatches and mitochondrial dysfunction.

  19. H3K4me1 marks DNA regions hypomethylated during aging in human stem and differentiated cells.

    PubMed

    Fernández, Agustín F; Bayón, Gustavo F; Urdinguio, Rocío G; Toraño, Estela G; García, María G; Carella, Antonella; Petrus-Reurer, Sandra; Ferrero, Cecilia; Martinez-Camblor, Pablo; Cubillo, Isabel; García-Castro, Javier; Delgado-Calle, Jesús; Pérez-Campo, Flor M; Riancho, José A; Bueno, Clara; Menéndez, Pablo; Mentink, Anouk; Mareschi, Katia; Claire, Fabian; Fagnani, Corrado; Medda, Emanuela; Toccaceli, Virgilia; Brescianini, Sonia; Moran, Sebastián; Esteller, Manel; Stolzing, Alexandra; de Boer, Jan; Nisticò, Lorenza; Stazi, Maria A; Fraga, Mario F

    2015-01-01

    In differentiated cells, aging is associated with hypermethylation of DNA regions enriched in repressive histone post-translational modifications. However, the chromatin marks associated with changes in DNA methylation in adult stem cells during lifetime are still largely unknown. Here, DNA methylation profiling of mesenchymal stem cells (MSCs) obtained from individuals aged 2 to 92 yr identified 18,735 hypermethylated and 45,407 hypomethylated CpG sites associated with aging. As in differentiated cells, hypermethylated sequences were enriched in chromatin repressive marks. Most importantly, hypomethylated CpG sites were strongly enriched in the active chromatin mark H3K4me1 in stem and differentiated cells, suggesting this is a cell type-independent chromatin signature of DNA hypomethylation during aging. Analysis of scedasticity showed that interindividual variability of DNA methylation increased during aging in MSCs and differentiated cells, providing a new avenue for the identification of DNA methylation changes over time. DNA methylation profiling of genetically identical individuals showed that both the tendency of DNA methylation changes and scedasticity depended on nongenetic as well as genetic factors. Our results indicate that the dynamics of DNA methylation during aging depend on a complex mixture of factors that include the DNA sequence, cell type, and chromatin context involved and that, depending on the locus, the changes can be modulated by genetic and/or external factors. © 2015 Fernández et al.; Published by Cold Spring Harbor Laboratory Press.

  20. A DNA methylation map of human cancer at single base-pair resolution

    PubMed Central

    Vidal, E; Sayols, S; Moran, S; Guillaumet-Adkins, A; Schroeder, M P; Royo, R; Orozco, M; Gut, M; Gut, I; Lopez-Bigas, N; Heyn, H; Esteller, M

    2017-01-01

    Although single base-pair resolution DNA methylation landscapes for embryonic and different somatic cell types provided important insights into epigenetic dynamics and cell-type specificity, such comprehensive profiling is incomplete across human cancer types. This prompted us to perform genome-wide DNA methylation profiling of 22 samples derived from normal tissues and associated neoplasms, including primary tumors and cancer cell lines. Unlike their invariant normal counterparts, cancer samples exhibited highly variable CpG methylation levels in a large proportion of the genome, involving progressive changes during tumor evolution. The whole-genome sequencing results from selected samples were replicated in a large cohort of 1112 primary tumors of various cancer types using genome-scale DNA methylation analysis. Specifically, we determined DNA hypermethylation of promoters and enhancers regulating tumor-suppressor genes, with potential cancer-driving effects. DNA hypermethylation events showed evidence of positive selection, mutual exclusivity and tissue specificity, suggesting their active participation in neoplastic transformation. Our data highlight the extensive changes in DNA methylation that occur in cancer onset, progression and dissemination. PMID:28581523

  1. A DNA methylation map of human cancer at single base-pair resolution.

    PubMed

    Vidal, E; Sayols, S; Moran, S; Guillaumet-Adkins, A; Schroeder, M P; Royo, R; Orozco, M; Gut, M; Gut, I; Lopez-Bigas, N; Heyn, H; Esteller, M

    2017-10-05

    Although single base-pair resolution DNA methylation landscapes for embryonic and different somatic cell types provided important insights into epigenetic dynamics and cell-type specificity, such comprehensive profiling is incomplete across human cancer types. This prompted us to perform genome-wide DNA methylation profiling of 22 samples derived from normal tissues and associated neoplasms, including primary tumors and cancer cell lines. Unlike their invariant normal counterparts, cancer samples exhibited highly variable CpG methylation levels in a large proportion of the genome, involving progressive changes during tumor evolution. The whole-genome sequencing results from selected samples were replicated in a large cohort of 1112 primary tumors of various cancer types using genome-scale DNA methylation analysis. Specifically, we determined DNA hypermethylation of promoters and enhancers regulating tumor-suppressor genes, with potential cancer-driving effects. DNA hypermethylation events showed evidence of positive selection, mutual exclusivity and tissue specificity, suggesting their active participation in neoplastic transformation. Our data highlight the extensive changes in DNA methylation that occur in cancer onset, progression and dissemination.

  2. Aberrant Promoter Hypermethylation of RASSF Family Members in Merkel Cell Carcinoma

    PubMed Central

    Richter, Antje M.; Haag, Tanja; Walesch, Sara; Herrmann-Trost, Peter; Marsch, Wolfgang C.; Kutzner, Heinz; Helmbold, Peter; Dammann, Reinhard H.

    2013-01-01

    Merkel cell carcinoma (MCC) is one of the most aggressive cancers of the skin. RASSFs are a family of tumor suppressors that are frequently inactivated by promoter hypermethylation in various cancers. We studied CpG island promoter hypermethylation in MCC of RASSF2, RASSF5A, RASSF5C and RASSF10 by combined bisulfite restriction analysis (COBRA) in MCC samples and control tissue. We found RASSF2 to be methylated in three out of 43 (7%), RASSF5A in 17 out of 39 (44%, but also 43% in normal tissue), RASSF5C in two out of 26 (8%) and RASSF10 in 19 out of 84 (23%) of the cancer samples. No correlation between the methylation status of the analyzed RASSFs or between RASSF methylation and MCC characteristics (primary versus metastatic, Merkel cell polyoma virus infection, age, sex) was found. Our results show that RASSF2, RASSF5C and RASSF10 are aberrantly hypermethylated in MCC to a varying degree and this might contribute to Merkel cell carcinogenesis. PMID:24252868

  3. Detection of Promoter DNA Methylation of APC, DAPK, and GSTP1 Genes in tissue Biopsy and Matched Serum of Advanced-Stage Lung Cancer Patients.

    PubMed

    Ali, Ashraf; Kumar, Sachin; Kakaria, Vinod Kumar; Mohan, Anant; Luthra, Kalpana; Upadhyay, Ashish Dutt; Guleria, Randeep

    2017-07-03

    Promoter DNA hypermethylation of APC, DAPK, and GSTP1 genes was evaluated in biopsy and matched serum of 160 lung cancer patients and 70 controls. In biopsy, 83.1, 83.1, and 78.1% of lung cancer patients and 72.9, 70, and 70% of controls, while in serum, 52.5, 30.6, and 65.6% of lung cancer patients and 14.3, 18.6, and 30% of controls were positive for APC, DAPK, and GSTP1 hypermethylation respectively. We couldn't find any significant role of DNA hypermethylation in lung cancer. However, long follow-up of methylation positive controls will be required to confirm its role for the prediction of lung cancer.

  4. DNA methylation profiling distinguishes histological subtypes of renal cell carcinoma

    PubMed Central

    Slater, Amy A.; Alokail, Majed; Gentle, Dean; Yao, Masahiro; Kovacs, Gyula; Maher, Eamonn R.

    2013-01-01

    Renal cell carcinoma (RCC) accounts for around 3% of cancers in the UK, and both incidence and mortality are increasing with the aging population. RCC can be divided into several subtypes: conventional RCC (the most common, comprising 75% of all cases), papillary RCC (15%) and chromophobe RCC (5%). Renal oncocytoma is a benign tumor and accounts for 5% of RCC. Cancer and epigenetics are closely associated, with DNA hypermethylation being widely accepted as a feature of many cancers. In this study the DNA methylation profiles of chromophobe RCC and renal oncocytomas were investigated by utilizing the Infinium HumanMethylation450 BeadChips. Cancer-specific hypermethylation was identified in 9.4% and 5.2% of loci in chromophobe RCC and renal oncocytoma samples, respectively, while the majority of the genome was hypomethylated. Thirty (hypermethylated) and 41 (hypomethylated) genes were identified as differentially methylated between chromophobe RCC and renal oncocytomas (p < 0.05). Pathway analysis identified some of the differentially hypermethylated genes to be involved in Wnt (EN2), MAPK (CACNG7) and TGFβ (AMH) signaling, Hippo pathway (NPHP4), and cell death and apoptosis (SPG20, NKX6-2, PAX3 and BAG2). In addition, we analyzed ccRCC and papillary RCC data available from The Cancer Genome Atlas portal to identify differentially methylated loci in chromophobe RCC and renal oncocytoma in relation to the other histological subtypes, providing insight into the pathology of RCC subtypes and classification of renal tumors. PMID:23428843

  5. Aberrantly methylated DNA as a biomarker in breast cancer.

    PubMed

    Kristiansen, Søren; Jørgensen, Lars M; Guldberg, Per; Sölétormos, György

    2013-01-01

    Aberrant DNA hypermethylation at gene promoters is a frequent event in human breast cancer. Recent genome-wide studies have identified hundreds of genes that exhibit differential methylation between breast cancer cells and normal breast tissue. Due to the tumor-specific nature of DNA hypermethylation events, their use as tumor biomarkers is usually not hampered by analytical signals from normal cells, which is a general problem for existing protein tumor markers used for clinical assessment of breast cancer. There is accumulating evidence that DNA-methylation changes in breast cancer patients occur early during tumorigenesis. This may open up for effective screening, and analysis of blood or nipple aspirate may later help in diagnosing breast cancer. As a more detailed molecular characterization of different types of breast cancer becomes available, the ability to divide patients into subgroups based on DNA biomarkers may improve prognosis. Serial monitoring of DNA-methylation markers in blood during treatment may be useful, particularly when the cancer burden is below the detection level for standard imaging techniques. Overall, aberrant DNA methylation has a great potential as a versatile biomarker tool for screening, diagnosis, prognosis and monitoring of breast cancer. Standardization of methods and biomarker panels will be required to fully exploit this clinical potential.

  6. Air pollution and DNA methylation alterations in lung cancer: A systematic and comparative study.

    PubMed

    Jiang, Cheng-Lan; He, Shui-Wang; Zhang, Yun-Dong; Duan, He-Xian; Huang, Tao; Huang, Yun-Chao; Li, Gao-Feng; Wang, Ping; Ma, Li-Ju; Zhou, Guang-Biao; Cao, Yi

    2017-01-03

    The lung cancer incidence in the Xuanwei and neighboring region, Yunnan, China, is among the highest in China and is attributed to severe air pollution with high benzo(a)pyrene levels. We systematically and comparatively analyzed DNA methylation alterations at genome and gene levels in Xuanwei lung cancer tissues and cell lines, as well as benzo(a)pyrene-treated cells and mouse samples. We obtained a comprehensive dataset of genome-wide cytosine-phosphate-guanine island methylation in air pollution-related lung cancer samples. Benzo(a)pyrene exposure induced multiple alterations in DNA methylation and in mRNA expressions of DNA methyltransferases and ten-11 translocation proteins; these alterations partially occurred in Xuanwei lung cancer. Furthermore, benzo(a)pyrene-induced DKK2 and EN1 promoter hypermethylation and LPAR2 promoter hypomethylation led to down-regulation and up-regulation of the genes, respectively; the down-regulation of DKK2 and EN1 promoted the cellular proliferation. Thus, DNA methylation alterations induced by benzo(a)pyrene contribute partially to abnormal DNA methylation in air pollution-related lung cancer, and these DNA methylation alterations may affect the development and progression of lung cancer. Additionally, vitamin C and B6 can reduce benzo(a)pyrene-induced DNA methylation alterations and may be used as chemopreventive agents for air pollution-related lung cancer.

  7. MET18 Connects the Cytosolic Iron-Sulfur Cluster Assembly Pathway to Active DNA Demethylation in Arabidopsis

    PubMed Central

    Tang, Kai; Zhang, Huiming; Mangrauthia, Satendra K.; Lei, Mingguang; Hsu, Chuan-Chih; Hou, Yueh-Ju; Wang, Chunguo; Li, Yan; Tao, W. Andy; Zhu, Jian-Kang

    2015-01-01

    DNA demethylation mediated by the DNA glycosylase ROS1 helps determine genomic DNA methylation patterns and protects active genes from being silenced. However, little is known about the mechanism of regulation of ROS1 enzymatic activity. Using a forward genetic screen, we identified an anti-silencing (ASI) factor, ASI3, the dysfunction of which causes transgene promoter hyper-methylation and silencing. Map-based cloning identified ASI3 as MET18, a component of the cytosolic iron-sulfur cluster assembly (CIA) pathway. Mutation in MET18 leads to hyper-methylation at thousands of genomic loci, the majority of which overlap with hypermethylated loci identified in ros1 and ros1dml2dml3 mutants. Affinity purification followed by mass spectrometry indicated that ROS1 physically associates with MET18 and other CIA components. Yeast two-hybrid and split luciferase assays showed that ROS1 can directly interact with MET18 and another CIA component, AE7. Site-directed mutagenesis of ROS1 indicated that the conserved iron-sulfur motif is indispensable for ROS1 enzymatic activity. Our results suggest that ROS1-mediated active DNA demethylation requires MET18-dependent transfer of the iron-sulfur cluster, highlighting an important role of the CIA pathway in epigenetic regulation. PMID:26492035

  8. Mitochondria DNA replication and DNA methylation in the metabolic memory associated with continued progression of diabetic retinopathy.

    PubMed

    Tewari, Shikha; Zhong, Qing; Santos, Julia M; Kowluru, Renu A

    2012-07-24

    Diabetic retinopathy fails to halt after cessation of hyperglycemic insult, and a vicious cycle of mitochondria damage continues. The aim of our study was to investigate the effect of termination of hyperglycemia on retinal mtDNA replication, and elucidate the mechanism responsible for the continued mtDNA damage. Polymerase gamma 1 (POLG1), the catalytic subunit of the mitochondrial DNA replication enzyme, and the damage to the displacement loop region of mtDNA (D-loop) were analyzed in the retina from streptozotocin-diabetic rats maintained in poor glycemic control (PC, glycated hemoglobin ∼11%) or in good glycemic control (GC, glycated hemoglobin ∼6%) for 6 months, or in PC for three months followed by GC for three months (Rev). To understand the mechanism DNA methylation status of POLG1 promoter was investigated by methylation-specific PCR. The key parameters were confirmed in the isolated retinal endothelial cells exposed to high glucose, followed by normal glucose. POLG1 continued to be down-regulated, the D-loop region damaged, and the CpG islands at the regulatory region of POLG hyper-methylated even after three months of GC that had followed three months of PC (Rev group). Similar results were observed in the retinal endothelial cells exposed to normal glucose after being exposed to high glucose. Continued hypermethylation of the CpG sites at the regulatory region of POLG affects its binding to the mtDNA, compromising the transcriptional activity. Modulation of DNA methylation using pharmaceutic or molecular means could help maintain mitochondria homeostasis, and prevent further progression of diabetic retinopathy.

  9. Cortical DNA methylation maintains remote memory.

    PubMed

    Miller, Courtney A; Gavin, Cristin F; White, Jason A; Parrish, R Ryley; Honasoge, Avinash; Yancey, Christopher R; Rivera, Ivonne M; Rubio, María D; Rumbaugh, Gavin; Sweatt, J David

    2010-06-01

    A behavioral memory's lifetime represents multiple molecular lifetimes, suggesting the necessity for a self-perpetuating signal. One candidate is DNA methylation, a transcriptional repression mechanism that maintains cellular memory throughout development. We found that persistent, gene-specific cortical hypermethylation was induced in rats by a single, hippocampus-dependent associative learning experience and pharmacologic inhibition of methylation 1 month after learning disrupted remote memory. We propose that the adult brain utilizes DNA methylation to preserve long-lasting memories.

  10. DNA methylation markers for oral pre-cancer progression: A critical review.

    PubMed

    Shridhar, Krithiga; Walia, Gagandeep Kaur; Aggarwal, Aastha; Gulati, Smriti; Geetha, A V; Prabhakaran, Dorairaj; Dhillon, Preet K; Rajaraman, Preetha

    2016-02-01

    Although oral cancers are generally preceded by a well-established pre-cancerous stage, there is a lack of well-defined clinical and morphological criteria to detect and signal progression from pre-cancer to malignant tumours. We conducted a critical review to summarize the evidence regarding aberrant DNA methylation patterns as a potential diagnostic biomarker predicting progression. We identified all relevant human studies published in English prior to 30th April 2015 that examined DNA methylation (%) in oral pre-cancer by searching PubMed, Web-of-Science and Embase databases using combined key-searches. Twenty-one studies (18-cross-sectional; 3-longitudinal) were eligible for inclusion in the review, with sample sizes ranging from 4 to 156 affected cases. Eligible studies examined promoter region hyper-methylation of tumour suppressor genes in pathways including cell-cycle-control (n=15), DNA-repair (n=7), cell-cycle-signalling (n=4) and apoptosis (n=3). Hyper-methylated loci reported in three or more studies included p16, p14, MGMT and DAPK. Two longitudinal studies reported greater p16 hyper-methylation in pre-cancerous lesions transformed to malignancy compared to lesions that regressed (57-63.6% versus 8-32.1%; p<0.01). The one study that explored epigenome-wide methylation patterns reported three novel hyper-methylated loci (TRHDE; ZNF454; KCNAB3). The majority of reviewed studies were small, cross-sectional studies with poorly defined control groups and lacking validation. Whilst limitations in sample size and study design preclude definitive conclusions, current evidence suggests a potential utility of DNA methylation patterns as a diagnostic biomarker for oral pre-cancer progression. Robust studies such as large epigenome-wide methylation explorations of oral pre-cancer with longitudinal tracking are needed to validate the currently reported signals and identify new risk-loci and the biological pathways of disease progression. Copyright © 2015 The

  11. DNA methylation markers for oral pre-cancer progression: A critical review

    PubMed Central

    Shridhar, Krithiga; Walia, Gagandeep Kaur; Aggarwal, Aastha; Gulati, Smriti; Geetha, A.V.; Prabhakaran, Dorairaj; Dhillon, Preet K.; Rajaraman, Preetha

    2016-01-01

    Summary Although oral cancers are generally preceded by a well-established pre-cancerous stage, there is a lack of well-defined clinical and morphological criteria to detect and signal progression from pre-cancer to malignant tumours. We conducted a critical review to summarize the evidence regarding aberrant DNA methylation patterns as a potential diagnostic biomarker predicting progression. We identified all relevant human studies published in English prior to 30th April 2015 that examined DNA methylation (%) in oral pre-cancer by searching PubMed, Web-of-Science and Embase databases using combined key-searches. Twenty-one studies (18-cross-sectional; 3-longitudinal) were eligible for inclusion in the review, with sample sizes ranging from 4 to 156 affected cases. Eligible studies examined promoter region hyper-methylation of tumour suppressor genes in pathways including cell-cycle-control (n = 15), DNA-repair (n = 7), cell-cycle-signalling (n = 4) and apoptosis (n = 3). Hyper-methylated loci reported in three or more studies included p16, p14, MGMT and DAPK. Two longitudinal studies reported greater p16 hyper-methylation in pre-cancerous lesions transformed to malignancy compared to lesions that regressed (57–63.6% versus 8–32.1%; p < 0.01). The one study that explored epigenome-wide methylation patterns reported three novel hyper-methylated loci (TRHDE; ZNF454; KCNAB3). The majority of reviewed studies were small, cross-sectional studies with poorly defined control groups and lacking validation. Whilst limitations in sample size and study design preclude definitive conclusions, current evidence suggests a potential utility of DNA methylation patterns as a diagnostic biomarker for oral pre-cancer progression. Robust studies such as large epigenome-wide methylation explorations of oral pre-cancer with longitudinal tracking are needed to validate the currently reported signals and identify new risk-loci and the biological pathways of disease

  12. DNA hypermethylation and X chromosome inactivation are major determinants of phenotypic variation in women heterozygous for G6PD mutations.

    PubMed

    Wang, Jin; Xiao, Qi-Zhi; Chen, You-Ming; Yi, Sheng; Liu, Dun; Liu, Yan-Hui; Zhang, Cui-Mei; Wei, Xiao-Feng; Zhou, Yu-Qiu; Zhong, Xing-Ming; Zhao, Cun-You; Xiong, Fu; Wei, Xiang-Cai; Xu, Xiang-Min

    2014-12-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked incompletely dominant enzyme deficiency that results from G6PD gene mutations. Women heterozygous for G6PD mutations exhibit variation in the loss of enzyme activity but the cause of this phenotypic variation is unclear. We determined DNA methylation and X-inactivation patterns in 71 G6PD-deficient female heterozygotes and 68 G6PD non-deficient controls with the same missense mutations (G6PD Canton c.1376G>T or Kaiping c.1388G>A) to correlate determinants with variable phenotypes. Specific CpG methylations within the G6PD promoter were significantly higher in G6PD-deficient heterozygotes than in controls. Preferential X-inactivation of the G6PD wild-type allele was determined in heterozygotes. The incidence of preferential X-inactivation was 86.2% in the deficient heterozygote group and 31.7% in the non-deficient heterozygote group. A significant negative correlation was observed between X-inactivation ratios of the wild-type allele and G6PD/6-phosphogluconate dehydrogenase (6PGD) ratios in heterozygous G6PD Canton (r=-0.657, p<0.001) or Kaiping (r=-0.668, p<0.001). Multivariate logistic regression indicated that heterozygotes with hypermethylation of specific CpG sites in the G6PD promoter and preferential X-inactivation of the wild-type allele were at risk of enzyme deficiency. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Epigenomic profiling of DNA methylation in paired prostate cancer versus adjacent benign tissue

    PubMed Central

    Geybels, Milan S.; Zhao, Shanshan; Wong, Chao-Jen; Bibikova, Marina; Klotzle, Brandy; Wu, Michael; Ostrander, Elaine A.; Fan, Jian-Bing; Feng, Ziding; Stanford, Janet L.

    2016-01-01

    Background Aberrant DNA methylation may promote prostate carcinogenesis. We investigated epigenome-wide DNA methylation profiles in prostate cancer (PCa) compared to adjacent benign tissue to identify differentially methylated CpG sites. Methods The study included paired PCa and adjacent benign tissue samples from 20 radical prostatectomy patients. Epigenetic profiling was done using the Infinium HumanMethylation450 BeadChip. Linear models that accounted for the paired study design and False Discovery Rate Q-values were used to evaluate differential CpG methylation. mRNA expression levels of the genes with the most differentially methylated CpG sites were analyzed. Results In total, 2,040 differentially methylated CpG sites were identified in PCa versus adjacent benign tissue (Q-value <0.001), the majority of which were hypermethylated (n = 1,946; 95%). DNA methylation profiles accurately distinguished between PCa and benign tissue samples. Twenty-seven top-ranked hypermethylated CpGs had a mean methylation difference of at least 40% between tissue types, which included 25 CpGs in 17 genes. Furthermore, for ten genes over 50% of promoter region CpGs were hypermethylated in PCa versus benign tissue. The top-ranked differentially methylated genes included three genes that were associated with both promoter hypermethylation and reduced gene expression: SCGB3A1, HIF3A, and AOX1. Analysis of The Cancer Genome Atlas (TCGA) data provided confirmatory evidence for our findings. Conclusions This study of PCa versus adjacent benign tissue showed many differentially methylated CpGs and regions in and outside gene promoter regions, which may potentially be used for the development of future epigenetic-based diagnostic tests or as therapeutic targets. PMID:26383847

  14. Epigenomic profiling of DNA methylation in paired prostate cancer versus adjacent benign tissue.

    PubMed

    Geybels, Milan S; Zhao, Shanshan; Wong, Chao-Jen; Bibikova, Marina; Klotzle, Brandy; Wu, Michael; Ostrander, Elaine A; Fan, Jian-Bing; Feng, Ziding; Stanford, Janet L

    2015-12-01

    Aberrant DNA methylation may promote prostate carcinogenesis. We investigated epigenome-wide DNA methylation profiles in prostate cancer (PCa) compared to adjacent benign tissue to identify differentially methylated CpG sites. The study included paired PCa and adjacent benign tissue samples from 20 radical prostatectomy patients. Epigenetic profiling was done using the Infinium HumanMethylation450 BeadChip. Linear models that accounted for the paired study design and False Discovery Rate Q-values were used to evaluate differential CpG methylation. mRNA expression levels of the genes with the most differentially methylated CpG sites were analyzed. In total, 2,040 differentially methylated CpG sites were identified in PCa versus adjacent benign tissue (Q-value < 0.001), the majority of which were hypermethylated (n = 1,946; 95%). DNA methylation profiles accurately distinguished between PCa and benign tissue samples. Twenty-seven top-ranked hypermethylated CpGs had a mean methylation difference of at least 40% between tissue types, which included 25 CpGs in 17 genes. Furthermore, for 10 genes over 50% of promoter region CpGs were hypermethylated in PCa versus benign tissue. The top-ranked differentially methylated genes included three genes that were associated with both promoter hypermethylation and reduced gene expression: SCGB3A1, HIF3A, and AOX1. Analysis of The Cancer Genome Atlas (TCGA) data provided confirmatory evidence for our findings. This study of PCa versus adjacent benign tissue showed many differentially methylated CpGs and regions in and outside gene promoter regions, which may potentially be used for the development of future epigenetic-based diagnostic tests or as therapeutic targets. © 2015 Wiley Periodicals, Inc.

  15. Nickel-induced Epithelial-Mesenchymal Transition by Reactive Oxygen Species Generation and E-cadherin Promoter Hypermethylation*

    PubMed Central

    Wu, Chih-Hsien; Tang, Sheau-Chung; Wang, Po-Hui; Lee, Huei; Ko, Jiunn-Liang

    2012-01-01

    Epithelial-mesenchymal transition (EMT) is considered a critical event in the pathogenesis of lung fibrosis and tumor metastasis. During EMT, the expression of differentiation markers switches from cell-cell junction proteins such as E-cadherin to mesenchymal markers such as fibronectin. Although nickel-containing compounds have been shown to be associated with lung carcinogenesis, the role of nickel in the EMT process in bronchial epithelial cells is not clear. The aim of this study was to examine whether nickel contributes to EMT in human bronchial epithelial cells. We also attempted to clarify the mechanisms involved in NiCl2-induced EMT. Our results showed that NiCl2 induced EMT phenotype marker alterations such as up-regulation of fibronectin and down-regulation of E-cadherin. In addition, the potent antioxidant N-acetylcysteine blocked EMT and expression of HIF-1α induced by NiCl2, whereas the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine restored the down-regulation of E-cadherin induced by NiCl2. Promoter hypermethylation of E-cadherin, determined by quantitative real time methyl-specific PCR and bisulfate sequencing, was also induced by NiCl2. These results shed new light on the contribution of NiCl2 to carcinogenesis. Specifically, NiCl2 induces down-regulation of E-cadherin by reactive oxygen species generation and promoter hypermethylation. This study demonstrates for the first time that nickel induces EMT in bronchial epithelial cells. PMID:22648416

  16. Associations of RASSF1A, RARβ, and CDH1 promoter hypermethylation with oral cancer risk

    PubMed Central

    Wen, Guohong; Wang, Huadong; Zhong, Zhaohui

    2018-01-01

    Abstract Background: Oral tumor is a heterogeneous group of tumors, in which it has several different histopathological and molecular features. Recently, genetic and epigenetic alterations are often detected in the development of oral cancer. Gene promoter hypermethylation leads to the silencing of cancer related genes without changes of genes sequence. To clarify the effect of RAS association domain family protein 1a (RASSF1A), retinoic acid receptor beta (RARβ), and E-cadherin (CDH1) promoter hypermethylation on the risk of oral cancer, we performed this meta-analysis. Methods: PubMed, Web of Science, Embase, and Chinese National Knowledge Infrastructure (CNKI) databases were retrieved to identify eligible articles. Stata 12.0 software was used to analyze extracted data of the included articles. Odds ratios (ORs) with the corresponding 95% confidence interval (95% CI) were calculated to evaluate the associations of RASSF1A, RARβ, and CDH1 promoter hypermethylation with oral cancer risk. Results: Around 23 literatures with 29 studies were included in the final meta-analysis, in which 12 studies were about RASSF1A promoter methylation, 4 studies were about RARβ promoter methylation, and 13 studies were about CDH1 promoter methylation. Overall, the results of this meta-analysis showed that there were significant associations between RASSF1A, RARβ, and CDH1 promoter hypermethylation and oral cancer risk (RASSF1A, OR = 11.8, 95% CI = 6.14–22.66; RARβ, OR = 20.35, 95% CI = 5.64–73.39; CDH1, OR = 13.46, 95% CI = 5.31–34.17). In addition, we found that RASSF1A promoter hypermethylation exerted higher frequency in the tongue tumor than other site tumor in mouth (RASSF1A, tongue tumor vs other site tumor in mouth, unmethylation vs methylation, OR = 0.65, 95%CI = 0.44–0.98). Conclusion: RASSF1A, RARβ, and CDH1 promoter hypermethylation might significantly increase the risk of oral cancer. PMID:29538221

  17. Reversal of hypermethylation and reactivation of glutathione S-transferase pi 1 gene by curcumin in breast cancer cell line.

    PubMed

    Kumar, Umesh; Sharma, Ujjawal; Rathi, Garima

    2017-02-01

    One of the mechanisms for epigenetic silencing of tumor suppressor genes is hypermethylation of cytosine residue at CpG islands at their promoter region that contributes to malignant progression of tumor. Therefore, activation of tumor suppressor genes that have been silenced by promoter methylation is considered to be very attractive molecular target for cancer therapy. Epigenetic silencing of glutathione S-transferase pi 1, a tumor suppressor gene, is involved in various types of cancers including breast cancer. Epigenetic silencing of tumor suppressor genes can be reversed by several molecules including natural compounds such as polyphenols that can act as a hypomethylating agent. Curcumin has been found to specifically target various tumor suppressor genes and alter their expression. To check the effect of curcumin on the methylation pattern of glutathione S-transferase pi 1 gene in MCF-7 breast cancer cell line in dose-dependent manner. To check the reversal of methylation pattern of hypermethylated glutathione S-transferase pi 1, MCF-7 breast cancer cell line was treated with different concentrations of curcumin for different time periods. DNA and proteins of treated and untreated cell lines were isolated, and methylation status of the promoter region of glutathione S-transferase pi 1 was analyzed using methylation-specific polymerase chain reaction assay, and expression of this gene was analyzed by immunoblotting using specific antibodies against glutathione S-transferase pi 1. A very low and a nontoxic concentration (10 µM) of curcumin treatment was able to reverse the hypermethylation and led to reactivation of glutathione S-transferase pi 1 protein expression in MCF-7 cells after 72 h of treatment, although the IC 50 value of curcumin was found to be at 20 µM. However, curcumin less than 3 µM of curcumin could not alter the promoter methylation pattern of glutathione S-transferase pi 1. Treatment of breast cancer MCF-7 cells with curcumin

  18. Genome-Wide Analysis of DNA Methylation before-and after Exercise in the Thoroughbred Horse with MeDIP-Seq

    PubMed Central

    Gim, Jeong-An; Hong, Chang Pyo; Kim, Dae-Soo; Moon, Jae-Woo; Choi, Yuri; Eo, Jungwoo; Kwon, Yun-Jeong; Lee, Ja-Rang; Jung, Yi-Deun; Bae, Jin-Han; Choi, Bong-Hwan; Ko, Junsu; Song, Sanghoon; Ahn, Kung; Ha, Hong-Seok; Yang, Young Mok; Lee, Hak-Kyo; Park, Kyung-Do; Do, Kyoung-Tag; Han, Kyudong; Yi, Joo Mi; Cha, Hee-Jae; Ayarpadikannan, Selvam; Cho, Byung-Wook; Bhak, Jong; Kim, Heui-Soo

    2015-01-01

    Athletic performance is an important criteria used for the selection of superior horses. However, little is known about exercise-related epigenetic processes in the horse. DNA methylation is a key mechanism for regulating gene expression in response to environmental changes. We carried out comparative genomic analysis of genome-wide DNA methylation profiles in the blood samples of two different thoroughbred horses before and after exercise by methylated-DNA immunoprecipitation sequencing (MeDIP-Seq). Differentially methylated regions (DMRs) in the pre-and post-exercise blood samples of superior and inferior horses were identified. Exercise altered the methylation patterns. After 30 min of exercise, 596 genes were hypomethylated and 715 genes were hypermethylated in the superior horse, whereas in the inferior horse, 868 genes were hypomethylated and 794 genes were hypermethylated. These genes were analyzed based on gene ontology (GO) annotations and the exercise-related pathway patterns in the two horses were compared. After exercise, gene regions related to cell division and adhesion were hypermethylated in the superior horse, whereas regions related to cell signaling and transport were hypermethylated in the inferior horse. Analysis of the distribution of methylated CpG islands confirmed the hypomethylation in the gene-body methylation regions after exercise. The methylation patterns of transposable elements also changed after exercise. Long interspersed nuclear elements (LINEs) showed abundance of DMRs. Collectively, our results serve as a basis to study exercise-based reprogramming of epigenetic traits. PMID:25666347

  19. Arachidonic and oleic acid exert distinct effects on the DNA methylome

    PubMed Central

    Silva-Martínez, Guillermo A.; Rodríguez-Ríos, Dalia; Alvarado-Caudillo, Yolanda; Vaquero, Alejandro; Esteller, Manel; Carmona, F. Javier; Moran, Sebastian; Nielsen, Finn C.; Wickström-Lindholm, Marie; Wrobel, Katarzyna; Wrobel, Kazimierz; Barbosa-Sabanero, Gloria; Zaina, Silvio; Lund, Gertrud

    2016-01-01

    ABSTRACT Abnormal fatty acid metabolism and availability are landmarks of metabolic diseases, which in turn are associated with aberrant DNA methylation profiles. To understand the role of fatty acids in disease epigenetics, we sought DNA methylation profiles specifically induced by arachidonic (AA) or oleic acid (OA) in cultured cells and compared those with published profiles of normal and diseased tissues. THP-1 monocytes were stimulated with AA or OA and analyzed using Infinium HumanMethylation450 BeadChip (Illumina) and Human Exon 1.0 ST array (Affymetrix). Data were corroborated in mouse embryonic fibroblasts. Comparisons with publicly available data were conducted by standard bioinformatics. AA and OA elicited a complex response marked by a general DNA hypermethylation and hypomethylation in the 1–200 μM range, respectively, with a maximal differential response at the 100 μM dose. The divergent response to AA and OA was prominent within the gene body of target genes, where it correlated positively with transcription. AA-induced DNA methylation profiles were similar to the corresponding profiles described for palmitic acid, atherosclerosis, diabetes, obesity, and autism, but relatively dissimilar from OA-induced profiles. Furthermore, human atherosclerosis grade-associated DNA methylation profiles were significantly enriched in AA-induced profiles. Biochemical evidence pointed to β-oxidation, PPAR-α, and sirtuin 1 as important mediators of AA-induced DNA methylation changes. In conclusion, AA and OA exert distinct effects on the DNA methylome. The observation that AA may contribute to shape the epigenome of important metabolic diseases, supports and expands current diet-based therapeutic and preventive efforts. PMID:27088456

  20. Eicosapentaenoic acid induces DNA demethylation in carcinoma cells through a TET1-dependent mechanism.

    PubMed

    Ceccarelli, Veronica; Valentini, Virginia; Ronchetti, Simona; Cannarile, Lorenza; Billi, Monia; Riccardi, Carlo; Ottini, Laura; Talesa, Vincenzo Nicola; Grignani, Francesco; Vecchini, Alba

    2018-05-14

    In cancer cells, global genomic hypomethylation is found together with localized hypermethylation of CpG islands within the promoters and regulatory regions of silenced tumor suppressor genes. Demethylating agents may reverse hypermethylation, thus promoting gene re-expression. Unfortunately, demethylating strategies are not efficient in solid tumor cells. DNA demethylation is mediated by ten-eleven translocation enzymes (TETs). They sequentially convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which is associated with active transcription; 5-formylcytosine; and finally, 5-carboxylcytosine. Although α-linolenic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid, the major n-3 polyunsaturated fatty acids, have anti-cancer effects, their action, as DNA-demethylating agents, has never been investigated in solid tumor cells. Here, we report that EPA demethylates DNA in hepatocarcinoma cells. EPA rapidly increases 5hmC on DNA, inducing p21 Waf1/Cip1 gene expression, which slows cancer cell-cycle progression. We show that the underlying molecular mechanism involves TET1. EPA simultaneously binds peroxisome proliferator-activated receptor γ (PPARγ) and retinoid X receptor α (RXRα), thus promoting their heterodimer and inducing a PPARγ-TET1 interaction. They generate a TET1-PPARγ-RXRα protein complex, which binds to a hypermethylated CpG island on the p21 gene, where TET1 converts 5mC to 5hmC. In an apparent shuttling motion, PPARγ and RXRα leave the DNA, whereas TET1 associates stably. Overall, EPA directly regulates DNA methylation levels, permitting TET1 to exert its anti-tumoral function.-Ceccarelli, V., Valentini, V., Ronchetti, S., Cannarile, L., Billi, M., Riccardi, C., Ottini, L., Talesa, V. N., Grignani, F., Vecchini, A., Eicosapentaenoic acid induces DNA demethylation in carcinoma cells through a TET1-dependent mechanism.

  1. Decoy receptor 1 (DCR1) promoter hypermethylation and response to irinotecan in metastatic colorectal cancer

    PubMed Central

    Bosch, Linda J.W.; Coupé, Veerle M.H.; Mongera, Sandra; Haan, Josien C.; Richman, Susan D.; Koopman, Miriam; Tol, Jolien; de Meyer, Tim; Louwagie, Joost; Dehaspe, Luc; van Grieken, Nicole C.T.; Ylstra, Bauke; Verheul, Henk M.W.; van Engeland, Manon; Nagtegaal, Iris D.; Herman, James G.; Quirke, Philip; Seymour, Matthew T.; Punt, Cornelis J.A.; van Criekinge, Wim; Carvalho, Beatriz; Meijer, Gerrit A.

    2017-01-01

    Diversity in colorectal cancer biology is associated with variable responses to standard chemotherapy. We aimed to identify and validate DNA hypermethylated genes as predictive biomarkers for irinotecan treatment of metastatic CRC patients. Candidate genes were selected from 389 genes involved in DNA Damage Repair by correlation analyses between gene methylation status and drug response in 32 cell lines. A large series of samples (n=818) from two phase III clinical trials was used to evaluate these candidate genes by correlating methylation status to progression-free survival after treatment with first-line single-agent fluorouracil (Capecitabine or 5-fluorouracil) or combination chemotherapy (Capecitabine or 5-fluorouracil plus irinotecan (CAPIRI/FOLFIRI)). In the discovery (n=185) and initial validation set (n=166), patients with methylated Decoy Receptor 1 (DCR1) did not benefit from CAPIRI over Capecitabine treatment (discovery set: HR=1.2 (95%CI 0.7-1.9, p=0.6), validation set: HR=0.9 (95%CI 0.6-1.4, p=0.5)), whereas patients with unmethylated DCR1 did (discovery set: HR=0.4 (95%CI 0.3-0.6, p=0.00001), validation set: HR=0.5 (95%CI 0.3-0.7, p=0.0008)). These results could not be replicated in the external data set (n=467), where a similar effect size was found in patients with methylated and unmethylated DCR1 for FOLFIRI over 5FU treatment (methylated DCR1: HR=0.7 (95%CI 0.5-0.9, p=0.01), unmethylated DCR1: HR=0.8 (95%CI 0.6-1.2, p=0.4)). In conclusion, DCR1 promoter hypermethylation status is a potential predictive biomarker for response to treatment with irinotecan, when combined with capecitabine. This finding could not be replicated in an external validation set, in which irinotecan was combined with 5FU. These results underline the challenge and importance of extensive clinical evaluation of candidate biomarkers in multiple trials. PMID:28968978

  2. Decoy receptor 1 (DCR1) promoter hypermethylation and response to irinotecan in metastatic colorectal cancer.

    PubMed

    Bosch, Linda J W; Trooskens, Geert; Snaebjornsson, Petur; Coupé, Veerle M H; Mongera, Sandra; Haan, Josien C; Richman, Susan D; Koopman, Miriam; Tol, Jolien; de Meyer, Tim; Louwagie, Joost; Dehaspe, Luc; van Grieken, Nicole C T; Ylstra, Bauke; Verheul, Henk M W; van Engeland, Manon; Nagtegaal, Iris D; Herman, James G; Quirke, Philip; Seymour, Matthew T; Punt, Cornelis J A; van Criekinge, Wim; Carvalho, Beatriz; Meijer, Gerrit A

    2017-09-08

    Diversity in colorectal cancer biology is associated with variable responses to standard chemotherapy. We aimed to identify and validate DNA hypermethylated genes as predictive biomarkers for irinotecan treatment of metastatic CRC patients. Candidate genes were selected from 389 genes involved in DNA Damage Repair by correlation analyses between gene methylation status and drug response in 32 cell lines. A large series of samples (n=818) from two phase III clinical trials was used to evaluate these candidate genes by correlating methylation status to progression-free survival after treatment with first-line single-agent fluorouracil (Capecitabine or 5-fluorouracil) or combination chemotherapy (Capecitabine or 5-fluorouracil plus irinotecan (CAPIRI/FOLFIRI)). In the discovery (n=185) and initial validation set (n=166), patients with methylated Decoy Receptor 1 ( DCR1) did not benefit from CAPIRI over Capecitabine treatment (discovery set: HR=1.2 (95%CI 0.7-1.9, p =0.6), validation set: HR=0.9 (95%CI 0.6-1.4, p =0.5)), whereas patients with unmethylated DCR1 did (discovery set: HR=0.4 (95%CI 0.3-0.6, p =0.00001), validation set: HR=0.5 (95%CI 0.3-0.7, p =0.0008)). These results could not be replicated in the external data set (n=467), where a similar effect size was found in patients with methylated and unmethylated DCR1 for FOLFIRI over 5FU treatment (methylated DCR1 : HR=0.7 (95%CI 0.5-0.9, p =0.01), unmethylated DCR1 : HR=0.8 (95%CI 0.6-1.2, p =0.4)). In conclusion, DCR1 promoter hypermethylation status is a potential predictive biomarker for response to treatment with irinotecan, when combined with capecitabine. This finding could not be replicated in an external validation set, in which irinotecan was combined with 5FU. These results underline the challenge and importance of extensive clinical evaluation of candidate biomarkers in multiple trials.

  3. Hypermethylation of Homeobox A10 by in Utero Diethylstilbestrol Exposure: An Epigenetic Mechanism for Altered Developmental Programming

    PubMed Central

    Bromer, Jason G.; Wu, Jie; Zhou, Yuping; Taylor, Hugh S.

    2009-01-01

    Diethylstilbestrol (DES) is a nonsteroidal estrogen that induces developmental anomalies of the female reproductive tract. The homeobox gene HOXA10 controls uterine organogenesis, and its expression is altered after in utero DES exposure. We hypothesized that an epigenetic mechanism underlies DES-mediated alterations in HOXA10 expression. We analyzed the expression pattern and methylation profile of HOXA10 after DES exposure. Expression of HOXA10 is increased in human endometrial cells after DES exposure, whereas Hoxa10 expression is repressed and shifted caudally from its normal location in mice exposed in utero. Cytosine guanine dinucleotide methylation frequency in the Hoxa10 intron was higher in DES-exposed offspring compared with controls (P = 0.017). The methylation level of Hoxa10 was also higher in the caudal portion of the uterus after DES exposure at the promoter and intron (P < 0.01). These changes were accompanied by increased expression of DNA methyltransferases 1 and 3b. No changes in methylation were observed after in vitro or adult DES exposure. DES has a dual mechanism of action as an endocrine disruptor; DES functions as a classical estrogen and directly stimulates HOXA10 expression with short-term exposure, however, in utero exposure results in hypermethylation of the HOXA10 gene and long-term altered HOXA10 expression. We identify hypermethylation as a novel mechanism of DES-induced altered developmental programming. PMID:19299448

  4. SOCS3 promoter hypermethylation is a favorable prognosticator and a novel indicator for G-CIMP-positive GBM patients.

    PubMed

    Feng, Ying; Wang, Zheng; Bao, Zhaoshi; Yan, Wei; You, Gan; Wang, Yinyan; Hu, Huimin; Zhang, Wei; Zhang, Quangeng; Jiang, Tao

    2014-01-01

    Hypermethylation of the suppressor of cytokine signaling 3(SOCS3) promoter has been reported to predict a poor prognosis in several cancers including glioblastoma multiforme (GBM). We explored the function of SOCS3 promoter hypermethylation in GBM cohorts, including analysis of the CpG island methylator phenotype (CIMP), when a large number of gene loci are simultaneously hypermethylated. A whole genome promoter methylation profile was performed in a cohort of 33 GBM samples, with 13 long-term survivors (LTS; overall survival ≥ 18 months) and 20 short-term survivors (STS; overall survival ≤ 9 months). The SOCS3 promoter methylation status was compared between the two groups. In addition, we investigated the relationship of SOCS3 promoter methylation and G-CIMP status. Interestingly, in our present study, we found that SOCS3 promoter methylation was statistically significantly higher in the 13 LTS than that in the 20 STS. Furthermore, high SOCS3 promoter methylation detected via pyro-sequencing predicted a better prognosis in an independent cohort containing 62 GBM patients. This correlation was validated by the dataset from the Cancer Genome Atlas(TCGA) and the Chinese Cancer Genome Atlas(CGGA). In addition, we found that hypermethylation of the SOCS3 promoter was tightly associated with the G-CIMP-positive GBM patients. Using a total of 359 clinical samples, we demonstrate that SOCS3 promoter hypermethylation status has a favorable prognostic value in GBM patients because of whole genome methylation status. Particularly, the hypermethylation of the SOCS3 promoter indicates positive G-CIMP status.

  5. Human native lipoprotein-induced de novo DNA methylation is associated with repression of inflammatory genes in THP-1 macrophages.

    PubMed

    Rangel-Salazar, Rubén; Wickström-Lindholm, Marie; Aguilar-Salinas, Carlos A; Alvarado-Caudillo, Yolanda; Døssing, Kristina B V; Esteller, Manel; Labourier, Emmanuel; Lund, Gertrud; Nielsen, Finn C; Rodríguez-Ríos, Dalia; Solís-Martínez, Martha O; Wrobel, Katarzyna; Wrobel, Kazimierz; Zaina, Silvio

    2011-11-25

    We previously showed that a VLDL- and LDL-rich mix of human native lipoproteins induces a set of repressive epigenetic marks, i.e. de novo DNA methylation, histone 4 hypoacetylation and histone 4 lysine 20 (H4K20) hypermethylation in THP-1 macrophages. Here, we: 1) ask what gene expression changes accompany these epigenetic responses; 2) test the involvement of candidate factors mediating the latter. We exploited genome expression arrays to identify target genes for lipoprotein-induced silencing, in addition to RNAi and expression studies to test the involvement of candidate mediating factors. The study was conducted in human THP-1 macrophages. Native lipoprotein-induced de novo DNA methylation was associated with a general repression of various critical genes for macrophage function, including pro-inflammatory genes. Lipoproteins showed differential effects on epigenetic marks, as de novo DNA methylation was induced by VLDL and to a lesser extent by LDL, but not by HDL, and VLDL induced H4K20 hypermethylation, while HDL caused H4 deacetylation. The analysis of candidate factors mediating VLDL-induced DNA hypermethylation revealed that this response was: 1) surprisingly, mediated exclusively by the canonical maintenance DNA methyltransferase DNMT1, and 2) independent of the Dicer/micro-RNA pathway. Our work provides novel insights into epigenetic gene regulation by native lipoproteins. Furthermore, we provide an example of DNMT1 acting as a de novo DNA methyltransferase independently of canonical de novo enzymes, and show proof of principle that de novo DNA methylation can occur independently of a functional Dicer/micro-RNA pathway in mammals.

  6. Procainamide Is a Specific Inhibitor of DNA Methyltransferase 1*

    PubMed Central

    Lee, Byron H.; Yegnasubramanian, Srinivasan; Lin, Xiaohui; Nelson, William G.

    2007-01-01

    CpG island hypermethylation occurs in most cases of cancer, typically resulting in the transcriptional silencing of critical cancer genes. Procainamide has been shown to inhibit DNA methyltransferase activity and reactivate silenced gene expression in cancer cells by reversing CpG island hypermethylation. We report here that procainamide specifically inhibits the hemimethylase activity of DNA methyltransferase 1 (DNMT1), the mammalian enzyme thought to be responsible for maintaining DNA methylation patterns during replication. At micromolar concentrations, procainamide was found to be a partial competitive inhibitor of DNMT1, reducing the affinity of the enzyme for its two substrates, hemimethylated DNA and S-adenosyl-l-methionine. By doing so, procainamide significantly decreased the processivity of DNMT1 on hemimethylated DNA. Procainamide was not a potent inhibitor of the de novo methyltransferases DNMT3a and DNMT3b2. As further evidence of the specificity of procainamide for DNMT1, procainamide failed to lower genomic 5-methyl-2′-deoxycytidine levels in HCT116 colorectal cancer cells when DNMT1 was genetically deleted but significantly reduced genomic 5-methyl-2′-deoxycyti-dine content in parental HCT116 cells and in HCT116 cells where DNMT3b was genetically deleted. Because many reports have strongly linked DNMT1 with epigenetic alterations in carcinogenesis, procainamide may be a useful drug in the prevention of cancer. PMID:16230360

  7. Chromosome and molecular abnormalities in myelodysplastic syndromes.

    PubMed

    Fenaux, Pierre

    2001-06-01

    Cytogenetic abnormalities are seen in approximately 50% of cases of myelodysplastic syndrome (MDS) and 80% of cases of secondary MDS (following chemotherapy or radiotherapy). These abnormalities generally consist of partial or complete chromosome deletion or addition (del5q, -7, +8, -Y, del20q), whereas balanced or unbalanced translocations are rarely found in MDS. Fluorescence hybridization techniques (fluorescence in situ hybridization [FISH], multiplex FISH, and spectral karyotyping) are useful in detecting chromosomal anomalies in cases in which few mitoses are obtained or rearrangements are complex. Ras mutations are the molecular abnormalities most frequently found in MDS, followed by p15 gene hypermethylation, FLT3 duplications, and p53 mutations, but none of these abnormalities are specific for MDS. The rare cases of balanced translocations in MDS have allowed the identification of genes whose rearrangements appear to play a role in the pathogenesis of some cases of MDS. These genes include MDS1-EVI1 in t(3;3) or t(3;21) translocations, TEL in t(5;12), HIP1 in t(5;7), MLF1 in t(3;5), and MEL1 in t(1;3). Genes more frequently implicated in the pathogenesis of MDS cases, such as those involving del5q, remain unknown, although some candidate genes are currently being studied. Cytogenetic and known molecular abnormalities generally carry a poor prognosis in MDS and can be incorporated into prognostic scoring systems such as the International Prognostic Scoring System.

  8. The Progeny of Arabidopsis thaliana Plants Exposed to Salt Exhibit Changes in DNA Methylation, Histone Modifications and Gene Expression

    PubMed Central

    Bilichak, Andriy; Ilnystkyy, Yaroslav; Hollunder, Jens; Kovalchuk, Igor

    2012-01-01

    Plants are able to acclimate to new growth conditions on a relatively short time-scale. Recently, we showed that the progeny of plants exposed to various abiotic stresses exhibited changes in genome stability, methylation patterns and stress tolerance. Here, we performed a more detailed analysis of methylation patterns in the progeny of Arabidopsis thaliana (Arabidopsis) plants exposed to 25 and 75 mM sodium chloride. We found that the majority of gene promoters exhibiting changes in methylation were hypermethylated, and this group was overrepresented by regulators of the chromatin structure. The analysis of DNA methylation at gene bodies showed that hypermethylation in the progeny of stressed plants was primarily due to changes in the 5′ and 3′ ends as well as in exons rather than introns. All but one hypermethylated gene tested had lower gene expression. The analysis of histone modifications in the promoters and coding sequences showed that hypermethylation and lower gene expression correlated with the enrichment of H3K9me2 and depletion of H3K9ac histones. Thus, our work demonstrated a high degree of correlation between changes in DNA methylation, histone modifications and gene expression in the progeny of salt-stressed plants. PMID:22291972

  9. DNA methylome profiling identifies novel methylated genes in African American patients with colorectal neoplasia.

    PubMed

    Ashktorab, Hassan; Daremipouran, M; Goel, Ajay; Varma, Sudhir; Leavitt, R; Sun, Xueguang; Brim, Hassan

    2014-04-01

    The identification of genes that are differentially methylated in colorectal cancer (CRC) has potential value for both diagnostic and therapeutic interventions specifically in high-risk populations such as African Americans (AAs). However, DNA methylation patterns in CRC, especially in AAs, have not been systematically explored and remain poorly understood. Here, we performed DNA methylome profiling to identify the methylation status of CpG islands within candidate genes involved in critical pathways important in the initiation and development of CRC. We used reduced representation bisulfite sequencing (RRBS) in colorectal cancer and adenoma tissues that were compared with DNA methylome from a healthy AA subject's colon tissue and peripheral blood DNA. The identified methylation markers were validated in fresh frozen CRC tissues and corresponding normal tissues from AA patients diagnosed with CRC at Howard University Hospital. We identified and validated the methylation status of 355 CpG sites located within 16 gene promoter regions associated with CpG islands. Fifty CpG sites located within CpG islands-in genes ATXN7L1 (2), BMP3 (7), EID3 (15), GAS7 (1), GPR75 (24), and TNFAIP2 (1)-were significantly hypermethylated in tumor vs. normal tissues (P<0.05). The methylation status of BMP3, EID3, GAS7, and GPR75 was confirmed in an independent, validation cohort. Ingenuity pathway analysis mapped three of these markers (GAS7, BMP3 and GPR) in the insulin and TGF-β1 network-the two key pathways in CRC. In addition to hypermethylated genes, our analysis also revealed that LINE-1 repeat elements were progressively hypomethylated in the normal-adenoma-cancer sequence. We conclude that DNA methylome profiling based on RRBS is an effective method for screening aberrantly methylated genes in CRC. While previous studies focused on the limited identification of hypermethylated genes, ours is the first study to systematically and comprehensively identify novel hypermethylated

  10. Effect of the one‑carbon unit cycle on overall DNA methylation in children with Down's syndrome.

    PubMed

    Song, Cui; He, Jingyi; Chen, Jie; Liu, Youxue; Xiong, Feng; Wang, Yutian; Li, Tingyu

    2015-12-01

    DNA methylation is a major epigenetic mechanism regulating gene expression. In order to analyze the impact of the one‑carbon unit cycle on the overall level of DNA methylation in children with Down's syndrome (DS), the levels of indicators associated with the one‑carbon unit cycle, including folic acid (FA), vitamin B12 (VB12) and homocysteine (Hcy), and the overall DNA methylation level of DS and healthy controls (HCs) were determined in the present study. A total of 36 DS children and 40 age‑ and gender‑matched HCs were included in the present study to determine the levels of FA, VB12, Hcy and overall DNA methylation. The effect of the one‑carbon unit cycle on the overall level of DNA methylation within the DS group was analyzed. The results demonstrated that the level of VB12 was decreased (P=0.008), while the Hcy level was increased (P=0.000) in DS patients compared with the HCs. FA and VB12 levels decreased with increasing age in DS patients (P<0.05). DNA hypermethylation and hypomethylation were observed in DS patients with VB12 deficiency and hyperhomocysteinemia, respectively (P=0.031, P=0.021). Abnormalities in the one‑carbon unit cycle tend to worsen with increasing age in DS children. Thus, one‑carbon unit cycle‑associated alterations in DNA methylation may be important in the neuropathological alterations observed in DS.

  11. DNA methylation of extracellular matrix remodeling genes in children exposed to arsenic.

    PubMed

    Gonzalez-Cortes, Tania; Recio-Vega, Rogelio; Lantz, Robert Clark; Chau, Binh T

    2017-08-15

    Several novel mechanistic findings regarding to arsenic's pathogenesis has been reported and some of them suggest that the etiology of some arsenic induced diseases are due in part to heritable changes to the genome via epigenetic processes such as DNA methylation, histone maintenance, and mRNA expression. Recently, we reported that arsenic exposure during in utero and early life was associated with impairment in the lung function and abnormal receptor for advanced glycation endproducts (RAGE), matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) sputum levels. Based on our results and the reported arsenic impacts on DNA methylation, we designed this study in our cohort of children exposed in utero and early childhood to arsenic with the aim to associate DNA methylation of MMP9, TIMP1 and RAGE genes with its protein sputum levels and with urinary and toenail arsenic levels. The results disclosed hypermethylation in MMP9 promotor region in the most exposed children; and an increase in the RAGE sputum levels among children with the mid methylation level; there were also positive associations between MMP9 DNA methylation with arsenic toenail concentrations; RAGE DNA methylation with iAs, and %DMA; and finally between TIMP1 DNA methylation with the first arsenic methylation. A negative correlation between MMP9 sputum levels with its DNA methylation was registered. In conclusion, arsenic levels were positive associated with the DNA methylation of extracellular matrix remodeling genes;, which in turn could modifies the biological process in which they are involved causing or predisposing to lung diseases. Copyright © 2017. Published by Elsevier Inc.

  12. DNA methylome signature in rheumatoid arthritis.

    PubMed

    Nakano, Kazuhisa; Whitaker, John W; Boyle, David L; Wang, Wei; Firestein, Gary S

    2013-01-01

    Epigenetics can influence disease susceptibility and severity. While DNA methylation of individual genes has been explored in autoimmunity, no unbiased systematic analyses have been reported. Therefore, a genome-wide evaluation of DNA methylation loci in fibroblast-like synoviocytes (FLS) isolated from the site of disease in rheumatoid arthritis (RA) was performed. Genomic DNA was isolated from six RA and five osteoarthritis (OA) FLS lines and evaluated using the Illumina HumanMethylation450 chip. Cluster analysis of data was performed and corrected using Benjamini-Hochberg adjustment for multiple comparisons. Methylation was confirmed by pyrosequencing and gene expression was determined by qPCR. Pathway analysis was performed using the Kyoto Encyclopedia of Genes and Genomes. RA and control FLS segregated based on DNA methylation, with 1859 differentially methylated loci. Hypomethylated loci were identified in key genes relevant to RA, such as CHI3L1, CASP1, STAT3, MAP3K5, MEFV and WISP3. Hypermethylation was also observed, including TGFBR2 and FOXO1. Hypomethylation of individual genes was associated with increased gene expression. Grouped analysis identified 207 hypermethylated or hypomethylated genes with multiple differentially methylated loci, including COL1A1, MEFV and TNF. Hypomethylation was increased in multiple pathways related to cell migration, including focal adhesion, cell adhesion, transendothelial migration and extracellular matrix interactions. Confirmatory studies with OA and normal FLS also demonstrated segregation of RA from control FLS based on methylation pattern. Differentially methylated genes could alter FLS gene expression and contribute to the pathogenesis of RA. DNA methylation of critical genes suggests that RA FLS are imprinted and implicate epigenetic contributions to inflammatory arthritis.

  13. DNA-Demethylase Regulated Genes Show Methylation-Independent Spatiotemporal Expression Patterns

    PubMed Central

    Schumann, Ulrike; Lee, Joanne; Kazan, Kemal; Ayliffe, Michael; Wang, Ming-Bo

    2017-01-01

    Recent research has indicated that a subset of defense-related genes is downregulated in the Arabidopsis DNA demethylase triple mutant rdd (ros1 dml2 dml3) resulting in increased susceptibility to the fungal pathogen Fusarium oxysporum. In rdd plants these downregulated genes contain hypermethylated transposable element sequences (TE) in their promoters, suggesting that this methylation represses gene expression in the mutant and that these sequences are actively demethylated in wild-type plants to maintain gene expression. In this study, the tissue-specific and pathogen-inducible expression patterns of rdd-downregulated genes were investigated and the individual role of ROS1, DML2, and DML3 demethylases in these spatiotemporal regulation patterns was determined. Large differences in defense gene expression were observed between pathogen-infected and uninfected tissues and between root and shoot tissues in both WT and rdd plants, however, only subtle changes in promoter TE methylation patterns occurred. Therefore, while TE hypermethylation caused decreased gene expression in rdd plants it did not dramatically effect spatiotemporal gene regulation, suggesting that this latter regulation is largely methylation independent. Analysis of ros1-3, dml2-1, and dml3-1 single gene mutant lines showed that promoter TE hypermethylation and defense-related gene repression was predominantly, but not exclusively, due to loss of ROS1 activity. These data demonstrate that DNA demethylation of TE sequences, largely by ROS1, promotes defense-related gene expression but does not control spatiotemporal expression in Arabidopsis. Summary: Ros1-mediated DNA demethylation of promoter transposable elements is essential for activation of defense-related gene expression in response to fungal infection in Arabidopsis thaliana. PMID:28894455

  14. DNA methylation and targeted sequencing of methyltransferases family genes in canine acute myeloid leukaemia, modelling human myeloid leukaemia.

    PubMed

    Bronzini, I; Aresu, L; Paganin, M; Marchioretto, L; Comazzi, S; Cian, F; Riondato, F; Marconato, L; Martini, V; Te Kronnie, G

    2017-09-01

    Tumours shows aberrant DNA methylation patterns, being hypermethylated or hypomethylated compared with normal tissues. In human acute myeloid leukaemia (hAML) mutations in DNA methyltransferase (DNMT3A) are associated to a more aggressive tumour behaviour. As AML is lethal in dogs, we defined global DNA methylation content, and screened the C-terminal domain of DNMT3 family of genes for sequence variants in 39 canine acute myeloid leukaemia (cAML) cases. A heterogeneous pattern of DNA methylation was found among cAML samples, with subsets of cases being hypermethylated or hypomethylated compared with healthy controls; four recurrent single nucleotide variations (SNVs) were found in DNMT3L gene. Although SNVs were not directly correlated to whole genome DNA methylation levels, all hypomethylated cAML cases were homozygous for the deleterious mutation at p.Arg222Trp. This study contributes to understand genetic modifications of cAML, leading up to studies that will elucidate the role of methylome alterations in the pathogenesis of AML in dogs. © 2016 John Wiley & Sons Ltd.

  15. Hypermethylation of gene promoters in peripheral blood leukocytes in humans long term after radiation exposure

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kuzmina, Nina S., E-mail: nin-kuzmin@youndex.ru; Lapteva, Nellya Sh.; Rubanovich, Alexander V.

    Some human genes known to undergo age-related promoter hypermethylation. These epigenetic modifications are similar to those occurring in the course of certain diseases, e.g. some types of cancer, which in turn may also associate with age. Given external genotoxic factors may additionally contribute to hypermethylation, this study was designed to analyzes, using methylation-sensitive polymerase chain reaction (PCR), the CpG island hypermethylation in RASSF1A, CDKN2A (including p16/INK4A and p14/ARF) and GSTP1 promoters in peripheral blood leukocytes of individuals exposed to ionizing radiation long time ago. One hundred and twenty-four irradiated subjects (24–77 years old at sampling: 83 Chernobyl Nuclear Power Plantmore » clean-up workers, 21 nuclear workers, 20 residents of territories with radioactive contamination) and 208 unirradiated volunteers (19–77 years old at sampling) were enrolled. In addition, 74 non-exposed offspring (2–51 years old at sampling) born to irradiated parents were examined. The frequency of individuals displaying promoter methylation of at least one gene in exposed group was significantly higher as compared to the control group (OR=5.44, 95% CI=2.62–11.76, p=3.9×10{sup −7}). No significant difference was found between the frequency of subjects with the revealed promoter methylation in the group of offspring born to irradiated parents and in the control group. The increase in the number of methylated loci of RASSF1A and p14/ARF was associated with age (β=0.242; p=1.7×10{sup −5}). In contrast, hypermethylation of p16/INK4A and GSTP1 genes correlated with the fact of radiation exposure only (β=0.290; p=1.7×10{sup −7}). The latter finding demonstrates that methylation changes in blood leukocytes of healthy subjects exposed to radiation resemble those reported in human malignancies. Additional studies are required to identify the dose-response of epigenetic markers specifically associating with radiation-induced premature

  16. MDS and secondary AML display unique patterns and abundance of aberrant DNA methylation

    PubMed Central

    Figueroa, Maria E.; Skrabanek, Lucy; Li, Yushan; Jiemjit, Anchalee; Fandy, Tamer E.; Paietta, Elisabeth; Fernandez, Hugo; Tallman, Martin S.; Greally, John M.; Carraway, Hetty; Licht, Jonathan D.; Gore, Steven D.

    2009-01-01

    Increasing evidence shows aberrant hypermethylation of genes occurring in and potentially contributing to pathogenesis of myeloid malignancies. Several of these diseases, such as myelodysplastic syndromes (MDSs), are responsive to DNA methyltransferase inhibitors. To determine the extent of promoter hypermethylation in such tumors, we compared the distribution of DNA methylation of 14 000 promoters in MDS and secondary acute myeloid leukemia (AML) patients enrolled in a phase 1 trial of 5-azacytidine and the histone deacetylase inhibitor entinostat against de novo AML patients and normal CD34+ bone marrow cells. The MDS and secondary AML patients displayed more extensive aberrant DNA methylation involving thousands of genes than did the normal CD34+ bone marrow cells or de novo AML blasts. Aberrant methylation in MDS and secondary AML tended to affect particular chromosomal regions, occurred more frequently in Alu-poor genes, and included prominent involvement of genes involved in the WNT and MAPK signaling pathways. DNA methylation was also measured at days 15 and 29 after the first treatment cycle. DNA methylation was reversed at day 15 in a uniform manner throughout the genome, and this effect persisted through day 29, even without continuous administration of the study drugs. This trial was registered at www.clinicaltrials.gov as J0443. PMID:19652201

  17. Analysis of the methylation status of the KCNQ1OT and H19 genes in leukocyte DNA for the diagnosis and prognosis of Beckwith-Wiedemann syndrome.

    PubMed

    Gaston, V; Le Bouc, Y; Soupre, V; Burglen, L; Donadieu, J; Oro, H; Audry, G; Vazquez, M P; Gicquel, C

    2001-06-01

    Beckwith-Wiedemann syndrome (BWS) is an overgrowth disorder involving developmental abnormalities, tissue and organ hyperplasia and an increased risk of embryonal tumours (most commonly Wilms tumour). This multigenic disorder is caused by dysregulation of the expression of imprinted genes in the 11p15 chromosomal region. Molecular diagnosis of BWS is currently difficult, mostly due to the large spectrum of genetic and epigenetic abnormalities. The other difficulty in managing BWS is the identification of patients at risk of tumour. An imprinted antisense transcript within KCNQ1, called KCNQ1OT (also known as LIT1), was recently shown to be normally expressed from the paternal allele. A loss of imprinting of the KCNQ1OT gene, associated with the loss of maternal allele-specific methylation of the differentially methylated region KvDMR1 has been described in BWS patients. The principal aim of this study was to evaluate the usefulness of KvDMR1 methylation analysis of leukocyte DNA for the diagnosis of BWS. The allelic status of the 11p15 region and the methylation status of the KCNQ1OT and H19 genes were investigated in leukocyte DNA from 97 patients referred for BWS and classified into two groups according to clinical data: complete BWS (CBWS) (n=61) and incomplete BWS (IBWS) (n=36). Fifty-eight (60%) patients (39/61 CBWS and 19/36 IBWS) displayed abnormal demethylation of KvDMR1. In 11 of the 56 informative cases, demethylation of KvDMR1 was related to 11p15 uniparental disomy (UPD) (nine CBWS and two IBWS). Thirteen of the 39 patients with normal methylation of KvDMR1 displayed hypermethylation of the H19 gene. These 13 patients included two siblings with 11p15 trisomy. These results show that analysis of the methylation status of KvDMR1 and the H19 gene in leukocyte DNA is useful in the diagnosis of 11p15-related overgrowth syndromes, resulting in the diagnosis of BWS in more than 70% of investigated patients. We also evaluated clinical and molecular features as

  18. HaCaT anchorage blockade leads to oxidative stress, DNA damage and DNA methylation changes.

    PubMed

    da Silva, Rodrigo A; Sammartino Mariano, Flavia; Planello, Aline C; Line, Sergio R P; de Souza, Ana Paula

    2015-07-01

    Cell adhesion plays an important role in neoplastic transformation. Thus, anchorage-independent growth and epithelial-mesenchymal transition, which are features associated to anoikis-resistance, are vital steps in cancer progression and metastatic colonization. Cell attachment loss may induce intracellular oxidative stress, which triggers DNA damage as methylation changes. HaCaT lineage cells were submitted to periods of 1, 3, 5 and 24 h of anchorage blockage with the purpose of study of oxidative stress effect on changes in the DNA methylation pattern, derived from attachment blockade. Through this study, HaCaT anchorage blockage-induced oxidative stress was reported to mediate alterations in global DNA methylation changes and into TP53 gene promoter pattern during anoikis-resistance acquisition. Furthermore, at the first experimental time-periods (1, 3 and 5 h), genome hypermethylation was found; however, genome hypomethylation was observed in later time-periods (24 h) of attachment impediment. The TP 53 methylation analyses were performed after 24 h of replated anoikis-resistance cells and same methylation pattern was observed, occurring an early (1 and 3 h) hypermethylation that was followed by late (5 and 24 h) hypomethylation. However, LINE-1, a marker of genomic instability, was perceived in time-dependent hypomethylation. The mRNA levels of the DNMTs enzymes were influenced by cell attachment blockage, but non-conclusive results were obtained in order to match DNMTs transcription to pattern methylation results. In conclusion, DNA damage was found, leaded by oxidative stress that has come up from HaCaT anchorage blockade, which rises a global genome hypomethylation tendency as consequence, which might denote genomic instability.

  19. Novel Multiplex MethyLight Protocol for Detection of DNA Methylation in Patient Tissues and Bodily Fluids

    PubMed Central

    Olkhov-Mitsel, Ekaterina; Zdravic, Darko; Kron, Ken; van der Kwast, Theodorus; Fleshner, Neil; Bapat, Bharati

    2014-01-01

    Aberrant DNA methylation is a hallmark of cancer and is an important potential biomarker. Particularly, combined analysis of a panel of hypermethylated genes shows the most promising clinical performance. Herein, we developed, optimized and standardized a multiplex MethyLight assay to simultaneously detect hypermethylation of APC, HOXD3 and TGFB2 in DNA extracted from prostate cancer (PCa) cell lines, archival tissue specimens, and urine samples. We established that the assay is capable of discriminating between fully methylated and unmethylated alleles with 100% specificity and demonstrated the assay as highly accurate and reproducible as the singleplex approach. For proof of principle, we analyzed the methylation status of these genes in tissue and urine samples of PCa patients as well as PCa-free controls. These data show that the multiplex MethyLight assay offers a significant advantage when working with limited quantities of DNA and has potential applications in research and clinical settings. PMID:24651255

  20. Microarray-based DNA methylation study of Ewing's sarcoma of the bone.

    PubMed

    Park, Hye-Rim; Jung, Woon-Won; Kim, Hyun-Sook; Park, Yong-Koo

    2014-10-01

    Alterations in DNA methylation patterns are a hallmark of malignancy. However, the majority of epigenetic studies of Ewing's sarcoma have focused on the analysis of only a few candidate genes. Comprehensive studies are thus lacking and are required. The aim of the present study was to identify novel methylation markers in Ewing's sarcoma using microarray analysis. The current study reports the microarray-based DNA methylation study of 1,505 CpG sites of 807 cancer-related genes from 69 Ewing's sarcoma samples. The Illumina GoldenGate Methylation Cancer Panel I microarray was used, and with the appropriate controls (n=14), a total of 92 hypermethylated genes were identified in the Ewing's sarcoma samples. The majority of the hypermethylated genes were associated with cell adhesion, cell regulation, development and signal transduction. The overall methylation mean values were compared between patients who survived and those that did not. The overall methylation mean was significantly higher in the patients who did not survive (0.25±0.03) than in those who did (0.22±0.05) (P=0.0322). However, the overall methylation mean was not found to significantly correlate with age, gender or tumor location. GDF10 , OSM , APC and HOXA11 were the most significant differentially-methylated genes, however, their methylation levels were not found to significantly correlate with the survival rate. The DNA methylation profile of Ewing's sarcoma was characterized and 92 genes that were significantly hypermethylated were detected. A trend towards a more aggressive behavior was identified in the methylated group. The results of this study indicated that methylation may be significant in the development of Ewing's sarcoma.

  1. Uncovering the Role of Hypermethylation by CTG Expansion in Myotonic Dystrophy Type 1 Using Mutant Human Embryonic Stem Cells

    PubMed Central

    Yanovsky-Dagan, Shira; Avitzour, Michal; Altarescu, Gheona; Renbaum, Paul; Eldar-Geva, Talia; Schonberger, Oshrat; Mitrani-Rosenbaum, Stella; Levy-Lahad, Ephrat; Birnbaum, Ramon Y.; Gepstein, Lior; Epsztejn-Litman, Silvina; Eiges, Rachel

    2015-01-01

    Summary CTG repeat expansion in DMPK, the cause of myotonic dystrophy type 1 (DM1), frequently results in hypermethylation and reduced SIX5 expression. The contribution of hypermethylation to disease pathogenesis and the precise mechanism by which SIX5 expression is reduced are unknown. Using 14 different DM1-affected human embryonic stem cell (hESC) lines, we characterized a differentially methylated region (DMR) near the CTGs. This DMR undergoes hypermethylation as a function of expansion size in a way that is specific to undifferentiated cells and is associated with reduced SIX5 expression. Using functional assays, we provide evidence for regulatory activity of the DMR, which is lost by hypermethylation and may contribute to DM1 pathogenesis by causing SIX5 haplo-insufficiency. This study highlights the power of hESCs in disease modeling and describes a DMR that functions both as an exon coding sequence and as a regulatory element whose activity is epigenetically hampered by a heritable mutation. PMID:26190529

  2. Skin score correlates with global DNA methylation and GSTO1 A140D polymorphism in arsenic-affected population of Eastern India.

    PubMed

    Majumder, Moumita; Dasgupta, Uma B; Guha Mazumder, D N; Das, Nilansu

    2017-07-01

    Arsenic is a potent environmental toxicant causing serious public health concerns in India, Bangladesh and other parts of the world. Gene- and promoter-specific hypermethylation has been reported in different arsenic-exposed cell lines, whereas whole genome DNA methylation study suggested genomic hypo- and hypermethylation after arsenic exposure in in vitro and in vivo studies. Along with other characteristic biomarkers, arsenic toxicity leads to typical skin lesions. The present study demonstrates significant correlation between severities of skin manifestations with their whole genome DNA methylation status as well as with a particular polymorphism (Ala 140 Asp) status in arsenic metabolizing enzyme Glutathione S-transferase Omega-1 (GSTO1) in arsenic-exposed population of the district of Nadia, West Bengal, India.

  3. Uncovering the Role of Hypermethylation by CTG Expansion in Myotonic Dystrophy Type 1 Using Mutant Human Embryonic Stem Cells.

    PubMed

    Yanovsky-Dagan, Shira; Avitzour, Michal; Altarescu, Gheona; Renbaum, Paul; Eldar-Geva, Talia; Schonberger, Oshrat; Mitrani-Rosenbaum, Stella; Levy-Lahad, Ephrat; Birnbaum, Ramon Y; Gepstein, Lior; Epsztejn-Litman, Silvina; Eiges, Rachel

    2015-08-11

    CTG repeat expansion in DMPK, the cause of myotonic dystrophy type 1 (DM1), frequently results in hypermethylation and reduced SIX5 expression. The contribution of hypermethylation to disease pathogenesis and the precise mechanism by which SIX5 expression is reduced are unknown. Using 14 different DM1-affected human embryonic stem cell (hESC) lines, we characterized a differentially methylated region (DMR) near the CTGs. This DMR undergoes hypermethylation as a function of expansion size in a way that is specific to undifferentiated cells and is associated with reduced SIX5 expression. Using functional assays, we provide evidence for regulatory activity of the DMR, which is lost by hypermethylation and may contribute to DM1 pathogenesis by causing SIX5 haplo-insufficiency. This study highlights the power of hESCs in disease modeling and describes a DMR that functions both as an exon coding sequence and as a regulatory element whose activity is epigenetically hampered by a heritable mutation. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  4. Hypermethylation of the breast cancer-associated gene 1 promoter does not predict cytologic atypia or correlate with surrogate end points of breast cancer risk.

    PubMed

    Bean, Gregory R; Ibarra Drendall, Catherine; Goldenberg, Vanessa K; Baker, Joseph C; Troch, Michelle M; Paisie, Carolyn; Wilke, Lee G; Yee, Lisa; Marcom, Paul K; Kimler, Bruce F; Fabian, Carol J; Zalles, Carola M; Broadwater, Gloria; Scott, Victoria; Seewaldt, Victoria L

    2007-01-01

    Mutation of the breast cancer-associated gene 1 (BRCA1) plays an important role in familial breast cancer. Although hypermethylation of the BRCA1 promoter has been observed in sporadic breast cancer, its exact role in breast cancer initiation and association with breast cancer risk is unknown. The frequency of BRCA1 promoter hypermethylation was tested in (a) 14 primary breast cancer biopsies and (b) the initial random periareolar fine-needle aspiration (RPFNA) cytologic samples obtained from 61 asymptomatic women who were at increased risk for breast cancer. BRCA1 promoter hypermethylation was assessed from nucleotide -150 to nucleotide +32 relative to the transcription start site. RPFNA specimens were stratified for cytologic atypia using the Masood cytology index. BRCA1 promoter hypermethylation was observed at similar frequency in nonproliferative (normal; Masood hypermethylation was not associated with (a) family history of breast or ovarian cancer or (b) calculated Gail or BRCAPRO risk score. BRCA1 promoter hypermethylation was associated with (a) age (P = 0.028) and (b) the combined frequency of promoter hypermethylation of the retinoic acid receptor-beta2 (RARB) gene, estrogen receptor-alpha (ESR1) gene, and p16 (INK4A) gene (P = 0.003). These observations show that BRCA1 promoter hypermethylation (a) is not associated with breast cancer risk as measured by mathematical risk models and (b) does not predict mammary atypia in RPFNA cytologic samples obtained from high-risk women.

  5. [Role and alterations of DNA methylation during the aging and cancer].

    PubMed

    Szigeti, Krisztina Andrea; Galamb, Orsolya; Kalmár, Alexandra; Barták, Barbara Kinga; Nagy, Zsófia Brigitta; Márkus, Eszter; Igaz, Péter; Tulassay, Zsolt; Molnár, Béla

    2018-01-01

    Besides the genetic research, increasing number of scientific studies focus on epigenetic phenomena - such as DNA methylation - regulating the expression of genes behind the phenotype, thus can be related to the pathomechanism of several diseases. In this review, we aim to summarize the current knowledge about the evolutionary appearance and functional diversity of DNA methylation as one of the epigenetic mechanisms and to demonstrate its role in aging and cancerous diseases. DNA methylation is also characteristic/also appear to prokaryotes, eukaryotes and viruses. In prokaryotes and viruses, it provides defence mechanisms against extragenous DNA. DNA methylation in prokaryotes plays a significant role in the regulation of transcription, the initiation of replication and in Dam-directed mismatch repair. In viruses, it participates not only in defence mechanisms, but in the assembly of capsids as well which is necessary for spreading. In eukaryotes, DNA methylation is involved in recombination, replication, X chromosome inactivation, transposon control, regulation of chromatin structure and transcription, and it also contributes to the imprinting phenomenon. Besides the above-mentioned aspects, DNA methylation also has an evolutionary role as it can change DNA mutation rate. Global hypomethylation appearing during aging and in cancerous diseases can lead to genetic instablility and spontaneous mutations through its role in the regulation of transposable elements. Local hypermethylated alterations such as hypermethylation of SFRP1, SFRP2, DKK1 and APC gene promoters can cause protein expression changes, thus contribute to development of cancer phenotype. DNA methylation alterations during aging in cancerous diseases support the importance of epigenetic research focusing on disease diagnostics and prognostics. Orv Hetil. 2018; 159(1): 3-15.

  6. Cell-free fetal DNA concentration in plasma of patients with abnormal uterine artery Doppler waveform and intrauterine growth restriction--a pilot study.

    PubMed

    Caramelli, Elisabetta; Rizzo, Nicola; Concu, Manuela; Simonazzi, Giuliana; Carinci, Paolo; Bondavalli, Corrado; Bovicelli, Luciano; Farina, Antonio

    2003-05-01

    To evaluate if an increased amount of fetal DNA concentration can be found in women screened positive for intrauterine growth restriction because of abnormal uterine artery Doppler waveforms. We enrolled eight pregnant women (each bearing a male fetus), with the evidence of abnormal uterine artery Doppler waveforms, and 16 control patients for a case-control study matched for gestational age (1 : 2). Uterine artery Doppler was carried out at 20 to 35 weeks' gestation (median 29). The mean uterine artery resistance index (RI) was subsequently calculated, and a value >0.6 was considered positive for the clinical features of pre-eclampsia. The SRY locus was used to determine the amount of male fetal DNA in the maternal plasma at the time of Doppler analysis. Two controls (normal Doppler) were excluded from the final analysis because they had a pre-term delivery. One case (abnormal Doppler) had evidence of intrauterine growth restriction at the time of enrolment. In four out of eight cases (abnormal Doppler), intrauterine growth restriction was subsequently observed. Multiples of median (MoM) conversion of the fetal DNA values showed an increase of 1.81 times in the cases when compared to the controls. An increase of 2.16 times was instead observed for the cases with a growth-restricted fetus (5 cases out of 8) in comparison with the controls (14 cases). In subjects positive to uterine artery Doppler velocimetry analysis (Doppler analysis for pre-eclampsia screening), the fetal DNA concentration is higher than expected, in the absence of any other clinical feature. Since the increase in fetal DNA seems to be related to the presence or to the future development of intrauterine growth restriction, this paper suggests a possible integration between ultrasound and molecular markers for predicting the disease in some cases. Copyright 2003 John Wiley & Sons, Ltd.

  7. DNA Methylation in Promoter Region as Biomarkers in Prostate Cancer

    PubMed Central

    Yang, Mihi; Park, Jong Y.

    2013-01-01

    The prostate gland is the most common site of cancer and the second leading cause of cancer death in American men. Recent emerging molecular biological technologies help us to know that epigenetic alterations such as DNA methylation within the regulatory (promoter) regions of genes are associated with transcriptional silencing in cancer. Promoter hypermethylation of critical pathway genes could be potential biomarkers and therapeutic targets for prostate cancer. In this chapter, we updated current information on methylated genes associated with the development and progression of prostate cancer. Over 40 genes have been investigated for methylation in promoter region in prostate cancer. These methylated genes are involved in critical pathways, such as DNA repair, metabolism, and invasion/metastasis. The role of hypermethylated genes in regulation of critical pathways in prostate cancer is discussed. These findings may provide new information of the pathogenesis, the exciting potential to be predictive and to provide personalized treatment of prostate cancer. Indeed, some epigenetic alterations in prostate tumors are being translated into clinical practice for therapeutic use. PMID:22359288

  8. Hypermethylation of the TSLC1 Gene Promoter in Primary Gastric Cancers and Gastric Cancer Cell Lines

    PubMed Central

    Honda, Teiichiro; Waki, Takayoshi; Jin, Zhe; Sato, Kiyoshi; Motoyama, Teiichi; Kawata, Sumio; Kimura, Wataru; Nishizuka, Satoshi; Murakami, Yoshinori

    2002-01-01

    The TSLC1 (tumor suppressor in lung cancer–1) gene is a novel tumor suppressor gene on chromosomal region 11q23.2, and is frequently inactivated by concordant promoter hypermethylation and loss of heterozygosity (LOH) in non‐small cell lung cancer (NSCLC). Because LOH on 11q has also been observed frequently in other human neoplasms including gastric cancer, we investigated the promoter methylation status of TSLC1 in 10 gastric cancer cell lines and 97 primary gastric cancers, as well as the corresponding non‐cancerous gastric tissues, by bisulfite‐SSCP analysis followed by direct sequencing. Allelic status of the TSLC1 gene was also investigated in these cell lines and primary gastric cancers. The TSLC1 promoter was methylated in two gastric cancer cell lines, KATO‐III and ECC10, and in 15 out of 97 (16%) primary gastric cancers. It was not methylated in non‐cancerous gastric tissues, suggesting that this hypermethylation is a cancer‐specific alteration. KATO‐III and ECC10 cells retained two alleles of TSLC1, both of which showed hypermethylation, associated with complete loss of gene expression. Most of the primary gastric cancers with promoter methylation also retained heterozygosity at the TSLC1 locus on 11q23.2. These data indicate that bi‐allelic hypermethylation of the TSLC1 promoter and resulting gene silencing occur in a subset of primary gastric cancers. PMID:12716461

  9. Replacing the combined test by cell-free DNA testing in screening for trisomies 21, 18 and 13: impact on the diagnosis of other chromosomal abnormalities.

    PubMed

    Syngelaki, Argyro; Pergament, Eugene; Homfray, Tessa; Akolekar, Ranjit; Nicolaides, Kypros H

    2014-01-01

    To estimate the proportion of other chromosomal abnormalities that could be missed if combined testing was replaced by cell-free (cf) DNA testing as the method of screening for trisomies 21, 18 and 13. The prevalence of trisomies 21, 18 or 13, sex chromosome aneuploidies, triploidy and other chromosomal abnormalities was examined in pregnancies undergoing first-trimester combined screening and chorionic villus sampling (CVS). In 1,831 clinically significant chromosomal abnormalities in pregnancies with combined risk for trisomies 21, 18 and 13≥1:100, the contribution of trisomies 21, 18 or 13, sex chromosome aneuploidies, triploidy and other chromosomal abnormalities at high risk of adverse outcome was 82.9, 8.2, 3.9 and 5.0%, respectively. Combined screening followed by CVS for risk≥1:10 and cfDNA testing for risk 1:11-1:2,500 could detect 97% of trisomy 21 and 98% of trisomies 18 and 13. Additionally, 86% of monosomy X, half of 47,XXY, 47,XYY or 47,XXX, half of other chromosomal abnormalities and one third of triploidies, which are currently detected by combined screening and CVS for risk≥1:100, could be detected. Screening by cfDNA testing, contingent on results of combined testing, improves detection of trisomies, but misses a few of the other chromosomal abnormalities detected by screening with the combined test. © 2014 S. Karger AG, Basel.

  10. Microarray-based DNA methylation study of Ewing’s sarcoma of the bone

    PubMed Central

    PARK, HYE-RIM; JUNG, WOON-WON; KIM, HYUN-SOOK; PARK, YONG-KOO

    2014-01-01

    Alterations in DNA methylation patterns are a hallmark of malignancy. However, the majority of epigenetic studies of Ewing’s sarcoma have focused on the analysis of only a few candidate genes. Comprehensive studies are thus lacking and are required. The aim of the present study was to identify novel methylation markers in Ewing’s sarcoma using microarray analysis. The current study reports the microarray-based DNA methylation study of 1,505 CpG sites of 807 cancer-related genes from 69 Ewing’s sarcoma samples. The Illumina GoldenGate Methylation Cancer Panel I microarray was used, and with the appropriate controls (n=14), a total of 92 hypermethylated genes were identified in the Ewing’s sarcoma samples. The majority of the hypermethylated genes were associated with cell adhesion, cell regulation, development and signal transduction. The overall methylation mean values were compared between patients who survived and those that did not. The overall methylation mean was significantly higher in the patients who did not survive (0.25±0.03) than in those who did (0.22±0.05) (P=0.0322). However, the overall methylation mean was not found to significantly correlate with age, gender or tumor location. GDF10, OSM, APC and HOXA11 were the most significant differentially-methylated genes, however, their methylation levels were not found to significantly correlate with the survival rate. The DNA methylation profile of Ewing’s sarcoma was characterized and 92 genes that were significantly hypermethylated were detected. A trend towards a more aggressive behavior was identified in the methylated group. The results of this study indicated that methylation may be significant in the development of Ewing’s sarcoma. PMID:25202378

  11. Effects of Trans-Resveratrol on hyperglycemia-induced abnormal spermatogenesis, DNA damage and alterations in poly (ADP-ribose) polymerase signaling in rat testis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Abdelali, Ala

    Diabetes induces oxidative stress, DNA damage and alters several intracellular signaling pathways in organ systems. This study investigated modulatory effects of Trans-Resveratrol on type 1 diabetes mellitus (T1DM)-induced abnormal spermatogenesis, DNA damage and alterations in poly (ADP-ribose) polymerase (PARP) signaling in rat testis. Trans-Resveratrol administration (5mg/kg/day, ip) to Streptozotocin-induced T1DM adult male Wistar rats from day 22–42 resulted in recovery of induced oxidative stress, abnormal spermatogenesis and inhibited DNA synthesis, and led to mitigation of 8-hydroxy-2'-deoxyguanosine formation in the testis and spermatozoa, and DNA double-strand breaks in the testis. Trans-Resveratrol aggravated T1DM-induced up-regulation of aminoacyl tRNA synthetase complex-interacting multifunctional proteinmore » 2 expression; however, it did not modify the up-regulated total PARP and down-regulated PARP1 expressions, but recovered the decreased SirT1 (Sirtuin 1) levels in T1DM rat testis. Trans-Resveratrol, when given alone, reduced the poly (ADP-ribosyl)ation (pADPr) process in the testis due to an increase in PAR glycohydrolase activity, but when given to T1DM rats it did not affect the pADPr levels. T1DM with or without Trans-Resveratrol did not induce nuclear translocation of apoptosis-inducing factor and the formation of 50 kb DNA breaks, suggesting to the lack of caspase-3-independent cell death called parthanatos. T1DM with or without Trans-Resveratrol did not increase necrotic cell death in the testis. Primary spermatocytes, Sertoli cells, Leydig cells and intra-testicular vessels showed the expression of PARP pathway related proteins. In conclusion, Trans-Resveratrol mitigates T1DM-induced sperm abnormality and DNA damage, but does not significantly modulate PARP signaling pathway, except the SirT1 expression, in the rat testis. - Highlights: • Resveratrol inhibits diabetes-induced abnormal sperm morphogenesis • Resveratrol recovers

  12. Thymidine kinase and mtDNA depletion in human cardiomyopathy: epigenetic and translational evidence for energy starvation

    PubMed Central

    Koczor, Christopher A.; Torres, Rebecca A.; Fields, Earl J.; Boyd, Amy; He, Stanley; Patel, Nilamkumar; Lee, Eva K.; Samarel, Allen M.

    2013-01-01

    This study addresses how depletion of human cardiac left ventricle (LV) mitochondrial DNA (mtDNA) and epigenetic nuclear DNA methylation promote cardiac dysfunction in human dilated cardiomyopathy (DCM) through regulation of pyrimidine nucleotide kinases. Samples of DCM LV and right ventricle (n = 18) were obtained fresh at heart transplant surgery. Parallel samples from nonfailing (NF) controls (n = 12) were from donor hearts found unsuitable for clinical use. We analyzed abundance of mtDNA and nuclear DNA (nDNA) using qPCR. LV mtDNA was depleted in DCM (50%, P < 0.05 each) compared with NF. No detectable change in RV mtDNA abundance occurred. DNA methylation and gene expression were determined using microarray analysis (GEO accession number: GSE43435). Fifty-seven gene promoters exhibited DNA hypermethylation or hypomethylation in DCM LVs. Among those, cytosolic thymidine kinase 1 (TK1) was hypermethylated. Expression arrays revealed decreased abundance of the TK1 mRNA transcript with no change in transcripts for other relevant thymidine metabolism enzymes. Quantitative immunoblots confirmed decreased TK1 polypeptide steady state abundance. TK1 activity remained unchanged in DCM samples while mitochondrial thymidine kinase (TK2) activity was significantly reduced. Compensatory TK activity was found in cardiac myocytes in the DCM LV. Diminished TK2 activity is mechanistically important to reduced mtDNA abundance and identified in DCM LV samples here. Epigenetic and genetic changes result in changes in mtDNA and in nucleotide substrates for mtDNA replication and underpin energy starvation in DCM. PMID:23695887

  13. Gene hypermethylation in blood leukocytes in humans long term after radiation exposure - Validation set.

    PubMed

    Kuzmina, Nina S; Lapteva, Nellya Sh; Rusinova, Galina G; Azizova, Tamara V; Vyazovskaya, Natalya S; Rubanovich, Alexander V

    2018-03-01

    Hypermethylation of СpG islands in the promoter regions of several genes with basic protective function in blood leukocytes of individuals exposed to ionizing radiation long time ago (2-46 years), and differential effects of age and radiation exposure on hypermethylation was reported in our previous work. To validate these results, epigenetic modifications were assessed in an independent series of 49 nuclear industry workers from the "Mayak" facility (67-84 years old at sampling) with documented individual accumulated doses from the prolonged external γ-radiation exposure (95.9-409.5 cGy, end of work with radiation:0.3-39 years ago), and in 50 non-exposed persons matched by age. In addition to the genes analyzed before (RASSF1A, p16/INK4A, p14/ARF, GSTP1), four additional loci were analyzed: TP53, ATM, SOD3, ESR1. The frequency of individuals displaying promoter methylation of at least one of the 8 genes (71.4%) was significantly higher in exposed group as compared to the control group (40%), p = .002, OR = 3.75. A significantly elevated frequency of individuals with hypermethylated СpG islands in GSTP1, TP53, SOD3 promoters was revealed among exposed subjects as compared to the control group (p = .012, OR = 8.41; p = .041, OR = 4.02 and p = .009, OR = 3.42, respectively). A similar trend (p = .12, OR = 3.06) was observed for the p16/INK4A gene. As a whole, p16/INK4A and GSTP1 promoter hypermethylation in irradiated subjects from both previously and currently analyzed groups was pronounced. Thus, the direction of the effects was fully confirmed, suggesting the result reproducibility. No statistically significant correlation between promoter methylation and individual radiation dose was found. Further studies are required to create an array of blood epigenetic markers of radiation exposure associating with premature aging and age-related diseases and to accurately evaluate radiation-added effect across the range of doses

  14. The cytosolic Fe-S cluster assembly component MET18 is required for the full enzymatic activity of ROS1 in active DNA demethylation.

    PubMed

    Wang, Xiaokang; Li, Qi; Yuan, Wei; Cao, Zhendong; Qi, Bei; Kumar, Suresh; Li, Yan; Qian, Weiqiang

    2016-05-19

    DNA methylation patterns in plants are dynamically regulated by DNA methylation and active DNA demethylation in response to both environmental changes and development of plant. Beginning with the removal of methylated cytosine by ROS1/DME family of 5-methylcytosine DNA glycosylases, active DNA demethylation in plants occurs through base excision repair. So far, many components involved in active DNA demethylation remain undiscovered. Through a forward genetic screening of Arabidopsis mutants showing DNA hypermethylation at the EPF2 promoter region, we identified the conserved iron-sulfur cluster assembly protein MET18. MET18 dysfunction caused DNA hypermethylation at more than 1000 loci as well as the silencing of reporter genes and some endogenous genes. MET18 can directly interact with ROS1 in vitro and in vivo. ROS1 activity was reduced in the met18 mutant plants and point mutation in the conserved Fe-S cluster binding motif of ROS1 disrupted its biological function. Interestingly, a large number of DNA hypomethylated loci, especially in the CHH context, were identified from the met18 mutants and most of the hypo-DMRs were from TE regions. Our results suggest that MET18 can regulate both active DNA demethylation and DNA methylation pathways in Arabidopsis.

  15. The cytosolic Fe-S cluster assembly component MET18 is required for the full enzymatic activity of ROS1 in active DNA demethylation

    PubMed Central

    Wang, Xiaokang; Li, Qi; Yuan, Wei; Cao, Zhendong; Qi, Bei; Kumar, Suresh; Li, Yan; Qian, Weiqiang

    2016-01-01

    DNA methylation patterns in plants are dynamically regulated by DNA methylation and active DNA demethylation in response to both environmental changes and development of plant. Beginning with the removal of methylated cytosine by ROS1/DME family of 5-methylcytosine DNA glycosylases, active DNA demethylation in plants occurs through base excision repair. So far, many components involved in active DNA demethylation remain undiscovered. Through a forward genetic screening of Arabidopsis mutants showing DNA hypermethylation at the EPF2 promoter region, we identified the conserved iron-sulfur cluster assembly protein MET18. MET18 dysfunction caused DNA hypermethylation at more than 1000 loci as well as the silencing of reporter genes and some endogenous genes. MET18 can directly interact with ROS1 in vitro and in vivo. ROS1 activity was reduced in the met18 mutant plants and point mutation in the conserved Fe-S cluster binding motif of ROS1 disrupted its biological function. Interestingly, a large number of DNA hypomethylated loci, especially in the CHH context, were identified from the met18 mutants and most of the hypo-DMRs were from TE regions. Our results suggest that MET18 can regulate both active DNA demethylation and DNA methylation pathways in Arabidopsis. PMID:27193999

  16. Constitutional H19 hypermethylation in a patient with isolated cardiac tumor.

    PubMed

    Descartes, Maria; Romp, Robb; Franklin, Judy; Biggio, Joseph R; Zehnbauer, Barbara

    2008-08-15

    Beckwith-Wiedemann syndrome (BWS) is clinically and molecularly very heterogenous. Molecular findings characteristic of BWS have been reported in individuals with no or few associated features. We report on a child with isolated cardiac tumor and a constitutional H19 hypermethylation with none of the features of BWS. Copyright 2008 Wiley-Liss, Inc.

  17. DNA methylation profiles of long- and short-term glioblastoma survivors

    PubMed Central

    Shinawi, Thoraia; Hill, Victoria K.; Krex, Dietmar; Schackert, Gabriele; Gentle, Dean; Morris, Mark R.; Wei, Wenbin; Cruickshank, Garth; Maher, Eamonn R.; Latif, Farida

    2013-01-01

    Glioblastoma (GBM) is the most common and malignant type of primary brain tumor in adults and prognosis of most GBM patients is poor. However, a small percentage of patients show a long term survival of 36 mo or longer after diagnosis. Epigenetic profiles can provide molecular markers for patient prognosis: recently, a G-CIMP positive phenotype associated with IDH1 mutations has been described for GBMs with good prognosis. In the present analysis we performed genome-wide DNA methylation profiling of short-term survivors (STS; overall survival < 1 y) and long-term survivors (LTS; overall survival > 3 y) by utilizing the HumanMethylation450K BeadChips to assess quantitative methylation at > 480,000 CpG sites. Cluster analysis has shown that a subset of LTS showed a G-CIMP positive phenotype that was tightly associated with IDH1 mutation status and was confirmed by analysis of the G-CIMP signature genes. Using high stringency criteria for differential hypermethylation between non-cancer brain and tumor samples, we identified 2,638 hypermethylated CpG loci (890 genes) in STS GBMs, 3,101 hypermethylated CpG loci (1,062 genes) in LTS (wild type IDH1) and 11,293 hypermethylated CpG loci in LTS (mutated for IDH1), reflecting the CIMP positive phenotype. The location of differentially hypermethylated CpG loci with respect to CpG content, neighborhood context and functional genomic distribution was similar in our sample set, with the majority of CpG loci residing in CpG islands and in gene promoters. Our preliminary study also identified a set of CpG loci differentially hypermethylated between STS and LTS cases, including members of the homeobox gene family (HOXD8, HOXD13 and HOXC4), the transcription factors NR2F2 and TFAP2A, and Dickkopf 2, a negative regulator of the wnt/β-catenin signaling pathway. PMID:23291739

  18. Quantitative methylation-sensitive arbitrarily primed PCR method to determine differential genomic DNA methylation in Down Syndrome.

    PubMed

    Chango, Abalo; Abdennebi-Najar, Latifa; Tessier, Frederic; Ferré, Séverine; Do, Sergio; Guéant, Jean-Louis; Nicolas, Jean Pierre; Willequet, Francis

    2006-10-20

    Relative levels of DNA hypermethylation were quantified in DS individuals using a new method based on a combination of methylation-sensitive arbitrarily primed polymerase chain reaction (MS-AP-PCR) and quantification of DNA fragments with the Agilent 2100 bioanalyzer. Four of the DS individuals had low plasma total homocysteine (tHcy) level (4.3 +/- 0.3 micromol/l) and 4 other had high-tHcy level (14.1 +/- 0.9 micromol/l). Eight healthy control individuals were matched to the DS cases for age, sex, and tHcy levels. We have identified and quantified six hypermethylated fragments. Their sizes ranged from 230-bp to 700-bp. In cases and controls, low-tHcy did not affect methylation level of identified fragments, mean methylation values were 68.0 +/- 39.7% and 52.1 +/- 40.3%, respectively. DNA methylation in DS individuals did not change significantly (59.7+/-34.5%) in response to high-tHcy level in contrast to controls (23.4 +/- 17.7%, P = 0.02). Further, the quantitative MS-AP-PCR using this microfludic system is a useful method for determining differential genomic DNA methylation.

  19. Quantitative DNA methylation analysis improves epigenotype-phenotype correlations in Beckwith-Wiedemann syndrome

    PubMed Central

    Calvello, Mariarosaria; Tabano, Silvia; Colapietro, Patrizia; Maitz, Silvia; Pansa, Alessandra; Augello, Claudia; Lalatta, Faustina; Gentilin, Barbara; Spreafico, Filippo; Calzari, Luciano; Perotti, Daniela; Larizza, Lidia; Russo, Silvia; Selicorni, Angelo; Sirchia, Silvia M; Miozzo, Monica

    2013-01-01

    Beckwith-Wiedemann syndrome (BWS) is a rare disorder characterized by overgrowth and predisposition to embryonal tumors. BWS is caused by various epigenetic and/or genetic alterations that dysregulate the imprinted genes on chromosome region 11p15.5. Molecular analysis is required to reinforce the clinical diagnosis of BWS and to identify BWS patients with cancer susceptibility. This is particularly crucial prenatally because most signs of BWS cannot be recognized in utero. We established a reliable molecular assay by pyrosequencing to quantitatively evaluate the methylation profiles of ICR1 and ICR2. We explored epigenotype-phenotype correlations in 19 patients that fulfilled the clinical diagnostic criteria for BWS, 22 patients with suspected BWS, and three fetuses with omphalocele. Abnormal methylation was observed in one prenatal case and 19 postnatal cases, including seven suspected BWS. Seven cases showed ICR1 hypermethylation, five cases showed ICR2 hypomethylation, and eight cases showed abnormal methylation of ICR1 and ICR2 indicating paternal uniparental disomy (UPD). More cases of ICR1 alterations and UPD were found than expected. This is likely due to the sensitivity of this approach, which can detect slight deviations in methylation from normal levels. There was a significant correlation (p < 0.001) between the percentage of ICR1 methylation and BWS features: severe hypermethylation (range: 75–86%) was associated with macroglossia, macrosomia, and visceromegaly, whereas mild hypermethylation (range: 55–59%) was associated with umbilical hernia and diastasis recti. Evaluation of ICR1 and ICR2 methylation by pyrosequencing in BWS can improve epigenotype-phenotype correlations, detection of methylation alterations in suspected cases, and identification of UPD. PMID:23917791

  20. DNA Hypomethylation Affects Cancer-Related Biological Functions and Genes Relevant in Neuroblastoma Pathogenesis

    PubMed Central

    Mayol, Gemma; Martín-Subero, José I.; Ríos, José; Queiros, Ana; Kulis, Marta; Suñol, Mariona; Esteller, Manel; Gómez, Soledad; Garcia, Idoia; de Torres, Carmen; Rodríguez, Eva; Galván, Patricia; Mora, Jaume; Lavarino, Cinzia

    2012-01-01

    Neuroblastoma (NB) pathogenesis has been reported to be closely associated with numerous genetic alterations. However, underlying DNA methylation patterns have not been extensively studied in this developmental malignancy. Here, we generated microarray-based DNA methylation profiles of primary neuroblastic tumors. Stringent supervised differential methylation analyses allowed us to identify epigenetic changes characteristic for NB tumors as well as for clinical and biological subtypes of NB. We observed that gene-specific loss of DNA methylation is more prevalent than promoter hypermethylation. Remarkably, such hypomethylation affected cancer-related biological functions and genes relevant to NB pathogenesis such as CCND1, SPRR3, BTC, EGF and FGF6. In particular, differential methylation in CCND1 affected mostly an evolutionary conserved functionally relevant 3′ untranslated region, suggesting that hypomethylation outside promoter regions may play a role in NB pathogenesis. Hypermethylation targeted genes involved in cell development and proliferation such as RASSF1A, POU2F2 or HOXD3, among others. The results derived from this study provide new candidate epigenetic biomarkers associated with NB as well as insights into the molecular pathogenesis of this tumor, which involves a marked gene-specific hypomethylation. PMID:23144874

  1. The Silencing of RECK Gene is Associated with Promoter Hypermethylation and Poor Survival in Hepatocellular Carcinoma

    PubMed Central

    Zhang, Changsong; Ling, Yang; Zhang, Chenghui; Xu, Yun; Gao, Lu; Li, Rong; Zhu, Jing; Fan, Lieying; Wei, Lixin

    2012-01-01

    Background: To evaluate the promoter methylation status of RECK gene and mRNA expression in patients with hepatocellular carcinoma (HCC). Methods: We analyzed RECK methylation by MSP, and RECK mRNA by real-time PCR in 74 HCC. The liver cell lines (7721, Chang and Hep-G2) were treated with 5-Aza-CdR and TSA. Results: RECK mRNA were lower in HCC tissues (Mean -∆Ct = -3.29) than that in Non-Hcc tissues (Mean -∆Ct = -2.42). Expression of RECK was elevated in only 24 (32.43%) of the 74 HCC patients but decreased (-∆∆Ct<0) in 50 (67.57%) of the patients. RECK promoter was hypermethylated in 55.4% (41/74) of HCCs, and in only 17.6% (13/74) of Non-Hcc samples. RECK mRNA were lower in HCC patients with hypermethylation (∆MI>=0.5) (Mean -∆∆Ct = -1.75) than those with demethylation (∆MI<0.5) (Mean -∆∆Ct = 0.05), and there is a decreased tendency for RECK mRNA in HCC patients with promoter hypermethylation (p = 0.002). There was a significantly correlation found between RECK mRNA and poor survival after surgery. After treated by 5-Aza-CdR and TSA, we found that RECK mRNA induced different changes in 7721, Chang and Hep-G2 cells. And RECK demethylation also induced by epigenetic inhibitors. Conclusion: The results suggested that the hypermethylation may lead to promoter silencing of RECK mRNA and associated with poor survival in HCC. PMID:22419890

  2. DNA Hypermethylation of the Serotonin Receptor Type-2A Gene Is Associated with a Worse Response to a Weight Loss Intervention in Subjects with Metabolic Syndrome

    PubMed Central

    Perez-Cornago, Aurora; Mansego, Maria L.; Zulet, María Angeles; Martinez, José Alfredo

    2014-01-01

    Understanding the regulation of gene activities depending on DNA methylation has been the subject of much recent study. However, although polymorphisms of the HTR2A gene have been associated with both obesity and psychiatric disorders, the role of HTR2A gene methylation in these illnesses remains uncertain. The aim of this study was to evaluate the association of HTR2A gene promoter methylation levels in white blood cells (WBC) with obesity traits and depressive symptoms in individuals with metabolic syndrome (MetS) enrolled in a behavioural weight loss programme. Analyses were based on 41 volunteers (mean age 49 ± 1 year) recruited within the RESMENA study. Depressive symptoms (as determined using the Beck Depression Inventory), anthropometric and biochemical measurements were analysed at the beginning and after six months of weight loss treatment. At baseline, DNA from WBC was isolated and cytosine methylation in the HTR2A gene promoter was quantified by a microarray approach. In the whole-study sample, a positive association of HTR2A gene methylation with waist circumference and insulin levels was detected at baseline. Obesity measures significantly improved after six months of dietary treatment, where a lower mean HTR2A gene methylation at baseline was associated with major reductions in body weight, BMI and fat mass after the treatment. Moreover, mean HTR2A gene methylation at baseline significantly predicted the decrease in depressive symptoms after the weight loss treatment. In conclusion, this study provides newer evidence that hypermethylation of the HTR2A gene in WBC at baseline is significantly associated with a worse response to a weight-loss intervention and with a lower decrease in depressive symptoms after the dietary treatment in subjects with MetS. PMID:24959950

  3. Histone modification alteration coordinated with acquisition of promoter DNA methylation during Epstein-Barr virus infection.

    PubMed

    Funata, Sayaka; Matsusaka, Keisuke; Yamanaka, Ryota; Yamamoto, Shogo; Okabe, Atsushi; Fukuyo, Masaki; Aburatani, Hiroyuki; Fukayama, Masashi; Kaneda, Atsushi

    2017-08-15

    Aberrant DNA hypermethylation is a major epigenetic mechanism to inactivate tumor suppressor genes in cancer. Epstein-Barr virus positive gastric cancer is the most frequently hypermethylated tumor among human malignancies. Herein, we performed comprehensive analysis of epigenomic alteration during EBV infection, by Infinium HumanMethylation 450K BeadChip for DNA methylation and ChIP-sequencing for histone modification alteration during EBV infection into gastric cancer cell line MKN7. Among 7,775 genes with increased DNA methylation in promoter regions, roughly half were "DNA methylation-sensitive" genes, which acquired DNA methylation in the whole promoter regions and thus were repressed. These included anti-oncogenic genes, e.g. CDKN2A . The other half were "DNA methylation-resistant" genes, where DNA methylation is acquired in the surrounding of promoter regions, but unmethylated status is protected in the vicinity of transcription start site. These genes thereby retained gene expression, and included DNA repair genes. Histone modification was altered dynamically and coordinately with DNA methylation alteration. DNA methylation-sensitive genes significantly correlated with loss of H3K27me3 pre-marks or decrease of active histone marks, H3K4me3 and H3K27ac. Apoptosis-related genes were significantly enriched in these epigenetically repressed genes. Gain of active histone marks significantly correlated with DNA methylation-resistant genes. Genes related to mitotic cell cycle and DNA repair were significantly enriched in these epigenetically activated genes. Our data show that orchestrated epigenetic alterations are important in gene regulation during EBV infection, and histone modification status in promoter regions significantly associated with acquisition of de novo DNA methylation or protection of unmethylated status at transcription start site.

  4. Ecstasy (MDMA) Alters Cardiac Gene Expression and DNA Methylation: Implications for Circadian Rhythm Dysfunction in the Heart

    PubMed Central

    Koczor, Christopher A.; Ludlow, Ivan; Hight, Robert S.; Jiao, Zhe; Fields, Earl; Ludaway, Tomika; Russ, Rodney; Torres, Rebecca A.; Lewis, William

    2015-01-01

    MDMA (ecstasy) is an illicit drug that stimulates monoamine neurotransmitter release and inhibits reuptake. MDMA’s acute cardiotoxicity includes tachycardia and arrhythmia which are associated with cardiomyopathy. MDMA acute cardiotoxicity has been explored, but neither long-term MDMA cardiac pathological changes nor epigenetic changes have been evaluated. Microarray analyses were employed to identify cardiac gene expression changes and epigenetic DNA methylation changes. To identify permanent MDMA-induced pathogenetic changes, mice received daily 10- or 35-day MDMA, or daily 10-day MDMA followed by 25-day saline washout (10 + 25 days). MDMA treatment caused differential gene expression (p < .05, fold change >1.5) in 752 genes following 10 days, 558 genes following 35 days, and 113 genes following 10-day MDMA + 25-day saline washout. Changes in MAPK and circadian rhythm gene expression were identified as early as 10 days. After 35 days, circadian rhythm genes (Per3, CLOCK, ARNTL, and NPAS2) persisted to be differentially expressed. MDMA caused DNA hypermethylation and hypomethylation that was independent of gene expression; hypermethylation of genes was found to be 71% at 10 days, 68% at 35 days, and 91% at 10 + 25 days washout. Differential gene expression paralleled DNA methylation in 22% of genes at 10-day treatment, 17% at 35 days, and 48% at 10 + 25 days washout. We show here that MDMA induced cardiac epigenetic changes in DNA methylation where hypermethylation predominated. Moreover, MDMA induced gene expression of key elements of circadian rhythm regulatory genes. This suggests a fundamental organism-level event to explain some of the etiologies of MDMA dysfunction in the heart. PMID:26251327

  5. A DEMETER-like DNA demethylase governs tomato fruit ripening.

    PubMed

    Liu, Ruie; How-Kit, Alexandre; Stammitti, Linda; Teyssier, Emeline; Rolin, Dominique; Mortain-Bertrand, Anne; Halle, Stefanie; Liu, Mingchun; Kong, Junhua; Wu, Chaoqun; Degraeve-Guibault, Charlotte; Chapman, Natalie H; Maucourt, Mickael; Hodgman, T Charlie; Tost, Jörg; Bouzayen, Mondher; Hong, Yiguo; Seymour, Graham B; Giovannoni, James J; Gallusci, Philippe

    2015-08-25

    In plants, genomic DNA methylation which contributes to development and stress responses can be actively removed by DEMETER-like DNA demethylases (DMLs). Indeed, in Arabidopsis DMLs are important for maternal imprinting and endosperm demethylation, but only a few studies demonstrate the developmental roles of active DNA demethylation conclusively in this plant. Here, we show a direct cause and effect relationship between active DNA demethylation mainly mediated by the tomato DML, SlDML2, and fruit ripening- an important developmental process unique to plants. RNAi SlDML2 knockdown results in ripening inhibition via hypermethylation and repression of the expression of genes encoding ripening transcription factors and rate-limiting enzymes of key biochemical processes such as carotenoid synthesis. Our data demonstrate that active DNA demethylation is central to the control of ripening in tomato.

  6. A DEMETER-like DNA demethylase governs tomato fruit ripening

    PubMed Central

    Liu, Ruie; How-Kit, Alexandre; Stammitti, Linda; Teyssier, Emeline; Rolin, Dominique; Mortain-Bertrand, Anne; Halle, Stefanie; Liu, Mingchun; Kong, Junhua; Wu, Chaoqun; Degraeve-Guibault, Charlotte; Chapman, Natalie H.; Maucourt, Mickael; Hodgman, T. Charlie; Tost, Jörg; Bouzayen, Mondher; Hong, Yiguo; Seymour, Graham B.; Giovannoni, James J.; Gallusci, Philippe

    2015-01-01

    In plants, genomic DNA methylation which contributes to development and stress responses can be actively removed by DEMETER-like DNA demethylases (DMLs). Indeed, in Arabidopsis DMLs are important for maternal imprinting and endosperm demethylation, but only a few studies demonstrate the developmental roles of active DNA demethylation conclusively in this plant. Here, we show a direct cause and effect relationship between active DNA demethylation mainly mediated by the tomato DML, SlDML2, and fruit ripening— an important developmental process unique to plants. RNAi SlDML2 knockdown results in ripening inhibition via hypermethylation and repression of the expression of genes encoding ripening transcription factors and rate-limiting enzymes of key biochemical processes such as carotenoid synthesis. Our data demonstrate that active DNA demethylation is central to the control of ripening in tomato. PMID:26261318

  7. Aberrant promoter hypermethylation of PBRM1, BAP1, SETD2, KDM6A and other chromatin-modifying genes is absent or rare in clear cell RCC

    PubMed Central

    Ibragimova, Ilsiya; Maradeo, Marie E.; Dulaimi, Essel; Cairns, Paul

    2013-01-01

    Recent sequencing studies of clear cell (conventional) renal cell carcinoma (ccRCC) have identified inactivating point mutations in the chromatin-modifying genes PBRM1, KDM6A/UTX, KDM5C/JARID1C, SETD2, MLL2 and BAP1. To investigate whether aberrant hypermethylation is a mechanism of inactivation of these tumor suppressor genes in ccRCC, we sequenced the promoter region within a bona fide CpG island of PBRM1, KDM6A, SETD2 and BAP1 in bisulfite-modified DNA of a representative series of 50 primary ccRCC, 4 normal renal parenchyma specimens and 5 RCC cell lines. We also interrogated the promoter methylation status of KDM5C and ARID1A in the Cancer Genome Atlas (TCGA) ccRCC Infinium data set. PBRM1, KDM6A, SETD2 and BAP1 were unmethylated in all tumor and normal specimens. KDM5C and ARID1A were unmethylated in the TCGA 219 ccRCC and 119 adjacent normal specimens. Aberrant promoter hypermethylation of PBRM1, BAP1 and the other chromatin-modifying genes examined here is therefore absent or rare in ccRCC. PMID:23644518

  8. HOXA9 inhibits migration of lung cancer cells and its hypermethylation is associated with recurrence in non-small cell lung cancer.

    PubMed

    Hwang, Jung-Ah; Lee, Bo Bin; Kim, Yujin; Hong, Seung-Hyun; Kim, Young-Ho; Han, Joungho; Shim, Young Mog; Yoon, Chae-Yeong; Lee, Yeon-Su; Kim, Duk-Hwan

    2015-06-01

    This study was aimed at understanding the clinicopathological significance of HOXA9 hypermethylation in non-small cell lung cancer (NSCLC). HOXA9 hypermethylation was characterized in six lung cancer cell lines, and its clinicopathological significance was analyzed using methylation-specific PCR in 271 formalin-fixed paraffin-embedded tissues and 27 fresh-frozen tumor and matched normal tissues from 298 NSCLC patients, and Ki-67 expression was analyzed using immunohistochemistry. The promoter region of HOXA9 was highly methylated in six lung cancer cell lines, but not in normal bronchial epithelial cells. The loss of expression was restored by treatment of the cells with a demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-dC). Transient transfection of HOXA9 into H23 lung cancer cells resulted in the inhibition of cell migration but not proliferation. Conversely, sequence-specific siRNA-mediated knockdown of HOXA9 enhanced cell migration. The mRNA levels of HOXA9 in 27 fresh-frozen tumor tissues were significantly lower than in matched normal tissues (P<0.0001; Wilcoxon signed-rank test). HOXA9 hypermethylation was found in 191 (70%) of 271 primary NSCLCs. HOXA9 hypermethylation was not associated with tumor size (P=0.12) and Ki-67 proliferation index (P=0.15). However, patients with HOXA9 hypermethylation had poor recurrence-free survival (hazard ratio=3.98, 95% confidence interval = 1.07-17.09, P=0.01) in never-smokers, after adjusting for age, sex, tumor size, adjuvant therapy, pathologic stage, and histology. In conclusion, the present study suggests that HOXA9 inhibits migration of lung cancer cells and its hypermethylation is an independent prognostic factor for recurrence-free survival in never-smokers with NSCLC. © 2014 Wiley Periodicals, Inc.

  9. REPRESSOR OF SILENCING5 Encodes a Member of the Small Heat Shock Protein Family and Is Required for DNA Demethylation in Arabidopsis[C][W

    PubMed Central

    Zhao, Yusheng; Xie, Shaojun; Li, Xiaojie; Wang, Chunlei; Chen, Zhongzhou; Lai, Jinsheng; Gong, Zhizhong

    2014-01-01

    In Arabidopsis thaliana, active DNA demethylation is initiated by the DNA glycosylase REPRESSOR OF SILENCING1 (ROS1) and its paralogs DEMETER, DEMETER-LIKE2 (DML2), and DML3. How these demethylation enzymes are regulated, however, is poorly understood. Here, using a transgenic Arabidopsis line harboring the stress-inducible RESPONSIVE TO DEHYDRATION29A (RD29A) promoter–LUCIFERASE (LUC) reporter gene and the cauliflower mosaic virus 35S promoter (35S)–NEOMYCIN PHOSPHOTRANSFERASE II (NPTII) antibiotic resistance marker gene, we characterize a ROS locus, ROS5, that encodes a protein in the small heat shock protein family. ROS5 mutations lead to the silencing of the 35S-NPTII transgene due to DNA hypermethylation but do not affect the expression of the RD29A-LUC transgene. ROS5 physically interacts with the histone acetyltransferase ROS4/INCREASED DNA METHYLATION1 (IDM1) and is required to prevent the DNA hypermethylation of some genes that are also regulated by ROS1 and IDM1. We propose that ROS5 regulates DNA demethylation by interacting with IDM1, thereby creating a chromatin environment that facilitates the binding of ROS1 to erase DNA methylation. PMID:24920332

  10. An epigenetically derived monoclonal origin for recurrent respiratory papillomatosis.

    PubMed

    Stephen, Josena Kunjoonju; Vaught, Lori E; Chen, Kang Mei; Shah, Veena; Schweitzer, Vanessa G; Gardner, Glendon; Benninger, Michael S; Worsham, Maria J

    2007-07-01

    To investigate the contribution of promoter methylation-mediated epigenetic events in recurrent respiratory papillomatosis tumorigenesis. Archival tissue DNA, extracted from microdissected papilloma lesions, was interrogated for methylation status by means of the novel, multigene methylation-specific multiplex ligation-dependent probe amplification assay. Fifteen subjects with recurrent respiratory papillomatosis, 3 females and 12 males, all with adult onset of illness (age range, 23-73 years) except for 1 female patient with juvenile onset (1 year old). Promoter hypermethylation was recorded in 14 of 15 cases, and 19 of 22 unique methylation-prone cancer genes in the multigene panel had altered DNA methylation in at least 1 laryngeal papilloma biopsy specimen. Identical abnormally methylated genes were found in 5 of 15 recurrent cases, of which the CDKN2B gene was hypermethylated in all 5 cases. Dissimilar epigenetic events were noted in the remaining cases. A clonal origin was derived for 5 of 15 recurrent respiratory papillomatosis biopsy specimens based on identical epigenetic events. The high frequency of epigenetic events, characterized by consistent promoter hypermethylation of multiple tumor suppressor genes, points to the use of gene silencing mechanisms in the pathogenesis of recurrent respiratory papillomatosis.

  11. Diagnostic value of secreted frizzled-related protein 2 gene promoter hypermethylation in stool for colorectal cancer: A meta-analysis.

    PubMed

    Zhou, Zhiran; Zhang, Huitian; Lei, Yunxia

    2016-10-01

    To evaluate the diagnostic value of secreted frizzled-related protein 2 (SFRP2) gene promoter hypermethylation in stool for colorectal cancer (CRC). Open published diagnostic study of SFRP2 gene promoter hypermethylation in stool for CRC detection was electronic searched in the databases of PubMed, EMBASE, Cochrane Library, Web of Science, and China National Knowledge Infrastructure. The data of true positive, false positive false negative, and true negative identified by stool SFRP2 gene hypermethylation was extracted and pooled for diagnostic sensitivity, specificity, and summary receiver operating characteristic (SROC) curve. According to the inclusion and exclusion criteria, we finally included nine publications with 792 cases in the meta-analysis. Thus, the diagnostic sensitivity was aggregated through random effect model. The pooled sensitivity was 0.82 with the corresponding 95% confidence interval (95% CI) of 0.79-0.85; the pooled specificity and its corresponding 95% CI were 0.47 and 0.40-0.53 by the random effect model; we pooled the SROC curve by sensitivity versus specificity according to data published in the nine studies. The area under the SROC curve was 0.70 (95% CI: 0.65-0.73). SFRP2 gene promoter hypermethylation in stool can was a potential biomarker for CRC diagnosis with relative high sensitivity.

  12. The development and validation of EpiComet-Chip, a modified high-throughput comet assay for the assessment of DNA methylation status.

    PubMed

    Townsend, Todd A; Parrish, Marcus C; Engelward, Bevin P; Manjanatha, Mugimane G

    2017-08-01

    DNA damage and alterations in global DNA methylation status are associated with multiple human diseases and are frequently correlated with clinically relevant information. Therefore, assessing DNA damage and epigenetic modifications, including DNA methylation, is critical for predicting human exposure risk of pharmacological and biological agents. We previously developed a higher-throughput platform for the single cell gel electrophoresis (comet) assay, CometChip, to assess DNA damage and genotoxic potential. Here, we utilized the methylation-dependent endonuclease, McrBC, to develop a modified alkaline comet assay, "EpiComet," which allows single platform evaluation of genotoxicity and global DNA methylation [5-methylcytosine (5-mC)] status of single-cell populations under user-defined conditions. Further, we leveraged the CometChip platform to create an EpiComet-Chip system capable of performing quantification across simultaneous exposure protocols to enable unprecedented speed and simplicity. This system detected global methylation alterations in response to exposures which included chemotherapeutic and environmental agents. Using EpiComet-Chip on 63 matched samples, we correctly identified single-sample hypermethylation (≥1.5-fold) at 87% (20/23), hypomethylation (≥1.25-fold) at 100% (9/9), with a 4% (2/54) false-negative rate (FNR), and 10% (4/40) false-positive rate (FPR). Using a more stringent threshold to define hypermethylation (≥1.75-fold) allowed us to correctly identify 94% of hypermethylation (17/18), but increased our FPR to 16% (7/45). The successful application of this novel technology will aid hazard identification and risk characterization of FDA-regulated products, while providing utility for investigating epigenetic modes of action of agents in target organs, as the assay is amenable to cultured cells or nucleated cells from any tissue. Environ. Mol. Mutagen. 58:508-521, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  13. Early de novo DNA methylation and prolonged demethylation in the muscle lineage.

    PubMed

    Tsumagari, Koji; Baribault, Carl; Terragni, Jolyon; Varley, Katherine E; Gertz, Jason; Pradhan, Sirharsa; Badoo, Melody; Crain, Charlene M; Song, Lingyun; Crawford, Gregory E; Myers, Richard M; Lacey, Michelle; Ehrlich, Melanie

    2013-03-01

    Myogenic cell cultures derived from muscle biopsies are excellent models for human cell differentiation. We report the first comprehensive analysis of myogenesis-specific DNA hyper- and hypo-methylation throughout the genome for human muscle progenitor cells (both myoblasts and myotubes) and skeletal muscle tissue vs. 30 non-muscle samples using reduced representation bisulfite sequencing. We also focused on four genes with extensive hyper- or hypo-methylation in the muscle lineage (PAX3, TBX1, MYH7B/MIR499 and OBSCN) to compare DNA methylation, DNaseI hypersensitivity, histone modification, and CTCF binding profiles. We found that myogenic hypermethylation was strongly associated with homeobox or T-box genes and muscle hypomethylation with contractile fiber genes. Nonetheless, there was no simple relationship between differential gene expression and myogenic differential methylation, rather only for subsets of these genes, such as contractile fiber genes. Skeletal muscle retained ~30% of the hypomethylated sites but only ~3% of hypermethylated sites seen in myogenic progenitor cells. By enzymatic assays, skeletal muscle was 2-fold enriched globally in genomic 5-hydroxymethylcytosine (5-hmC) vs. myoblasts or myotubes and was the only sample type enriched in 5-hmC at tested myogenic hypermethylated sites in PAX3/CCDC140 andTBX1. TET1 and TET2 RNAs, which are involved in generation of 5-hmC and DNA demethylation, were strongly upregulated in myoblasts and myotubes. Our findings implicate de novo methylation predominantly before the myoblast stage and demethylation before and after the myotube stage in control of transcription and co-transcriptional RNA processing. They also suggest that, in muscle, TET1 or TET2 are involved in active demethylation and in formation of stable 5-hmC residues.

  14. Early de novo DNA methylation and prolonged demethylation in the muscle lineage

    PubMed Central

    Tsumagari, Koji; Baribault, Carl; Terragni, Jolyon; Varley, Katherine E.; Gertz, Jason; Pradhan, Sirharsa; Badoo, Melody; Crain, Charlene M.; Song, Lingyun; Crawford, Gregory E.; Myers, Richard M.; Lacey, Michelle; Ehrlich, Melanie

    2013-01-01

    Myogenic cell cultures derived from muscle biopsies are excellent models for human cell differentiation. We report the first comprehensive analysis of myogenesis-specific DNA hyper- and hypo-methylation throughout the genome for human muscle progenitor cells (both myoblasts and myotubes) and skeletal muscle tissue vs. 30 non-muscle samples using reduced representation bisulfite sequencing. We also focused on four genes with extensive hyper- or hypo-methylation in the muscle lineage (PAX3, TBX1, MYH7B/MIR499 and OBSCN) to compare DNA methylation, DNaseI hypersensitivity, histone modification, and CTCF binding profiles. We found that myogenic hypermethylation was strongly associated with homeobox or T-box genes and muscle hypomethylation with contractile fiber genes. Nonetheless, there was no simple relationship between differential gene expression and myogenic differential methylation, rather only for subsets of these genes, such as contractile fiber genes. Skeletal muscle retained ~30% of the hypomethylated sites but only ~3% of hypermethylated sites seen in myogenic progenitor cells. By enzymatic assays, skeletal muscle was 2-fold enriched globally in genomic 5-hydroxymethylcytosine (5-hmC) vs. myoblasts or myotubes and was the only sample type enriched in 5-hmC at tested myogenic hypermethylated sites in PAX3/CCDC140 andTBX1. TET1 and TET2 RNAs, which are involved in generation of 5-hmC and DNA demethylation, were strongly upregulated in myoblasts and myotubes. Our findings implicate de novo methylation predominantly before the myoblast stage and demethylation before and after the myotube stage in control of transcription and co-transcriptional RNA processing. They also suggest that, in muscle, TET1 or TET2 are involved in active demethylation and in formation of stable 5-hmC residues. PMID:23417056

  15. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kishigami, Satoshi; Mizutani, Eiji; Ohta, Hiroshi

    The low success rate of animal cloning by somatic cell nuclear transfer (SCNT) is believed to be associated with epigenetic errors including abnormal DNA hypermethylation. Recently, we elucidated by using round spermatids that, after nuclear transfer, treatment of zygotes with trichostatin A (TSA), an inhibitor of histone deacetylase, can remarkably reduce abnormal DNA hypermethylation depending on the origins of transferred nuclei and their genomic regions [S. Kishigami, N. Van Thuan, T. Hikichi, H. Ohta, S. Wakayama. E. Mizutani, T. Wakayama, Epigenetic abnormalities of the mouse paternal zygotic genome associated with microinsemination of round spermatids, Dev. Biol. (2005) in press]. Here,more » we found that 5-50 nM TSA-treatment for 10 h following oocyte activation resulted in more efficient in vitro development of somatic cloned embryos to the blastocyst stage from 2- to 5-fold depending on the donor cells including tail tip cells, spleen cells, neural stem cells, and cumulus cells. This TSA-treatment also led to more than 5-fold increase in success rate of mouse cloning from cumulus cells without obvious abnormality but failed to improve ES cloning success. Further, we succeeded in establishment of nuclear transfer-embryonic stem (NT-ES) cells from TSA-treated cloned blastocyst at a rate three times higher than those from untreated cloned blastocysts. Thus, our data indicate that TSA-treatment after SCNT in mice can dramatically improve the practical application of current cloning techniques.« less

  16. Significant improvement of mouse cloning technique by treatment with trichostatin A after somatic nuclear transfer.

    PubMed

    Kishigami, Satoshi; Mizutani, Eiji; Ohta, Hiroshi; Hikichi, Takafusa; Thuan, Nguyen Van; Wakayama, Sayaka; Bui, Hong-Thuy; Wakayama, Teruhiko

    2006-02-03

    The low success rate of animal cloning by somatic cell nuclear transfer (SCNT) is believed to be associated with epigenetic errors including abnormal DNA hypermethylation. Recently, we elucidated by using round spermatids that, after nuclear transfer, treatment of zygotes with trichostatin A (TSA), an inhibitor of histone deacetylase, can remarkably reduce abnormal DNA hypermethylation depending on the origins of transferred nuclei and their genomic regions [S. Kishigami, N. Van Thuan, T. Hikichi, H. Ohta, S. Wakayama. E. Mizutani, T. Wakayama, Epigenetic abnormalities of the mouse paternal zygotic genome associated with microinsemination of round spermatids, Dev. Biol. (2005) in press]. Here, we found that 5-50 nM TSA-treatment for 10 h following oocyte activation resulted in more efficient in vitro development of somatic cloned embryos to the blastocyst stage from 2- to 5-fold depending on the donor cells including tail tip cells, spleen cells, neural stem cells, and cumulus cells. This TSA-treatment also led to more than 5-fold increase in success rate of mouse cloning from cumulus cells without obvious abnormality but failed to improve ES cloning success. Further, we succeeded in establishment of nuclear transfer-embryonic stem (NT-ES) cells from TSA-treated cloned blastocyst at a rate three times higher than those from untreated cloned blastocysts. Thus, our data indicate that TSA-treatment after SCNT in mice can dramatically improve the practical application of current cloning techniques.

  17. Abnormal XPD-induced nuclear receptor transactivation in DNA repair disorders: trichothiodystrophy and xeroderma pigmentosum.

    PubMed

    Zhou, Xiaolong; Khan, Sikandar G; Tamura, Deborah; Ueda, Takahiro; Boyle, Jennifer; Compe, Emmanuel; Egly, Jean-Marc; DiGiovanna, John J; Kraemer, Kenneth H

    2013-08-01

    XPD (ERCC2) is a DNA helicase involved in nucleotide excision repair and in transcription as a structural bridge tying the transcription factor IIH (TFIIH) core with the cdk-activating kinase complex, which phosphorylates nuclear receptors. Mutations in XPD are associated with several different phenotypes, including trichothiodystrophy (TTD), with sulfur-deficient brittle hair, bone defects, and developmental abnormalities without skin cancer, xeroderma pigmentosum (XP), with pigmentary abnormalities and increased skin cancer, or XP/TTD with combined features, including skin cancer. We describe the varied clinical features and mutations in nine patients examined at the National Institutes of Health who were compound heterozygotes for XPD mutations but had different clinical phenotypes: four TTD, three XP, and two combined XP/TTD. We studied TFIIH-dependent transactivation by nuclear receptor for vitamin D (VDR) and thyroid in cells from these patients. The vitamin D stimulation ratio of CYP24 and osteopontin was associated with specific pairs of mutations (reduced in 5, elevated in 1) but not correlated with distinct clinical phenotypes. Thyroid receptor stimulation ratio for KLF9 was not significantly different from normal. XPD mutations frequently were associated with abnormal VDR stimulation in compound heterozygote patients with TTD, XP, or XP/TTD.

  18. Abnormal XPD-induced nuclear receptor transactivation in DNA repair disorders: trichothiodystrophy and xeroderma pigmentosum

    PubMed Central

    Zhou, Xiaolong; Khan, Sikandar G; Tamura, Deborah; Ueda, Takahiro; Boyle, Jennifer; Compe, Emmanuel; Egly, Jean-Marc; DiGiovanna, John J; Kraemer, Kenneth H

    2013-01-01

    XPD (ERCC2) is a DNA helicase involved in nucleotide excision repair and in transcription as a structural bridge tying the transcription factor IIH (TFIIH) core with the cdk-activating kinase complex, which phosphorylates nuclear receptors. Mutations in XPD are associated with several different phenotypes, including trichothiodystrophy (TTD), with sulfur-deficient brittle hair, bone defects, and developmental abnormalities without skin cancer, xeroderma pigmentosum (XP), with pigmentary abnormalities and increased skin cancer, or XP/TTD with combined features, including skin cancer. We describe the varied clinical features and mutations in nine patients examined at the National Institutes of Health who were compound heterozygotes for XPD mutations but had different clinical phenotypes: four TTD, three XP, and two combined XP/TTD. We studied TFIIH-dependent transactivation by nuclear receptor for vitamin D (VDR) and thyroid in cells from these patients. The vitamin D stimulation ratio of CYP24 and osteopontin was associated with specific pairs of mutations (reduced in 5, elevated in 1) but not correlated with distinct clinical phenotypes. Thyroid receptor stimulation ratio for KLF9 was not significantly different from normal. XPD mutations frequently were associated with abnormal VDR stimulation in compound heterozygote patients with TTD, XP, or XP/TTD. PMID:23232694

  19. Down-regulation of TCF21 by hypermethylation induces cell proliferation, migration and invasion in colorectal cancer

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Dai, Youyi; Duan, Huaxin; The First Affiliated Hospital of Hunan Normal University

    Epigenetic alteration induced loss function of the transcription factor 21 (TCF21) has been associated with different types of human cancers. However, the epigenetic regulation and molecular functions of TCF21 in colorectal cancer (CRC) remain unknown. In this study, TCF21 expression levels and methylation status of its promoter region in CRC cell lines (n = 5) and CRC tissues (n = 151) as well as normal colorectal mucosa (n = 30) were assessed by RTq-PCR and methylation analysis (methylation specific PCR, MSP and bisulfite sequencing PCR, BSP), respectively. The cellular functions of TCF21 on CRC cell proliferation, apoptosis, invasion and migration were investigated in vitro. Our data revealedmore » that TCF21 was frequently silenced by promoter hypermethylation in both tested CRC cell lines and primary CRC, and correlation analysis between methylation status and clinicopathologic parameters found that TCF21 methylation was significantly correlated with lymph node invasion (P = 0.013), while no significant correlation was found in other parameters. In addition, demethylation treatment resulted in re-expression of TCF21 in CRC cell lines, and cellular function experiments revealed that restoration of TCF21 inhibited CRC cell proliferation, promoted apoptosis and suppressed cell invasion and migration, suggesting that TCF21 may function as a tumor suppressor gene, which is downregulated through promoter hypermethylation in CRC development. - Highlights: • TCF21 was frequently silenced by promoter DNA methylation in CRC cells. • TCF21 was frequently methylated in primary CRC and significantly correlated with metastasis. • Restoration of TCF21 promotes cell apoptosis of CRC cells. • Restoration of TCF21 inhibits cell invasion and migration of CRC cells.« less

  20. Recurrent patterns of DNA methylation in the ZNF154, CASP8, and VHL promoters across a wide spectrum of human solid epithelial tumors and cancer cell lines

    PubMed Central

    Sánchez-Vega, Francisco; Gotea, Valer; Petrykowska, Hanna M; Margolin, Gennady; Krivak, Thomas C; DeLoia, Julie A; Bell, Daphne W; Elnitski, Laura

    2013-01-01

    The study of aberrant DNA methylation in cancer holds the key to the discovery of novel biological markers for diagnostics and can help to delineate important mechanisms of disease. We have identified 12 loci that are differentially methylated in serous ovarian cancers and endometrioid ovarian and endometrial cancers with respect to normal control samples. The strongest signal showed hypermethylation in tumors at a CpG island within the ZNF154 promoter. We show that hypermethylation of this locus is recurrent across solid human epithelial tumor samples for 15 of 16 distinct cancer types from TCGA. Furthermore, ZNF154 hypermethylation is strikingly present across a diverse panel of ENCODE cell lines, but only in those derived from tumor cells. By extending our analysis from the Illumina 27K Infinium platform to the 450K platform, to sequencing of PCR amplicons from bisulfite treated DNA, we demonstrate that hypermethylation extends across the breadth of the ZNF154 CpG island. We have also identified recurrent hypomethylation in two genomic regions associated with CASP8 and VHL. These three genes exhibit significant negative correlation between methylation and gene expression across many cancer types, as well as patterns of DNaseI hypersensitivity and histone marks that reflect different chromatin accessibility in cancer vs. normal cell lines. Our findings emphasize hypermethylation of ZNF154 as a biological marker of relevance for tumor identification. Epigenetic modifications affecting the promoters of ZNF154, CASP8, and VHL are shared across a vast array of tumor types and may therefore be important for understanding the genomic landscape of cancer. PMID:24149212

  1. Recurrent patterns of DNA methylation in the ZNF154, CASP8, and VHL promoters across a wide spectrum of human solid epithelial tumors and cancer cell lines.

    PubMed

    Sánchez-Vega, Francisco; Gotea, Valer; Petrykowska, Hanna M; Margolin, Gennady; Krivak, Thomas C; DeLoia, Julie A; Bell, Daphne W; Elnitski, Laura

    2013-12-01

    The study of aberrant DNA methylation in cancer holds the key to the discovery of novel biological markers for diagnostics and can help to delineate important mechanisms of disease. We have identified 12 loci that are differentially methylated in serous ovarian cancers and endometrioid ovarian and endometrial cancers with respect to normal control samples. The strongest signal showed hypermethylation in tumors at a CpG island within the ZNF154 promoter. We show that hypermethylation of this locus is recurrent across solid human epithelial tumor samples for 15 of 16 distinct cancer types from TCGA. Furthermore, ZNF154 hypermethylation is strikingly present across a diverse panel of ENCODE cell lines, but only in those derived from tumor cells. By extending our analysis from the Illumina 27K Infinium platform to the 450K platform, to sequencing of PCR amplicons from bisulfite treated DNA, we demonstrate that hypermethylation extends across the breadth of the ZNF154 CpG island. We have also identified recurrent hypomethylation in two genomic regions associated with CASP8 and VHL. These three genes exhibit significant negative correlation between methylation and gene expression across many cancer types, as well as patterns of DNaseI hypersensitivity and histone marks that reflect different chromatin accessibility in cancer vs. normal cell lines. Our findings emphasize hypermethylation of ZNF154 as a biological marker of relevance for tumor identification. Epigenetic modifications affecting the promoters of ZNF154, CASP8, and VHL are shared across a vast array of tumor types and may therefore be important for understanding the genomic landscape of cancer.

  2. Comprehensive DNA methylation analysis of human neuroblastoma cells treated with blonanserin.

    PubMed

    Murata, Yui; Nishioka, Masaki; Bundo, Miki; Sunaga, Fumiko; Kasai, Kiyoto; Iwamoto, Kazuya

    2014-03-20

    Blonanserin is a second-generation antipsychotic drug for schizophrenia. The pharmacological actions of blonanserin are shown to be the antagonism of dopamine receptor 2 and serotonin receptors. However, its molecular mechanisms in brain cells have not been fully characterized. Accumulating evidence suggests that antipsychotic drugs and mood stabilizers show epigenetic effects on a wide range of genes in animal and cellular models. We performed genome-wide DNA methylation analysis targeting 479,814 CpG sites of cultured human neuroblastoma cells administered with blonanserin. We found that 3,057 CpG sites showed statistically significant changes in DNA methylation at two different doses of blonanserin (1.36 nM and 13.6 nM). These included hypermethylated CpG sites that were enriched in genes related to axonogenesis and cell morphogenesis involved in neuron differentiation. We also showed that the global effect on DNA methylome depends on the concentration of the drug. With a high dose of blonanserin, the overall methylation levels across all CpG sites significantly increased. These increases in DNA methylation were prominent in the CpG sites distant from promoter regions. We further examined DNA methylation changes in specific genes implicated for the actions of antipsychotic drugs, such as the dopamine receptor 2 (DRD2) gene and the serotonin receptor 2A (HTR2A) gene. We observed that CpG sites that were located within DRD2 and HTR2A genes were significantly hypermethylated by blonanserin. The DNA methylation changes induced by the treatment with blonanserin will be useful for understanding its pharmacological actions at the cellular level. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  3. Histone modification alteration coordinated with acquisition of promoter DNA methylation during Epstein-Barr virus infection

    PubMed Central

    Funata, Sayaka; Matsusaka, Keisuke; Yamanaka, Ryota; Yamamoto, Shogo; Okabe, Atsushi; Fukuyo, Masaki; Aburatani, Hiroyuki; Fukayama, Masashi; Kaneda, Atsushi

    2017-01-01

    Aberrant DNA hypermethylation is a major epigenetic mechanism to inactivate tumor suppressor genes in cancer. Epstein-Barr virus positive gastric cancer is the most frequently hypermethylated tumor among human malignancies. Herein, we performed comprehensive analysis of epigenomic alteration during EBV infection, by Infinium HumanMethylation 450K BeadChip for DNA methylation and ChIP-sequencing for histone modification alteration during EBV infection into gastric cancer cell line MKN7. Among 7,775 genes with increased DNA methylation in promoter regions, roughly half were “DNA methylation-sensitive” genes, which acquired DNA methylation in the whole promoter regions and thus were repressed. These included anti-oncogenic genes, e.g. CDKN2A. The other half were “DNA methylation-resistant” genes, where DNA methylation is acquired in the surrounding of promoter regions, but unmethylated status is protected in the vicinity of transcription start site. These genes thereby retained gene expression, and included DNA repair genes. Histone modification was altered dynamically and coordinately with DNA methylation alteration. DNA methylation-sensitive genes significantly correlated with loss of H3K27me3 pre-marks or decrease of active histone marks, H3K4me3 and H3K27ac. Apoptosis-related genes were significantly enriched in these epigenetically repressed genes. Gain of active histone marks significantly correlated with DNA methylation-resistant genes. Genes related to mitotic cell cycle and DNA repair were significantly enriched in these epigenetically activated genes. Our data show that orchestrated epigenetic alterations are important in gene regulation during EBV infection, and histone modification status in promoter regions significantly associated with acquisition of de novo DNA methylation or protection of unmethylated status at transcription start site. PMID:28903418

  4. Genome-wide DNA methylation analysis identifies MEGF10 as a novel epigenetically repressed candidate tumor suppressor gene in neuroblastoma.

    PubMed

    Charlet, Jessica; Tomari, Ayumi; Dallosso, Anthony R; Szemes, Marianna; Kaselova, Martina; Curry, Thomas J; Almutairi, Bader; Etchevers, Heather C; McConville, Carmel; Malik, Karim T A; Brown, Keith W

    2017-04-01

    Neuroblastoma is a childhood cancer in which many children still have poor outcomes, emphasising the need to better understand its pathogenesis. Despite recent genome-wide mutation analyses, many primary neuroblastomas do not contain recognizable driver mutations, implicating alternate molecular pathologies such as epigenetic alterations. To discover genes that become epigenetically deregulated during neuroblastoma tumorigenesis, we took the novel approach of comparing neuroblastomas to neural crest precursor cells, using genome-wide DNA methylation analysis. We identified 93 genes that were significantly differentially methylated of which 26 (28%) were hypermethylated and 67 (72%) were hypomethylated. Concentrating on hypermethylated genes to identify candidate tumor suppressor loci, we found the cell engulfment and adhesion factor gene MEGF10 to be epigenetically repressed by DNA hypermethylation or by H3K27/K9 methylation in neuroblastoma cell lines. MEGF10 showed significantly down-regulated expression in neuroblastoma tumor samples; furthermore patients with the lowest-expressing tumors had reduced relapse-free survival. Our functional studies showed that knock-down of MEGF10 expression in neuroblastoma cell lines promoted cell growth, consistent with MEGF10 acting as a clinically relevant, epigenetically deregulated neuroblastoma tumor suppressor gene. © 2016 The Authors. Molecular Carcinogenesis Published by Wiley Periodicals, Inc. © 2016 The Authors. Molecular Carcinogenesis Published by Wiley Periodicals, Inc.

  5. Genome‐wide DNA methylation analysis identifies MEGF10 as a novel epigenetically repressed candidate tumor suppressor gene in neuroblastoma

    PubMed Central

    Charlet, Jessica; Tomari, Ayumi; Dallosso, Anthony R.; Szemes, Marianna; Kaselova, Martina; Curry, Thomas J.; Almutairi, Bader; Etchevers, Heather C.; McConville, Carmel; Malik, Karim T. A.

    2016-01-01

    Neuroblastoma is a childhood cancer in which many children still have poor outcomes, emphasising the need to better understand its pathogenesis. Despite recent genome‐wide mutation analyses, many primary neuroblastomas do not contain recognizable driver mutations, implicating alternate molecular pathologies such as epigenetic alterations. To discover genes that become epigenetically deregulated during neuroblastoma tumorigenesis, we took the novel approach of comparing neuroblastomas to neural crest precursor cells, using genome‐wide DNA methylation analysis. We identified 93 genes that were significantly differentially methylated of which 26 (28%) were hypermethylated and 67 (72%) were hypomethylated. Concentrating on hypermethylated genes to identify candidate tumor suppressor loci, we found the cell engulfment and adhesion factor gene MEGF10 to be epigenetically repressed by DNA hypermethylation or by H3K27/K9 methylation in neuroblastoma cell lines. MEGF10 showed significantly down‐regulated expression in neuroblastoma tumor samples; furthermore patients with the lowest‐expressing tumors had reduced relapse‐free survival. Our functional studies showed that knock‐down of MEGF10 expression in neuroblastoma cell lines promoted cell growth, consistent with MEGF10 acting as a clinically relevant, epigenetically deregulated neuroblastoma tumor suppressor gene. © 2016 The Authors. Molecular Carcinogenesis Published by Wiley Periodicals, Inc. PMID:27862318

  6. Ecstasy (MDMA) Alters Cardiac Gene Expression and DNA Methylation: Implications for Circadian Rhythm Dysfunction in the Heart.

    PubMed

    Koczor, Christopher A; Ludlow, Ivan; Hight, Robert S; Jiao, Zhe; Fields, Earl; Ludaway, Tomika; Russ, Rodney; Torres, Rebecca A; Lewis, William

    2015-11-01

    MDMA (ecstasy) is an illicit drug that stimulates monoamine neurotransmitter release and inhibits reuptake. MDMA's acute cardiotoxicity includes tachycardia and arrhythmia which are associated with cardiomyopathy. MDMA acute cardiotoxicity has been explored, but neither long-term MDMA cardiac pathological changes nor epigenetic changes have been evaluated. Microarray analyses were employed to identify cardiac gene expression changes and epigenetic DNA methylation changes. To identify permanent MDMA-induced pathogenetic changes, mice received daily 10- or 35-day MDMA, or daily 10-day MDMA followed by 25-day saline washout (10 + 25 days). MDMA treatment caused differential gene expression (p < .05, fold change >1.5) in 752 genes following 10 days, 558 genes following 35 days, and 113 genes following 10-day MDMA + 25-day saline washout. Changes in MAPK and circadian rhythm gene expression were identified as early as 10 days. After 35 days, circadian rhythm genes (Per3, CLOCK, ARNTL, and NPAS2) persisted to be differentially expressed. MDMA caused DNA hypermethylation and hypomethylation that was independent of gene expression; hypermethylation of genes was found to be 71% at 10 days, 68% at 35 days, and 91% at 10 + 25 days washout. Differential gene expression paralleled DNA methylation in 22% of genes at 10-day treatment, 17% at 35 days, and 48% at 10 + 25 days washout. We show here that MDMA induced cardiac epigenetic changes in DNA methylation where hypermethylation predominated. Moreover, MDMA induced gene expression of key elements of circadian rhythm regulatory genes. This suggests a fundamental organism-level event to explain some of the etiologies of MDMA dysfunction in the heart. © The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  7. Evaluation of p16 hypermethylation in oral submucous fibrosis: A quantitative and comparative analysis in buccal cells and saliva using real-time methylation-specific polymerase chain reaction.

    PubMed

    Kaliyaperumal, Subadra; Sankarapandian, Sathasivasubramanian

    2016-01-01

    The aim of this study was to quantitatively investigate the hypermethylation of p16 gene in buccal cells and saliva of oral submucous fibrosis (OSMF) patients using real-time quantitative methylation-specific polymerase chain reaction (PCR) and to compare the values of two methods. A total of 120 samples were taken from 60 subjects selected for this study, of which 30 were controls and 30 patients were clinically and histopathologically diagnosed with OSMF. In both groups, two sets of samples were collected, one directly from the buccal cells through cytobrush technique and the other through salivary rinse. We analyzed the samples for the presence of p16 hypermethylation using quantitative real-time PCR. In OSMF, the hypermethylation status of p16 in buccal cells was very high (93.3%) and in salivary samples, it was partially methylated (50%). However, no hypermethylation was found in controls suggesting that significant quantity of p16 hypermethylation was present in buccal cells and saliva in OSMF. This study indicates that buccal cell sampling may be a better method for evaluation than the salivary samples. It signifies that hypermethylation of p16 is an important factor to be considered in epigenetic alterations of normal cells to oral precancer, i.e. OSMF.

  8. Loss of maintenance DNA methylation results in abnormal DNA origin firing during DNA replication.

    PubMed

    Haruta, Mayumi; Shimada, Midori; Nishiyama, Atsuya; Johmura, Yoshikazu; Le Tallec, Benoît; Debatisse, Michelle; Nakanishi, Makoto

    2016-01-22

    The mammalian maintenance methyltransferase DNMT1 [DNA (cytosine-5-)-methyltransferase 1] mediates the inheritance of the DNA methylation pattern during replication. Previous studies have shown that depletion of DNMT1 causes a severe growth defect and apoptosis in differentiated cells. However, the detailed mechanisms behind this phenomenon remain poorly understood. Here we show that conditional ablation of Dnmt1 in murine embryonic fibroblasts (MEFs) resulted in an aberrant DNA replication program showing an accumulation of late-S phase replication and causing severely defective growth. Furthermore, we found that the catalytic activity and replication focus targeting sequence of DNMT1 are required for a proper DNA replication program. Taken together, our findings suggest that the maintenance of DNA methylation by DNMT1 plays a critical role in proper regulation of DNA replication in mammalian cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. EZH2 mutations and promoter hypermethylation in childhood acute lymphoblastic leukemia.

    PubMed

    Schäfer, Vivien; Ernst, Jana; Rinke, Jenny; Winkelmann, Nils; Beck, James F; Hochhaus, Andreas; Gruhn, Bernd; Ernst, Thomas

    2016-07-01

    Acute lymphoblastic leukemia (ALL) is the most common malignancy in children and young adults. The polycomb repressive complex 2 (PRC2) has been identified as one of the most frequently mutated epigenetic protein complexes in hematologic cancers. PRC2 acts as an epigenetic repressor through histone H3 lysine 27 trimethylation (H3K27me3), catalyzed by the histone methyltransferase enhancer of zeste homolog 2 protein (EZH2). To study the prevalence and clinical impact of PRC2 aberrations in an unselected childhood ALL cohort (n = 152), we performed PRC2 mutational screenings by Sanger sequencing and promoter methylation analyses by quantitative pyrosequencing for the three PRC2 core component genes EZH2, suppressor of zeste 12 (SUZ12), and embryonic ectoderm development (EED). Targeted deep next-generation sequencing of 30 frequently mutated genes in leukemia was performed to search for cooperating mutations in patients harboring PRC2 aberrations. Finally, the functional consequence of EZH2 promoter hypermethylation on H3K27me3 was studied by Western blot analyses of primary cells. Loss-of-function EZH2 mutations were detected in 2/152 (1.3 %) patients with common-ALL and early T-cell precursor (ETP)-ALL, respectively. In one patient, targeted deep sequencing identified cooperating mutations in ASXL1 and TET2. EZH2 promoter hypermethylation was found in one patient with ETP-ALL which led to reduced H3K27me3. In comparison with healthy children, the EZH2 promoter was significantly higher methylated in T-ALL patients. No mutations or promoter methylation changes were identified for SUZ12 or EED genes, respectively. Although PRC2 aberrations seem to be rare in childhood ALL, our findings indicate that EZH2 aberrations might contribute to the disease in specific cases. Hereby, EZH2 promoter hypermethylation might have functionally similar consequences as loss-of-function mutations.

  10. Altered DNA Methylation and Expression Profiles of 8-Oxoguanine DNA Glycosylase 1 in Lens Tissue from Age-related Cataract Patients.

    PubMed

    Wang, Yong; Li, Fei; Zhang, Guowei; Kang, Lihua; Qin, Bai; Guan, Huaijin

    2015-01-01

    Oxidative stress and DNA damage contribute to the pathogenesis of age-related cataract (ARC). Most oxidative DNA lesions are repaired via the base excision repair (BER) proteins including 8-oxoguanine DNA glycosylase 1 (OGG1). This study examined DNA methylation of CpG islands upstream of OGG1 and their relation to the gene expression in lens cortex from ARC patients. The clinical case-control study consisted of 15 cortical type of ARC patients and 15 age-matched non-ARC controls who received transparent lens extraction due to vitreoretinal diseases. OGG1 expression in lens cortex was analyzed by qRT-PCR and Western blot. The localization and the proportion of cells positive for OGG1 were determined by immunofluorescence. Bisulfite-sequencing PCR (BSP) was performed to evaluate the methylation status of CpG islands near OGG1 in DNA extracted from lens cortex. To test relationship between the methylation and the expression of the gene of interest, 5-Aza-2'-deoxycytidine (5-Aza-dC) was used to induce demethylation of cultured human lens epithelium B-3 (HLE B-3). To test the role of OGG1 in the repair of cellular damage, HLE B-3 was transfected with OGG1 vector, followed by ultraviolet radiation b (UVB) exposure to induce apoptosis. The mRNA and protein levels of OGG1 were significantly reduced in the lens cortex of ARC. Immunofluorescence showed that the proportion of OGG1-positive cells decreased significantly in ARC cortex in comparison with the control. The CpG island in first exon of OGG1 displayed hypermethylation in the DNA extracted from the lens cortex of ARC. Treatment of HLEB-3 cells with 5-Aza-dC upregulated OGG1 expression. UVB-induced apoptosis was attenuated after transfection with OGG1. A reduced OGG1 expression was correlated with hypermethylation of a CpG island of OGG1 in lens cortex of ARC. The role of epigenetic change in OGG1 gene in the susceptibility to oxidative stress induced cortical ARC is warranted to further study.

  11. ESR1 Promoter Hypermethylation Does Not Predict Atypia in RPFNA nor Persistent Atypia after 12 Months Tamoxifen Chemoprevention

    PubMed Central

    Baker, Joseph C.; Ostrander, Julie H.; Lem, Siya; Broadwater, Gloria; Bean, Gregory R.; D'Amato, Nicholas C.; Goldenberg, Vanessa K.; Rowell, Craig; Ibarra-Drendall, Catherine; Grant, Tracey; Pilie, Patrick G.; Vasilatos, Shauna N.; Troch, Michelle M.; Scott, Victoria; Wilke, Lee G.; Paisie, Carolyn; Rabiner, Sarah M.; Torres-Hernandez, Alejandro; Zalles, Carola M.; Seewaldt, Victoria L.

    2009-01-01

    Purpose Currently, we lack biomarkers to predict whether high-risk women with mammary atypia will respond to tamoxifen chemoprevention. Experimental Design Thirty-four women with cytologic mammary atypia from the Duke University High-Risk clinic were offered tamoxifen chemoprevention. We tested whether ESR1 promoter hypermethylation and/or estrogen receptor (ER) protein expression by immunohistochemistry predicted persistent atypia in 18 women who were treated with tamoxifen for 12 months and in 16 untreated controls. Results We observed a statistically significant decrease in the Masood score of women on tamoxifen chemoprevention for 12 months compared with control women. This was a significant interaction effect of time (0, 6, and 12 months) and treatment group (tamoxifen versus control) P = 0.0007. However, neither ESR1 promoter hypermethylation nor low ER expression predicted persistent atypia in Random Periareolar Fine Needle Aspiration after 12 months tamoxifen prevention. Conclusions Results from this single institution pilot study provide evidence that, unlike for invasive breast cancer, ESR1 promoter hypermethylation and/or low ER expression is not a reliable marker of tamoxifen-resistant atypia. PMID:18708376

  12. DNA methylation of phosphatase and actin regulator 3 detects colorectal cancer in stool and complements FIT.

    PubMed

    Bosch, Linda J W; Oort, Frank A; Neerincx, Maarten; Khalid-de Bakker, Carolina A J; Terhaar sive Droste, Jochim S; Melotte, Veerle; Jonkers, Daisy M A E; Masclee, Ad A M; Mongera, Sandra; Grooteclaes, Madeleine; Louwagie, Joost; van Criekinge, Wim; Coupé, Veerle M H; Mulder, Chris J; van Engeland, Manon; Carvalho, Beatriz; Meijer, Gerrit A

    2012-03-01

    Using a bioinformatics-based strategy, we set out to identify hypermethylated genes that could serve as biomarkers for early detection of colorectal cancer (CRC) in stool. In addition, the complementary value to a Fecal Immunochemical Test (FIT) was evaluated. Candidate genes were selected by applying cluster alignment and computational analysis of promoter regions to microarray-expression data of colorectal adenomas and carcinomas. DNA methylation was measured by quantitative methylation-specific PCR on 34 normal colon mucosa, 71 advanced adenoma, and 64 CRC tissues. The performance as biomarker was tested in whole stool samples from in total 193 subjects, including 19 with advanced adenoma and 66 with CRC. For a large proportion of these series, methylation data for GATA4 and OSMR were available for comparison. The complementary value to FIT was measured in stool subsamples from 92 subjects including 44 with advanced adenoma or CRC. Phosphatase and Actin Regulator 3 (PHACTR3) was identified as a novel hypermethylated gene showing more than 70-fold increased DNA methylation levels in advanced neoplasia compared with normal colon mucosa. In a stool training set, PHACTR3 methylation showed a sensitivity of 55% (95% CI: 33-75) for CRC and a specificity of 95% (95% CI: 87-98). In a stool validation set, sensitivity reached 66% (95% CI: 50-79) for CRC and 32% (95% CI: 14-57) for advanced adenomas at a specificity of 100% (95% CI: 86-100). Adding PHACTR3 methylation to FIT increased sensitivity for CRC up to 15%. PHACTR3 is a new hypermethylated gene in CRC with a good performance in stool DNA testing and has complementary value to FIT.

  13. Multi-step aberrant CpG island hyper-methylation is associated with the progression of adult T-cell leukemia/lymphoma.

    PubMed

    Sato, Hiaki; Oka, Takashi; Shinnou, Yoko; Kondo, Takami; Washio, Kana; Takano, Masayuki; Takata, Katsuyoshi; Morito, Toshiaki; Huang, Xingang; Tamura, Maiko; Kitamura, Yuta; Ohara, Nobuya; Ouchida, Mamoru; Ohshima, Koichi; Shimizu, Kenji; Tanimoto, Mitsune; Takahashi, Kiyoshi; Matsuoka, Masao; Utsunomiya, Atae; Yoshino, Tadashi

    2010-01-01

    Aberrant CpG island methylation contributes to the pathogenesis of various malignancies. However, little is known about the association of epigenetic abnormalities with multistep tumorigenic events in adult T cell leukemia/lymphoma (ATLL). To determine whether epigenetic abnormalities induce the progression of ATLL, we analyzed the methylation profiles of the SHP1, p15, p16, p73, HCAD, DAPK, hMLH-1, and MGMT genes by methylation specific PCR assay in 65 cases with ATLL patients. The number of CpG island methylated genes increased with disease progression and aberrant hypermethylation in specific genes was detected even in HTLV-1 carriers and correlated with progression to ATLL. The CpG island methylator phenotype (CIMP) was observed most frequently in lymphoma type ATLL and was also closely associated with the progression and crisis of ATLL. The high number of methylated genes and increase of CIMP incidence were shown to be unfavorable prognostic factors and correlated with a shorter overall survival by Kaplan-Meyer analysis. The present findings strongly suggest that the multistep accumulation of aberrant CpG methylation in specific target genes and the presence of CIMP are deeply involved in the crisis, progression, and prognosis of ATLL, as well as indicate the value of CpG methylation and CIMP for new diagnostic and prognostic biomarkers.

  14. Multi-Step Aberrant CpG Island Hyper-Methylation Is Associated with the Progression of Adult T–Cell Leukemia/Lymphoma

    PubMed Central

    Sato, Hiaki; Oka, Takashi; Shinnou, Yoko; Kondo, Takami; Washio, Kana; Takano, Masayuki; Takata, Katsuyoshi; Morito, Toshiaki; Huang, Xingang; Tamura, Maiko; Kitamura, Yuta; Ohara, Nobuya; Ouchida, Mamoru; Ohshima, Koichi; Shimizu, Kenji; Tanimoto, Mitsune; Takahashi, Kiyoshi; Matsuoka, Masao; Utsunomiya, Atae; Yoshino, Tadashi

    2010-01-01

    Aberrant CpG island methylation contributes to the pathogenesis of various malignancies. However, little is known about the association of epigenetic abnormalities with multistep tumorigenic events in adult T cell leukemia/lymphoma (ATLL). To determine whether epigenetic abnormalities induce the progression of ATLL, we analyzed the methylation profiles of the SHP1, p15, p16, p73, HCAD, DAPK, hMLH-1, and MGMT genes by methylation specific PCR assay in 65 cases with ATLL patients. The number of CpG island methylated genes increased with disease progression and aberrant hypermethylation in specific genes was detected even in HTLV-1 carriers and correlated with progression to ATLL. The CpG island methylator phenotype (CIMP) was observed most frequently in lymphoma type ATLL and was also closely associated with the progression and crisis of ATLL. The high number of methylated genes and increase of CIMP incidence were shown to be unfavorable prognostic factors and correlated with a shorter overall survival by Kaplan-Meyer analysis. The present findings strongly suggest that the multistep accumulation of aberrant CpG methylation in specific target genes and the presence of CIMP are deeply involved in the crisis, progression, and prognosis of ATLL, as well as indicate the value of CpG methylation and CIMP for new diagnostic and prognostic biomarkers. PMID:20019193

  15. Genital shedding of human immunodeficiency virus type 1 DNA during pregnancy: association with immunosuppression, abnormal cervical or vaginal discharge, and severe vitamin A deficiency.

    PubMed

    John, G C; Nduati, R W; Mbori-Ngacha, D; Overbaugh, J; Welch, M; Richardson, B A; Ndinya-Achola, J; Bwayo, J; Krieger, J; Onyango, F; Kreiss, J K

    1997-01-01

    The presence of human immunodeficiency virus type 1 (HIV-1) in genital secretions may be a determinant of vertical HIV-1 transmission. Cervical and vaginal secretions from HIV-1-seropositive pregnant women were evaluated to determine prevalence and correlates of HIV-1-infected cells in the genital tract. HIV-1 DNA was detected by polymerase chain reaction in 32% of 212 cervical and 10% of 215 vaginal specimens. Presence of HIV-1 DNA in the cervix was associated with cervical mucopus and a significantly lower absolute CD4 cell count (354 vs. 469, P < .001). An absolute CD4 cell count <200 was associated with a 9.6-fold increased odds of cervical HIV-1 DNA detection compared with a count > or = 500 (95% confidence interval, 2.8-34.2). Detection of vaginal HIV- 1 DNA was associated with abnormal vaginal discharge, lower absolute CD4 cell count, and severe vitamin A deficiency. Presence of HIV-1-infected cells in genital secretions was associated with immunosuppression and abnormal cervical or vaginal discharge.

  16. Genital Shedding of Human Immunodeficiency Virus Type 1 DNA during Pregnancy: Association with Immunosuppression, Abnormal Cervical or Vaginal Discharge, and Severe Vitamin A Deficiency

    PubMed Central

    John, Grace C.; Nduati, Ruth W.; Mbori-Ngacha, Dorothy; Overbaugh, Julie; Welch, Mary; Richardson, Barbra A.; Ndinya-Achola, Jeckoniah; Bwayo, Job; Krieger, John; Onyango, Francis; Kreiss, Joan K.

    2012-01-01

    The presence of human immunodeficiency virus type 1 (HIV-1) in genital secretions may be a determinant of vertical HIV-1 transmission. Cervical and vaginal secretions from HIV-1–seropositive pregnant women were evaluated to determine prevalence and correlates of HIV-1–infected cells in the genital tract. HIV-1 DNA was detected by polymerase chain reaction in 32% of 212 cervical and 10% of 215 vaginal specimens. Presence of HIV-1 DNA in the cervix was associated with cervical mucopus and a significantly lower absolute CD4 cell count (354 vs. 469, P < .001). An absolute CD4 cell count <200 was associated with a 9.6-fold increased odds of cervical HIV-1 DNA detection compared with a count ≥500 (95% confidence interval, 2.8–34.2). Detection of vaginal HIV-1 DNA was associated with abnormal vaginal discharge, lower absolute CD4 cell count, and severe vitamin A deficiency. Presence of HIV-1–infected cells in genital secretions was associated with immunosuppression and abnormal cervical or vaginal discharge. PMID:8985196

  17. Protection of cisplatin-induced spermatotoxicity, DNA damage and chromatin abnormality by selenium nano-particles.

    PubMed

    Rezvanfar, Mohammad Amin; Rezvanfar, Mohammad Ali; Shahverdi, Ahmad Reza; Ahmadi, Abbas; Baeeri, Maryam; Mohammadirad, Azadeh; Abdollahi, Mohammad

    2013-02-01

    Cisplatin (CIS), an anticancer alkylating agent, induces DNA adducts and effectively cross links the DNA strands and so affects spermatozoa as a male reproductive toxicant. The present study investigated the cellular/biochemical mechanisms underlying possible protective effect of selenium nano-particles (Nano-Se) as an established strong antioxidant with more bioavailability and less toxicity, on reproductive toxicity of CIS by assessment of sperm characteristics, sperm DNA integrity, chromatin quality and spermatogenic disorders. To determine the role of oxidative stress (OS) in the pathogenesis of CIS gonadotoxicity, the level of lipid peroxidation (LPO), antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) and peroxynitrite (ONOO) as a marker of nitrosative stress (NS) and testosterone (T) concentration as a biomarker of testicular function were measured in the blood and testes. Thirty-two male Wistar rats were equally divided into four groups. A single IP dose of CIS (7 mg/kg) and protective dose of Nano-Se (2 mg/kg/day) were administered alone or in combination. The CIS-exposed rats showed a significant increase in testicular and serum LPO and ONOO level, along with a significant decrease in enzymatic antioxidants levels, diminished serum T concentration and abnormal histologic findings with impaired sperm quality associated with increased DNA damage and decreased chromatin quality. Coadministration of Nano-Se significantly improved the serum T, sperm quality, and spermatogenesis and reduced CIS-induced free radical toxic stress and spermatic DNA damage. In conclusion, the current study demonstrated that Nano-Se may be useful to prevent CIS-induced gonadotoxicity through its antioxidant potential. Copyright © 2012 Elsevier Inc. All rights reserved.

  18. Genome-wide DNA methylation profile identified a unique set of differentially methylated immune genes in oral squamous cell carcinoma patients in India.

    PubMed

    Basu, Baidehi; Chakraborty, Joyeeta; Chandra, Aditi; Katarkar, Atul; Baldevbhai, Jadav Ritesh Kumar; Dhar Chowdhury, Debjit; Ray, Jay Gopal; Chaudhuri, Keya; Chatterjee, Raghunath

    2017-01-01

    Oral squamous cell carcinoma (OSCC) is one of the common malignancies in Southeast Asia. Epigenetic changes, mainly the altered DNA methylation, have been implicated in many cancers. Considering the varied environmental and genotoxic exposures among the Indian population, we conducted a genome-wide DNA methylation study on paired tumor and adjacent normal tissues of ten well-differentiated OSCC patients and validated in an additional 53 well-differentiated OSCC and adjacent normal samples. Genome-wide DNA methylation analysis identified several novel differentially methylated regions associated with OSCC. Hypermethylation is primarily enriched in the CpG-rich regions, while hypomethylation is mainly in the open sea. Distinct epigenetic drifts for hypo- and hypermethylation across CpG islands suggested independent mechanisms of hypo- and hypermethylation in OSCC development. Aberrant DNA methylation in the promoter regions are concomitant with gene expression. Hypomethylation of immune genes reflect the lymphocyte infiltration into the tumor microenvironment. Comparison of methylome data with 312 TCGA HNSCC samples identified a unique set of hypomethylated promoters among the OSCC patients in India. Pathway analysis of unique hypomethylated promoters indicated that the OSCC patients in India induce an anti-tumor T cell response, with mobilization of T lymphocytes in the neoplastic environment. Survival analysis of these epigenetically regulated immune genes suggested their prominent role in OSCC progression. Our study identified a unique set of hypomethylated regions, enriched in the promoters of immune response genes, and indicated the presence of a strong immune component in the tumor microenvironment. These methylation changes may serve as potential molecular markers to define risk and to monitor the prognosis of OSCC patients in India.

  19. Arsenic exposure from drinking water is associated with decreased gene expression and increased DNA methylation in peripheral blood

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ameer, Syeda Shegufta

    Background: Exposure to inorganic arsenic increases the risk of cancer and non-malignant diseases. Inefficient arsenic metabolism is a marker for susceptibility to arsenic toxicity. Arsenic may alter gene expression, possibly by altering DNA methylation. Objectives: To elucidate the associations between arsenic exposure, gene expression, and DNA methylation in peripheral blood, and the modifying effects of arsenic metabolism. Methods: The study participants, women from the Andes, Argentina, were exposed to arsenic via drinking water. Arsenic exposure was assessed as the sum of arsenic metabolites in urine (U-As), using high performance liquid-chromatography hydride-generation inductively-coupled-plasma-mass-spectrometry, and arsenic metabolism efficiency was assessed by themore » urinary fractions (%) of the individual metabolites. Genome-wide gene expression (N = 80 women) and DNA methylation (N = 93; 80 overlapping with gene expression) in peripheral blood were measured using Illumina DirectHyb HumanHT-12 v4.0 and Infinium Human-Methylation 450K BeadChip, respectively. Results: U-As concentrations, ranging 10–1251 μg/L, was associated with decreased gene expression: 64% of the top 1000 differentially expressed genes were down-regulated with increasing U-As. U-As was also associated with hypermethylation: 87% of the top 1000 CpGs were hypermethylated with increasing U-As. The expression of six genes and six individual CpG sites were significantly associated with increased U-As concentration. Pathway analyses revealed enrichment of genes related to cell death and cancer. The pathways differed somewhat depending on arsenic metabolism efficiency. We found no overlap between arsenic-related gene expression and DNA methylation for individual genes. Conclusions: Increased arsenic exposure was associated with lower gene expression and hypermethylation in peripheral blood, but with no evident overlap. - Highlights: • Women exposed to inorganic arsenic were studied

  20. Association of the hypermethylation status of PTEN tumor suppressor gene with the risk of breast cancer among Kurdish population from Western Iran.

    PubMed

    Yari, Kheirollah; Payandeh, Mehrdad; Rahimi, Zohreh

    2016-06-01

    Breast cancer is the most common cancer with high morbidity and mortality among women worldwide. Aberrant hypermethylation in promoter regions of the tumor suppressor genes such as PTEN gene is a key event in the progression and development of breast cancer. The aim of the present study was to evaluate an association between PTEN gene methylation status with the risk of breast cancer in an Iranian population. We studied 255 individuals, including 103 patients with breast cancer, 102 first-degree female relatives of patients (mother, sister, or daughter of patients), and 50 healthy individuals as a control group. Genomic DNA was extracted from peripheral blood leukocytes, and the PTEN promoter methylation status was detected using methylation-specific PCR (MSP) method with specific methylated and unmethylated primers. In some samples, direct DNA sequencing was used to confirm the results obtained by the MSP method. The frequency of PTEN-methylated (MM) genotype was 6 % in the healthy control group, 23.3 % in relatives of patients, and 41.7 % in patients (χ (2) = 24.62, p < 0.001). There were significant differences in the frequency of PTEN-methylated genotype between healthy control compared to that in patients (χ (2) = 15.1, p < 0.001) and also compared to that in relatives of patients (χ (2) = 6.9, p = 0.009). In the presence of PTEN MM genotype, there was a 3.1-fold susceptibility to breast cancer compared to the UU genotype (p < 0.001). Also, in the presence of PTEN M allele, the risk of breast cancer was 2.71-fold compared to the presence of U allele (p < 0.001). Our findings indicated increased frequency of hypermethylation of PTEN promoter in the studied patients and their relatives that could be considered as one of the epigenetic factors affecting the risk of breast cancer in Iranians.

  1. Associations of P16INK4a promoter hypermethylation with squamous intra-epithelial lesion, cervical cancer and their clinicopathological features: a meta-analysis

    PubMed Central

    Cui, Ning-hua; Zhang, Shuai; Wang, Chen; Zheng, Fang

    2017-01-01

    To assess the associations of P16INK4a methylation status with low-grade squamous intra-epithelial lesion (LSIL), high-grade squamous intra-epithelial lesion (HSIL), cervical cancer (CC) and their clinicopathological features, a meta-analysis with 29 eligible studies was conducted. Pooled odds ratios (ORs) with their 95% confidence intervals (CIs) were estimated to assess the strength of the associations. Heterogeneity, sensitivity of pooled results and publication bias were also evaluated. Overall, there was an increasing trend of P16INK4a hypermethylation rates among LSIL (21.4%), HSIL (30.9%) and CC (35.0%) specimens. P16INK4a hypermethylation was significantly associated with the increased risk of LSIL, HSIL and CC, with the pooled ORs of 3.26 (95% CI: 1.86-5.71), 5.80 (95% CI: 3.80-8.84) and 12.17 (95% CI: 5.86-25.27), respectively. A significant association was also found between P16INK4a hypermethylation and smoking habit (OR = 3.88, 95% CI: 2.13-7.08). Taken together, meta-analysis results support P16INK4a hypermethylation as an epigenetic marker for the progression of cervical carcinogenesis. PMID:27669738

  2. Protection of cisplatin-induced spermatotoxicity, DNA damage and chromatin abnormality by selenium nano-particles

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Rezvanfar, Mohammad Amin; Rezvanfar, Mohammad Ali; Shahverdi, Ahmad Reza

    Cisplatin (CIS), an anticancer alkylating agent, induces DNA adducts and effectively cross links the DNA strands and so affects spermatozoa as a male reproductive toxicant. The present study investigated the cellular/biochemical mechanisms underlying possible protective effect of selenium nano-particles (Nano-Se) as an established strong antioxidant with more bioavailability and less toxicity, on reproductive toxicity of CIS by assessment of sperm characteristics, sperm DNA integrity, chromatin quality and spermatogenic disorders. To determine the role of oxidative stress (OS) in the pathogenesis of CIS gonadotoxicity, the level of lipid peroxidation (LPO), antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidasemore » (GSH-Px) and peroxynitrite (ONOO) as a marker of nitrosative stress (NS) and testosterone (T) concentration as a biomarker of testicular function were measured in the blood and testes. Thirty-two male Wistar rats were equally divided into four groups. A single IP dose of CIS (7 mg/kg) and protective dose of Nano-Se (2 mg/kg/day) were administered alone or in combination. The CIS-exposed rats showed a significant increase in testicular and serum LPO and ONOO level, along with a significant decrease in enzymatic antioxidants levels, diminished serum T concentration and abnormal histologic findings with impaired sperm quality associated with increased DNA damage and decreased chromatin quality. Coadministration of Nano-Se significantly improved the serum T, sperm quality, and spermatogenesis and reduced CIS-induced free radical toxic stress and spermatic DNA damage. In conclusion, the current study demonstrated that Nano-Se may be useful to prevent CIS-induced gonadotoxicity through its antioxidant potential. Highlights: ► Cisplatin (CIS) affects spermatozoa as a male reproductive toxicant. ► Effect of Nano-Se on CIS-induced spermatotoxicity was investigated. ► CIS-exposure induces oxidative sperm DNA

  3. Hypermethylation of gene promoters in peripheral blood leukocytes in humans long term after radiation exposure.

    PubMed

    Kuzmina, Nina S; Lapteva, Nellya Sh; Rubanovich, Alexander V

    2016-04-01

    Some human genes known to undergo age-related promoter hypermethylation. These epigenetic modifications are similar to those occurring in the course of certain diseases, e.g. some types of cancer, which in turn may also associate with age. Given external genotoxic factors may additionally contribute to hypermethylation, this study was designed to analyzes, using methylation-sensitive polymerase chain reaction (PCR), the CpG island hypermethylation in RASSF1A, CDKN2A (including p16/INK4A and p14/ARF) and GSTP1 promoters in peripheral blood leukocytes of individuals exposed to ionizing radiation long time ago. One hundred and twenty-four irradiated subjects (24-77 years old at sampling: 83 Chernobyl Nuclear Power Plant clean-up workers, 21 nuclear workers, 20 residents of territories with radioactive contamination) and 208 unirradiated volunteers (19-77 years old at sampling) were enrolled. In addition, 74 non-exposed offspring (2-51 years old at sampling) born to irradiated parents were examined. The frequency of individuals displaying promoter methylation of at least one gene in exposed group was significantly higher as compared to the control group (OR=5.44, 95% CI=2.62-11.76, p=3.9×10(-7)). No significant difference was found between the frequency of subjects with the revealed promoter methylation in the group of offspring born to irradiated parents and in the control group. The increase in the number of methylated loci of RASSF1A and p14/ARF was associated with age (β=0.242; p=1.7×10(-5)). In contrast, hypermethylation of p16/INK4A and GSTP1 genes correlated with the fact of radiation exposure only (β=0.290; p=1.7×10(-7)). The latter finding demonstrates that methylation changes in blood leukocytes of healthy subjects exposed to radiation resemble those reported in human malignancies. Additional studies are required to identify the dose-response of epigenetic markers specifically associating with radiation-induced premature aging and/or with the development

  4. Global Epigenetic Changes May Underlie Ethnic Differences and susceptibility to Prostate Cancer

    DTIC Science & Technology

    2012-09-01

    tissues; in the prostate, hypermethylation of the GSTP1 CpG has been detected in PIA lesions [8]. DNA methylation occurs at CpG sites in the human...that the GSTP1 CpG island was frequently hypermethylated in PCa, more than 40 genes have been reported to be targets of DNA hypermethylation-associated...One study demonstrated that GSTP1 hypermethylation was significantly higher in PCa samples from AA men in comparison with EA and Asians [12]. Another

  5. Basic Mechanics of DNA Methylation and the Unique Landscape of the DNA Methylome in Metal-Induced Carcinogenesis

    PubMed Central

    Brocato, Jason; Costa, Max

    2013-01-01

    DNA methylation plays an intricate role in the regulation of gene expression and events that compromise the integrity of the methylome may potentially contribute to disease development. DNA methylation is a reversible and regulatory modification that elicits a cascade of events leading to chromatin condensation and gene silencing. In general, normal cells are characterized by gene-specific hypomethylation and global hypermethylation, while cancer cells portray a reverse profile to this norm. The unique methylome displayed in cancer cells is induced after exposure to carcinogenic metals such as nickel, arsenic, cadmium, and chromium (VI). These metals alter the DNA methylation profile by provoking both hyper- and hypomethylation events. The metal-stimulated deviations to the methylome are possible mechanisms for metal-induced carcinogenesis and may provide potential biomarkers for cancer detection. Development of therapies based on the cancer methylome requires further research including human studies that supply results with larger impact and higher human relevance. PMID:23844698

  6. Basic mechanics of DNA methylation and the unique landscape of the DNA methylome in metal-induced carcinogenesis.

    PubMed

    Brocato, Jason; Costa, Max

    2013-07-01

    DNA methylation plays an intricate role in the regulation of gene expression and events that compromise the integrity of the methylome may potentially contribute to disease development. DNA methylation is a reversible and regulatory modification that elicits a cascade of events leading to chromatin condensation and gene silencing. In general, normal cells are characterized by gene-specific hypomethylation and global hypermethylation, while cancer cells portray a reverse profile to this norm. The unique methylome displayed in cancer cells is induced after exposure to carcinogenic metals such as nickel, arsenic, cadmium, and chromium (VI). These metals alter the DNA methylation profile by provoking both hyper- and hypo-methylation events. The metal-stimulated deviations to the methylome are possible mechanisms for metal-induced carcinogenesis and may provide potential biomarkers for cancer detection. Development of therapies based on the cancer methylome requires further research including human studies that supply results with larger impact and higher human relevance.

  7. Characterization of three different clusters of 18S-26S ribosomal DNA genes in the sea urchin P. lividus: Genetic and epigenetic regulation synchronous to 5S rDNA.

    PubMed

    Bellavia, Daniele; Dimarco, Eufrosina; Caradonna, Fabio

    2016-04-15

    We previously reported the characterization 5S ribosomal DNA (rDNA) clusters in the common sea urchin Paracentrotus lividus and demonstrated the presence of DNA methylation-dependent silencing of embryo specific 5S rDNA cluster in adult tissue. In this work, we show genetic and epigenetic characterization of 18S-26S rDNA clusters in this specie. The results indicate the presence of three different 18S-26S rDNA clusters with different Non-Transcribed Spacer (NTS) regions that have different chromosomal localizations. Moreover, we show that the two largest clusters are hyper-methylated in the promoter-containing NTS regions in adult tissues, as in the 5S rDNA. These findings demonstrate an analogous epigenetic regulation in small and large rDNA clusters and support the logical synchronism in building ribosomes. In fact, all the ribosomal RNA genes must be synchronously and equally transcribed to perform their unique final product. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Enhanced GSH synthesis by Bisphenol A exposure promoted DNA methylation process in the testes of adult rare minnow Gobiocypris rarus.

    PubMed

    Yuan, Cong; Zhang, Yingying; Liu, Yan; Zhang, Ting; Wang, Zaizhao

    2016-09-01

    DNA methylation is a commonly studied epigenetic modification. The mechanism of BPA on DNA methylation is poorly understood. The present study aims to explore whether GSH synthesis affects DNA methylation in the testes of adult male rare minnow Gobiocypris rarus in response to Bisphenol A (BPA). Male G. rarus was exposed to 1, 15 and 225μgL(-1) BPA for 7 days. The levels of global DNA methylation, hydrogen peroxide (H2O2) and glutathione (GSH) in the testes were analyzed. Meanwhile, the levels of enzymes involved in DNA methylation and de novo GSH synthesis, and the substrate contents for GSH production were measured. Furthermore, gene expression profiles of the corresponding genes of all studied enzymes were analyzed. Results indicated that BPA at 15 and 225μgL(-1) caused hypermethylation of global DNA in the testes. The 15μgL(-1) BPA resulted in significant decrease of ten-eleven translocation proteins (TETs) while 225μgL(-1) BPA caused significant increase of DNA methyltransferase proteins (DNMTs). Moreover, 225μgL(-1) BPA caused significant increase of H2O2 and GSH levels, and the de novo GSH synthesis was enhanced. These results indicated that the significant decrease of the level of TETs may be sufficient to cause the DNA hypermethylation by 15μgL(-1) BPA. However, the significantly increased of DNMTs contributed to the significant increase of DNA methylation levels by 225μgL(-1) BPA. Moreover, the elevated de novo GSH synthesis may promote the DNA methylation process. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Comparison of Promoter Hypermethylation Pattern in Salivary Rinses Collected with and without an Exfoliating Brush from Patients with HNSCC

    PubMed Central

    Sun, Wenyue; Zaboli, David; Liu, Yan; Arnaoutakis, Demetri; Khan, Tanbir; Wang, Hao; Koch, Wayne; Khan, Zubair; Califano, Joseph A.

    2012-01-01

    Background Salivary rinses have been recently proposed as a valuable resource for the development of epigenetic biomarkers for detection and monitoring of head and neck squamous cell carcinoma (HNSCC). Both salivary rinses collected with and without an exfoliating brush from patients with HNSCC are used in detection of promoter hypermethylation, yet their correlation of promoter hypermethylation has not been evaluated. This study was to evaluate the concordance of promoter hypermethylation between salivary rinses collected with and without an exfoliating brush from patients with HNSCC. Methodolgy 57 paired salivary rinses collected with or without an exfoliating brush from identical HNSCC patients were evaluated for promoter hypermethylation status using Quantitative Methylation-Specific PCR. Target tumor suppressor gene promoter regions were selected based on our previous studies describing a panel for HNSCC screening and surveillance, including P16, CCNA1, DCC, TIMP3, MGMT, DAPK and MINT31. Principal Findings In salivary rinses collected with and without brush, frequent methylation was detected in P16 (8.8% vs. 5.2%), CCNA1 (26.3% vs. 22.8%), DCC (33.3% vs. 29.8%), TIMP3 (31.6% vs. 36.8%), MGMT (29.8% vs. 38.6%), DAPK (14.0% vs. 19.2%), and MINT31 (10.5% vs. 8.8%). Spearman's rank correlation coefficient showed a positive correlation between salivary rinses collected with and without brush for P16 (ρ = 0.79), CCNA1 (ρ = 0.61), DCC (ρ = 0.58), TIMP3 (ρ = 0.10), MGMT (ρ = 0.70), DAPK (ρ = 0.51) and MINT31 (ρ = 0.72) (P<0.01). The percent agreement of promoter methylation between salivary rinses with brush and without brush were 96.5% for P16, 82.5% for CCNA1, 78.9% for DCC, 59.7% for TIMP3, 84.2% for MGMT, 84.2% for DAPK, and 94.7% for MINT31. Conclusions Our study demonstrated strong correlations of gene promoter hypermethylation between salivary rinses collected with and without an exfoliating brush. Salivary rinse collection

  10. The dynamic DNA methylation landscape of the mutL homolog 1 shore is altered by MLH1-93G>A polymorphism in normal tissues and colorectal cancer.

    PubMed

    Savio, Andrea J; Mrkonjic, Miralem; Lemire, Mathieu; Gallinger, Steven; Knight, Julia A; Bapat, Bharat

    2017-01-01

    Colorectal cancers (CRCs) undergo distinct genetic and epigenetic alterations. Expression of mutL homolog 1 ( MLH1 ), a mismatch repair gene that corrects DNA replication errors, is lost in up to 15% of sporadic tumours due to mutation or, more commonly, due to DNA methylation of its promoter CpG island. A single nucleotide polymorphism (SNP) in the CpG island of MLH1 ( MLH1 -93G>A or rs1800734) is associated with CpG island hypermethylation and decreased MLH1 expression in CRC tumours. Further, in peripheral blood mononuclear cell (PBMC) DNA of both CRC cases and non-cancer controls, the variant allele of rs1800734 is associated with hypomethylation at the MLH1 shore, a region upstream of its CpG island that is less dense in CpG sites . To determine whether this genotype-epigenotype association is present in other tissue types, including colorectal tumours, we assessed DNA methylation in matched normal colorectal tissue, tumour, and PBMC DNA from 349 population-based CRC cases recruited from the Ontario Familial Colorectal Cancer Registry. Using the semi-quantitative real-time PCR-based MethyLight assay, MLH1 shore methylation was significantly higher in tumour tissue than normal colon or PBMCs ( P  < 0.01). When shore methylation levels were stratified by SNP genotype, normal colorectal DNA and PBMC DNA were significantly hypomethylated in association with variant SNP genotype ( P  < 0.05). However, this association was lost in tumour DNA. Among distinct stages of CRC, metastatic stage IV CRC tumours incurred significant hypomethylation compared to stage I-III cases, irrespective of genotype status. Shore methylation of MLH1 was not associated with MSI status or promoter CpG island hypermethylation, regardless of genotype. To confirm these results, bisulfite sequencing was performed in matched tumour and normal colorectal specimens from six CRC cases, including two cases per genotype (wildtype, heterozygous, and homozygous variant). Bisulfite sequencing

  11. [Dynamics of LINE-1 Retrotransposon Methylation Levels in Circulating DNA from Lung Cancer Patients Undergoing Antitumor Therapy].

    PubMed

    Ponomaryova, A A; Cherdyntseva, N V; Bondar, A A; Dobrodeev, A Y; Zavyalov, A A; Tuzikov, S A; Vlassov, V V; Choinzonov, E L; Laktionov, P P; Rykova, E Y

    2017-01-01

    Malignant cell transformation is accompanied with abnormal DNA methylation, such as the hypermethylation of certain gene promoters and hypomethylation of retrotransposons. In particular, the hypomethylation of the human-specific family of LINE-1 retrotransposons was observed in lung cancer tissues. It is also known that the circulating DNA (cirDNA) of blood plasma and cell-surface-bound circulating DNA (csb-cirDNA) of cancer patients accumulate tumor-specific aberrantly methylated DNA fragments, which are currently considered to be valuable cancer markers. This work compares LINE-1 retrotransposon methylation patterns in cirDNA of 16 lung cancer patients before and after treatment. CirDNA was isolated from blood plasma, and csb-cirDNA fractions were obtained by successive elution with EDTA-containing phosphate buffered saline and trypsin. Concentrations of methylated LINE-1 region 1 copies (LINE-1-met) were assayed by real-time methylation-specific PCR. LINE-1 methylation levels were normalized to the concentration of LINE-1 region 2, which was independent of the methylation status (LINE-1-Ind). The concentrations of LINE-1-met and LINE-1-Ind in csb-cirDNA of lung cancer patients exhibited correlations before treatment (r = 0.54), after chemotherapy (r = 0.72), and after surgery (r = 0.83) (P < 0.05, Spearman rank test). In the total group of patients, the level of LINE-1 methylation (determined as the LINE-1-met/LINE-1-Ind ratio) was shown to increase significantly during the follow-up after chemotherapy (P < 0.05, paired t test) and after surgery compared to the level of methylation before treatment (P < 0.05, paired t test). The revealed association between the level of LINE-1 methylation and the effect of antitumor therapy was more pronounced in squamous cell lung cancer than in adenocarcinoma (P < 0.05 and P > 0.05, respectively). These results suggest a need for the further investigation of dynamic changes in levels of LINE-1 methylation depending on the

  12. Genome-wide DNA methylation analysis reveals estrogen-mediated epigenetic repression of metallothionein-1 gene cluster in breast cancer.

    PubMed

    Jadhav, Rohit R; Ye, Zhenqing; Huang, Rui-Lan; Liu, Joseph; Hsu, Pei-Yin; Huang, Yi-Wen; Rangel, Leticia B; Lai, Hung-Cheng; Roa, Juan Carlos; Kirma, Nameer B; Huang, Tim Hui-Ming; Jin, Victor X

    2015-01-01

    Recent genome-wide analysis has shown that DNA methylation spans long stretches of chromosome regions consisting of clusters of contiguous CpG islands or gene families. Hypermethylation of various gene clusters has been reported in many types of cancer. In this study, we conducted methyl-binding domain capture (MBDCap) sequencing (MBD-seq) analysis on a breast cancer cohort consisting of 77 patients and 10 normal controls, as well as a panel of 38 breast cancer cell lines. Bioinformatics analysis determined seven gene clusters with a significant difference in overall survival (OS) and further revealed a distinct feature that the conservation of a large gene cluster (approximately 70 kb) metallothionein-1 (MT1) among 45 species is much lower than the average of all RefSeq genes. Furthermore, we found that DNA methylation is an important epigenetic regulator contributing to gene repression of MT1 gene cluster in both ERα positive (ERα+) and ERα negative (ERα-) breast tumors. In silico analysis revealed much lower gene expression of this cluster in The Cancer Genome Atlas (TCGA) cohort for ERα + tumors. To further investigate the role of estrogen, we conducted 17β-estradiol (E2) and demethylating agent 5-aza-2'-deoxycytidine (DAC) treatment in various breast cancer cell types. Cell proliferation and invasion assays suggested MT1F and MT1M may play an anti-oncogenic role in breast cancer. Our data suggests that DNA methylation in large contiguous gene clusters can be potential prognostic markers of breast cancer. Further investigation of these clusters revealed that estrogen mediates epigenetic repression of MT1 cluster in ERα + breast cancer cell lines. In all, our studies identify thousands of breast tumor hypermethylated regions for the first time, in particular, discovering seven large contiguous hypermethylated gene clusters.

  13. Dental Pulp Stem Cells Model Early Life and Imprinted DNA Methylation Patterns.

    PubMed

    Dunaway, Keith; Goorha, Sarita; Matelski, Lauren; Urraca, Nora; Lein, Pamela J; Korf, Ian; Reiter, Lawrence T; LaSalle, Janine M

    2017-04-01

    Early embryonic stages of pluripotency are modeled for epigenomic studies primarily with human embryonic stem cells (ESC) or induced pluripotent stem cells (iPSCs). For analysis of DNA methylation however, ESCs and iPSCs do not accurately reflect the DNA methylation levels found in preimplantation embryos. Whole genome bisulfite sequencing (WGBS) approaches have revealed the presence of large partially methylated domains (PMDs) covering 30%-40% of the genome in oocytes, preimplantation embryos, and placenta. In contrast, ESCs and iPSCs show abnormally high levels of DNA methylation compared to inner cell mass (ICM) or placenta. Here we show that dental pulp stem cells (DPSCs), derived from baby teeth and cultured in serum-containing media, have PMDs and mimic the ICM and placental methylome more closely than iPSCs and ESCs. By principal component analysis, DPSC methylation patterns were more similar to two other neural stem cell types of human derivation (EPI-NCSC and LUHMES) and placenta than were iPSCs, ESCs or other human cell lines (SH-SY5Y, B lymphoblast, IMR90). To test the suitability of DPSCs in modeling epigenetic differences associated with disease, we compared methylation patterns of DPSCs derived from children with chromosome 15q11.2-q13.3 maternal duplication (Dup15q) to controls. Differential methylation region (DMR) analyses revealed the expected Dup15q hypermethylation at the imprinting control region, as well as hypomethylation over SNORD116, and novel DMRs over 147 genes, including several autism candidate genes. Together these data suggest that DPSCs are a useful model for epigenomic and functional studies of human neurodevelopmental disorders. Stem Cells 2017;35:981-988. © 2016 AlphaMed Press.

  14. DNA methylation in sugarcane somaclonal variants assessed through methylation-sensitive amplified polymorphism.

    PubMed

    Francischini, J H M B; Kemper, E L; Costa, J B; Manechini, J R V; Pinto, L R

    2017-05-04

    Micropropagation is an important tool for large-scale multiplication of plant superior genotypes. However, somaclonal variation is one of the drawbacks of this process. Changes in DNA methylation have been widely reported as one of the main causes of somaclonal variations in plants. In order to investigate the occurrence of changes in the methylation pattern of sugarcane somaclonal variants, the MSAP (methylation-sensitive amplified polymorphism) technique was applied to micro-propagated plantlets sampled at the third subculture phase. The mother plant, in vitro normal plantlets, and in vitro abnormal plantlets (somaclonal variants) of four sugarcane clones were screened against 16 MSAP selective primers for EcoRI/MspI and EcoRI/HpaII restriction enzymes. A total of 1005 and 1200 MSAP-derived markers with polymorphism percentages of 28.36 and 40.67 were obtained for EcoRI/HpaII and EcoRI/MspI restriction enzyme combinations, respectively. The genetic similarity between the mother plant and the somaclonal variants ranged from 0.877 to 0.911 (EcoRI/MspI) and from 0.928 to 0.955 (EcoRI/HpaII). Most of the MASPs among mother plant and micro-propagated plantlets were derived from EcoRI/MspI restriction enzymes suggesting alteration due to gain or loss of internal cytosine methylation. A higher rate of loss of methylation (hypomethylation) than gain of methylation (hypermethylation) was observed in the abnormal in vitro sugarcane plantlets. Although changes in the methylation pattern were also observed in the in vitro normal plantlets, they were lower than those observed for the in vitro abnormal plantlets. The MASP technique proved to be a promising tool to early assessment of genetic fidelity of micro-propagated sugarcane plants.

  15. Methylation analysis of plasma cell-free DNA for breast cancer early detection using bisulfite next-generation sequencing.

    PubMed

    Li, Zibo; Guo, Xinwu; Tang, Lili; Peng, Limin; Chen, Ming; Luo, Xipeng; Wang, Shouman; Xiao, Zhi; Deng, Zhongping; Dai, Lizhong; Xia, Kun; Wang, Jun

    2016-10-01

    Circulating cell-free DNA (cfDNA) has been considered as a potential biomarker for non-invasive cancer detection. To evaluate the methylation levels of six candidate genes (EGFR, GREM1, PDGFRB, PPM1E, SOX17, and WRN) in plasma cfDNA as biomarkers for breast cancer early detection, quantitative analysis of the promoter methylation of these genes from 86 breast cancer patients and 67 healthy controls was performed by using microfluidic-PCR-based target enrichment and next-generation bisulfite sequencing technology. The predictive performance of different logistic models based on methylation status of candidate genes was investigated by means of the area under the ROC curve (AUC) and odds ratio (OR) analysis. Results revealed that EGFR, PPM1E, and 8 gene-specific CpG sites showed significantly hypermethylation in cancer patients' plasma and significantly associated with breast cancer (OR ranging from 2.51 to 9.88). The AUC values for these biomarkers were ranging from 0.66 to 0.75. Combinations of multiple hypermethylated genes or CpG sites substantially improved the predictive performance for breast cancer detection. Our study demonstrated the feasibility of quantitative measurement of candidate gene methylation in cfDNA by using microfluidic-PCR-based target enrichment and bisulfite next-generation sequencing, which is worthy of further validation and potentially benefits a broad range of applications in clinical oncology practice. Quantitative analysis of methylation pattern of plasma cfDNA by next-generation sequencing might be a valuable non-invasive tool for early detection of breast cancer.

  16. Frequent silencing of RASSF1A by DNA methylation in thymic neuroendocrine tumours.

    PubMed

    Kajiura, Koichiro; Takizawa, Hiromitsu; Morimoto, Yuki; Masuda, Kiyoshi; Tsuboi, Mitsuhiro; Kishibuchi, Reina; Wusiman, Nuliamina; Sawada, Toru; Kawakita, Naoya; Toba, Hiroaki; Yoshida, Mitsuteru; Kawakami, Yukikiyo; Naruto, Takuya; Imoto, Issei; Tangoku, Akira; Kondo, Kazuya

    2017-09-01

    Aberrant methylation of promoter CpG islands (CGIs) of tumour suppressor genes is a common epigenetic mechanism underlying cancer pathogenesis. The methylation patterns of thymic tumours have not been studied in detail since such tumours are rare. Herein, we sought to identify genes that could serve as epigenetic targets for thymic neuroendocrine tumour (NET) therapy. Genome-wide screening for aberrantly methylated CGIs was performed in three NET samples, seven thymic carcinoma (TC) samples, and eight type-B3 thymoma samples. The methylation status of thymic epithelial tumours (TETs) samples was validated by pyrosequencing in a larger cohort. The expression status was analysed by quantitative polymerase chain reaction (PCR) and immunohistochemistry. We identified a CGI on a novel gene, RASSF1A, which was strongly hypermethylated in NET, but not in thymic carcinoma or B3 thymoma. RASSF1A was identified as a candidate gene statistically and bibliographically, as it showed frequent CGI hypermethylation in NET by genome-wide screening. Pyrosequencing confirmed significant hypermethylation of a RASSF1A CGI in NET. Low-grade NET tissue was more strongly methylated than high-grade NET. Quantitative PCR and immunohistochemical staining revealed that RASSF1A mRNA and protein expression levels were negatively regulated by DNA methylation. RASSF1A is a tumour suppressor gene epigenetically dysregulated in NET. Aberrant methylation of RASSF1A has been reported in various tumours, but this is the first report of RASSF1A hypermethylation in TETs. RASSF1A may represent an epigenetic therapeutic target in thymic NET. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. APC alterations are frequently involved in the pathogenesis of acinar cell carcinoma of the pancreas, mainly through gene loss and promoter hypermethylation.

    PubMed

    Furlan, Daniela; Sahnane, Nora; Bernasconi, Barbara; Frattini, Milo; Tibiletti, Maria Grazia; Molinari, Francesca; Marando, Alessandro; Zhang, Lizhi; Vanoli, Alessandro; Casnedi, Selenia; Adsay, Volkan; Notohara, Kenji; Albarello, Luca; Asioli, Sofia; Sessa, Fausto; Capella, Carlo; La Rosa, Stefano

    2014-05-01

    Genetic and epigenetic alterations involved in the pathogenesis of pancreatic acinar cell carcinomas (ACCs) are poorly characterized, including the frequency and role of gene-specific hypermethylation, chromosome aberrations, and copy number alterations (CNAs). A subset of ACCs is known to show alterations in the APC/β-catenin pathway which includes mutations of APC gene. However, it is not known whether, in addition to mutation, loss of APC gene function can occur through alternative genetic and epigenetic mechanisms such as gene loss or promoter methylation. We investigated the global methylation profile of 34 tumor suppressor genes, CNAs of 52 chromosomal regions, and APC gene alterations (mutation, methylation, and loss) together with APC mRNA level in 45 ACCs and related peritumoral pancreatic tissues using methylation-specific multiplex ligation probe amplification (MS-MLPA), fluorescence in situ hybridization (FISH), mutation analysis, and reverse transcription-droplet digital PCR. ACCs did not show an extensive global gene hypermethylation profile. RASSF1 and APC were the only two genes frequently methylated. APC mutations were found in only 7 % of cases, while APC loss and methylation were more frequently observed (48 and 56 % of ACCs, respectively). APC mRNA low levels were found in 58 % of cases and correlated with CNAs. In conclusion, ACCs do not show extensive global gene hypermethylation. APC alterations are frequently involved in the pathogenesis of ACCs mainly through gene loss and promoter hypermethylation, along with reduction of APC mRNA levels.

  18. DNA Methylation Mediated Downregulation of miR-449c Controls Osteosarcoma Cell Cycle Progression by Directly Targeting Oncogene c-Myc

    PubMed Central

    Li, Qing; Li, Hua; Zhao, Xueling; Wang, Bing; Zhang, Lin; Zhang, Caiguo; Zhang, Fan

    2017-01-01

    MicroRNAs (miRNAs) are critical regulators of gene expression, and they have broad roles in the pathogenesis of different diseases including cancer. Limited studies and expression profiles of miRNAs are available in human osteosarcoma cells. By applying a miRNA microarray analysis, we observed a number of miRNAs with abnormal expression in cancerous tissues from osteosarcoma patients. Of particular interest in this study was miR-449c, which was significantly downregulated in osteosarcoma cells and patients, and its expression was negatively correlated with tumor size and tumor MSTS stages. Ectopic expression of miR-449c significantly inhibited osteosarcoma cell proliferation and colony formation ability, and caused cell cycle arrest at the G1 phase. Further analysis identified that miR-449c was able to directly target the oncogene c-Myc and negatively regulated its expression. Overexpression of c-Myc partially reversed miR-449c-mimic-inhibited cell proliferation and colony formation. Moreover, DNA hypermethylation was observed in two CpG islands adjacent to the genomic locus of miR-449c in osteosarcoma cells. Conversely, treatment with the DNA methylation inhibitor AZA caused induction of miR-449c. In conclusion, our results support a model that DNA methylation mediates downregulation of miR-449c, diminishing miR-449c mediated inhibition of c-Myc and thus leading to the activation of downstream targets, eventually contributing to osteosarcoma tumorigenesis. PMID:28924385

  19. CXCR4 expression varies significantly among different subtypes of glioblastoma multiforme (GBM) and its low expression or hypermethylation might predict favorable overall survival.

    PubMed

    Ma, Xinlong; Shang, Feng; Zhu, Weidong; Lin, Qingtang

    2017-09-01

    CXCR4 is an oncogene in glioblastoma multiforme (GBM) but the mechanism of its dysregulation and its prognostic value in GBM have not been fully understood. Bioinformatic analysis was performed by using R2 and the UCSC Xena browser based on data from GSE16011 in GEO datasets and in GBM cohort in TCGA database (TCGA-GBM). Kaplan Meier curves of overall survival (OS) were generated to assess the association between CXCR4 expression/methylation and OS in patients with GBM. GBM patients with high CXCR4 expression had significantly worse 5 and 10 yrs OS (p < 0.05). Across different GBM subtypes, there was an inverse relationship between overall DNA methylation and CXCR4 expression. CXCR4 expression was significantly lower in CpG island methylation phenotype (CIMP) group than in non CIMP group. Log rank test results showed that patients with high CXCR4 methylation (first tertile) had significantly better 5 yrs OS (p = 0.038). CXCR4 expression is regulated by DNA methylation in GBM and its low expression or hypermethylation might indicate favorable OS in GBM patients.

  20. Overlapping DNA Methylation Dynamics in Mouse Intestinal Cell Differentiation and Early Stages of Malignant Progression

    PubMed Central

    Forn, Marta; Díez-Villanueva, Anna; Merlos-Suárez, Anna; Muñoz, Mar; Lois, Sergi; Carriò, Elvira; Jordà, Mireia; Bigas, Anna; Batlle, Eduard; Peinado, Miguel A.

    2015-01-01

    Mouse models of intestinal crypt cell differentiation and tumorigenesis have been used to characterize the molecular mechanisms underlying both processes. DNA methylation is a key epigenetic mark and plays an important role in cell identity and differentiation programs and cancer. To get insights into the dynamics of cell differentiation and malignant transformation we have compared the DNA methylation profiles along the mouse small intestine crypt and early stages of tumorigenesis. Genome-scale analysis of DNA methylation together with microarray gene expression have been applied to compare intestinal crypt stem cells (EphB2high), differentiated cells (EphB2negative), ApcMin/+ adenomas and the corresponding non-tumor adjacent tissue, together with small and large intestine samples and the colon cancer cell line CT26. Compared with late stages, small intestine crypt differentiation and early stages of tumorigenesis display few and relatively small changes in DNA methylation. Hypermethylated loci are largely shared by the two processes and affect the proximities of promoter and enhancer regions, with enrichment in genes associated with the intestinal stem cell signature and the PRC2 complex. The hypermethylation is progressive, with minute levels in differentiated cells, as compared with intestinal stem cells, and reaching full methylation in advanced stages. Hypomethylation shows different signatures in differentiation and cancer and is already present in the non-tumor tissue adjacent to the adenomas in ApcMin/+ mice, but at lower levels than advanced cancers. This study provides a reference framework to decipher the mechanisms driving mouse intestinal tumorigenesis and also the human counterpart. PMID:25933092

  1. Non-invasive prenatal testing for fetal chromosomal abnormalities by low-coverage whole-genome sequencing of maternal plasma DNA: review of 1982 consecutive cases in a single center.

    PubMed

    Lau, T K; Cheung, S W; Lo, P S S; Pursley, A N; Chan, M K; Jiang, F; Zhang, H; Wang, W; Jong, L F J; Yuen, O K C; Chan, H Y C; Chan, W S K; Choy, K W

    2014-03-01

    To review the performance of non-invasive prenatal testing (NIPT) by low-coverage whole-genome sequencing of maternal plasma DNA at a single center. The NIPT result and pregnancy outcome of 1982 consecutive cases were reviewed. NIPT was based on low coverage (0.1×) whole-genome sequencing of maternal plasma DNA. All subjects were contacted for pregnancy and fetal outcome. Of the 1982 NIPT tests, a repeat blood sample was required in 23 (1.16%). In one case, a conclusive report could not be issued, probably because of an abnormal vanished twin fetus. NIPT was positive for common trisomies in 29 cases (23 were trisomy 21, four were trisomy 18 and two were trisomy 13); all were confirmed by prenatal karyotyping (specificity=100%). In addition, 11 cases were positive for sex-chromosomal abnormalities (SCA), and nine cases were positive for other aneuploidies or deletion/duplication. Fourteen of these 20 subjects agreed to undergo further investigations, and the abnormality was found to be of fetal origin in seven, confined placental mosaicism (CPM) in four, of maternal origin in two and not confirmed in one. Overall, 85.7% of the NIPT-suspected SCA were of fetal origin, and 66.7% of the other abnormalities were caused by CPM. Two of the six cases suspected or confirmed to have CPM were complicated by early-onset growth restriction requiring delivery before 34 weeks. Fetal outcome of the NIPT-negative cases was ascertained in 1645 (85.15%). Three chromosomal abnormalities were not detected by NIPT, including one case each of a balanced translocation, unbalanced translocation and triploidy. There were no known false negatives involving the common trisomies (sensitivity=100%). Low-coverage whole-genome sequencing of maternal plasma DNA was highly accurate in detecting common trisomies. It also enabled the detection of other aneuploidies and structural chromosomal abnormalities with high positive predictive value. Copyright © 2013 ISUOG. Published by John Wiley & Sons

  2. Ras regulation of DNA-methylation and cancer

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Patra, Samir Kumar

    2008-04-01

    Genome wide hypomethylation and regional hypermethylation of cancer cells and tissues remain a paradox, though it has received a convincing confirmation that epigenetic switching systems, including DNA-methylation represent a fundamental regulatory mechanism that has an impact on genome maintenance and gene transcription. Methylated cytosine residues of vertebrate DNA are transmitted by clonal inheritance through the strong preference of DNA methyltransferase, DNMT1, for hemimethylated-DNA. Maintenance of methylation patterns is necessary for normal development of mice, and aberrant methylation patterns are associated with many human tumours. DNMT1 interacts with many proteins during cell cycle progression, including PCNA, p53, EZH2 and HP1. Rasmore » family of GTPases promotes cell proliferation by its oncogenic nature, which transmits signals by multiple pathways in both lipid raft dependent and independent fashion. DNA-methylation-mediated repression of DNA-repair protein O6-methylguanine DNA methyltransferase (MGMT) gene and increased rate of K-Ras mutation at codon for amino acids 12 and 13 have been correlated with a secondary role for Ras-effector homologues (RASSFs) in tumourigenesis. Lines of evidence suggest that DNA-methylation associated repression of tumour suppressors and apoptotic genes and ceaseless proliferation of tumour cells are regulated in part by Ras-signaling. Control of Ras GTPase signaling might reduce the aberrant methylation and accordingly may reduce the risk of cancer development.« less

  3. DNA methylation similarities in genes of black South Africans with systemic lupus erythematosus and systemic sclerosis.

    PubMed

    Matatiele, Puleng; Tikly, Mohamed; Tarr, Gareth; Gulumian, Mary

    2015-05-20

    Systemic lupus erythematosus (SLE) and systemic sclerosis (SSc) are systemic autoimmune connective tissue diseases that share overlapping clinico-pathological features. It is highly probable that there is an overlap in epigenetic landscapes of both diseases. This study aimed to identify similarities in DNA methylation changes in genes involved in SLE and SSc. Global DNA methylation and twelve genes selected on the basis of their involvement in inflammation, autoimmunity and/or fibrosis were analyzed using PCR arrays in three groups, each of 30 Black South Africans with SLE and SSc, plus 40 healthy control subjects. Global methylation in both diseases was significantly lower (<25 %) than in healthy subjects (>30 %, p = 0.0000001). In comparison to healthy controls, a similar gene-specific methylation pattern was observed in both SLE and SSc. Three genes, namely; PRF1, ITGAL and FOXP3 were consistently hypermethylated while CDKN2A and CD70 were hypomethylated in both diseases. The other genes (SOCS1, CTGF, THY1, CXCR4, MT1-G, FLI1, and DNMT1) were generally hypomethylated in SLE whereas they were neither hyper- nor hypo-methylated in SSc. SSc and SLE patients have a higher global hypomethylation than healthy subjects with specific genes being hypomethylated and others hypermethylated. The majority of genes studied were hypomethylated in SLE compared to SSc. In addition to the commonly known hypomethylated genes in SLE and SSc, there are other hypomethylated genes (such as MT-1G and THY-1) that have not previously been investigated in SLE and SSc though are known to be hypermethylated in cancer.

  4. DNA methylation of retrotransposons, DNA transposons and genes in sugar beet (Beta vulgaris L.).

    PubMed

    Zakrzewski, Falk; Schmidt, Martin; Van Lijsebettens, Mieke; Schmidt, Thomas

    2017-06-01

    The methylation of cytosines shapes the epigenetic landscape of plant genomes, coordinates transgenerational epigenetic inheritance, represses the activity of transposable elements (TEs), affects gene expression and, hence, can influence the phenotype. Sugar beet (Beta vulgaris ssp. vulgaris), an important crop that accounts for 30% of worldwide sugar needs, has a relatively small genome size (758 Mbp) consisting of approximately 485 Mbp repetitive DNA (64%), in particular satellite DNA, retrotransposons and DNA transposons. Genome-wide cytosine methylation in the sugar beet genome was studied in leaves and leaf-derived callus with a focus on repetitive sequences, including retrotransposons and DNA transposons, the major groups of repetitive DNA sequences, and compared with gene methylation. Genes showed a specific methylation pattern for CG, CHG (H = A, C, and T) and CHH sites, whereas the TE pattern differed, depending on the TE class (class 1, retrotransposons and class 2, DNA transposons). Along genes and TEs, CG and CHG methylation was higher than that of adjacent genomic regions. In contrast to the relatively low CHH methylation in retrotransposons and genes, the level of CHH methylation in DNA transposons was strongly increased, pointing to a functional role of asymmetric methylation in DNA transposon silencing. Comparison of genome-wide DNA methylation between sugar beet leaves and callus revealed a differential methylation upon tissue culture. Potential epialleles were hypomethylated (lower methylation) at CG and CHG sites in retrotransposons and genes and hypermethylated (higher methylation) at CHH sites in DNA transposons of callus when compared with leaves. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  5. Review: Diagnosis and impact of sperm DNA alterations in assisted reproduction.

    PubMed

    Simon, Luke; Emery, Benjamin R; Carrell, Douglas T

    2017-10-01

    Sperm nuclear and chromatin abnormalities are common among infertile men and are known to influence natural reproduction. These abnormalities are also considered detrimental to normal fertilization, embryo development, and successful implantation and pregnancies following assisted reproductive treatment (ART). Abnormalities in the sperm nucleus can be broadly classified into sperm chromosomal abnormalities (aneuploidies) and sperm DNA abnormalities such as abnormal packing, DNA integrity, or DNA fragmentation. For the past 30 years, numerous tests have been developed to quantify these abnormalities in sperm. In this chapter, we review the causes of sperm DNA and chromosomal abnormalities, describe the commonly used tests to evaluate these abnormalities, and finally review the impact of these abnormalities on male fertility and ART outcomes. We also performed a comprehensive meta-analysis and systematic review from the existing literature to summarize the effect of sperm DNA fragmentation on ART outcomes such as fertilization rate, embryo quality, and clinical pregnancies. A review of the literature presented in this chapter suggests that sperm nuclear and chromatin abnormalities are associated with male infertility, and they reduce the probability of a successful pregnancy following ART. Copyright © 2017. Published by Elsevier Ltd.

  6. DNA methylation patterns in tissues from mid-gestation bovine foetuses produced by somatic cell nuclear transfer show subtle abnormalities in nuclear reprogramming.

    PubMed

    Couldrey, Christine; Lee, Rita Sf

    2010-03-07

    Cloning of cattle by somatic cell nuclear transfer (SCNT) is associated with a high incidence of pregnancy failure characterized by abnormal placental and foetal development. These abnormalities are thought to be due, in part, to incomplete re-setting of the epigenetic state of DNA in the donor somatic cell nucleus to a state that is capable of driving embryonic and foetal development to completion. Here, we tested the hypothesis that DNA methylation patterns were not appropriately established during nuclear reprogramming following SCNT. A panel of imprinted, non-imprinted genes and satellite repeat sequences was examined in tissues collected from viable and failing mid-gestation SCNT foetuses and compared with similar tissues from gestation-matched normal foetuses generated by artificial insemination (AI). Most of the genomic regions examined in tissues from viable and failing SCNT foetuses had DNA methylation patterns similar to those in comparable tissues from AI controls. However, statistically significant differences were found between SCNT and AI at specific CpG sites in some regions of the genome, particularly those associated with SNRPN and KCNQ1OT1, which tended to be hypomethylated in SCNT tissues. There was a high degree of variation between individuals in methylation levels at almost every CpG site in these two regions, even in AI controls. In other genomic regions, methylation levels at specific CpG sites were tightly controlled with little variation between individuals. Only one site (HAND1) showed a tissue-specific pattern of DNA methylation. Overall, DNA methylation patterns in tissues of failing foetuses were similar to apparently viable SCNT foetuses, although there were individuals showing extreme deviant patterns. These results show that SCNT foetuses that had developed to mid-gestation had largely undergone nuclear reprogramming and that the epigenetic signature at this stage was not a good predictor of whether the foetus would develop to term

  7. Epigenomic profiling reveals an association between persistence of DNA methylation and metabolic memory in the DCCT/EDIC type 1 diabetes cohort.

    PubMed

    Chen, Zhuo; Miao, Feng; Paterson, Andrew D; Lachin, John M; Zhang, Lingxiao; Schones, Dustin E; Wu, Xiwei; Wang, Jinhui; Tompkins, Joshua D; Genuth, Saul; Braffett, Barbara H; Riggs, Arthur D; Natarajan, Rama

    2016-05-24

    We examined whether persistence of epigenetic DNA methylation (DNA-me) alterations at specific loci over two different time points in people with diabetes are associated with metabolic memory, the prolonged beneficial effects of intensive vs. conventional therapy during the Diabetes Control and Complications Trial (DCCT) on the progression of microvascular outcomes in the long-term follow-up Epidemiology of Diabetes Interventions and Complications (EDIC) Study. We compared DNA-me profiles in genomic DNA of whole blood (WB) isolated at EDIC Study baseline from 32 cases (DCCT conventional therapy group subjects showing retinopathy or albuminuria progression by EDIC Study year 10) vs. 31 controls (DCCT intensive therapy group subjects without complication progression by EDIC year 10). DNA-me was also profiled in blood monocytes (Monos) of the same patients obtained during EDIC Study years 16-17. In WB, 153 loci depicted hypomethylation, and 225 depicted hypermethylation, whereas in Monos, 155 hypomethylated loci and 247 hypermethylated loci were found (fold change ≥1.3; P < 0.005; cases vs. controls). Twelve annotated differentially methylated loci were common in both WB and Monos, including thioredoxin-interacting protein (TXNIP), known to be associated with hyperglycemia and related complications. A set of differentially methylated loci depicted similar trends of associations with prior HbA1c in both WB and Monos. In vitro, high glucose induced similar persistent hypomethylation at TXNIP in cultured THP1 Monos. These results show that DNA-me differences during the DCCT persist at certain loci associated with glycemia for several years during the EDIC Study and support an epigenetic explanation for metabolic memory.

  8. Overexpression of Human-Derived DNMT3A Induced Intergenerational Inheritance of Active DNA Methylation Changes in Rat Sperm

    PubMed Central

    Zheng, Xiaoguo; Li, Zhenhua; Wang, Guishuan; Li, Zhengzheng; Liang, Ajuan; Wang, Hanshu; Dai, Yubing; Huang, Xingxu; Chen, Xuejin; Ma, Yuanwu; Sun, Fei

    2017-01-01

    DNA methylation is the major focus of studies on paternal epigenetic inheritance in mammals, but most previous studies about inheritable DNA methylation changes are passively induced by environmental factors. However, it is unclear whether the active changes mediated by variations in DNA methyltransferase activity are heritable. Here, we established human-derived DNMT3A (hDNMT3A) transgenic rats to study the effect of hDNMT3A overexpression on the DNA methylation pattern of rat sperm and to investigate whether this actively altered DNA methylation status is inheritable. Our results revealed that hDNMT3A was overexpressed in the testis of transgenic rats and induced genome-wide alterations in the DNA methylation pattern of rat sperm. Among 5438 reliable loci identified with 64 primer-pair combinations using a methylation-sensitive amplification polymorphism method, 28.01% showed altered amplified band types. Among these amplicons altered loci, 68.42% showed an altered DNA methylation status in the offspring of transgenic rats compared with wild-type rats. Further analysis based on loci which had identical DNA methylation status in all three biological replicates revealed that overexpression of hDNMT3A in paternal testis induced hypermethylation in sperm of both genotype-negative and genotype-positive offspring. Among the differentially methylated loci, 34.26% occurred in both positive and negative offspring of transgenic rats, indicating intergenerational inheritance of active DNA methylation changes in the absence of hDNM3A transmission. Furthermore, 75.07% of the inheritable loci were hyper-methylated while the remaining were hypomethylated. Distribution analysis revealed that the DNA methylation variations mainly occurred in introns and intergenic regions. Functional analysis revealed that genes related to differentially methylated loci were involved in a wide range of functions. Finally, this study demonstrated that active DNA methylation changes induced by h

  9. High fat diet and exercise lead to a disrupted and pathogenic DNA methylome in mouse liver.

    PubMed

    Zhou, Dan; Hlady, Ryan A; Schafer, Marissa J; White, Thomas A; Liu, Chen; Choi, Jeong-Hyeon; Miller, Jordan D; Roberts, Lewis R; LeBrasseur, Nathan K; Robertson, Keith D

    2017-01-02

    High-fat diet consumption and sedentary lifestyle elevates risk for obesity, non-alcoholic fatty liver disease, and cancer. Exercise training conveys health benefits in populations with or without these chronic conditions. Diet and exercise regulate gene expression by mediating epigenetic mechanisms in many tissues; however, such effects are poorly documented in the liver, a central metabolic organ. To dissect the consequences of diet and exercise on the liver epigenome, we measured DNA methylation, using reduced representation bisulfite sequencing, and transcription, using RNA-seq, in mice maintained on a fast food diet with sedentary lifestyle or exercise, compared with control diet with and without exercise. Our analyses reveal that genome-wide differential DNA methylation and expression of gene clusters are induced by diet and/or exercise. A combination of fast food and exercise triggers extensive gene alterations, with enrichment of carbohydrate/lipid metabolic pathways and muscle developmental processes. Through evaluation of putative protective effects of exercise on diet-induced DNA methylation, we show that hypermethylation is effectively prevented, especially at promoters and enhancers, whereas hypomethylation is only partially attenuated. We assessed diet-induced DNA methylation changes associated with liver cancer-related epigenetic modifications and identified significant increases at liver-specific enhancers in fast food groups, suggesting partial loss of liver cell identity. Hypermethylation at a subset of gene promoters was associated with inhibition of tissue development and promotion of carcinogenic processes. Our study demonstrates extensive reprogramming of the epigenome by diet and exercise, emphasizing the functional relevance of epigenetic mechanisms as an interface between lifestyle modifications and phenotypic alterations.

  10. DNA methylation mediated control of gene expression is critical for development of crown gall tumors.

    PubMed

    Gohlke, Jochen; Scholz, Claus-Juergen; Kneitz, Susanne; Weber, Dana; Fuchs, Joerg; Hedrich, Rainer; Deeken, Rosalia

    2013-01-01

    Crown gall tumors develop after integration of the T-DNA of virulent Agrobacterium tumefaciens strains into the plant genome. Expression of the T-DNA-encoded oncogenes triggers proliferation and differentiation of transformed plant cells. Crown gall development is known to be accompanied by global changes in transcription, metabolite levels, and physiological processes. High levels of abscisic acid (ABA) in crown galls regulate expression of drought stress responsive genes and mediate drought stress acclimation, which is essential for wild-type-like tumor growth. An impact of epigenetic processes such as DNA methylation on crown gall development has been suggested; however, it has not yet been investigated comprehensively. In this study, the methylation pattern of Arabidopsis thaliana crown galls was analyzed on a genome-wide scale as well as at the single gene level. Bisulfite sequencing analysis revealed that the oncogenes Ipt, IaaH, and IaaM were unmethylated in crown galls. Nevertheless, the oncogenes were susceptible to siRNA-mediated methylation, which inhibited their expression and subsequently crown gall growth. Genome arrays, hybridized with methylated DNA obtained by immunoprecipitation, revealed a globally hypermethylated crown gall genome, while promoters were rather hypomethylated. Mutants with reduced non-CG methylation developed larger tumors than the wild-type controls, indicating that hypermethylation inhibits plant tumor growth. The differential methylation pattern of crown galls and the stem tissue from which they originate correlated with transcriptional changes. Genes known to be transcriptionally inhibited by ABA and methylated in crown galls became promoter methylated upon treatment of A. thaliana with ABA. This suggests that the high ABA levels in crown galls may mediate DNA methylation and regulate expression of genes involved in drought stress protection. In summary, our studies provide evidence that epigenetic processes regulate gene

  11. Global changes in DNA methylation in Alzheimer's disease peripheral blood mononuclear cells.

    PubMed

    Di Francesco, Andrea; Arosio, Beatrice; Falconi, Anastasia; Micioni Di Bonaventura, Maria Vittoria; Karimi, Mohsen; Mari, Daniela; Casati, Martina; Maccarrone, Mauro; D'Addario, Claudio

    2015-03-01

    Changes in epigenetic marks may help explain the late onset of Alzheimer's disease (AD). In this study we measured genome-wide DNA methylation by luminometric methylation assay, a quantitative measurement of genome-wide DNA methylation, on DNA isolated from peripheral blood mononuclear cells of 37 subjects with late-onset AD (LOAD) and 44 healthy controls (CT). We found an increase in global DNA methylation in LOAD subjects compared to CT (p=0.0122), associated with worse cognitive performances (p=0.0002). DNA hypermethylation in LOAD group was paralleled by higher DNA methyltransferase 1 (DNMT1) gene expression and protein levels. When data were stratified on the basis of the APOE polymorphisms, higher DNA methylation levels were associated with the presence of APOE ε4 allele (p=0.0043) in the global population. Among the APOE ε3 carriers, a significant increase of DNA methylation was still observed in LOAD patients compared to healthy controls (p=0.05). Our data suggest global DNA methylation in peripheral samples as a useful marker for screening individuals at risk of developing AD. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. EG-09EPIGENETIC PROFILING REVEALS A CpG HYPERMETHYLATION PHENOTYPE (CIMP) ASSOCIATED WITH WORSE PROGRESSION-FREE SURVIVAL IN MENINGIOMA

    PubMed Central

    Olar, Adriana; Wani, Khalida; Mansouri, Alireza; Zadeh, Gelareh; Wilson, Charmaine; DeMonte, Franco; Fuller, Gregory; Jones, David; Pfister, Stefan; von Deimling, Andreas; Sulman, Erik; Aldape, Kenneth

    2014-01-01

    BACKGROUND: Methylation profiling of solid tumors has revealed biologic subtypes, often with clinical implications. Methylation profiles of meningioma and their clinical implications are not well understood. METHODS: Ninety-two meningioma samples (n = 44 test set and n = 48 validation set) were profiled using the Illumina HumanMethylation450 BeadChip. Unsupervised clustering and analyses for recurrence-free survival (RFS) were performed. RESULTS: Unsupervised clustering of the test set using approximately 900 highly variable markers identified two clearly defined methylation subgroups. One of the groups (n = 19) showed global hypermethylation of a set of markers, analogous to CpG island methylator phenotype (CIMP). These findings were reproducible in the validation set, with 18/48 samples showing the CIMP-positive phenotype. Importantly, of 347 highly variable markers common to both the test and validation set analyses, 107 defined CIMP in the test set and 94 defined CIMP in the validation set, with an overlap of 83 markers between the two datasets. This number is much greater than expected by chance indicating reproducibly of the hypermethylated markers that define CIMP in meningioma. With respect to clinical correlation, the 37 CIMP-positive cases displayed significantly shorter RFS compared to the 55 non-CIMP cases (hazard ratio 2.9, p = 0.013). In an effort to develop a preliminary outcome predictor, a 155-marker subset correlated with RFS was identified in the test dataset. When interrogated in the validation dataset, this 155-marker subset showed a statistical trend (p < 0.1) towards distinguishing survival groups. CONCLUSIONS: This study defines the existence of a CIMP phenotype in meningioma, which involves a substantial proportion (37/92, 40%) of samples with clinical implications. Ongoing work will expand this cohort and examine identification of additional biologic differences (mutational and DNA copy number analysis) to further characterize the aberrant

  13. Aberrant methylation accounts for cell adhesion-related gene silencing during 3-methylcholanthrene and diethylnitrosamine induced multistep rat lung carcinogenesis associated with overexpression of DNA methyltransferases 1 and 3a

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Liu Wenbin; Cui Zhihong; Ao Lin

    To evaluate the significance of alterations in cell adhesion-related genes methylation during lung multistep carcinogenesis induced by the genotoxic carcinogens 3-methylcholanthrene (MCA) and diethylnitrosamine (DEN), tissue samples microdissected from MCA/DEN-induced rat lung carcinogenesis model were subjected to methylation-specific PCR to evaluate the DNA methylation status of CADM1, TIMP3, E-cadherin and N-cadherin. Immunohistochemistry was used to determine protein expression of CADM1, TIMP3, N-cadherin and the DNA methyltransferases (DNMTs) 1, 3a and 3b. E-cadherin hypermethylation was not detected in any tissue. CADM1, TIMP3 and N-cadherin hypermethylation was correlated with the loss of their protein expression during the progression of pathologic lesions. Themore » prevalence of DNA methylation of at least one gene and the average number of methylated genes increased with the histological progression. DNMT1 and DNMT3a protein expression increased progressively during the stages of lung carcinogenesis, whereas DNMT3b overexpression was only found in several samples. Furthermore, DNMT1 protein expression levels were correlated with CADM1 methylation, and DNMT3a protein expression levels were correlated with CADM1, TIMP3 and N-cadherin methylation. The average number of methylated genes during carcinogenesis was significantly correlated with DNMT1 and DNMT3a protein expression levels. Moreover, mRNA expression of CADM1 significantly increased after treatment with DNMT inhibitor 5-aza-2'-deoxycytidine in CADM1-methylated primary tumor cell lines. Our findings suggest that an accumulation of hypermethylation accounts for cell adhesion-related gene silencing is associated with dynamic changes in the progression of MCA/DEN-induced rat lung carcinogenesis. We suggest that DNMT1 and DNMT3a protein overexpression may be responsible for this aberrant DNA methylation.« less

  14. The DNA methylation landscape of human melanoma.

    PubMed

    Jin, Seung-Gi; Xiong, Wenying; Wu, Xiwei; Yang, Lu; Pfeifer, Gerd P

    2015-12-01

    Using MIRA-seq, we have characterized the DNA methylome of metastatic melanoma and normal melanocytes. Individual tumors contained several thousand hypermethylated regions. We discovered 179 tumor-specific methylation peaks present in all (27/27) melanomas that may be effective disease biomarkers, and 3113 methylation peaks were seen in >40% of the tumors. We found that 150 of the approximately 1200 tumor-associated methylation peaks near transcription start sites (TSSs) were marked by H3K27me3 in melanocytes. DNA methylation in melanoma was specific for distinct H3K27me3 peaks rather than for broadly covered regions. However, numerous H3K27me3 peak-associated TSS regions remained devoid of DNA methylation in tumors. There was no relationship between BRAF mutations and the number of methylation peaks. Gene expression analysis showed upregulated immune response genes in melanomas presumably as a result of lymphocyte infiltration. Down-regulated genes were enriched for melanocyte differentiation factors; e.g., KIT, PAX3 and SOX10 became methylated and downregulated in melanoma. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Genome-wide hypermethylation coupled with promoter hypomethylation in the chorioamniotic membranes of early onset pre-eclampsia

    PubMed Central

    Ching, Travers; Song, Min-Ae; Tiirikainen, Maarit; Molnar, Janos; Berry, Marla; Towner, Dena; Garmire, Lana X.

    2014-01-01

    Pre-eclampsia is the leading cause of fetal and maternal morbidity and mortality. Early onset pre-eclampsia (EOPE) is a disorder that has severe maternal and fetal outcomes, whilst its etiology is poorly understood. We hypothesize that epigenetics plays an important role to mediate the development of EOPE and conducted a case–control study to compare the genome-wide methylome difference between chorioamniotic membranes from 30 EOPE and 17 full-term pregnancies using the Infinium Human Methylation 450 BeadChip arrays. Bioinformatics analysis tested differential methylation (DM) at CpG site level, gene level, and pathway and network level. A striking genome-wide hypermethylation pattern coupled with hypomethylation in promoters was observed. Out of 385 184 CpG sites, 9995 showed DM (2.6%). Of those DM sites, 91.9% showed hypermethylation (9186 of 9995). Over 900 genes had DM associated with promoters. Promoter-based DM analysis revealed that genes in canonical cancer-related pathways such as Rac, Ras, PI3K/Akt, NFκB and ErBB4 were enriched, and represented biological functional alterations that involve cell cycle, apoptosis, cancer signaling and inflammation. A group of genes previously found to be up-regulated in pre-eclampsia, including GRB2, ATF3, NFKB2, as well as genes in proteasome subunits (PSMA1, PMSE1, PSMD1 and PMSD8), harbored hypomethylated promoters. Contrarily, a cluster of microRNAs, including mir-519a1, mir-301a, mir-487a, mir-185, mir-329, mir-194, mir-376a1, mir-486 and mir-744 were all hypermethylated in their promoters in the EOPE samples. These findings collectively reveal new avenues of research regarding the vast epigenetic modifications in EOPE. PMID:24944161

  16. An integrated genomics analysis of epigenetic subtypes in human breast tumors links DNA methylation patterns to chromatin states in normal mammary cells.

    PubMed

    Holm, Karolina; Staaf, Johan; Lauss, Martin; Aine, Mattias; Lindgren, David; Bendahl, Pär-Ola; Vallon-Christersson, Johan; Barkardottir, Rosa Bjork; Höglund, Mattias; Borg, Åke; Jönsson, Göran; Ringnér, Markus

    2016-02-29

    Aberrant DNA methylation is frequently observed in breast cancer. However, the relationship between methylation patterns and the heterogeneity of breast cancer has not been comprehensively characterized. Whole-genome DNA methylation analysis using Illumina Infinium HumanMethylation450 BeadChip arrays was performed on 188 human breast tumors. Unsupervised bootstrap consensus clustering was performed to identify DNA methylation epigenetic subgroups (epitypes). The Cancer Genome Atlas data, including methylation profiles of 669 human breast tumors, was used for validation. The identified epitypes were characterized by integration with publicly available genome-wide data, including gene expression levels, DNA copy numbers, whole-exome sequencing data, and chromatin states. We identified seven breast cancer epitypes. One epitype was distinctly associated with basal-like tumors and with BRCA1 mutations, one epitype contained a subset of ERBB2-amplified tumors characterized by multiple additional amplifications and the most complex genomes, and one epitype displayed a methylation profile similar to normal epithelial cells. Luminal tumors were stratified into the remaining four epitypes, with differences in promoter hypermethylation, global hypomethylation, proliferative rates, and genomic instability. Specific hyper- and hypomethylation across the basal-like epitype was rare. However, we observed that the candidate genomic instability drivers BRCA1 and HORMAD1 displayed aberrant methylation linked to gene expression levels in some basal-like tumors. Hypomethylation in luminal tumors was associated with DNA repeats and subtelomeric regions. We observed two dominant patterns of aberrant methylation in breast cancer. One pattern, constitutively methylated in both basal-like and luminal breast cancer, was linked to genes with promoters in a Polycomb-repressed state in normal epithelial cells and displayed no correlation with gene expression levels. The second pattern

  17. High fructose consumption induces DNA methylation at PPARα and CPT1A promoter regions in the rat liver

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ohashi, Koji; Munetsuna, Eiji; Yamada, Hiroya, E-mail: hyamada@fujita-hu.ac.jp

    DNA methylation status is affected by environmental factors, including nutrition. Fructose consumption is considered a risk factor for the conditions that make up metabolic syndrome such as dyslipidemia. However, the pathogenetic mechanism by which fructose consumption leads to metabolic syndrome is unclear. Based on observations that epigenetic modifications are closely related to induction of metabolic syndrome, we hypothesized that fructose-induced metabolic syndrome is caused by epigenetic alterations. Male SD rats were designated to receive water or 20% fructose solution for 14 weeks. mRNA levels for peroxisome proliferator-activated receptor alpha (PPARα) and carnitine palmitoyltransferase 1A (CPT1A) was analyzed using Real-time PCR.more » Restriction digestion and real-time PCR (qAMP) was used for the analysis of DNA methylation status. Hepatic lipid accumulation was also observed by fructose intake. Fructose feeding also significantly decreased mRNA levels for PPARα and CPT1A. qAMP analysis demonstrated the hypermethylation of promoter regions of PPARα and CTP1A genes. Fructose-mediated attenuated gene expression may be mediated by alterations of DNA methylation status, and pathogenesis of metabolic syndrome induced by fructose relates to DNA methylation status. - Highlights: • No general consensus has been reached regarding the molecular mechanisms of the pathogenesis of fructose-induced diseases. • Significant increase in hepatic total methylation level was observed after fructose-supplemented feeding. • Fructose feeding significantly decreased mRNA levels for PPARα and CPT1A. • qAMP analysis demonstrated the hypermethylation of promoter regions of PPARα and CTP1A genes. • Fructose-mediated attenuated gene expression may be mediated by alterations of DNA methylation status in rat liver.« less

  18. Profiling the genome-wide DNA methylation pattern of porcine ovaries using reduced representation bisulfite sequencing.

    PubMed

    Yuan, Xiao-Long; Gao, Ning; Xing, Yan; Zhang, Hai-Bin; Zhang, Ai-Ling; Liu, Jing; He, Jin-Long; Xu, Yuan; Lin, Wen-Mian; Chen, Zan-Mou; Zhang, Hao; Zhang, Zhe; Li, Jia-Qi

    2016-02-25

    Substantial evidence has shown that DNA methylation regulates the initiation of ovarian and sexual maturation. Here, we investigated the genome-wide profile of DNA methylation in porcine ovaries at single-base resolution using reduced representation bisulfite sequencing. The biological variation was minimal among the three ovarian replicates. We found hypermethylation frequently occurred in regions with low gene abundance, while hypomethylation in regions with high gene abundance. The DNA methylation around transcriptional start sites was negatively correlated with their own CpG content. Additionally, the methylation level in the bodies of genes was higher than that in their 5' and 3' flanking regions. The DNA methylation pattern of the low CpG content promoter genes differed obviously from that of the high CpG content promoter genes. The DNA methylation level of the porcine ovary was higher than that of the porcine intestine. Analyses of the genome-wide DNA methylation in porcine ovaries would advance the knowledge and understanding of the porcine ovarian methylome.

  19. Detection of Prostate Cancer Progression by Serum DNA Integrity

    DTIC Science & Technology

    2009-04-01

    DNA was assayed for AI of 6 genome microsatellites. We assessed meth- ylation of RASSF1, RARB2, and GSTP1 using a methylation-specific PCR assay and...microsatellites. The epigenetic biomarkers evaluated were 3 tumor suppressor genes that are frequently hypermethylated in PCa: GSTP1 (glutathione S...8p22, D8S262 at 8p23, D9S171 at 9p21, D10S591 at 10p15, and D18S70 at 18q23. Forward primer sets were labeled with WellRed 8 Human genes: GSTP1

  20. Glutathione-S-transferase pi 1(GSTP1) gene silencing in prostate cancer cells is reversed by the histone deacetylase inhibitor depsipeptide.

    PubMed

    Hauptstock, Vera; Kuriakose, Sapuna; Schmidt, Doris; Düster, Robert; Müller, Stefan C; von Ruecker, Alexander; Ellinger, Jörg

    2011-09-09

    Gene silencing by epigenetic mechanisms is frequent in prostate cancer (PCA). The link between DNA hypermethylation and histone modifications is not completely understood. We chose the GSTP1 gene which is silenced by hypermethylation to analyze the effect of the histone deacetylase inhibitor depsipeptide on DNA methylation and histone modifications at the GSTP1 promoter site. Prostate cell lines (PC-3, LNCaP, and BPH-1) were treated with depsipeptide; apoptosis (FACS analysis), GSTP1 mRNA levels (quantitative real-time PCR), DNA hypermethylation (methylation-specific PCR), and histone modifications (chromatin immunoprecipitation) were studied. Depsipeptide induced apoptosis in PCA cells, but not a cell cycle arrest. Depispeptide reversed DNA hypermethylation and repressive histone modifications (reduction of H3K9me2/3 and H3K27me2/3; increase of H3K18Ac), thereby inducing GSTP1 mRNA re-expression. Successful therapy requires both, DNA demethylation and activating histone modifications, to induce complete gene expression of epigenetically silenced genes and depsipeptide fulfils both criteria. Copyright © 2011 Elsevier Inc. All rights reserved.

  1. Epigenomic profiling reveals an association between persistence of DNA methylation and metabolic memory in the DCCT/EDIC type 1 diabetes cohort

    PubMed Central

    Chen, Zhuo; Miao, Feng; Paterson, Andrew D.; Lachin, John M.; Zhang, Lingxiao; Schones, Dustin E.; Wu, Xiwei; Wang, Jinhui; Tompkins, Joshua D.; Genuth, Saul; Braffett, Barbara H.; Riggs, Arthur D.; Natarajan, Rama

    2016-01-01

    We examined whether persistence of epigenetic DNA methylation (DNA-me) alterations at specific loci over two different time points in people with diabetes are associated with metabolic memory, the prolonged beneficial effects of intensive vs. conventional therapy during the Diabetes Control and Complications Trial (DCCT) on the progression of microvascular outcomes in the long-term follow-up Epidemiology of Diabetes Interventions and Complications (EDIC) Study. We compared DNA-me profiles in genomic DNA of whole blood (WB) isolated at EDIC Study baseline from 32 cases (DCCT conventional therapy group subjects showing retinopathy or albuminuria progression by EDIC Study year 10) vs. 31 controls (DCCT intensive therapy group subjects without complication progression by EDIC year 10). DNA-me was also profiled in blood monocytes (Monos) of the same patients obtained during EDIC Study years 16–17. In WB, 153 loci depicted hypomethylation, and 225 depicted hypermethylation, whereas in Monos, 155 hypomethylated loci and 247 hypermethylated loci were found (fold change ≥1.3; P < 0.005; cases vs. controls). Twelve annotated differentially methylated loci were common in both WB and Monos, including thioredoxin-interacting protein (TXNIP), known to be associated with hyperglycemia and related complications. A set of differentially methylated loci depicted similar trends of associations with prior HbA1c in both WB and Monos. In vitro, high glucose induced similar persistent hypomethylation at TXNIP in cultured THP1 Monos. These results show that DNA-me differences during the DCCT persist at certain loci associated with glycemia for several years during the EDIC Study and support an epigenetic explanation for metabolic memory. PMID:27162351

  2. DNA methylation patterns in tissues from mid-gestation bovine foetuses produced by somatic cell nuclear transfer show subtle abnormalities in nuclear reprogramming

    PubMed Central

    2010-01-01

    Background Cloning of cattle by somatic cell nuclear transfer (SCNT) is associated with a high incidence of pregnancy failure characterized by abnormal placental and foetal development. These abnormalities are thought to be due, in part, to incomplete re-setting of the epigenetic state of DNA in the donor somatic cell nucleus to a state that is capable of driving embryonic and foetal development to completion. Here, we tested the hypothesis that DNA methylation patterns were not appropriately established during nuclear reprogramming following SCNT. A panel of imprinted, non-imprinted genes and satellite repeat sequences was examined in tissues collected from viable and failing mid-gestation SCNT foetuses and compared with similar tissues from gestation-matched normal foetuses generated by artificial insemination (AI). Results Most of the genomic regions examined in tissues from viable and failing SCNT foetuses had DNA methylation patterns similar to those in comparable tissues from AI controls. However, statistically significant differences were found between SCNT and AI at specific CpG sites in some regions of the genome, particularly those associated with SNRPN and KCNQ1OT1, which tended to be hypomethylated in SCNT tissues. There was a high degree of variation between individuals in methylation levels at almost every CpG site in these two regions, even in AI controls. In other genomic regions, methylation levels at specific CpG sites were tightly controlled with little variation between individuals. Only one site (HAND1) showed a tissue-specific pattern of DNA methylation. Overall, DNA methylation patterns in tissues of failing foetuses were similar to apparently viable SCNT foetuses, although there were individuals showing extreme deviant patterns. Conclusion These results show that SCNT foetuses that had developed to mid-gestation had largely undergone nuclear reprogramming and that the epigenetic signature at this stage was not a good predictor of whether the

  3. qPCR-based mitochondrial DNA quantification: Influence of template DNA fragmentation on accuracy

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jackson, Christopher B., E-mail: Christopher.jackson@insel.ch; Gallati, Sabina, E-mail: sabina.gallati@insel.ch; Schaller, Andre, E-mail: andre.schaller@insel.ch

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer Serial qPCR accurately determines fragmentation state of any given DNA sample. Black-Right-Pointing-Pointer Serial qPCR demonstrates different preservation of the nuclear and mitochondrial genome. Black-Right-Pointing-Pointer Serial qPCR provides a diagnostic tool to validate the integrity of bioptic material. Black-Right-Pointing-Pointer Serial qPCR excludes degradation-induced erroneous quantification. -- Abstract: Real-time PCR (qPCR) is the method of choice for quantification of mitochondrial DNA (mtDNA) by relative comparison of a nuclear to a mitochondrial locus. Quantitative abnormal mtDNA content is indicative of mitochondrial disorders and mostly confines in a tissue-specific manner. Thus handling of degradation-prone bioptic material is inevitable. We established a serialmore » qPCR assay based on increasing amplicon size to measure degradation status of any DNA sample. Using this approach we can exclude erroneous mtDNA quantification due to degraded samples (e.g. long post-exicision time, autolytic processus, freeze-thaw cycles) and ensure abnormal DNA content measurements (e.g. depletion) in non-degraded patient material. By preparation of degraded DNA under controlled conditions using sonification and DNaseI digestion we show that erroneous quantification is due to the different preservation qualities of the nuclear and the mitochondrial genome. This disparate degradation of the two genomes results in over- or underestimation of mtDNA copy number in degraded samples. Moreover, as analysis of defined archival tissue would allow to precise the molecular pathomechanism of mitochondrial disorders presenting with abnormal mtDNA content, we compared fresh frozen (FF) with formalin-fixed paraffin-embedded (FFPE) skeletal muscle tissue of the same sample. By extrapolation of measured decay constants for nuclear DNA ({lambda}{sub nDNA}) and mtDNA ({lambda}{sub mtDNA}) we present an approach to possibly correct

  4. CpG site hypermethylation of E-cadherin and Connexin26 genes in hepatocellular carcinomas induced by a choline-deficient L-Amino Acid-defined diet in rats.

    PubMed

    Tsujiuchi, Toshifumi; Shimizu, Kyoko; Itsuzaki, Yumi; Onishi, Mariko; Sugata, Eriko; Fujii, Hiromasa; Honoki, Kanya

    2007-04-01

    We investigated DNA methylation patterns of E-cadherin and Connexin26 (Cx26) genes in rat hepatocellular carcinomas (HCCs) induced by a choline-deficient L-Amino Acid-defined (CDAA) diet. Six-wks-old F344 male rats were continuously fed with a CDAA diet for 75 wks, and were then killed. A total of five HCCs were obtained, and genomic DNA was extracted from each HCC for assessment of methylation status in the 5' upstream regions of E-cadherin and Cx26 genes by bisulfite sequencing, comparing to two normal liver tissues. The five HCCs showed highly methylated E-cadherin and Cx26 genes, while these genes in two normal liver tissues were all unmethylated. For analysis of gene expression, real-time quantitative reverse transcription (RT)-polymerase chain reaction (PCR) was performed. Expressions of E-cadherin and Cx26 genes were significantly reduced in the five HCCs (P < 0.0001 and P < 0.001, respectively) compared to normal liver tissues, correlating with their methylation statuses. These results suggested that hypermethylation of E-cadherin and Cx26 genes may be involved in the development of HCCs induced by a CDAA diet in rats.

  5. DNA methylation patterns of genes related to immune response in the different clinical forms of oral lichen planus.

    PubMed

    Cruz, Aline Fernanda; de Resende, Renata Gonçalves; de Lacerda, Júlio César Tanos; Pereira, Núbia Braga; Melo, Leonardo Augusto; Diniz, Marina Gonçalves; Gomes, Carolina Cavalieri; Gomez, Ricardo Santiago

    2018-01-01

    The oral lichen planus is a chronic inflammatory disease. Although its aetiology is not well understood, the role of T lymphocytes in its inflammatory events is recognised. Identifying the epigenetic mechanisms involved in the pathogenesis of this immune-mediated condition is fundamental for understanding the inflammatory reaction that occurs in the disease. The purpose of this work was to evaluate the methylation pattern of 21 immune response-related genes in the different clinical forms of oral lichen planus. A cross-sectional study was performed to analyse the DNA methylation patterns in three distinct groups of oral lichen planus: (i) reticular/plaque lesions; (ii) erosive lesions; (iii) normal oral mucosa (control group). After DNA extraction from biopsies, the samples were submitted to digestions by methylation-sensitive and methylation-dependent enzymes and double digestion. The relative percentage of methylated DNA for each gene was provided using real-time polymerase chain reaction arrays. Hypermethylation of the STAT5A gene was observed only in the control group (59.0%). A higher hypermethylation of the ELANE gene was found in reticular/plaque lesions (72.1%) compared to the erosive lesions (50.0%). Our results show variations in the methylation profile of immune response-related genes, according to the clinical type of oral lichen planus after comparing with the normal oral mucosa. Further studies are necessary to validate these findings using gene expression analysis. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  6. Age-Related DNA Methylation Changes and Neoplastic Transformation of the Human Prostate

    DTIC Science & Technology

    2009-07-01

    transcriptional silencing by aberrant CpG m ethylation of C pG-rich promoter regions. 5, 6 Aberrant promoter methylation of GSTP1 , e ncoding the π-class...during prostate cancer developm ent.7 Since the recogni tion that the GSTP1 Cp G was frequently hypermethylated in prostate cancer, more than 40 genes...8 genes; SPARC, RARb2, AR, TIMP3, GSTP1 , NKX2 .5, RASSF1 A and CYP27B1 in DNA sa mples fro m African American (AA) and Caucasian (C au) m en as a

  7. Hypermethylation of CDH13, DKK3 and FOXL2 promoters and the expression of EZH2 in ovary granulosa cell tumors.

    PubMed

    Xu, Yanmei; Li, Xia; Wang, Hongtao; Xie, Pengmu; Yan, Xun; Bai, Yu; Zhang, Tingguo

    2016-09-01

    Aberrant epigenetic modification is associated with the development and progression of cancer. Hypermethylation of tumor suppressor gene promoters and cooperative histone modification have been considered to be the primary mechanisms of epigenetic modification. Ovary granulosa cell tumors (GCTs) are relatively rare, accounting for ~3% of all ovarian malignancies. The present study assessed hypermethylation of the cadherin 13 (CDH13), dickkopf WNT signaling pathway inhibitor 3 (DKK3) and forkhead box L2 (FOXL2) promoters in 30 GCT tissues and 30 healthy control tissues using methylation-specific polymerase chain reaction analysis. The data showed that the frequencies of CDH13, DKK3 and FOXL2 promoter methylation were significantly higher in the GCT tissues, compared with the healthy control tissues (86.67, vs. 23.33%; 80, vs. 26.67% and 66.67, vs. 20%, respectively; P<0.001). Immunostaining of enhancer of zeste homolog 2 (EZH2), a histone H3K27 methyltransferase, showed that the EZH2 protein was expressed in 11 of the 30 GCT tissue samples, whereas no EZH2 protein was expressed in the 30 healthy control tissues (P<0.01). These data suggested that hypermethylation of the CDH13, DKK3 and FOXL2 gene promoters, and overexpression of the EZH2 protein were involved in the development of GCT.

  8. Epigenetic modulations in early endothelial cells and DNA hypermethylation in human skin after sulfur mustard exposure.

    PubMed

    Steinritz, Dirk; Schmidt, Annette; Balszuweit, Frank; Thiermann, Horst; Simons, Thilo; Striepling, Enno; Bölck, Birgit; Bloch, Wilhelm

    2016-02-26

    Victims that were exposed to the chemical warfare agent sulfur mustard (SM) suffer from chronic dermal and ocular lesions, severe pulmonary problems and cancer development. It has been proposed that epigenetic perturbations might be involved in that process but this has not been investigated so far. In this study, we investigated epigenetic modulations in vitro using early endothelial cells (EEC) that were exposed to different SM concentrations (0.5, 1.0, 23.5 and 50μM). A comprehensive analysis of 78 genes related to epigenetic pathways (i.e., DNA-methylation and post-translational histone modifications) was performed. Moreover, we analyzed global DNA methylation in vitro in EEC after SM exposure as a maker for epigenetic modulations and in vivo using human skin samples that were obtained from a patient 1 year after an accidently exposure to pure SM. SM exposure resulted in a complex regulation pattern of epigenetic modulators which was accompanied by a global increase of DNA methylation in vitro. Examination of the SM exposed human skin samples also revealed a significant increase of global DNA methylation in vivo, underlining the biological relevance of our findings. Thus, we demonstrated for the first time that SM affects epigenetic pathways and causes epigenetic modulations both in vivo and in vitro. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  9. DNA Methylation Mediated Control of Gene Expression Is Critical for Development of Crown Gall Tumors

    PubMed Central

    Kneitz, Susanne; Weber, Dana; Fuchs, Joerg; Hedrich, Rainer; Deeken, Rosalia

    2013-01-01

    Crown gall tumors develop after integration of the T-DNA of virulent Agrobacterium tumefaciens strains into the plant genome. Expression of the T-DNA–encoded oncogenes triggers proliferation and differentiation of transformed plant cells. Crown gall development is known to be accompanied by global changes in transcription, metabolite levels, and physiological processes. High levels of abscisic acid (ABA) in crown galls regulate expression of drought stress responsive genes and mediate drought stress acclimation, which is essential for wild-type-like tumor growth. An impact of epigenetic processes such as DNA methylation on crown gall development has been suggested; however, it has not yet been investigated comprehensively. In this study, the methylation pattern of Arabidopsis thaliana crown galls was analyzed on a genome-wide scale as well as at the single gene level. Bisulfite sequencing analysis revealed that the oncogenes Ipt, IaaH, and IaaM were unmethylated in crown galls. Nevertheless, the oncogenes were susceptible to siRNA–mediated methylation, which inhibited their expression and subsequently crown gall growth. Genome arrays, hybridized with methylated DNA obtained by immunoprecipitation, revealed a globally hypermethylated crown gall genome, while promoters were rather hypomethylated. Mutants with reduced non-CG methylation developed larger tumors than the wild-type controls, indicating that hypermethylation inhibits plant tumor growth. The differential methylation pattern of crown galls and the stem tissue from which they originate correlated with transcriptional changes. Genes known to be transcriptionally inhibited by ABA and methylated in crown galls became promoter methylated upon treatment of A. thaliana with ABA. This suggests that the high ABA levels in crown galls may mediate DNA methylation and regulate expression of genes involved in drought stress protection. In summary, our studies provide evidence that epigenetic processes regulate gene

  10. DNA methylation patterns in ulcerative colitis-associated cancer: a systematic review.

    PubMed

    Emmett, Ruth A; Davidson, Katherine L; Gould, Nicholas J; Arasaradnam, Ramesh P

    2017-07-01

    Evidence points to the role of DNA methylation in ulcerative colitis (UC)-associated cancer (UCC), the most serious complication of ulcerative colitis. A better understanding of the etiology of UCC may facilitate the development of new therapeutic targets and help to identify biomarkers of the disease risk. A search was performed in three databases following PRISMA protocol. DNA methylation in UCC was compared with sporadic colorectal cancer (SCRC), and individual genes differently methylated in UCC identified. While there were some similarities in the methylation patterns of UCC compared with SCRC, generally lower levels of hypermethylation in promoter regions of individual genes was evident in UCC. Certain individual genes are, however, highly methylated in colitis-associated cancer: RUNX3, MINT1, MYOD and p16 exon1 and the promoter regions of EYA4 and ESR. Patterns of DNA methylation differ between UCC and SCRC. Seven genes appear to be promising putative biomarkers.

  11. Stimulation of ribosomal RNA gene promoter by transcription factor Sp1 involves active DNA demethylation by Gadd45-NER pathway.

    PubMed

    Rajput, Pallavi; Pandey, Vijaya; Kumar, Vijay

    2016-08-01

    The well-studied Pol II transcription factor Sp1 has not been investigated for its regulatory role in rDNA transcription. Here, we show that Sp1 bound to specific sites on rDNA and localized into the nucleoli during the G1 phase of cell cycle to activate rDNA transcription. It facilitated the recruitment of Pol I pre-initiation complex and impeded the binding of nucleolar remodeling complex (NoRC) to rDNA resulting in the formation of euchromatin active state. More importantly, Sp1 also orchestrated the site-specific binding of Gadd45a-nucleotide excision repair (NER) complex resulting in active demethylation and transcriptional activation of rDNA. Interestingly, knockdown of Sp1 impaired rDNA transcription due to reduced engagement of the Gadd45a-NER complex and hypermethylation of rDNA. Thus, the present study unveils a novel role of Sp1 in rDNA transcription involving promoter demethylation. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. EG-07CELL CYCLE SIGNATURE AND TUMOR PHYLOGENY ARE ENCODED IN THE EVOLUTIONARY DYNAMICS OF DNA METHYLATION IN GLIOMA

    PubMed Central

    Mazor, Tali; Pankov, Aleksandr; Johnson, Brett E.; Hong, Chibo; Bell, Robert J.A.; Smirnov, Ivan V.; Reis, Gerald F.; Phillips, Joanna J.; Barnes, Michael; Bollen, Andrew W.; Taylor, Barry S.; Molinaro, Annette M.; Olshen, Adam B.; Song, Jun S.; Berger, Mitchel S.; Chang, Susan M.; Costello, Joseph F.

    2014-01-01

    The clonal evolution of tumor cell populations can be reconstructed from patterns of genetic alterations. In contrast, tumor epigenetic states, including DNA methylation, are reversible and sensitive to the tumor microenvironment, presumably precluding the use of epigenetics to discover tumor phylogeny. Here we examined the spatial and temporal dynamics of DNA methylation in a clinically and genetically characterized cohort of IDH1-mutant low-grade gliomas and their patient-matched recurrences. WHO grade II gliomas are diffuse, infiltrative tumors that frequently recur and may undergo malignant progression to a higher grade with a worse prognosis. The extent to which epigenetic alterations contribute to the evolution of low-grade gliomas, including malignant progression, is unknown. While all gliomas in the cohort exhibited the hypermethylation signature associated with IDH1 mutation, low-grade gliomas that underwent malignant progression to high-grade glioblastoma (GBM) had a unique signature of DNA hypomethylation enriched for active enhancers, as well as sites of age-related hypermethylation in the brain. Genes with promoter hypomethylation and concordant transcriptional upregulation during evolution to GBM were enriched in cell cycle function, evolving in concert with genetic alterations that deregulate the G1/S cell cycle checkpoint. Despite the plasticity of tumor epigenetic states, phyloepigenetic trees robustly recapitulated phylogenetic trees derived from somatic mutations in the same patients. These findings highlight widespread co-dependency of genetic and epigenetic events throughout the clonal evolution of initial and recurrent glioma.

  13. Regulatory link between DNA methylation and active demethylation in Arabidopsis

    PubMed Central

    Lei, Mingguang; Zhang, Huiming; Julian, Russell; Tang, Kai; Xie, Shaojun; Zhu, Jian-Kang

    2015-01-01

    De novo DNA methylation through the RNA-directed DNA methylation (RdDM) pathway and active DNA demethylation play important roles in controlling genome-wide DNA methylation patterns in plants. Little is known about how cells manage the balance between DNA methylation and active demethylation activities. Here, we report the identification of a unique RdDM target sequence, where DNA methylation is required for maintaining proper active DNA demethylation of the Arabidopsis genome. In a genetic screen for cellular antisilencing factors, we isolated several REPRESSOR OF SILENCING 1 (ros1) mutant alleles, as well as many RdDM mutants, which showed drastically reduced ROS1 gene expression and, consequently, transcriptional silencing of two reporter genes. A helitron transposon element (TE) in the ROS1 gene promoter negatively controls ROS1 expression, whereas DNA methylation of an RdDM target sequence between ROS1 5′ UTR and the promoter TE region antagonizes this helitron TE in regulating ROS1 expression. This RdDM target sequence is also targeted by ROS1, and defective DNA demethylation in loss-of-function ros1 mutant alleles causes DNA hypermethylation of this sequence and concomitantly causes increased ROS1 expression. Our results suggest that this sequence in the ROS1 promoter region serves as a DNA methylation monitoring sequence (MEMS) that senses DNA methylation and active DNA demethylation activities. Therefore, the ROS1 promoter functions like a thermostat (i.e., methylstat) to sense DNA methylation levels and regulates DNA methylation by controlling ROS1 expression. PMID:25733903

  14. DNA methylation and soy phytoestrogens: quantitative study in DU-145 and PC-3 human prostate cancer cell lines.

    PubMed

    Adjakly, Mawussi; Bosviel, Rémy; Rabiau, Nadège; Boiteux, Jean-Paul; Bignon, Yves-Jean; Guy, Laurent; Bernard-Gallon, Dominique

    2011-12-01

    DNA hypermethylation is an epigenetic mechanism which induces silencing of tumor-suppressor genes in prostate cancer. Many studies have reported that specific components of food plants like soy phytoestrogens may have protective effects against prostate carcinogenesis or progression. Genistein and daidzein, the major phytoestrogens, have been reported to have the ability to reverse DNA hypermethylation in cancer cell lines. The aim of this study was to investigate the potential demethylating effects of these two soy compounds on BRCA1, GSTP1, EPHB2 and BRCA2 promoter genes. Prostate cell lines DU-145 and PC-3 were treated with genistein 40 µM, daidzein 110 µM, budesonide (methylating agent) 2 µM and 5-azacytidine (demethylating agent) 2 µM. In these two human prostate cancer cell lines we performed methylation quantification by using Methyl Profiler DNA methylation analysis. This technique is based on a methylation-specific digestion followed by quantitative PCR. We analyzed the corresponding protein expression by western blotting. Soy phytoestrogens induced a demethylation of all promoter regions studied except for BRCA2, which is not methylated in control cell lines. An increase in their protein expression was also demonstrated by western blot analysis and corroborated the potential demethylating effect of soy phytoestrogens. This study showed that the soy phytoestrogens, genistein and daidzein, induce a decrease of methylation of BRCA1, GSTP1 and EPHB2 promoters. Therefore, soy phytoestrogens may have a protective effect on prostate cancer. However, more studies are needed in order to understand the mechanism by which genistein and daidzein have an inhibiting action on DNA methylation.

  15. A DNA methylation fingerprint of 1628 human samples

    PubMed Central

    Fernandez, Agustin F.; Assenov, Yassen; Martin-Subero, Jose Ignacio; Balint, Balazs; Siebert, Reiner; Taniguchi, Hiroaki; Yamamoto, Hiroyuki; Hidalgo, Manuel; Tan, Aik-Choon; Galm, Oliver; Ferrer, Isidre; Sanchez-Cespedes, Montse; Villanueva, Alberto; Carmona, Javier; Sanchez-Mut, Jose V.; Berdasco, Maria; Moreno, Victor; Capella, Gabriel; Monk, David; Ballestar, Esteban; Ropero, Santiago; Martinez, Ramon; Sanchez-Carbayo, Marta; Prosper, Felipe; Agirre, Xabier; Fraga, Mario F.; Graña, Osvaldo; Perez-Jurado, Luis; Mora, Jaume; Puig, Susana; Prat, Jaime; Badimon, Lina; Puca, Annibale A.; Meltzer, Stephen J.; Lengauer, Thomas; Bridgewater, John; Bock, Christoph; Esteller, Manel

    2012-01-01

    Most of the studies characterizing DNA methylation patterns have been restricted to particular genomic loci in a limited number of human samples and pathological conditions. Herein, we present a compromise between an extremely comprehensive study of a human sample population with an intermediate level of resolution of CpGs at the genomic level. We obtained a DNA methylation fingerprint of 1628 human samples in which we interrogated 1505 CpG sites. The DNA methylation patterns revealed show this epigenetic mark to be critical in tissue-type definition and stemness, particularly around transcription start sites that are not within a CpG island. For disease, the generated DNA methylation fingerprints show that, during tumorigenesis, human cancer cells underwent a progressive gain of promoter CpG-island hypermethylation and a loss of CpG methylation in non-CpG-island promoters. Although transformed cells are those in which DNA methylation disruption is more obvious, we observed that other common human diseases, such as neurological and autoimmune disorders, had their own distinct DNA methylation profiles. Most importantly, we provide proof of principle that the DNA methylation fingerprints obtained might be useful for translational purposes by showing that we are able to identify the tumor type origin of cancers of unknown primary origin (CUPs). Thus, the DNA methylation patterns identified across the largest spectrum of samples, tissues, and diseases reported to date constitute a baseline for developing higher-resolution DNA methylation maps and provide important clues concerning the contribution of CpG methylation to tissue identity and its changes in the most prevalent human diseases. PMID:21613409

  16. Fluorescence-labeled methylation-sensitive amplified fragment length polymorphism (FL-MS-AFLP) analysis for quantitative determination of DNA methylation and demethylation status.

    PubMed

    Kageyama, Shinji; Shinmura, Kazuya; Yamamoto, Hiroko; Goto, Masanori; Suzuki, Koichi; Tanioka, Fumihiko; Tsuneyoshi, Toshihiro; Sugimura, Haruhiko

    2008-04-01

    The PCR-based DNA fingerprinting method called the methylation-sensitive amplified fragment length polymorphism (MS-AFLP) analysis is used for genome-wide scanning of methylation status. In this study, we developed a method of fluorescence-labeled MS-AFLP (FL-MS-AFLP) analysis by applying a fluorescence-labeled primer and fluorescence-detecting electrophoresis apparatus to the existing method of MS-AFLP analysis. The FL-MS-AFLP analysis enables quantitative evaluation of more than 350 random CpG loci per run. It was shown to allow evaluation of the differences in methylation level of blood DNA of gastric cancer patients and evaluation of hypermethylation and hypomethylation in DNA from gastric cancer tissue in comparison with adjacent non-cancerous tissue.

  17. Xeroderma pigmentosum neurological abnormalities correlate with colony-forming ability after ultraviolet radiation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Andrews, A.D.; Barrett, S.F.; Robbins, J.H.

    1978-04-01

    Xeroderma pigmentosum is an autosomal recessive disease in which DNA repair processes are defective. All xeroderma pigmentosum patients develop premature aging of sun exposed skin, and some develop neurological abnormalities due to premature death of nerve cells. Sensitivity to ultraviolet radiation of 24 xeroderma pigmentosum fibroblast strains was studied in vitro by measuring each strain's ability to divide and form colonies after irradiation. The most sensitive strains were derived from patients who had an early onset of neurological abnormalities; less sensitive strains were from patients with a later onset; and the most resistant strains were from patients without neurological abnormalities.more » The uv sensitivities of strains from each member of a sibling pair with xeroderma pigmentosum were identical, indicating that uv sensitivity of xeroderma pigmentosum strains is determined by the patient's inherited DNA repair defect. The results suggest that effective DNA repair is required to maintain the functional integrity of the human nervous system by preventing premature death of neurons.« less

  18. Differential DNA hypermethylation of critical genes mediates the stage-specific tobacco smoke-induced neoplastic progression of lung cancer.

    PubMed

    Russo, Andrea L; Thiagalingam, Arunthathi; Pan, Hongjie; Califano, Joseph; Cheng, Kuang-hung; Ponte, Jose F; Chinnappan, Dharmaraj; Nemani, Pratima; Sidransky, David; Thiagalingam, Sam

    2005-04-01

    Promoter DNA methylation status of six genes in samples derived from 27 bronchial epithelial cells and matching blood samples from 22 former/current smokers and five nonsmokers as well as 49 primary non-small cell lung cancer samples with corresponding blood controls was determined using methylation-specific PCR (MSP). Lung tumor tissues showed a significantly higher frequency of promoter DNA methylation in p16, MGMT, and DAPK (P < 0.05; Fisher's exact test). p16 promoter DNA methylation in tumors was observed at consistently higher levels when compared with all the other samples analyzed (P = 0.001; Fisher's exact test). ECAD and DAPK exhibited statistically insignificant differences in their levels of DNA methylation among the tumors and bronchial epithelial cells from the smokers. Interestingly, similar levels of methylation were observed in bronchial epithelial cells and corresponding blood from smokers for all four genes (ECAD, p16, MGMT, and DAPK) that showed smoking/lung cancer-associated methylation changes. In summary, our data suggest that targeted DNA methylation silencing of ECAD and DAPK occurs in the early stages and that of p16 and MGMT in the later stages of lung cancer progression. We also provide preliminary evidence that peripheral lymphocytes could potentially be used as a surrogate for bronchial epithelial cells to detect altered DNA methylation in smokers.

  19. Methyl-donor deficiency in adolescence affects memory and epigenetic status in the mouse hippocampus.

    PubMed

    Tomizawa, H; Matsuzawa, D; Ishii, D; Matsuda, S; Kawai, K; Mashimo, Y; Sutoh, C; Shimizu, E

    2015-03-01

    DNA methylation is one of the essential factors in the control of gene expression. Alteration of the DNA methylation pattern has been linked to various neurological, behavioral and neurocognitive dysfunctions. Recent studies have pointed out the importance of epigenetics in brain development and functions including learning and memory. Nutrients related to one-carbon metabolism are known to play important roles in the maintenance of genomic DNA methylation. Previous studies have shown that the long-term administration of a diet lacking essential one-carbon nutrients such as methionine, choline and folic acid (methyl donors) caused global DNA hypermethylation in the brain. Therefore, the long-term feeding of a methyl-donor-deficient diet may cause abnormal brain development including learning and memory. To confirm this hypothesis, 3-week-old mice were maintained on a folate-, methionine- and choline-deficient (FMCD) or control (CON) diet for 3 weeks. We found that the methyl-donor deficiency impaired both novel object recognition and fear extinction after 3 weeks of treatment. The FMCD group showed spontaneous recovery of fear that differed from that in CON. In addition, we found decreased Gria1 gene expression and specific CpG hypermethylation of the Gria1 promoter region in the FMCD hippocampus. Our data suggest that a chronic dietary lack of methyl donors in the developmental period affects learning, memory and gene expressions in the hippocampus. © 2015 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

  20. Effect of different doses of nandrolone decanoate on lipid peroxidation, DNA fragmentation, sperm abnormality and histopathology of testes of male Wister rats.

    PubMed

    Mohamed, Hanaa Mahmoud; Mohamed, Manal Abdul-Hamid

    2015-01-01

    The present study aims of to investigate the effects of low and high doses of nandrolone decanoate (ND) on histopathology and apoptosis of the spermatogenic cells as well as lipid peroxidation, antioxidant enzyme activities, sperm abnormality and DNA fragmentation. Eighteen animals were divided into three groups each group contain six animals. The rats were divided into three groups as following: Group 1 was administered saline (control). Group 2, received nandrolone decanoate (3 mg/kg/weekly) (low dose) with intramuscular injection. Group 3, received intramuscular injection dose of nandrolone decanoate (10 mg/kg/weekly) (high dose). After 8 weeks, caspase-3 assay was used to determine the apoptotic cells. The sperm parameters, lipid peroxidation, antioxidant enzyme activities and testosterone concentration were also investigated in the experimental groups of both low and high dose compared to the control groups. Treated group with high dose showed degenerated germinal epithelial cells sloughed in the lumina of seminiferous tubules, where almost seminiferous tubules were devoid of spermatids and spermatozoa compared to control and group treated with low dose. Also, a significant increase of lipid peroxidation levels and heat shock proteins was observed in two groups administrated with two different doses of ND while, antioxidant enzyme activities, and testosterone concentration was significantly decreased in two treated group when compared with control. Administration of ND at high and low doses leads to deteriorated sperm parameters, DNA fragmentation and testicular apoptosis. In conclusion, the administration ND at high doses more effective on lipid peroxidation, antioxidant enzyme activities, sperm abnormality, histopathology, apoptotic and DNA changes compared to low dose group and to control group. Published by Elsevier GmbH.

  1. Prostate cancer molecular detection in plasma samples by glutathione S-transferase P1 (GSTP1) methylation analysis.

    PubMed

    Dumache, Raluca; Puiu, Maria; Motoc, Marilena; Vernic, Corina; Dumitrascu, Victor

    2014-01-01

    Prostate cancer (PCa) represents the most commonly diagnosed type of malignancy among men in Western European countries and the second cause of cancer-related deaths among men worldwide. Methylation of the CpG island has an important role in prostate carcinogenesis and progression. The purpose of the study was to analyse the diagnostic value of aberrant promoter hypermethylation of the gene for glutathione S-transferase P1 (GSTP1) in plasma DNA to discriminate between prostate cancer (PCa) and benign prostatic hyperplasia (BPH) patients by minimally invasive methods. Aberrant promoter hypermethylation was investigated in DNA isolated from plasma samples of 31 patients with diagnostic of PCa and 44 cancer-free males (control subjects). Extracted genomic DNA was bisulfite treated and analyzed using methylation-specific polymerase chain reaction (MS-PCR) technique. Hypermethylation of the GSTP1 gene was detected in plasma samples from 27 of 31 (92.86%) patients with PCa. Genomic DNA from plasma samples from the 44 controls without genitourinary cancer revealed promoter hypermethylation of GSTP1 gene in 3 (10.6%) of the 44 patients. Receiver operating curve (ROC) included clinico-pathological parameters such as: serum PSA levels, pathological stage, Gleason score, hypermethylation status of GSTP1 gene, and it gave a predictive accuracy of 93% with a sensitivity and specificity of 95% and 87%, respectively. In this study, we have evaluated the ability of GSTP1 gene to discriminate between PCa and BPH patients in genomic DNA from plasma samples by non-invasive methods.

  2. Parental vitamin D deficiency during pregnancy is associated with increased blood pressure in offspring via Panx1 hypermethylation.

    PubMed

    Meems, Laura M G; Mahmud, Hasan; Buikema, Hendrik; Tost, Jörg; Michel, Sven; Takens, Janny; Verkaik-Schakel, Rikst N; Vreeswijk-Baudoin, Inge; Mateo-Leach, Irene V; van der Harst, Pim; Plösch, Torsten; de Boer, Rudolf A

    2016-12-01

    Vitamin D deficiency is one of the most common nutritional deficiencies worldwide. Maternal vitamin D deficiency is associated with increased susceptibility to hypertension in offspring, but the reasons for this remain unknown. The aim of this study was to determine if parental vitamin D deficiency leads to altered DNA methylation in offspring that may relate to hypertension. Male and female Sprague-Dawley rats were fed a standard or vitamin D-depleted diet. After 10 wk, nonsibling rats were mated. The conceived pups received standard chow. We observed an increased systolic and diastolic blood pressure in the offspring from depleted parents (F1-depl). Genome-wide methylation analyses in offspring identified hypermethylation of the promoter region of the Pannexin-1 (Panx1) gene in F1-depl rats. Panx1 encodes a hemichannel known to be involved in endothelial-dependent relaxation, and we demonstrated that in F1-depl rats the increase in blood pressure was associated with impaired endothelial relaxation of the large vessels, suggesting an underlying biological mechanism of increased blood pressure in children from parents with vitamin deficiency. Parental vitamin D deficiency is associated with epigenetic changes and increased blood pressure levels in offspring. Copyright © 2016 the American Physiological Society.

  3. A lincRNA connected to cell mortality and epigenetically-silenced in most common human cancers

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Vrba, Lukas; Garbe, James C.; Stampfer, Martha R.

    Immortality is an essential characteristic of human carcinoma cells. We recently developed an efficient, reproducible method that immortalizes human mammary epithelial cells (HMEC) in the absence of gross genomic changes by targeting 2 critical senescence barriers. Consistent transcriptomic changes associated with immortality were identified using microarray analysis of isogenic normal finite pre-stasis, abnormal finite post-stasis, and immortal HMECs from 4 individuals. A total of 277 genes consistently changed in cells that transitioned from post-stasis to immortal. Gene ontology analysis of affected genes revealed biological processes significantly altered in the immortalization process. These immortalization-associated changes showed striking similarity to the genemore » expression changes seen in The Cancer Genome Atlas (TCGA) clinical breast cancer data. The most dramatic change in gene expression seen during the immortalization step was the downregulation of an unnamed, incompletely annotated transcript that we called MORT, for mortality, since its expression was closely associated with the mortal, finite lifespan phenotype. We show here that MORT (ZNF667-AS1) is expressed in all normal finite lifespan human cells examined to date and is lost in immortalized HMEC. MORT gene silencing at the mortal/immortal boundary was due to DNA hypermethylation of its CpG island promoter. This epigenetic silencing is also seen in human breast cancer cell lines and in a majority of human breast tumor tissues. The functional importance of DNA hypermethylation in MORT gene silencing is supported by the ability of 5-aza-2'- deoxycytidine to reactivate MORT expression. Analysis of TCGA data revealed deregulation of MORT expression due to DNA hypermethylation in 15 out of the 17 most common human cancers. In conclusion, the epigenetic silencing of MORT in a large majority of the common human cancers suggests a potential fundamental role in cellular immortalization during human

  4. A lincRNA connected to cell mortality and epigenetically-silenced in most common human cancers

    DOE PAGES

    Vrba, Lukas; Garbe, James C.; Stampfer, Martha R.; ...

    2015-10-19

    Immortality is an essential characteristic of human carcinoma cells. We recently developed an efficient, reproducible method that immortalizes human mammary epithelial cells (HMEC) in the absence of gross genomic changes by targeting 2 critical senescence barriers. Consistent transcriptomic changes associated with immortality were identified using microarray analysis of isogenic normal finite pre-stasis, abnormal finite post-stasis, and immortal HMECs from 4 individuals. A total of 277 genes consistently changed in cells that transitioned from post-stasis to immortal. Gene ontology analysis of affected genes revealed biological processes significantly altered in the immortalization process. These immortalization-associated changes showed striking similarity to the genemore » expression changes seen in The Cancer Genome Atlas (TCGA) clinical breast cancer data. The most dramatic change in gene expression seen during the immortalization step was the downregulation of an unnamed, incompletely annotated transcript that we called MORT, for mortality, since its expression was closely associated with the mortal, finite lifespan phenotype. We show here that MORT (ZNF667-AS1) is expressed in all normal finite lifespan human cells examined to date and is lost in immortalized HMEC. MORT gene silencing at the mortal/immortal boundary was due to DNA hypermethylation of its CpG island promoter. This epigenetic silencing is also seen in human breast cancer cell lines and in a majority of human breast tumor tissues. The functional importance of DNA hypermethylation in MORT gene silencing is supported by the ability of 5-aza-2'- deoxycytidine to reactivate MORT expression. Analysis of TCGA data revealed deregulation of MORT expression due to DNA hypermethylation in 15 out of the 17 most common human cancers. In conclusion, the epigenetic silencing of MORT in a large majority of the common human cancers suggests a potential fundamental role in cellular immortalization during human

  5. Sensorineural hearing loss in a pediatric population: association of congenital cytomegalovirus infection with intracranial abnormalities.

    PubMed

    Kimani, Jane W; Buchman, Craig A; Booker, Jessica K; Huang, Benjamin Y; Castillo, Mauricio; Powell, Cynthia M; Weck, Karen E

    2010-10-01

    To examine the incidence of congenital cytomegalovirus (CMV) infection relative to common genetic etiologies of hearing loss in a pediatric population with sensorineural hearing loss (SNHL), and to characterize intracranial radiological abnormalities in patients with CMV-associated hearing loss. Retrospective study. Academic tertiary care center. A total of 112 pediatric patients with confirmed SNHL. The association of congenital CMV infection status with abnormal brain magnetic resonance imaging (MRI) scans and the frequencies of congenital CMV infection, gap junction β-2 (GJB2) mutations, and the mitochondrial DNA (mtDNA) 1555A>G mutation in children with SNHL. Of 109 patients, 11 (10%) had positive results for CMV DNA; 10 of the 11 had normal GJB2 sequence and had negative test results for the mtDNA 1555A>G mutation. Brain MRI scans for 97 patients demonstrated a higher proportion of abnormalities in patients with positive CMV test results (80%) compared with those with no detectable CMV DNA (33%) (P = .006). GJB2 mutations and the mtDNA 1555A>G mutation were seen in 10 of 88 patients (11%) and 1 of 97 patients (1%) with SNHL, respectively. The presence of brain abnormalities in most patients with congenital CMV infection suggests that neurological damage in otherwise asymptomatic patients may not be limited to SNHL. Congenital CMV infection accounted for a significant proportion of patients with SNHL, with an incidence rate comparable with that of GJB2-related SNHL.

  6. DNA methylation biomarkers for head and neck squamous cell carcinoma.

    PubMed

    Zhou, Chongchang; Ye, Meng; Ni, Shumin; Li, Qun; Ye, Dong; Li, Jinyun; Shen, Zhishen; Deng, Hongxia

    2018-06-21

    DNA methylation plays an important role in the etiology and pathogenesis of head and neck squamous cell carcinoma (HNSCC). The current study aimed to identify aberrantly methylated-differentially expressed genes (DEGs) by a comprehensive bioinformatics analysis. In addition, we screened for DEGs affected by DNA methylation modification and further investigated their prognostic values for HNSCC. We included microarray data of DNA methylation (GSE25093 and GSE33202) and gene expression (GSE23036 and GSE58911) from Gene Expression Omnibus. Aberrantly methylated-DEGs were analyzed with R software. The Cancer Genome Atlas (TCGA) RNA sequencing and DNA methylation (Illumina HumanMethylation450) databases were utilized for validation. In total, 27 aberrantly methylated genes accompanied by altered expression were identified. After confirmation by The Cancer Genome Atlas (TCGA) database, 2 hypermethylated-low-expression genes (FAM135B and ZNF610) and 2 hypomethylated-high-expression genes (HOXA9 and DCC) were identified. A receiver operating characteristic (ROC) curve confirmed the diagnostic value of these four methylated genes for HNSCC. Multivariate Cox proportional hazards analysis showed that FAM135B methylation was a favorable independent prognostic biomarker for overall survival of HNSCC patients.

  7. DNA methylation profiling of esophageal adenocarcinoma using Methylation Ligation-dependent Macroarray (MLM).

    PubMed

    Guilleret, Isabelle; Losi, Lorena; Chelbi, Sonia T; Fonda, Sergio; Bougel, Stéphanie; Saponaro, Sara; Gozzi, Gaia; Alberti, Loredana; Braunschweig, Richard; Benhattar, Jean

    2016-10-14

    Most types of cancer cells are characterized by aberrant methylation of promoter genes. In this study, we described a rapid, reproducible, and relatively inexpensive approach allowing the detection of multiple human methylated promoter genes from many tissue samples, without the need of bisulfite conversion. The Methylation Ligation-dependent Macroarray (MLM), an array-based analysis, was designed in order to measure methylation levels of 58 genes previously described as putative biomarkers of cancer. The performance of the design was proven by screening the methylation profile of DNA from esophageal cell lines, as well as microdissected formalin-fixed and paraffin-embedded (FFPE) tissues from esophageal adenocarcinoma (EAC). Using the MLM approach, we identified 32 (55%) hypermethylated promoters in EAC, and not or rarely methylated in normal tissues. Among them, 21promoters were found aberrantly methylated in more than half of tumors. Moreover, seven of them (ADAMTS18, APC, DKK2, FOXL2, GPX3, TIMP3 and WIF1) were found aberrantly methylated in all or almost all the tumor samples, suggesting an important role for these genes in EAC. In addition, dysregulation of the Wnt pathway with hypermethylation of several Wnt antagonist genes was frequently observed. MLM revealed a homogeneous pattern of methylation for a majority of tumors which were associated with an advanced stage at presentation and a poor prognosis. Interestingly, the few tumors presenting less methylation changes had a lower pathological stage. In conclusion, this study demonstrated the feasibility and accuracy of MLM for DNA methylation profiling of FFPE tissue samples. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. SMYD2 promoter DNA methylation is associated with abdominal aortic aneurysm (AAA) and SMYD2 expression in vascular smooth muscle cells.

    PubMed

    Toghill, Bradley J; Saratzis, Athanasios; Freeman, Peter J; Sylvius, Nicolas; Bown, Matthew J

    2018-01-01

    Abdominal aortic aneurysm (AAA) is a deadly cardiovascular disease characterised by the gradual, irreversible dilation of the abdominal aorta. AAA is a complex genetic disease but little is known about the role of epigenetics. Our objective was to determine if global DNA methylation and CpG-specific methylation at known AAA risk loci is associated with AAA, and the functional effects of methylation changes. We assessed global methylation in peripheral blood mononuclear cell DNA from 92 individuals with AAA and 93 controls using enzyme-linked immunosorbent assays, identifying hyper-methylation in those with large AAA and a positive linear association with AAA diameter ( P  < 0.0001, R 2  = 0.3175).We then determined CpG methylation status of regulatory regions in genes located at AAA risk loci identified in genome-wide association studies, using bisulphite next-generation sequencing (NGS) in vascular smooth muscle cells (VSMCs) taken from aortic tissues of 44 individuals (24 AAAs and 20 controls). In IL6R , 2 CpGs were hyper-methylated ( P  = 0.0145); in ERG , 13 CpGs were hyper-methylated ( P  = 0.0005); in SERPINB9 , 6 CpGs were hypo-methylated ( P  = 0.0037) and 1 CpG was hyper-methylated ( P  = 0.0098); and in SMYD2 , 4 CpGs were hypo-methylated ( P  = 0.0012).RT-qPCR was performed for each differentially methylated gene on mRNA from the same VSMCs and compared with methylation. This analysis revealed downregulation of SMYD2 and SERPINB9 in AAA, and a direct linear relationship between SMYD2 promoter methylation and SMYD2 expression ( P  = 0.038). Furthermore, downregulation of SMYD2 at the site of aneurysm in the aortic wall was further corroborated in 6 of the same samples used for methylation and gene expression analysis with immunohistochemistry. This study is the first to assess DNA methylation in VSMCs from individuals with AAA using NGS, and provides further evidence there is an epigenetic basis to AAA. Our study shows that

  9. MicroRNA-145 is regulated by DNA methylation and p53 gene mutation in prostate cancer

    PubMed Central

    Suh, Seong O.; Chen, Yi; Zaman, Mohd Saif; Hirata, Hiroshi; Yamamura, Soichiro; Shahryari, Varahram; Liu, Jan; Tabatabai, Z.Laura; Kakar, Sanjay; Deng, Guoren; Tanaka, Yuichiro; Dahiya, Rajvir

    2011-01-01

    MiR-145 is downregulated in various cancers including prostate cancer. However, the underlying mechanisms of miR-145 downregulation are not fully understood. Here, we reported that miR-145 was silenced through DNA hypermethylation and p53 mutation status in laser capture microdissected (LCM) prostate cancer and matched adjacent normal tissues. In 22 of 27 (81%) prostate tissues, miR-145 was significantly downregulated in the cancer compared with the normal tissues. Further studies on miR-145 downregulation mechanism showed that miR-145 is methylated at the promoter region in both prostate cancer tissues and 50 different types of cancer cell lines. In seven cancer cell lines with miR-145 hypermethylation, 5-aza-2′-deoxycytidine treatment dramatically induced miR-145 expression. Interestingly, we also found a significant correlation between miR-145 expression and the status of p53 gene in both LCM prostate tissues and 47 cancer cell lines. In 29 cell lines with mutant p53, miR-145 levels were downregulated in 28 lines (97%), whereas in 18 cell lines with wild-type p53 (WT p53), miR-145 levels were downregulated in only 6 lines (33%, P < 0.001). Electrophoretic mobility shift assay showed that p53 binds to the p53 response element upstream of miR-145, but the binding was inhibited by hypermethylation. To further confirm that p53 binding to miR-145 could regulate miR-145 expression, we transfected WT p53 and MUT p53 into PC-3 cells and found that miR-145 is upregulated by WT p53 but not with MUTp53. The apoptotic cells are increased after WT p53 transfection. In summary, this is the first report documenting that downregulation of miR-145 is through DNA methylation and p53 mutation pathways in prostate cancer. PMID:21349819

  10. Distinct DNA methylation patterns associated with active and inactive centromeres of the maize B chromosome.

    PubMed

    Koo, Dal-Hoe; Han, Fangpu; Birchler, James A; Jiang, Jiming

    2011-06-01

    Centromeres are determined by poorly understood epigenetic mechanisms. Centromeres can be activated or inactivated without changing the underlying DNA sequences. However, virtually nothing is known about the epigenetic transition of a centromere from an active to an inactive state because of the lack of examples of the same centromere exhibiting alternative forms and being distinguishable from other centromeres. The centromere of the supernumerary B chromosome of maize provides such an opportunity because its functional core can be cytologically tracked, and an inactive version of the centromere is available. We developed a DNA fiber-based technique that can be used to assess the levels of cytosine methylation associated with repetitive DNA sequences. We report that DNA sequences in the normal B centromere exhibit hypomethylation. This methylation pattern is not affected by the genetic background or structural rearrangement of the B chromosome, but is slightly changed when the B chromosome is transferred to oat as an addition chromosome. In contrast, an inactive version of this same centromere exhibits hypermethylation, indicating that the inactive centromere was modified into a different epigenetic state at the DNA level.

  11. Increasing methylation of the calcitonin gene during disease progression in sequential samples from CML patients.

    PubMed

    Mills, K I; Guinn, B A; Walsh, V A; Burnett, A K

    1996-09-01

    In chronic myeloid leukaemia (CML), disease progression from the initial chronic phase to the acute phase or blast crisis has previously been shown to be correlated with progressive increases in hyper-methylation of the calcitonin gene, located at chromosome 11p15. However, sequential studies of individual patients were not performed in these investigations. We have analysed 44 samples from nine patients with typical Philadelphia chromosome positive CML throughout their disease progression to determine the methylation state of the calcitonin gene at these time points. Densitometry was used to quantitate the ratio of the normal 2.0 kb Hpa II fragments, indicating normal methylation status of the gene, compared to the intensity of the abnormal, hyper-methylated, 2.6-3.1 kb Hpa II fragments. We found a gradual increase in the ratio of methylated:unmethylated calcitonin gene during chronic phase with a dramatic rise at blast crisis. Further, the ratio of the abnormal hypermethylated 3.1 kb fragments to the methylated 2.6 kb fragment resulted in the identification of a clonal expansion of abnormally methylated cells. This expansion of cells with hypermethylation of the calcitonin gene during chronic phase was shown to coincide with the presence of a mutation in the p53 gene. The data presented in this study would suggest that an increased methylation status of the calcitonin gene during disease progression may indicate the expansion of abnormal blast cell populations and subsequent progression to blast crisis.

  12. Ribosomal DNA stability is supported by many 'buffer genes'-introduction to the Yeast rDNA Stability Database.

    PubMed

    Kobayashi, Takehiko; Sasaki, Mariko

    2017-01-01

    The ribosomal RNA gene (rDNA) is the most abundant gene in yeast and other eukaryotic organisms. Due to its heavy transcription, repetitive structure and programmed replication fork pauses, the rDNA is one of the most unstable regions in the genome. Thus, the rDNA is the best region to study the mechanisms responsible for maintaining genome integrity. Recently, we screened a library of ∼4800 budding yeast gene knockout strains to identify mutants defective in the maintenance of rDNA stability. The results of this screen are summarized in the Yeast rDNA Stability (YRS) Database, in which the stability and copy number of rDNA in each mutant are presented. From this screen, we identified ∼700 genes that may contribute to the maintenance of rDNA stability. In addition, ∼50 mutants had abnormally high or low rDNA copy numbers. Moreover, some mutants with unstable rDNA displayed abnormalities in another chromosome. In this review, we introduce the YRS Database and discuss the roles of newly identified genes that contribute to rDNA maintenance and genome integrity. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  13. Unique cell-type-specific patterns of DNA methylation in the root meristem.

    PubMed

    Kawakatsu, Taiji; Stuart, Tim; Valdes, Manuel; Breakfield, Natalie; Schmitz, Robert J; Nery, Joseph R; Urich, Mark A; Han, Xinwei; Lister, Ryan; Benfey, Philip N; Ecker, Joseph R

    2016-04-29

    DNA methylation is an epigenetic modification that differs between plant organs and tissues, but the extent of variation between cell types is not known. Here, we report single-base-resolution whole-genome DNA methylomes, mRNA transcriptomes and small RNA transcriptomes for six cell populations covering the major cell types of the Arabidopsis root meristem. We identify widespread cell-type-specific patterns of DNA methylation, especially in the CHH sequence context, where H is A, C or T. The genome of the columella root cap is the most highly methylated Arabidopsis cell characterized so far. It is hypermethylated within transposable elements (TEs), accompanied by increased abundance of transcripts encoding RNA-directed DNA methylation (RdDM) pathway components and 24-nt small RNAs (smRNAs). The absence of the nucleosome remodeller DECREASED DNA METHYLATION 1 (DDM1), required for maintenance of DNA methylation, and low abundance of histone transcripts involved in heterochromatin formation suggests that a loss of heterochromatin may occur in the columella, thus allowing access of RdDM factors to the whole genome, and producing an excess of 24-nt smRNAs in this tissue. Together, these maps provide new insights into the epigenomic diversity that exists between distinct plant somatic cell types.

  14. Densely ionizing radiation affects DNA methylation of selective LINE-1 elements1

    PubMed Central

    Prior, Sara; Miousse, Isabelle R.; Nzabarushimana, Etienne; Pathak, Rupak; Skinner, Charles; Kutanzi, Kristy R.; Allen, Antiño R.; Raber, Jacob; Tackett, Alan J.; Hauer-Jensen, Martin; Nelson, Gregory A.; Koturbash, Igor

    2016-01-01

    Long Interspersed Nucleotide Element 1 (LINE-1) retrotransposons are heavily methylated and are the most abundant transposable elements in mammalian genomes. Here, we investigated the differential DNA methylation within the LINE-1 under normal conditions and in response to environmentally relevant doses of sparsely and densely ionizing radiation. We demonstrate that DNA methylation of LINE-1 elements in the lungs of C57BL6 mice is dependent on their evolutionary age, where the elder age of the element is associated with the lower extent of DNA methylation. Exposure to 5-aza-2′-deoxycytidine and methionine-deficient diet affected DNA methylation of selective LINE-1 elements in an age- and promoter type-dependent manner. Exposure to densely IR, but not sparsely IR, resulted in DNA hypermethylation of older LINE-1 elements, while the DNA methylation of evolutionary younger elements remained mostly unchanged. We also demonstrate that exposure to densely IR increased mRNA and protein levels of LINE-1 via the loss of the histone H3K9 dimethylation and an increase in the H3K4 trimethylation at the LINE-1 5′-untranslated region, independently of DNA methylation. Our findings suggest that DNA methylation is important for regulation of LINE-1 expression under normal conditions, but histone modifications may dictate the transcriptional activity of LINE-1 in response to exposure to densely IR. PMID:27419368

  15. Absolute quantification of DNA methylation using microfluidic chip-based digital PCR.

    PubMed

    Wu, Zhenhua; Bai, Yanan; Cheng, Zule; Liu, Fangming; Wang, Ping; Yang, Dawei; Li, Gang; Jin, Qinghui; Mao, Hongju; Zhao, Jianlong

    2017-10-15

    Hypermethylation of CpG islands in the promoter region of many tumor suppressor genes downregulates their expression and in a result promotes tumorigenesis. Therefore, detection of DNA methylation status is a convenient diagnostic tool for cancer detection. Here, we reported a novel method for the integrative detection of methylation by the microfluidic chip-based digital PCR. This method relies on methylation-sensitive restriction enzyme HpaII, which cleaves the unmethylated DNA strands while keeping the methylated ones intact. After HpaII treatment, the DNA methylation level is determined quantitatively by the microfluidic chip-based digital PCR with the lower limit of detection equal to 0.52%. To validate the applicability of this method, promoter methylation of two tumor suppressor genes (PCDHGB6 and HOXA9) was tested in 10 samples of early stage lung adenocarcinoma and their adjacent non-tumorous tissues. The consistency was observed in the analysis of these samples using our method and a conventional bisulfite pyrosequencing. Combining high sensitivity and low cost, the microfluidic chip-based digital PCR method might provide a promising alternative for the detection of DNA methylation and early diagnosis of epigenetics-related diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Comprehensive analyses of imprinted differentially methylated regions reveal epigenetic and genetic characteristics in hepatoblastoma

    PubMed Central

    2013-01-01

    Background Aberrant methylation at imprinted differentially methylated regions (DMRs) in human 11p15.5 has been reported in many tumors including hepatoblastoma. However, the methylation status of imprinted DMRs in imprinted loci scattered through the human genome has not been analyzed yet in any tumors. Methods The methylation statuses of 33 imprinted DMRs were analyzed in 12 hepatoblastomas and adjacent normal liver tissue by MALDI-TOF MS and pyrosequencing. Uniparental disomy (UPD) and copy number abnormalities were investigated with DNA polymorphisms. Results Among 33 DMRs analyzed, 18 showed aberrant methylation in at least 1 tumor. There was large deviation in the incidence of aberrant methylation among the DMRs. KvDMR1 and IGF2-DMR0 were the most frequently hypomethylated DMRs. INPP5Fv2-DMR and RB1-DMR were hypermethylated with high frequencies. Hypomethylation was observed at certain DMRs not only in tumors but also in a small number of adjacent histologically normal liver tissue, whereas hypermethylation was observed only in tumor samples. The methylation levels of long interspersed nuclear element-1 (LINE-1) did not show large differences between tumor tissue and normal liver controls. Chromosomal abnormalities were also found in some tumors. 11p15.5 and 20q13.3 loci showed the frequent occurrence of both genetic and epigenetic alterations. Conclusions Our analyses revealed tumor-specific aberrant hypermethylation at some imprinted DMRs in 12 hepatoblastomas with additional suggestion for the possibility of hypomethylation prior to tumor development. Some loci showed both genetic and epigenetic alterations with high frequencies. These findings will aid in understanding the development of hepatoblastoma. PMID:24373183

  17. Genome-wide methylation sequencing of paired primary and metastatic cell lines identifies common DNA methylation changes and a role for EBF3 as a candidate epigenetic driver of melanoma metastasis

    PubMed Central

    Chatterjee, Aniruddha; Stockwell, Peter A; Ahn, Antonio; Rodger, Euan J; Leichter, Anna L; Eccles, Michael R

    2017-01-01

    Epigenetic alterations are increasingly implicated in metastasis, whereas very few genetic mutations have been identified as authentic drivers of cancer metastasis. Yet, to date, few studies have identified metastasis-related epigenetic drivers, in part because a framework for identifying driver epigenetic changes in metastasis has not been established. Using reduced representation bisulfite sequencing (RRBS), we mapped genome-wide DNA methylation patterns in three cutaneous primary and metastatic melanoma cell line pairs to identify metastasis-related epigenetic drivers. Globally, metastatic melanoma cell lines were hypomethylated compared to the matched primary melanoma cell lines. Using whole genome RRBS we identified 75 shared (10 hyper- and 65 hypomethylated) differentially methylated fragments (DMFs), which were associated with 68 genes showing significant methylation differences. One gene, Early B Cell Factor 3 (EBF3), exhibited promoter hypermethylation in metastatic cell lines, and was validated with bisulfite sequencing and in two publicly available independent melanoma cohorts (n = 40 and 458 melanomas, respectively). We found that hypermethylation of the EBF3 promoter was associated with increased EBF3 mRNA levels in metastatic melanomas and subsequent inhibition of DNA methylation reduced EBF3 expression. RNAi-mediated knockdown of EBF3 mRNA levels decreased proliferation, migration and invasion in primary and metastatic melanoma cell lines. Overall, we have identified numerous epigenetic changes characterising metastatic melanoma cell lines, including EBF3-induced aggressive phenotypic behaviour with elevated EBF3 expression in metastatic melanoma, suggesting that EBF3 promoter hypermethylation may be a candidate epigenetic driver of metastasis. PMID:28030832

  18. Aberrant DNA methylation of WNT pathway genes in the development and progression of CIMP-negative colorectal cancer.

    PubMed

    Galamb, Orsolya; Kalmár, Alexandra; Péterfia, Bálint; Csabai, István; Bodor, András; Ribli, Dezső; Krenács, Tibor; Patai, Árpád V; Wichmann, Barnabás; Barták, Barbara Kinga; Tóth, Kinga; Valcz, Gábor; Spisák, Sándor; Tulassay, Zsolt; Molnár, Béla

    2016-08-02

    The WNT signaling pathway has an essential role in colorectal carcinogenesis and progression, which involves a cascade of genetic and epigenetic changes. We aimed to analyze DNA methylation affecting the WNT pathway genes in colorectal carcinogenesis in promoter and gene body regions using whole methylome analysis in 9 colorectal cancer, 15 adenoma, and 6 normal tumor adjacent tissue (NAT) samples by methyl capture sequencing. Functional methylation was confirmed on 5-aza-2'-deoxycytidine-treated colorectal cancer cell line datasets. In parallel with the DNA methylation analysis, mutations of WNT pathway genes (APC, β-catenin/CTNNB1) were analyzed by 454 sequencing on GS Junior platform. Most differentially methylated CpG sites were localized in gene body regions (95% of WNT pathway genes). In the promoter regions, 33 of the 160 analyzed WNT pathway genes were differentially methylated in colorectal cancer vs. normal, including hypermethylated AXIN2, CHP1, PRICKLE1, SFRP1, SFRP2, SOX17, and hypomethylated CACYBP, CTNNB1, MYC; 44 genes in adenoma vs. NAT; and 41 genes in colorectal cancer vs. adenoma comparisons. Hypermethylation of AXIN2, DKK1, VANGL1, and WNT5A gene promoters was higher, while those of SOX17, PRICKLE1, DAAM2, and MYC was lower in colon carcinoma compared to adenoma. Inverse correlation between expression and methylation was confirmed in 23 genes, including APC, CHP1, PRICKLE1, PSEN1, and SFRP1. Differential methylation affected both canonical and noncanonical WNT pathway genes in colorectal normal-adenoma-carcinoma sequence. Aberrant DNA methylation appears already in adenomas as an early event of colorectal carcinogenesis.

  19. Overexpression of Tet3 in donor cells enhances goat somatic cell nuclear transfer efficiency.

    PubMed

    Han, Chengquan; Deng, Ruizhi; Mao, Tingchao; Luo, Yan; Wei, Biao; Meng, Peng; Zhao, Lu; Zhang, Qing; Quan, Fusheng; Liu, Jun; Zhang, Yong

    2018-05-23

    Ten-eleven translocation 3 (TET3) mediates active DNA demethylation of paternal genomes during mouse embryonic development. However, the mechanism of DNA demethylation in goat embryos remains unknown. In addition, aberrant DNA methylation reprogramming prevalently occurs in embryos cloned by somatic cell nuclear transfer (SCNT). In this study, we reported that TET3 is a key factor in DNA demethylation in goat pre-implantation embryos. Knockdown of Tet3 hindered DNA demethylation at the two- to four-cell stage in goat embryos and decreased Nanog expression in blastocysts. Overexpression of Tet3 in somatic cells can initiate DNA demethylation, reduce 5-methylcytosine level, increase 5-hydroxymethylcytosine level and promote the expression of key pluripotency genes. After SCNT, overexpression of Tet3 in donor cells corrected abnormal DNA hypermethylation of cloned embryos and significantly enhanced in vitro and in vivo developmental rate (P < 0.05). We conclude that overexpression of Tet3 in donor cells significantly improves goat SCNT efficiency. © 2018 Federation of European Biochemical Societies.

  20. Changes in DNA Methylation of Oocytes and Granulosa Cells Assessed by HELMET during Folliculogenesis in Mouse Ovary

    PubMed Central

    Liu, Jin; Zhang, Wenchang; Wu, Zhiren; Dai, Lei; Koji, Takehiko

    2018-01-01

    For a better understanding of epigenetic regulation of cell differentiation, it is important to analyze DNA methylation at a specific site. In this study, we examined changes in the methylation level of CCGG and GATCG sites during mouse folliculogenesis in paraffin-embedded sections of mouse ovaries. For the purpose, we used a new method, histo endonuclease-linked detection of methylation sites of DNA (HELMET), designed to detect methylation sites of DNA with a specific sequence in a tissue section. Unlike the global level of DNA methylation, which was no change in immunohistochemical staining of 5-methylcytosine throughout folliculogenesis, we found that there were hypermethylation of CCGG and GATCG sites in most of the granulosa cells of tertiary follicles compared to that of primary and secondary follicles. Interestingly, TUNEL-positive granulosa cells, which were frequent in mammalian folliculogenesis, became markedly Hpa II-reactive and Sau3A I-reactive, indicating that the CCGG and GATCG sites may be preferentially demethylated during apoptosis. PMID:29867282

  1. Changes in DNA Methylation of Oocytes and Granulosa Cells Assessed by HELMET during Folliculogenesis in Mouse Ovary.

    PubMed

    Liu, Jin; Zhang, Wenchang; Wu, Zhiren; Dai, Lei; Koji, Takehiko

    2018-04-27

    For a better understanding of epigenetic regulation of cell differentiation, it is important to analyze DNA methylation at a specific site. In this study, we examined changes in the methylation level of CCGG and GATCG sites during mouse folliculogenesis in paraffin-embedded sections of mouse ovaries. For the purpose, we used a new method, histo endonuclease-linked detection of methylation sites of DNA (HELMET), designed to detect methylation sites of DNA with a specific sequence in a tissue section. Unlike the global level of DNA methylation, which was no change in immunohistochemical staining of 5-methylcytosine throughout folliculogenesis, we found that there were hypermethylation of CCGG and GATCG sites in most of the granulosa cells of tertiary follicles compared to that of primary and secondary follicles. Interestingly, TUNEL-positive granulosa cells, which were frequent in mammalian folliculogenesis, became markedly Hpa II-reactive and Sau 3A I-reactive, indicating that the CCGG and GATCG sites may be preferentially demethylated during apoptosis.

  2. Vitrification of neat semen alters sperm parameters and DNA integrity.

    PubMed

    Khalili, Mohammad Ali; Adib, Maryam; Halvaei, Iman; Nabi, Ali

    2014-05-06

    Our aim was to evaluate the effect of neat semen vitrification on human sperm vital parameters and DNA integrity in men with normal and abnormal sperm parameters. Semen samples were 17 normozoospermic samples and 17 specimens with abnormal sperm parameters. Semen analysis was performed according to World Health Organization (WHO) criteria. Then, the smear was provided from each sample and fixed for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Vitrification of neat semen was done by plunging cryoloops directly into liquid nitrogen and preserved for 7 days. The samples were warmed and re-evaluated for sperm parameters as well as DNA integrity. Besides, the correlation between sperm parameters and DNA fragmentation was assessed pre- and post vitrification. Cryopreserved spermatozoa showed significant decrease in sperm motility, viability and normal morphology after thawing in both normal and abnormal semen. Also, the rate of sperm DNA fragmentation was significantly higher after vitrification compared to fresh samples in normal (24.76 ± 5.03 and 16.41 ± 4.53, P = .002) and abnormal (34.29 ± 10.02 and 23.5 ± 8.31, P < .0001), respectively. There was negative correlation between sperm motility and sperm DNA integrity in both groups after vitrification. Vitrification of neat ejaculates has negative impact on sperm parameters as well as DNA integrity, particularly among abnormal semen subjects. It is, therefore, recommend to process semen samples and vitrify the sperm pellets.

  3. Patients with colorectal cancer associated with Lynch syndrome and MLH1 promoter hypermethylation have similar prognoses.

    PubMed

    Haraldsdottir, Sigurdis; Hampel, Heather; Wu, Christina; Weng, Daniel Y; Shields, Peter G; Frankel, Wendy L; Pan, Xueliang; de la Chapelle, Albert; Goldberg, Richard M; Bekaii-Saab, Tanios

    2016-09-01

    Mismatch repair-deficient (dMMR) colorectal cancer (CRC) is caused by Lynch syndrome (LS) in 3% and sporadic inactivation of MLH1 by hypermethylation (MLH1-hm) in 12% of cases. It is not clear whether outcomes between LS-associated and MLH1-hm CRC differ. The objective of this study was to explore differences in clinical factors and outcomes in these two groups. Patients with dMMR CRC identified by immunohistochemistry staining and treated at a single institution from 1998 to 2012 were included. MLH1-hm was established with BRAF mutational analysis or hypermethylation testing. Patients' charts were accessed for information on pathology, germ-line MMR mutation testing, and clinical course. A total of 143 patients had CRC associated with LS (37 patients, 26%) or MLH1-hm (106 patients, 74%). Patients with LS were younger, more often male, presented more often with stage III disease, and had more metachronous disease than patients with MLH1-hm tumors. There was no difference in cancer-specific survival (CSS) between the groups; overall survival was longer in patients with LS, but this difference was minimal after adjusting for age and stage at diagnosis. CSS did not differ in LS-associated CRC compared with MLH1-hm CRC, suggesting that they carry a similar prognosis.Genet Med 18 9, 863-868.

  4. Effect of cryopreservation on sperm DNA integrity in patients with teratospermia.

    PubMed

    Kalthur, Guruprasad; Adiga, Satish Kumar; Upadhya, Dinesh; Rao, Satish; Kumar, Pratap

    2008-06-01

    To test whether sperm with abnormal head morphology are more likely to undergo DNA damage and/or chromatin modification during the process of freeze-thawing. In this prospective study, the semen samples from forty-four men attending the infertility clinic were included. Samples were divided into aliquots to allow direct comparison of fresh and frozen spermatozoa from the same ejaculate. The sperm morphology and the sperm DNA damage were evaluated before and after cryopreservation. The relationship between sperm head abnormalities and freeze-thaw-induced DNA modification was assessed. University hospital fertility center. Men attending infertility clinic for semen analysis. The normospermic and teratospermic semen samples were evaluated for DNA damage before and after cryopreservation by comet assay and acridine orange bindability test. Elucidation of association between sperm morphologic defect and cryodamage. A threefold increase in the amount of DNA damage was observed in teratospermic samples compared with their normospermic counterparts, indicating a higher susceptibility of morphologically abnormal sperm to cryodamage. The susceptibility of morphologically abnormal sperm to DNA damage/chromatin modification during the freeze-thaw process is significantly higher than that of sperm with normal morphology.

  5. Dietary betaine supplementation to gestational sows enhances hippocampal IGF2 expression in newborn piglets with modified DNA methylation of the differentially methylated regions.

    PubMed

    Li, Xi; Sun, Qinwei; Li, Xian; Cai, Demin; Sui, Shiyan; Jia, Yimin; Song, Haogang; Zhao, Ruqian

    2015-10-01

    The adequate supply of methyl donors is critical for the normal development of brain. The purpose of the present study was to investigate the effects of maternal betaine supplementation on hippocampal gene expression in neonatal piglets and to explore the possible mechanisms. Gestational sows were fed control or betaine-supplemented (3 g/kg) diets throughout the pregnancy. Immediately after birth, male piglets were killed, and the hippocampus was dissected for analyses. The mRNA abundance was determined by reverse transcription real-time polymerase chain reaction. Protein content was measured by Western blot, and DNA methylation was detected by methylated DNA immunoprecipitation assay. Prenatal betaine supplementation did not alter the body weight or the hippocampus weight, but increased the hippocampal DNA content as well as the mRNA expression of proliferation-related genes. Prenatal betaine supplementation increased serum level of methionine (P < 0.05) and up-regulated (P < 0.05) the mRNA and protein expression of betaine-homocysteine methyltransferase, glycine N-methyltransferase and DNA methyltransferase 1 in the neonatal hippocampus. Hippocampal expression of insulin growth factor II (IGF2) and its receptors IGF1R and IGF2R were all significantly up-regulated (P < 0.05) in betaine-treated group, together with a significant activation (P < 0.01) of the downstream extracellular signal-regulated kinase 1/2. Moreover, the differentially methylated region (DMR) 1 and 2 on IGF2 locus was found to be hypermethylated (P < 0.05) in the hippocampus of betaine-treated piglets. These results indicate that maternal betaine supplementation enhances betaine/methionine metabolism and DNA methyltransferase expression, causes hypermethylation of DMR on IGF2 gene, which was associated with augmented expression of IGF2 and cell proliferation/anti-apoptotic markers in the hippocampus of neonatal piglets.

  6. The expression of hematopoietic progenitor cell antigen CD34 is regulated by DNA methylation in a site-dependent manner in gastrointestinal stromal tumours.

    PubMed

    Bure, Irina; Braun, Alexander; Kayser, Claudia; Geddert, Helene; Schaefer, Inga-Marie; Cameron, Silke; Ghadimi, Michael B; Ströbel, Philipp; Werner, Martin; Hartmann, Arndt; Wiemann, Stefan; Agaimy, Abbas; Haller, Florian; Moskalev, Evgeny A

    2017-12-01

    The anatomic site-dependent expression of hematopoietic progenitor cell antigen CD34 is a feature of gastrointestinal stromal tumours (GISTs). The basis for the differential CD34 expression is only incompletely understood. This study aimed at understanding the regulation of CD34 in GISTs and clarification of its site-dependent expression. Two sample sets of primary GISTs were interrogated including 52 fresh-frozen and 134 paraffin-embedded and formalin-fixed specimens. DNA methylation analysis was performed by HumanMethylation450 BeadChip array in three cell lines derived from gastric and intestinal GISTs, and differentially methylated CpG sites were established upstream of CD34. The methylation degree was further quantified by pyrosequencing, and inverse correlation with CD34 mRNA and protein abundance was revealed. The gene's expression could be activated upon induction of DNA hypomethylation with 5-aza-2'-deoxycytidine in GIST-T1 cells. In patient samples, a strong inverse correlation of DNA methylation degree with immunohistochemically evaluated CD34 expression was documented. Both CD34 expression and DNA methylation levels were specific to the tumours' anatomic location and mutation status. A constant decrease in methylation levels was observed ranging from almost 100% hypermethylation in intestinal GISTs from duodenum to hypomethylation in rectum. CD34 was heavily methylated in gastric PDGFRA-mutant GISTs in comparison to hypomethylated KIT-mutant counterparts. Next to CD34 hypermethylation, miR-665 was predicted and experimentally confirmed to target CD34 mRNA in GIST-T1 cells. Our results suggest that CD34 expression in GISTs may undergo a complex control by DNA methylation and miR-665. Differential methylation and expression of CD34 in GISTs along the gastrointestinal tract axis and in tumours that harbour different gain-of-function mutations suggest the origin from different cell populations in the gastrointestinal tract. © 2017 UICC.

  7. Ablation of XP-V gene causes adipose tissue senescence and metabolic abnormalities

    PubMed Central

    Chen, Yih-Wen; Harris, Robert A.; Hatahet, Zafer; Chou, Kai-ming

    2015-01-01

    Obesity and the metabolic syndrome have evolved to be major health issues throughout the world. Whether loss of genome integrity contributes to this epidemic is an open question. DNA polymerase η (pol η), encoded by the xeroderma pigmentosum (XP-V) gene, plays an essential role in preventing cutaneous cancer caused by UV radiation-induced DNA damage. Herein, we demonstrate that pol η deficiency in mice (pol η−/−) causes obesity with visceral fat accumulation, hepatic steatosis, hyperleptinemia, hyperinsulinemia, and glucose intolerance. In comparison to WT mice, adipose tissue from pol η−/− mice exhibits increased DNA damage and a greater DNA damage response, indicated by up-regulation and/or phosphorylation of ataxia telangiectasia mutated (ATM), phosphorylated H2AX (γH2AX), and poly[ADP-ribose] polymerase 1 (PARP-1). Concomitantly, increased cellular senescence in the adipose tissue from pol η−/− mice was observed and measured by up-regulation of senescence markers, including p53, p16Ink4a, p21, senescence-associated (SA) β-gal activity, and SA secretion of proinflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) as early as 4 wk of age. Treatment of pol η−/− mice with a p53 inhibitor, pifithrin-α, reduced adipocyte senescence and attenuated the metabolic abnormalities. Furthermore, elevation of adipocyte DNA damage with a high-fat diet or sodium arsenite exacerbated adipocyte senescence and metabolic abnormalities in pol η−/− mice. In contrast, reduction of adipose DNA damage with N-acetylcysteine or metformin ameliorated cellular senescence and metabolic abnormalities. These studies indicate that elevated DNA damage is a root cause of adipocyte senescence, which plays a determining role in the development of obesity and insulin resistance. PMID:26240351

  8. Heterogeneity of DNA methylation in multifocal prostate cancer.

    PubMed

    Serenaite, Inga; Daniunaite, Kristina; Jankevicius, Feliksas; Laurinavicius, Arvydas; Petroska, Donatas; Lazutka, Juozas R; Jarmalaite, Sonata

    2015-01-01

    Most prostate cancer (PCa) cases are multifocal, and separate foci display histological and molecular heterogeneity. DNA hypermethylation is a frequent alteration in PCa, but interfocal heterogeneity of these changes has not been extensively investigated. Ten pairs of foci from multifocal PCa and 15 benign prostatic hyperplasia (BPH) samples were obtained from prostatectomy specimens, resulting altogether in 35 samples. Methylation-specific PCR (MSP) was used to evaluate methylation status of nine tumor suppressor genes (TSGs), and a set of selected TSGs was quantitatively analyzed for methylation intensity by pyrosequencing. Promoter sequences of the RASSF1 and ESR1 genes were methylated in all paired PCa foci, and frequent (≥75 %) DNA methylation was detected in RARB, GSTP1, and ABCB1 genes. MSP revealed different methylation status of at least one gene in separate foci in 8 out of 10 multifocal tumors. The mean methylation level of ESR1, GSTP1, RASSF1, and RARB differed between the paired foci of all PCa cases. The intensity of DNA methylation in these TSGs was significantly higher in PCa cases than in BPH (p < 0.001). Hierarchical cluster analysis revealed a divergent methylation profile of paired PCa foci, while the foci from separate cases with biochemical recurrence showed similar methylation profile and the highest mean levels of DNA methylation. Our findings suggest that PCa tissue is heterogeneous, as between paired foci differences in DNA methylation status were found. Common epigenetic profile of recurrent tumors can be inferred from our data.

  9. Existence of host-related DNA sequences in the schistosome genome.

    PubMed

    Iwamura, Y; Irie, Y; Kominami, R; Nara, T; Yasuraoka, K

    1991-06-01

    DNA sequences homologous to the mouse intracisternal A particle and endogenous type C retrovirus were detected in the DNAs of Schistosoma japonicum adults and S. mansoni eggs. Furthermore, other kinds of repetitive sequences in the host genome such as mouse type 1 Alu sequence (B1), mouse type 2 Alu sequence (B2) and mo-2 sequence, a mouse mini-satellite, were also detected in the DNAs from adults and eggs of S. japonicum and eggs of S. mansoni. Almost all of the sequences described above were absent in the DNAs of S. mansoni adults. The DNA fingerprints of schistosomes, using the mo-2 sequence, were indistinguishable from each other and resembled those of their murine hosts. Moreover, the mo-2 sequence was hypermethylated in the DNAs of schistosomes and its amount was variable in them. These facts indicate that host-related sequences are actually present in schistosomes and that the mo-2 repetitive sequence exists probably in extra-chromosome.

  10. Chronic radiation exposure as an ecological factor: Hypermethylation and genetic differentiation in irradiated Scots pine populations.

    PubMed

    Volkova, P Yu; Geras'kin, S A; Horemans, N; Makarenko, E S; Saenen, E; Duarte, G T; Nauts, R; Bondarenko, V S; Jacobs, G; Voorspoels, S; Kudin, M

    2018-01-01

    Genetic and epigenetic changes were investigated in chronically irradiated Scots pine (Pinus sylvestris L.) populations from territories that were heavily contaminated by radionuclides as result of the Chernobyl Nuclear Power Plant accident. In comparison to the reference site, the genetic diversity revealed by electrophoretic mobility of AFLPs was found to be significantly higher at the radioactively contaminated areas. In addition, the genome of pine trees was significantly hypermethylated at 4 of the 7 affected sites. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. DNA Methylation Suppresses Expression of the Urea Cycle Enzyme Carbamoyl Phosphate Synthetase 1 (CPS1) in Human Hepatocellular Carcinoma

    PubMed Central

    Liu, Hongyan; Dong, Huijia; Robertson, Keith; Liu, Chen

    2011-01-01

    Carbamoyl phosphate synthetase 1 (CPS1) is a liver-specific, intramitochondrial, rate-limiting enzyme in the urea cycle. A previous study showed that CPS1 is the antigen for hepatocyte paraffin 1 antibody, a commonly used antibody in surgical pathology practice; and CPS1 expression appears to be down-regulated in liver cancer tissue and cell lines. The aim of this study is to understand how the CPS1 gene is regulated in liver carcinogenesis. In this report, we show that human hepatocellular carcinoma (HCC) cells do not express CPS1, whereas cultured human primary hepatocytes express abundant levels. In addition, CPS1 was silenced or down-regulated in liver tumor tissues compared with the matched noncancerous tissues. The expression of CPS1 in HCC cells was restored with a demethylation agent, 5-azacytidine. We show that two CpG dinucleotides, located near the transcription start site, and a CpG-rich region in the first intron were hypermethylated in HCC cells. The hypermethylation of the two CpG dinucleotides was also detected in HCC tumor tissues compared with noncancerous tissues. Further molecular analysis with mutagenesis indicated that the two CpG dinucleotides play a role in promoter activity of the CPS1 gene. In conclusion, our study demonstrates that DNA methylation is a key mechanism of silencing CPS1 expression in human HCC cells, and CPS1 gene hypermethylation of the two CpG dinucleotides is a potential biomarker for HCC. PMID:21281797

  12. Analysis of DNA methylation related to rice adult plant resistance to bacterial blight based on methylation-sensitive AFLP (MSAP) analysis.

    PubMed

    Sha, A H; Lin, X H; Huang, J B; Zhang, D P

    2005-07-01

    DNA methylation is known to play an important role in the regulation of gene expression in eukaryotes. The rice cultivar Wase Aikoku 3 becomes resistant to the blight pathogen Xanthomonas oryzae pv. oryzae at the adult stage. Using methylation-sensitive amplified polymorphism (MSAP) analysis, we compared the patterns of cytosine methylation in seedlings and adult plants of the rice cultivar Wase Aikoku 3 that had been inoculated with the pathogen Xanthomonas oryzae pv. oryzae, subjected to mock inoculation or left untreated. In all, 2000 DNA fragments, each representing a recognition site cleaved by either or both of two isoschizomers, were amplified using 60 pairs of selective primers. A total of 380 sites were found to be methylated. Of these, 45 showed differential cytosine methylation among the seedlings and adult plants subjected to different treatments, and overall levels of methylation were higher in adult plants than in seedlings. All polymorphic fragments were sequenced, and six showed homology to genes that code for products of known function. Northern analysis of three fragments indicated that their expression varied with methylation pattern, with hypermethylation being correlated with repression of transcription, as expected. The results suggest that significant differences in cytosine methylation exist between seedlings and adult plants, and that hypermethylation or hypomethylation of specific genes may be involved in the development of adult plant resistance (APR) in rice plants.

  13. Exome sequencing for prenatal diagnosis of fetuses with sonographic abnormalities.

    PubMed

    Drury, Suzanne; Williams, Hywel; Trump, Natalie; Boustred, Christopher; Lench, Nicholas; Scott, Richard H; Chitty, Lyn S

    2015-10-01

    In the absence of aneuploidy or other pathogenic cytogenetic abnormality, fetuses with increased nuchal translucency (NT ≥ 3.5 mm) and/or other sonographic abnormalities have a greater incidence of genetic syndromes, but defining the underlying pathology can be challenging. Here, we investigate the value of whole exome sequencing in fetuses with sonographic abnormalities but normal microarray analysis. Whole exome sequencing was performed on DNA extracted from chorionic villi or amniocytes in 24 fetuses with unexplained ultrasound findings. In the first 14 cases sequencing was initially performed on fetal DNA only. For the remaining 10, the trio of fetus, mother and father was sequenced simultaneously. In 21% (5/24) cases, exome sequencing provided definitive diagnoses (Milroy disease, hypophosphatasia, achondrogenesis type 2, Freeman-Sheldon syndrome and Baraitser-Winter Syndrome). In a further case, a plausible diagnosis of orofaciodigital syndrome type 6 was made. In two others, a single mutation in an autosomal recessive gene was identified, but incomplete sequencing coverage precluded exclusion of the presence of a second mutation. Whole exome sequencing improves prenatal diagnosis in euploid fetuses with abnormal ultrasound scans. In order to expedite interpretation of results, trio sequencing should be employed, but interpretation can still be compromised by incomplete coverage of relevant genes. © 2015 John Wiley & Sons, Ltd.

  14. Polycomb repressive complex 2 epigenomic signature defines age-associated hypermethylation and gene expression changes

    PubMed Central

    Dozmorov, Mikhail G

    2015-01-01

    Although age-associated gene expression and methylation changes have been reported throughout the literature, the unifying epigenomic principles of aging remain poorly understood. Recent explosion in availability and resolution of functional/regulatory genome annotation data (epigenomic data), such as that provided by the ENCODE and Roadmap Epigenomics projects, provides an opportunity for the identification of epigenomic mechanisms potentially altered by age-associated differentially methylated regions (aDMRs) and regulatory signatures in the promoters of age-associated genes (aGENs). In this study we found that aDMRs and aGENs identified in multiple independent studies share a common Polycomb Repressive Complex 2 signature marked by EZH2, SUZ12, CTCF binding sites, repressive H3K27me3, and activating H3K4me1 histone modification marks, and a “poised promoter” chromatin state. This signature is depleted in RNA Polymerase II-associated transcription factor binding sites, activating H3K79me2, H3K36me3, H3K27ac marks, and an “active promoter” chromatin state. The PRC2 signature was shown to be generally stable across cell types. When considering the directionality of methylation changes, we found the PRC2 signature to be associated with aDMRs hypermethylated with age, while hypomethylated aDMRs were associated with enhancers. In contrast, aGENs were associated with the PRC2 signature independently of the directionality of gene expression changes. In this study we demonstrate that the PRC2 signature is the common epigenomic context of genomic regions associated with hypermethylation and gene expression changes in aging. PMID:25880792

  15. Gata3 hypermethylation and Foxp3 hypomethylation are associated with sustained protection and bystander effect following epicutaneous immunotherapy in peanut-sensitized mice.

    PubMed

    Mondoulet, Lucie; Dioszeghy, Vincent; Busato, Florence; Plaquet, Camille; Dhelft, Véronique; Bethune, Kevin; Leclere, Laurence; Daviaud, Christian; Ligouis, Mélanie; Sampson, Hugh; Dupont, Christophe; Tost, Jörg

    2018-05-19

    Epicutaneous immunotherapy (EPIT) is a promising method for treating food allergies. In animal models, EPIT induces sustained unresponsiveness and prevents further sensitization mediated by Tregs. Here, we elucidate the mechanisms underlying the therapeutic effect of EPIT, by characterizing the kinetics of DNA methylation changes in sorted cells from spleen and blood and by evaluating its persistence and bystander effect compared to oral immunotherapy (OIT). BALB/c mice orally sensitized to peanut proteins (PPE) were treated by EPIT using a PPE-patch or by PPE-OIT. Another set of peanut-sensitized mice treated by EPIT or OIT were sacrificed following a protocol of sensitization to OVA. DNA methylation was analysed during immunotherapy and 8 weeks after the end of treatment in sorted cells from spleen and blood by pyrosequencing. Humoral and cellular responses were measured during and after immunotherapy. Analyses showed a significant hypermethylation of the Gata3 promoter detectable only in Th2 cells for EPIT from the 4 th week and a significant hypomethylation of the Foxp3 promoter in CD62L + Tregs, which was sustained only for EPIT. In addition, mice treated with EPIT were protected from subsequent sensitization and maintained the epigenetic signature characteristic for EPIT. Our study demonstrates that EPIT leads to a unique and stable epigenetic signature in specific T cell compartments with down regulation of Th2 key regulators and upregulation of Treg transcription factors, likely explaining the sustainability of protection and the observed bystander effect. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  16. DNA Gyrase Is Involved in Chloroplast Nucleoid Partitioning

    PubMed Central

    Cho, Hye Sun; Lee, Sang Sook; Kim, Kwang Dong; Hwang, Inhwan; Lim, Jong-Seok; Park, Youn-Il; Pai, Hyun-Sook

    2004-01-01

    DNA gyrase, which catalyzes topological transformation of DNA, plays an essential role in replication and transcription in prokaryotes. Virus-induced gene silencing of NbGyrA or NbGyrB, which putatively encode DNA gyrase subunits A and B, respectively, resulted in leaf yellowing phenotypes in Nicotiana benthamiana. NbGyrA and NbGyrB complemented the gyrA and gyrB temperature-sensitive mutations of Escherichia coli, respectively, which indicates that the plant and bacterial subunits are functionally similar. NbGyrA and NbGyrB were targeted to both chloroplasts and mitochondria, and depletion of these subunits affected both organelles by reducing chloroplast numbers and inducing morphological and physiological abnormalities in both organelles. Flow cytometry analysis revealed that the average DNA content in the affected chloroplasts and mitochondria was significantly higher than in the control organelles. Furthermore, 4′,6-diamidino-2-phenylindole staining revealed that the abnormal chloroplasts contained one or a few large nucleoids instead of multiple small nucleoids dispersed throughout the stroma. Pulse-field gel electrophoresis analyses of chloroplasts demonstrated that the sizes and/or structure of the DNA molecules in the abnormal chloroplast nucleoids are highly aberrant. Based on these results, we propose that DNA gyrase plays a critical role in chloroplast nucleoid partitioning by regulating DNA topology. PMID:15367714

  17. Slow Joining of Newly Replicated DNA Chains in DNA Polymerase I-Deficient Escherichia coli Mutants*

    PubMed Central

    Okazaki, Reiji; Arisawa, Mikio; Sugino, Akio

    1971-01-01

    In Escherichia coli mutants deficient in DNA polymerase I, newly replicated short DNA is joined at about 10% of the rate in the wild-type strains. It is postulated that DNA polymerase I normally functions in filling gaps between the nascent short segments synthesized by the replication complex. Possible implications of the finding are discussed in relation to other abnormal properties of these mutants. PMID:4943548

  18. Effect of Methylation on Local Mechanics and Hydration Structure of DNA.

    PubMed

    Teng, Xiaojing; Hwang, Wonmuk

    2018-04-24

    Cytosine methylation affects mechanical properties of DNA and potentially alters the hydration fingerprint for recognition by proteins. The atomistic origin for these effects is not well understood, and we address this via all-atom molecular dynamics simulations. We find that the stiffness of the methylated dinucleotide step changes marginally, whereas the neighboring steps become stiffer. Stiffening is further enhanced for consecutively methylated steps, providing a mechanistic origin for the effect of hypermethylation. Steric interactions between the added methyl groups and the nonpolar groups of the neighboring nucleotides are responsible for the stiffening in most cases. By constructing hydration maps, we found that methylation also alters the surface hydration structure in distinct ways. Its resistance to deformation may contribute to the stiffening of DNA for deformational modes lacking steric interactions. These results highlight the sequence- and deformational-mode-dependent effects of cytosine methylation. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  19. Frequent MGMT (06-methylguanine-DNA methyltransferase) hypermethylation in long-term survivors of glioblastoma: a single institution experience

    PubMed Central

    Baur, Martina; Preusser, Matthias; Piribauer, Maria; Elandt, Katarzyna; Hassler, Marco; Hudec, Marcus; Dittrich, Christian; Marosi, Christine

    2010-01-01

    Background The aim of this retrospective study was to analyse the MGMT (06-methylguanine-DNA methyltransferase) promoter methylation status in long-term surviving (≥ 3 years) patients with glioblastoma multiforme (GBM). Methods The methylation status of the MGMT promoter was determined by bisulfite modification of the DNA and subsequent methylation-specific polymerase-chain-reaction (MSP). DNA was extracted from routinely formalin-fixed and paraffin-embedded tumour tissue samples. Results MSP yielded interpretable results in only 14 of 33 (42%) long-term surviving patients with GBM. A methylated band was seen in 3 of 14, methylated as well as unmethylated bands in 8 of 14 and an only unmethylated band in 3 of 14 patients, thus, yielding MGMT promoter methylation in 11 of 14 patients. The two groups of patients with methylated and unmethylated MGMT promoter status were too small to draw any firm statistical conclusions. Conclusions Long-term surviving patients with GBM have very frequently intratumoural MGMT promoter methylation. This phenomenon discriminates long-term survivors from a non-selected group of patients with GBM. The standardization of the MSP for the determination of the MGMT promoter methylation status seems to be necessary in order to make this methodology a more reliable one. PMID:22933901

  20. Epigenetic variants of a transgenic petunia line show hypermethylation in transgene DNA: an indication for specific recognition of foreign DNA in transgenic plants.

    PubMed

    Meyer, P; Heidmann, I

    1994-05-25

    We analysed de novo DNA methylation occurring in plants obtained from the transgenic petunia line R101-17. This line contains one copy of the maize A1 gene that leads to the production of brick-red pelargonidin pigment in the flowers. Due to its integration into an unmethylated genomic region the A1 transgene is hypomethylated and transcriptionally active. Several epigenetic variants of line 17 were selected that exhibit characteristic and somatically stable pigmentation patterns, displaying fully coloured, marbled or colourless flowers. Analysis of the DNA methylation patterns revealed that the decrease in pigmentation among the epigenetic variants was correlated with an increase in methylation, specifically of the transgene DNA. No change in methylation of the hypomethylated integration region could be detected. A similar increase in methylation, specifically in the transgene region, was also observed among progeny of R101-17del, a deletion derivative of R101-17 that no longer produces pelargonidin pigments due to a deletion in the A1 coding region. Again de novo methylation is specifically directed to the transgene, while the hypomethylated character of neighbouring regions is not affected. Possible mechanisms for transgene-specific methylation and its consequences for long-term use of transgenic material are discussed.

  1. Brief Guide to Genomics: DNA, Genes and Genomes

    MedlinePlus

    ... Sheets A Brief Guide to Genomics About NHGRI Research About the International HapMap Project Biological Pathways Chromosome Abnormalities Chromosomes Cloning Comparative Genomics DNA Microarray Technology DNA Sequencing Deoxyribonucleic Acid ( ...

  2. Promoter DNA methylation regulates progranulin expression and is altered in FTLD

    PubMed Central

    2013-01-01

    Background Frontotemporal lobar degeneration (FTLD) is a heterogeneous group of neurodegenerative diseases associated with personality changes and progressive dementia. Loss-of-function mutations in the growth factor progranulin (GRN) cause autosomal dominant FTLD, but so far the pathomechanism of sporadic FTLD is unclear. Results We analyzed whether DNA methylation in the GRN core promoter restricts GRN expression and, thus, might promote FTLD in the absence of GRN mutations. GRN expression in human lymphoblast cell lines is negatively correlated with methylation at several CpG units within the GRN promoter. Chronic treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (DAC) strongly induces GRN mRNA and protein levels. In a reporter assay, CpG methylation blocks transcriptional activity of the GRN core promoter. In brains of FTLD patients several CpG units in the GRN promoter are significantly hypermethylated compared to age-matched healthy controls, Alzheimer and Parkinson patients. These CpG motifs are critical for GRN promoter activity in reporter assays. Furthermore, DNA methyltransferase 3a (DNMT3a) is upregulated in FTLD patients and overexpression of DNMT3a reduces GRN promoter activity and expression. Conclusion These data suggest that altered DNA methylation is a novel pathomechanism for FTLD that is potentially amenable to targeted pharmacotherapy. PMID:24252647

  3. Relationships between human sperm protamines, DNA damage and assisted reproduction outcomes.

    PubMed

    Simon, Luke; Castillo, Judit; Oliva, Rafael; Lewis, Sheena E M

    2011-12-01

    The exchange of histones with protamines in sperm DNA results in sperm chromatin compaction and protection. Variations in sperm protamine expression are associated with male infertility. The aim of this study was to investigate relationships between DNA fragmentation, sperm protamines and assisted reproduction treatment. Semen and spermatozoa prepared by density-gradient centrifugation (DGC) from 73 men undergoing IVF and 24 men undergoing intracytoplasmic sperm injection (ICSI) were included in the study. Nuclear DNA fragmentation was assessed using the alkaline Comet assay and protamines were separated by acid-urea polyacrylamide gels. Sperm DNA fragmentation and protamine content (P1-DNA, P2-DNA, P1+P2-DNA) decreased in spermatozoa after DGC. Abnormally high and low P1/P2 ratios were associated with increased sperm DNA fragmentation. Couples with idiopathic infertility had abnormally high P1/P2 ratios. Fertilization rates and embryo quality decreased as sperm DNA fragmentation or protamines increased. Sperm DNA fragmentation was lower in couples achieving pregnancies after IVF, but not after ICSI. There was no correlation between protamine content (P1-DNA, P2-DNA, P1+P2-DNA) or P1/P2 ratios and IVF or ICSI pregnancies. Increased sperm DNA fragmentation was associated with abnormal protamination and resulted in lower fertilization rates, poorer embryo quality and reduced pregnancy rates. Copyright © 2011 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  4. Interrupted E2F1-miR-34c-SCF negative feedback loop by hyper-methylation promotes colorectal cancer cell proliferation

    PubMed Central

    Yang, Shu; Wu, Bo; Sun, Haimei; Ji, Fengqing; Sun, Tingyi; Zhao, Yan; Zhou, Deshan

    2015-01-01

    Tumour suppressor miR-34c deficiency resulted from hyper-methylation in its promoter is believed to be one of the main causes of colorectal cancer (CRC). Till date, miR-34c has been validated as a direct target of p53; but previous evidence suggested other transcription factor(s) must be involved in miR-34c transcription. In the present study, we in the first place identified a core promoter region (−1118 to −883 bp) of pre-miR-34c which was embedded within a hyper-methylated CpG island. Secondly, E2F1 promoted miR-34c transcription by physical interaction with the miR-34c promoter at site −897 to −889 bp. The transcriptional activating effect of E2F1 on miR-34c was in a p53 independent manner but profoundly promoted in the presence of p53 with exposure to 5-aza-2′-deoxycytidine (DAC). Thirdly, stem cell factor (SCF), a miR-34c target, was specifically reduced upon an introduction of E2F1 which lead to suppression of CRC cell proliferation. The E2F1-suppressed cell proliferation was partially abrogated by additional miR-34c inhibitor, indicating that the anti-proliferation effect of E2F1 was probably through activating miR-34c-SCF axis. Finally, SCF/KIT signalling increased E2F1 production by reducing its proteosomal degradation dependent on PI3K/Akt-GSK3β pathway. In conclusion, our results suggested the existence of E2F1-miR-34c-SCF negative feedback loop which was interrupted by the hyper-methylation of miR-34c promoter in CRC cells and increased cell proliferation. PMID:26704889

  5. Lynch syndrome-associated endometrial carcinoma with MLH1 germline mutation and MLH1 promoter hypermethylation: a case report and literature review.

    PubMed

    Yokoyama, Takanori; Takehara, Kazuhiro; Sugimoto, Nao; Kaneko, Keika; Fujimoto, Etsuko; Okazawa-Sakai, Mika; Okame, Shinichi; Shiroyama, Yuko; Yokoyama, Takashi; Teramoto, Norihiro; Ohsumi, Shozo; Saito, Shinya; Imai, Kazuho; Sugano, Kokichi

    2018-05-21

    Lynch syndrome is an autosomal dominant inherited disease caused by germline mutations in mismatch repair genes. Analysis for microsatellite instability (MSI) and immunohistochemistry (IHC) of protein expressions of disease-associated genes is used to screen for Lynch syndrome in endometrial cancer patients. When losses of both MLH1 and PMS2 proteins are observed by IHC, MLH1 promoter methylation analysis is conducted to distinguish Lynch syndrome-associated endometrial cancer from sporadic cancer. Here we report a woman who developed endometrial cancer at the age of 49 years. She had a family history of colorectal cancer (first-degree relative aged 52 years) and stomach cancer (second-degree relative with the age of onset unknown). No other family history was present, and she failed to meet the Amsterdam II criteria for the diagnosis of Lynch syndrome. Losses of MLH1 and PMS2, but not MSH2 and MSH6, proteins were observed by IHC in endometrial cancer tissues. Because MLH1 promoter hypermethylation was detected in endometrial cancer tissue samples, the epigenetic silencing of MLH1 was suspected as the cause of the protein loss. However, because of the early onset of endometrial cancer and the positive family history, a diagnosis of Lynch syndrome was also suspected. Therefore, we provided her with genetic counseling. After obtaining her consent, MLH1 promoter methylation testing and genetic testing of peripheral blood were performed. MLH1 promoter methylation was not observed in peripheral blood. However, genetic testing revealed a large deletion of exon 5 in MLH1; thus, we diagnosed the presence of Lynch syndrome. Both MLH1 germline mutation and MLH1 promoter hypermethylation may be observed in endometrial cancer. Therefore, even if MLH1 promoter hypermethylation is detected, a diagnosis of Lynch syndrome cannot be excluded.

  6. DNA Methyl Transferase 1 Reduces Expression of SRD5A2 in the Aging Adult Prostate

    PubMed Central

    Ge, Rongbin; Wang, Zongwei; Bechis, Seth K.; Otsetov, Alexander G.; Hua, Shengyu; Wu, Shulin; Wu, Chin-Lee; Tabatabaei, Shahin; Olumi, Aria F.

    2016-01-01

    5-α Reductase type 2 (SRD5A2) is a critical enzyme for prostatic development and growth. Inhibition of SRD5A2 by finasteride is used commonly for the management of urinary obstruction caused by benign prostatic hyperplasia. Contrary to common belief, we have found that expression of SRD5A2 is variable and absent in one third of benign adult prostates. In human samples, absent SRD5A2 expression is associated with hypermethylation of the SRD5A2 promoter, and in vitro SRD5A2 promoter activity is suppressed by methylation. We show that methylation of SRD5A2 is regulated by DNA methyltransferase 1, and inflammatory mediators such as tumor necrosis factor α, NF-κB, and IL-6 regulate DNA methyltransferase 1 expression and thereby affect SRD5A2 promoter methylation and gene expression. Furthermore, we show that increasing age in mice and humans is associated with increased methylation of the SRD5A2 promoter and concomitantly decreased protein expression. Artificial induction of inflammation in prostate primary epithelial cells leads to hypermethylation of the SRD5A2 promoter and silencing of SRD5A2, whereas inhibition with tumor necrosis factor α inhibitor reactivates SRD5A2 expression. Therefore, expression of SRD5A2 is not static and ubiquitous in benign adult prostate tissues. Methylation and expression of SRD5A2 may be used as a gene signature to tailor therapies for more effective treatment of prostatic diseases. PMID:25700986

  7. DNA methylation at a bovine alpha satellite I repeat CpG site during development following fertilization and somatic cell nuclear transfer.

    PubMed

    Couldrey, Christine; Wells, David N

    2013-01-01

    Incomplete epigenetic reprogramming is postulated to contribute to the low developmental success following somatic cell nuclear transfer (SCNT). Here, we describe the epigenetic reprogramming of DNA methylation at an alpha satellite I CpG site (αsatI-5) during development of cattle generated either by artificial insemination (AI) or in vitro fertilization (IVF) and SCNT. Quantitative methylation analysis identified that SCNT donor cells were highly methylated at αsatI-5 and resulting SCNT blastocysts showed significantly more methylation than IVF blastocysts. At implantation, no difference in methylation was observed between SCNT and AI in trophoblast tissue at αsatI-5, however, SCNT embryos were significantly hyper-methylated compared to AI controls at this time point. Following implantation, DNA methylation at αsatI-5 decreased in AI but not SCNT placental tissues. In contrast to placenta, the proportion of methylation at αsatI-5 remained high in adrenal, kidney and muscle tissues during development. Differences in the average proportion of methylation were smaller in somatic tissues than placental tissues but, on average, SCNT somatic tissues were hyper-methylated at αsatI-5. Although sperm from all bulls was less methylated than somatic tissues at αsatI-5, on average this site remained hyper-methylated in sperm from cloned bulls compared with control bulls. This developmental time course confirms that epigenetic reprogramming does occur, at least to some extent, following SCNT. However, the elevated methylation levels observed in SCNT blastocysts and cellular derivatives implies that there is either insufficient time or abundance of appropriate reprogramming factors in oocytes to ensure complete reprogramming. Incomplete reprogramming at this CpG site may be a contributing factor to low SCNT success rates, but more likely represents the tip of the iceberg in terms of incompletely reprogramming. Until protocols ensure the epigenetic signature of a

  8. DNA Methylation at a Bovine Alpha Satellite I Repeat CpG Site during Development following Fertilization and Somatic Cell Nuclear Transfer

    PubMed Central

    Couldrey, Christine; Wells, David N.

    2013-01-01

    Incomplete epigenetic reprogramming is postulated to contribute to the low developmental success following somatic cell nuclear transfer (SCNT). Here, we describe the epigenetic reprogramming of DNA methylation at an alpha satellite I CpG site (αsatI-5) during development of cattle generated either by artificial insemination (AI) or in vitro fertilization (IVF) and SCNT. Quantitative methylation analysis identified that SCNT donor cells were highly methylated at αsatI-5 and resulting SCNT blastocysts showed significantly more methylation than IVF blastocysts. At implantation, no difference in methylation was observed between SCNT and AI in trophoblast tissue at αsatI-5, however, SCNT embryos were significantly hyper-methylated compared to AI controls at this time point. Following implantation, DNA methylation at αsatI-5 decreased in AI but not SCNT placental tissues. In contrast to placenta, the proportion of methylation at αsatI-5 remained high in adrenal, kidney and muscle tissues during development. Differences in the average proportion of methylation were smaller in somatic tissues than placental tissues but, on average, SCNT somatic tissues were hyper-methylated at αsatI-5. Although sperm from all bulls was less methylated than somatic tissues at αsatI-5, on average this site remained hyper-methylated in sperm from cloned bulls compared with control bulls. This developmental time course confirms that epigenetic reprogramming does occur, at least to some extent, following SCNT. However, the elevated methylation levels observed in SCNT blastocysts and cellular derivatives implies that there is either insufficient time or abundance of appropriate reprogramming factors in oocytes to ensure complete reprogramming. Incomplete reprogramming at this CpG site may be a contributing factor to low SCNT success rates, but more likely represents the tip of the iceberg in terms of incompletely reprogramming. Until protocols ensure the epigenetic signature of a

  9. Isogenic mice exhibit sexually-dimorphic DNA methylation patterns across multiple tissues.

    PubMed

    McCormick, Helen; Young, Paul E; Hur, Suzy S J; Booher, Keith; Chung, Hunter; Cropley, Jennifer E; Giannoulatou, Eleni; Suter, Catherine M

    2017-12-13

    Cytosine methylation is a stable epigenetic modification of DNA that plays an important role in both normal physiology and disease. Most diseases exhibit some degree of sexual dimorphism, but the extent to which epigenetic states are influenced by sex is understudied and poorly understood. To address this deficit we studied DNA methylation patterns across multiple reduced representation bisulphite sequencing datasets (from liver, heart, brain, muscle and spleen) derived from isogenic male and female mice. DNA methylation patterns varied significantly from tissue to tissue, as expected, but they also varied between the sexes, with thousands of sexually dimorphic loci identified. The loci affected were largely autonomous to each tissue, even within tissues derived from the same germ layer. At most loci, differences between genders were driven by females exhibiting hypermethylation relative to males; a proportion of these differences were independent of the presence of testosterone in males. Loci harbouring gender differences were clustered in ontologies related to tissue function. Our findings suggest that gender is underwritten in the epigenome in a tissue-specific and potentially sex hormone-independent manner. Gender-specific epigenetic states are likely to have important implications for understanding sexually dimorphic phenotypes in health and disease.

  10. Site-specificity of abnormal excision: the mechanism of formation of a specialized transducing bacteriophage lambda plac5.

    PubMed Central

    Shpakovski, G V; Berlin, Y A

    1984-01-01

    Molecular mechanism of the specialized transducing bacteriophage lambda plac5 formation has been studied. Phage-bacterial DNA junctions in lambda plac5 DNA are localized and primary structure of regions of the abnormal excisional recombination leading to the phage formation is elucidated; the crossover region proved to be comparable with the central part of attP and attB sites (the core and the adjacent tetranucleotide) in length and degree of homology. Bacterial insert in lambda plac5 DNA is shown to end immediately after Z-Y spacer, the DNA not containing lacY gene segments. The data obtained led to the conclusion of site-specific (homologous) character of abnormal excision upon formation of lambda transducing bacteriophages. Possible mechanisms of the excision are discussed. Images PMID:6091038

  11. Development of TRAIL Resistance by Radiation-Induced Hypermethylation of DR4 CpG Island in Recurrent Laryngeal Squamous Cell Carcinoma

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lee, Jong Cheol; Department of Biomedical Research Center, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan; Lee, Won Hyeok

    2014-04-01

    Purpose: There are limited therapeutic options for patients with recurrent head and neck cancer after radiation therapy failure. To assess the use of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) as a salvage chemotherapeutic agent for recurrent cancer after radiation failure, we investigated the effect of clinically relevant cumulative irradiation on TRAIL-induced apoptosis. Methods and Materials: Using a previously established HN3 cell line from a laryngeal carcinoma patient, we generated a chronically irradiated HN3R isogenic cell line. Viability and apoptosis in HN3 and HN3R cells treated with TRAIL were analyzed with MTS and PI/annexin V-FITC assays. Western blotting and flow cytometry weremore » used to determine the underlying mechanism of TRAIL resistance. DR4 expression was semiquantitatively scored in a tissue microarray with 107 laryngeal cancer specimens. Methylation-specific polymerase chain reaction and bisulfite sequencing for DR4 were performed for genomic DNA isolated from each cell line. Results: HN3R cells were more resistant than HN3 cells to TRAIL-induced apoptosis because of significantly reduced levels of the DR4 receptor. The DR4 staining score in 37 salvage surgical specimens after radiation failure was lower in 70 surgical specimens without radiation treatment (3.03 ± 2.75 vs 5.46 ± 3.30, respectively; P<.001). HN3R cells had a methylated DR4 CpG island that was partially demethylated by the DNA demethylating agent 5-aza-2′-deoxycytidine. Conclusion: Epigenetic silencing of the TRAIL receptor by hypermethylation of a DR4 CpG island might be an underlying mechanism for TRAIL resistance in recurrent laryngeal carcinoma treated with radiation.« less

  12. Differential DNA Methylation of MicroRNA Genes in Temporal Cortex from Alzheimer's Disease Individuals.

    PubMed

    Villela, Darine; Ramalho, Rodrigo F; Silva, Aderbal R T; Brentani, Helena; Suemoto, Claudia K; Pasqualucci, Carlos Augusto; Grinberg, Lea T; Krepischi, Ana C V; Rosenberg, Carla

    2016-01-01

    This study investigated for the first time the genomewide DNA methylation changes of noncoding RNA genes in the temporal cortex samples from individuals with Alzheimer's disease (AD). The methylome of 10 AD individuals and 10 age-matched controls were obtained using Illumina 450 K methylation array. A total of 2,095 among the 15,258 interrogated noncoding RNA CpG sites presented differential methylation, 161 of which were associated with miRNA genes. In particular, 10 miRNA CpG sites that were found to be hypermethylated in AD compared to control brains represent transcripts that have been previously associated with the disease. This miRNA set is predicted to target 33 coding genes from the neuregulin receptor complex (ErbB) signaling pathway, which is required for the neurons myelination process. For 6 of these miRNA genes (MIR9-1, MIR9-3, MIR181C, MIR124-1, MIR146B, and MIR451), the hypermethylation pattern is in agreement with previous results from literature that shows downregulation of miR-9, miR-181c, miR-124, miR-146b, and miR-451 in the AD brain. Our data implicate dysregulation of miRNA methylation as contributor to the pathogenesis of AD.

  13. The methylation of nuclear and mitochondrial DNA in ageing phenotypes and longevity.

    PubMed

    Bacalini, Maria Giulia; D'Aquila, Patrizia; Marasco, Elena; Nardini, Christine; Montesanto, Alberto; Franceschi, Claudio; Passarino, Giuseppe; Garagnani, Paolo; Bellizzi, Dina

    2017-07-01

    An increasing body of data is progressively indicating that the comprehension of the epigenetic landscape, actively integrated with the genetic elements, is crucial to delineate the molecular basis of the inter-individual complexity of ageing process. Indeed, it has emerged that DNA methylation changes occur during ageing, consisting mainly in a progressive process of genome demethylation, in a hypermethylation of gene-specific CpG dinucleotides, as well as in an inter-individual divergence of the epigenome due to stochastic events and environmental exposures throughout life, namely as epigenetic drift. Additionally, it has also come to light an implication of the mitochondrial genome in the regulation of the intracellular epigenetic landscape, as demonstrated by the being itself object of epigenetic modifications. An overview of DNA methylation changes occurring during ageing process at both nuclear and mitochondrial level will be described in this review, also taking into account the recent and promising data available on the 5-hydroxymethylcytosine. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Differential DNA methylation profiles of coding and non-coding genes define hippocampal sclerosis in human temporal lobe epilepsy

    PubMed Central

    Miller-Delaney, Suzanne F.C.; Bryan, Kenneth; Das, Sudipto; McKiernan, Ross C.; Bray, Isabella M.; Reynolds, James P.; Gwinn, Ryder; Stallings, Raymond L.

    2015-01-01

    Temporal lobe epilepsy is associated with large-scale, wide-ranging changes in gene expression in the hippocampus. Epigenetic changes to DNA are attractive mechanisms to explain the sustained hyperexcitability of chronic epilepsy. Here, through methylation analysis of all annotated C-phosphate-G islands and promoter regions in the human genome, we report a pilot study of the methylation profiles of temporal lobe epilepsy with or without hippocampal sclerosis. Furthermore, by comparative analysis of expression and promoter methylation, we identify methylation sensitive non-coding RNA in human temporal lobe epilepsy. A total of 146 protein-coding genes exhibited altered DNA methylation in temporal lobe epilepsy hippocampus (n = 9) when compared to control (n = 5), with 81.5% of the promoters of these genes displaying hypermethylation. Unique methylation profiles were evident in temporal lobe epilepsy with or without hippocampal sclerosis, in addition to a common methylation profile regardless of pathology grade. Gene ontology terms associated with development, neuron remodelling and neuron maturation were over-represented in the methylation profile of Watson Grade 1 samples (mild hippocampal sclerosis). In addition to genes associated with neuronal, neurotransmitter/synaptic transmission and cell death functions, differential hypermethylation of genes associated with transcriptional regulation was evident in temporal lobe epilepsy, but overall few genes previously associated with epilepsy were among the differentially methylated. Finally, a panel of 13, methylation-sensitive microRNA were identified in temporal lobe epilepsy including MIR27A, miR-193a-5p (MIR193A) and miR-876-3p (MIR876), and the differential methylation of long non-coding RNA documented for the first time. The present study therefore reports select, genome-wide DNA methylation changes in human temporal lobe epilepsy that may contribute to the molecular architecture of the epileptic brain. PMID

  15. Mechanisms and consequences of paternally transmitted chromosomal abnormalities

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Marchetti, F; Wyrobek, A J

    Paternally transmitted chromosomal damage has been associated with pregnancy loss, developmental and morphological defects, infant mortality, infertility, and genetic diseases in the offspring including cancer. There is epidemiological evidence linking paternal exposure to occupational or environmental agents with an increased risk of abnormal reproductive outcomes. There is also a large body of literature on germ cell mutagenesis in rodents showing that treatment of male germ cells with mutagens has dramatic consequences on reproduction producing effects such as those observed in human epidemiological studies. However, we know very little about the etiology, transmission and early embryonic consequences of paternally-derived chromosomal abnormalities.more » The available evidence suggests that: (1) there are distinct patterns of germ cell-stage differences in the sensitivity of induction of transmissible genetic damage with male postmeiotic cells being the most sensitive; (2) cytogenetic abnormalities at first metaphase after fertilization are critical intermediates between paternal exposure and abnormal reproductive outcomes; and, (3) there are maternally susceptibility factors that may have profound effects on the amount of sperm DNA damage that is converted into chromosomal aberrations in the zygote and directly affect the risk for abnormal reproductive outcomes.« less

  16. The Role of DNA Methylation in Aging, Rejuvenation, and Age-Related Disease

    PubMed Central

    Johnson, Adiv A.; Akman, Kemal; Calimport, Stuart R.G.; Wuttke, Daniel; de Magalhães, João Pedro

    2012-01-01

    Abstract DNA methylation is a major control program that modulates gene expression in a plethora of organisms. Gene silencing through methylation occurs through the activity of DNA methyltransferases, enzymes that transfer a methyl group from S-adenosyl-l-methionine to the carbon 5 position of cytosine. DNA methylation patterns are established by the de novo DNA methyltransferases (DNMTs) DNMT3A and DNMT3B and are subsequently maintained by DNMT1. Aging and age-related diseases include defined changes in 5-methylcytosine content and are generally characterized by genome-wide hypomethylation and promoter-specific hypermethylation. These changes in the epigenetic landscape represent potential disease biomarkers and are thought to contribute to age-related pathologies, such as cancer, osteoarthritis, and neurodegeneration. Some diseases, such as a hereditary form of sensory neuropathy accompanied by dementia, are directly caused by methylomic changes. Epigenetic modifications, however, are reversible and are therefore a prime target for therapeutic intervention. Numerous drugs that specifically target DNMTs are being tested in ongoing clinical trials for a variety of cancers, and data from finished trials demonstrate that some, such as 5-azacytidine, may even be superior to standard care. DNMTs, demethylases, and associated partners are dynamically shaping the methylome and demonstrate great promise with regard to rejuvenation. PMID:23098078

  17. [Novel Approaches in DNA Methylation Studies - MS-HRM Analysis and Electrochemistry].

    PubMed

    Bartošík, M; Ondroušková, E

    Cytosine methylation in DNA is an epigenetic mechanism regulating gene expression and plays a vital role in cell differentiation or proliferation. Tumor cells often exhibit aberrant DNA methylation, e.g. hypermethylation of tumor suppressor gene promoters. New methods, capable of determining methylation status of specific DNA sequences, are thus being developed. Among them, MS-HRM (methylation-specific high resolution melting) and electrochemistry offer relatively inexpensive instrumentation, fast assay times and possibility of screening multiple samples/DNA regions simultaneously. MS-HRM is due to its sensitivity and simplicity an interesting alternative to already established techniques, including methylation-specific PCR or bisulfite sequencing. Electrochemistry, when combined with suitable electroactive labels and electrode surfaces, has been applied in several unique strategies for discrimination of cytosines and methylcytosines. Both techniques were successfully tested in analysis of DNA methylation within promoters of important tumor suppressor genes and could thus help in achieving more precise diagnostics and prognostics of cancer. Aberrant methylation of promoters has already been described in hundreds of genes associated with tumorigenesis and could serve as important biomarker if new methods applicable into clinical practice are sufficiently advanced.Key words: DNA methylation - 5-methylcytosine - HRM analysis - melting temperature - DNA duplex - electrochemistry - nucleic acid hybridizationThis work was supported by MEYS - NPS I - LO1413.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 6. 5. 2016Accepted: 16. 5. 2016.

  18. Densely ionizing radiation affects DNA methylation of selective LINE-1 elements

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Prior, Sara; Miousse, Isabelle R.

    Long Interspersed Nucleotide Element 1 (LINE-1) retrotransposons are heavily methylated and are the most abundant transposable elements in mammalian genomes. Here, we investigated the differential DNA methylation within the LINE-1 under normal conditions and in response to environmentally relevant doses of sparsely and densely ionizing radiation. We demonstrate that DNA methylation of LINE-1 elements in the lungs of C57BL6 mice is dependent on their evolutionary age, where the elder age of the element is associated with the lower extent of DNA methylation. Exposure to 5-aza-2′-deoxycytidine and methionine-deficient diet affected DNA methylation of selective LINE-1 elements in an age- and promotermore » type-dependent manner. Exposure to densely IR, but not sparsely IR, resulted in DNA hypermethylation of older LINE-1 elements, while the DNA methylation of evolutionary younger elements remained mostly unchanged. We also demonstrate that exposure to densely IR increased mRNA and protein levels of LINE-1 via the loss of the histone H3K9 dimethylation and an increase in the H3K4 trimethylation at the LINE-1 5′-untranslated region, independently of DNA methylation. Our findings suggest that DNA methylation is important for regulation of LINE-1 expression under normal conditions, but histone modifications may dictate the transcriptional activity of LINE-1 in response to exposure to densely IR. - Highlights: • DNA methylation of LINE-1 elements is dependent on their evolutionary age. • Densely ionizing radiation affects DNA methylation of selective LINE-1 elements. • Radiation-induced reactivation of LINE-1 is DNA methylation-independent. • Histone modifications dictate the transcriptional activity of LINE-1.« less

  19. Global DNA hypomethylation coupled to repressive chromatin domain formation and gene silencing in breast cancer

    PubMed Central

    Hon, Gary C.; Hawkins, R. David; Caballero, Otavia L.; Lo, Christine; Lister, Ryan; Pelizzola, Mattia; Valsesia, Armand; Ye, Zhen; Kuan, Samantha; Edsall, Lee E.; Camargo, Anamaria Aranha; Stevenson, Brian J.; Ecker, Joseph R.; Bafna, Vineet; Strausberg, Robert L.; Simpson, Andrew J.; Ren, Bing

    2012-01-01

    While genetic mutation is a hallmark of cancer, many cancers also acquire epigenetic alterations during tumorigenesis including aberrant DNA hypermethylation of tumor suppressors, as well as changes in chromatin modifications as caused by genetic mutations of the chromatin-modifying machinery. However, the extent of epigenetic alterations in cancer cells has not been fully characterized. Here, we describe complete methylome maps at single nucleotide resolution of a low-passage breast cancer cell line and primary human mammary epithelial cells. We find widespread DNA hypomethylation in the cancer cell, primarily at partially methylated domains (PMDs) in normal breast cells. Unexpectedly, genes within these regions are largely silenced in cancer cells. The loss of DNA methylation in these regions is accompanied by formation of repressive chromatin, with a significant fraction displaying allelic DNA methylation where one allele is DNA methylated while the other allele is occupied by histone modifications H3K9me3 or H3K27me3. Our results show a mutually exclusive relationship between DNA methylation and H3K9me3 or H3K27me3. These results suggest that global DNA hypomethylation in breast cancer is tightly linked to the formation of repressive chromatin domains and gene silencing, thus identifying a potential epigenetic pathway for gene regulation in cancer cells. PMID:22156296

  20. Identification of body fluid-specific DNA methylation markers for use in forensic science.

    PubMed

    Park, Jong-Lyul; Kwon, Oh-Hyung; Kim, Jong Hwan; Yoo, Hyang-Sook; Lee, Han-Chul; Woo, Kwang-Man; Kim, Seon-Young; Lee, Seung-Hwan; Kim, Yong Sung

    2014-11-01

    DNA methylation, which occurs at the 5'-position of the cytosine in CpG dinucleotides, has great potential for forensic identification of body fluids, because tissue-specific patterns of DNA methylation have been demonstrated, and DNA is less prone to degradation than proteins or RNA. Previous studies have reported several body fluid-specific DNA methylation markers, but DNA methylation differences are sometimes low in saliva and vaginal secretions. Moreover, specific DNA methylation markers in four types of body fluids (blood, saliva, semen, and vaginal secretions) have not been investigated with genome-wide profiling. Here, we investigated novel DNA methylation markers for identification of body fluids for use in forensic science using the Illumina HumanMethylation 450K bead array, which contains over 450,000 CpG sites. Using methylome data from 16 samples of blood, saliva, semen, and vaginal secretions, we first selected 2986 hypermethylated or hypomethylated regions that were specific for each type of body fluid. We then selected eight CpG sites as novel, forensically relevant DNA methylation markers: cg06379435 and cg08792630 for blood, cg26107890 and cg20691722 for saliva, cg23521140 and cg17610929 for semen, and cg01774894 and cg14991487 for vaginal secretions. These eight selected markers were evaluated in 80 body fluid samples using pyrosequencing, and all showed high sensitivity and specificity for identification of the target body fluid. We suggest that these eight DNA methylation markers may be good candidates for developing an effective molecular assay for identification of body fluids in forensic science. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  1. Global Epigenetic Changes May Underlie Ethnic Differences and Susceptibility to Prostate Cancer

    DTIC Science & Technology

    2013-09-01

    malignancy and can often be found in non-cancerous tissues; in the prostate, hypermethylation of the GSTP1 CpG has been detected in PIA lesions [8]. DNA...methylcytosine (5-meC; [9, 10]). Since the recognition that the GSTP1 CpG island was frequently hypermethylated in PCa, more than 40 genes have been reported to...has also been identified for several genes. One study demonstrated that GSTP1 hypermethylation was significantly higher in PCa samples from AA men

  2. Genome-wide DNA methylation profiling identifies ALDH1A3 promoter methylation as a prognostic predictor in G-CIMP- primary glioblastoma.

    PubMed

    Zhang, Wei; Yan, Wei; You, Gan; Bao, Zhaoshi; Wang, Yongzhi; Liu, Yanwei; You, Yongping; Jiang, Tao

    2013-01-01

    To date, the aberrations in the DNA methylation patterns that are associated with different prognoses of G-CIMP- primary GBMs remain to be elucidated. Here, DNA methylation profiling of primary GBM tissues from 13 long-term survivors (LTS; overall survival ⩾18months) and 20 short-term survivors (STS; overall survival ⩽9months) was performed. Then G-CIMP+ samples were excluded. The differentially expressed CpG loci were identified between residual 18 STS and 9 LTS G-CIMP- samples. Methylation levels of 11 CpG loci (10genes) were statistically significantly lower, and 43 CpG loci (40genes) were statistically significantly higher in the tumor tissues of LTS than those of STS G-CIMP- samples (P<0.01). Of the 43 CpG loci that were hypermethylated in LTS G-CIMP- samples, 3 CpG loci localized in the promoter of ALDH1A3. Furthermore, using an independent validation cohort containing 37 primary GBM samples without IDH1 mutation and MGMT promoter methylation, the hypermethylation status of ALDH1A3 promoter predicted a better prognosis with an accompanied low expression of ALDH1A3 protein. Taken together, our results defined prognosis-related methylation signatures systematically for the first time in G-CIMP- primary GBMs. ALDH1A3 promoter methylation conferred a favorable prognosis in G-CIMP- primary GBMs. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  3. Antipsychotic drugs attenuate aberrant DNA methylation of DTNBP1 (dysbindin) promoter in saliva and post-mortem brain of patients with schizophrenia and Psychotic bipolar disorder.

    PubMed

    Abdolmaleky, Hamid M; Pajouhanfar, Sara; Faghankhani, Masoomeh; Joghataei, Mohammad Taghi; Mostafavi, Ashraf; Thiagalingam, Sam

    2015-12-01

    Due to the lack of genetic association between individual genes and schizophrenia (SCZ) pathogenesis, the current consensus is to consider both genetic and epigenetic alterations. Here, we report the examination of DNA methylation status of DTNBP1 promoter region, one of the most credible candidate genes affected in SCZ, assayed in saliva and post-mortem brain samples. The Illumina DNA methylation profiling and bisulfite sequencing of representative samples were used to identify methylation status of the DTNBP1 promoter region. Quantitative methylation specific PCR (qMSP) was employed to assess methylation of DTNBP1 promoter CpGs flanking a SP1 binding site in the saliva of SCZ patients, their first-degree relatives and control subjects (30, 15, and 30/group, respectively) as well as in post-mortem brains of patients with SCZ and bipolar disorder (BD) versus controls (35/group). qRT-PCR was used to assess DTNBP1 expression. We found DNA hypermethylation of DTNBP1 promoter in the saliva of SCZ patients (∼12.5%, P = 0.036), particularly in drug-naïve patients (∼20%, P = 0.011), and a trend toward hypermethylation in their first-degree relatives (P = 0.085) versus controls. Analysis of post-mortem brain samples revealed an inverse correlation between DTNBP1 methylation and expression, and normalization of this epigenetic change by classic antipsychotic drugs. Additionally, BD patients with psychotic depression exhibited higher degree of methylation versus other BD patients (∼80%, P = 0.025). DTNBP1 promoter DNA methylation may become a key element in a panel of biomarkers for diagnosis, prevention, or therapy in SCZ and at risk individuals pending confirmatory studies with larger sample sizes to attain a higher degree of significance. © 2015 Wiley Periodicals, Inc.

  4. Similar DNA methylation pattern in lung tumours from smokers and never-smokers with second-hand tobacco smoke exposure.

    PubMed

    Scesnaite, Asta; Jarmalaite, Sonata; Mutanen, Pertti; Anttila, Sisko; Nyberg, Fredrik; Benhamou, Simone; Boffetta, Paolo; Husgafvel-Pursiainen, Kirsti

    2012-07-01

    Tobacco smoke causes lung cancer in smokers and in never-smokers exposed to second-hand tobacco smoke (SHS). Nonetheless, molecular mechanisms of lung cancer in SHS-exposed never-smokers are still elusive. We studied lung cancers from current smokers (n = 109), former smokers (n = 56) and never-smokers (n = 47) for promoter hypermethylation of five tumour suppressor genes--p16, RARB, RASSF1, MGMT and DAPK1--using methylation-specific polymerase chain reaction. Lung tumours from ever-smokers suggested an increased risk of p16 hypermethylation as compared to never-smokers (P = 0.073), with former smokers having the highest frequency of p16 hypermethylation (P = 0.044 versus current smokers and P = 0.009 versus never-smokers). In the never-smoking group, p16 hypermethylation was seen in lung tumours from SHS-exposed individuals (4/33; 12%) but in none of the non-exposed individuals (0/9). The overall occurrence of hypermethylation (measured both as methylation index and as number of genes affected) was similar in those ever exposed to tobacco smoke (smokers, SHS-exposed never-smokers) and differed from non-exposed never-smokers. In multivariate analysis, p16 hypermethylation was more prevalent in lung tumours from male than female patients (P = 0.018) and in squamous cell carcinomas than in adenocarcinomas (P = 0.025). Occurrence of TP53 mutation in the tumour was associated with hypermethylation of at least one gene (P = 0.027). In all, our data suggest that promoter hypermethylation pattern in SHS-exposed never-smokers resembles that observed in smokers. Association between TP53 mutation, a hallmark of smokers' lung cancer, and methylation of one or more of the lung cancer-related genes studied, provides further evidence for common tobacco smoke-related origin for both types of molecular alterations.

  5. Plasticity of DNA methylation and gene expression under zinc deficiency in Arabidopsis roots.

    PubMed

    Chen, Xiaochao; Schönberger, Brigitte; Menz, Jochen; Ludewig, Uwe

    2018-05-25

    DNA methylation is a heritable chromatin modification that maintains chromosome stability, regulates transposon silencing and appears to be involved in gene expression in response to environmental conditions. Environmental stress alters DNA methylation patterns that are correlated with gene expression differences. Here, genome-wide differential DNA-methylation was identified upon prolonged Zn deficiency, leading to hypo- and hyper-methylated chromosomal regions. Preferential CpG methylation changes occurred in gene promoters and gene bodies, but did not overlap with transcriptional start sites. Methylation changes were also prominent in transposable elements. By contrast, non-CG methylation differences were exclusively found in promoters of protein coding genes and in transposable elements. Strongly Zn deficiency-induced genes and their promoters were mostly non-methylated, irrespective of Zn supply. Differential DNA methylation in the CpG and CHG, but not in the CHH context, was found close to a few up-regulated Zn-deficiency genes. However, the transcriptional Zn-deficiency response in roots appeared little correlated with associated DNA methylation changes in promoters or gene bodies. Furthermore, under Zn deficiency, developmental defects were identified in an Arabidopsis mutant lacking non-CpG methylation. The root methylome thus responds specifically to a micro-nutrient deficiency and is important for efficient Zn utilization at low availability, but the relationship of differential methylation and differentially expressed genes is surprisingly poor.

  6. Arsenicals produce stable progressive changes in DNA methylation patterns that are linked to malignant transformation of immortalized urothelial cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jensen, Taylor J.; Arizona Cancer Center, University of Arizona, Tucson, AZ 85724; Novak, Petr

    2009-12-01

    Aberrant DNA methylation participates in carcinogenesis and is a molecular hallmark of a tumor cell. Tumor cells generally exhibit a redistribution of DNA methylation resulting in global hypomethylation with regional hypermethylation; however, the speed in which these changes emerge has not been fully elucidated and may depend on the temporal location of the cell in the path from normal, finite lifespan to malignant transformation. We used a model of arsenical-induced malignant transformation of immortalized human urothelial cells and DNA methylation microarrays to examine the extent and temporal nature of changes in DNA methylation that occur during the transition from immortalmore » to malignantly transformed. Our data presented herein suggest that during arsenical-induced malignant transformation, aberrant DNA methylation occurs non-randomly, progresses gradually at hundreds of gene promoters, and alters expression of the associated gene, and these changes are coincident with the acquisition of malignant properties, such as anchorage independent growth and tumor formation in immunocompromised mice. The DNA methylation changes appear stable, since malignantly transformed cells removed from the transforming arsenical exhibited no reversion in DNA methylation levels, associated gene expression, or malignant phenotype. These data suggest that arsenicals act as epimutagens and directly link their ability to induce malignant transformation to their actions on the epigenome.« less

  7. GFRA3 promoter methylation may be associated with decreased postoperative survival in gastric cancer.

    PubMed

    Eftang, Lars Lohne; Klajic, Jovana; Kristensen, Vessela N; Tost, Jörg; Esbensen, Qin Ying; Blom, Gustav Peter; Bukholm, Ida Rashida Khan; Bukholm, Geir

    2016-03-16

    A large number of epigenetic alterations has been found to be implicated in the etiology of gastric cancer. We have studied the DNA methylation status of 27 500 gene promoter regions in 24 gastric adenocarcinomas from a Norwegian cohort, and aimed at identifying the hypermethylated regions. We have compared our findings to the gene expression in the same tissue, and linked our results to prognosis and survival. Biopsies from gastric adenocarcinomas and adjacent normal gastric mucosa were obtained from 24 patients following surgical resection of the tumor. Genome-wide DNA methylation profiling of the tumor and matched non-cancerous mucosa was performed. The results were compared to whole transcriptome cDNA microarray analysis of the same material. Most of the gene promoter regions in both types of tissue showed a low degree of methylation, however there was a small, but significant hypermethylation of the tumors. Hierarchical clustering showed separate grouping of the tumor and normal tissue. Hypermethylation of the promoter region of the GFRA3 gene showed a strong correlation to post-operative survival and several of the clinicopathological parameters, however no difference was found between the two main histological types of gastric cancer. There was only a modest correlation between the DNA methylation status and gene expression. The different DNA methylation clusters of the tumors and normal tissue indicate that aberrant DNA methylation is a distinct feature of gastric cancer, although there is little difference in the overall, and low, methylation levels between the two tissue types. The GFRA3 promoter region showed marked hypermethylation in almost all tumors, and its correlation with survival and other clinicopathological parameters may have important prognostic significance.

  8. Curcumin-induced mitotic arrest is characterized by spindle abnormalities, defects in chromosomal congression and DNA damage

    PubMed Central

    Manson, Margaret M.

    2013-01-01

    The chemopreventive agent curcumin has anti-proliferative effects in many tumour types, but characterization of cell cycle arrest, particularly with physiologically relevant concentrations, is still incomplete. Following oral ingestion, the highest concentrations of curcumin are achievable in the gut. Although it has been established that curcumin induces arrest at the G2/M stage of the cell cycle in colorectal cancer lines, it is not clear whether arrest occurs at the G2/M transition or in mitosis. To elucidate the precise stage of arrest, we performed a direct comparison of the levels of curcumin-induced G2/M boundary and mitotic arrest in eight colorectal cancer lines (Caco-2, DLD-1, HCA-7, HCT116p53+/+, HCT116p53–/–, HCT116p21–/–, HT-29 and SW480). Flow cytometry confirmed that these lines underwent G2/M arrest following treatment for 12h with clinically relevant concentrations of curcumin (5–10 μM). In all eight lines, the majority of this arrest occurred at the G2/M transition, with a proportion of cells arresting in mitosis. Examination of the mitotic index using fluorescence microscopy showed that the HCT116 and Caco-2 lines exhibited the highest levels of curcumin-induced mitotic arrest. Image analysis revealed impaired mitotic progression in all lines, exemplified by mitotic spindle abnormalities and defects in chromosomal congression. Pre-treatment with inhibitors of the DNA damage signalling pathway abrogated curcumin-induced mitotic arrest, but had little effect at the G2/M boundary. Moreover, pH2A.X staining seen in mitotic, but not interphase, cells suggests that this aberrant mitosis results in DNA damage. PMID:23125222

  9. Monozygotic twins discordant for common variable immunodeficiency reveal impaired DNA demethylation during naïve-to-memory B-cell transition

    PubMed Central

    Rodríguez-Cortez, Virginia C.; del Pino-Molina, Lucia; Rodríguez-Ubreva, Javier; Ciudad, Laura; Gómez-Cabrero, David; Company, Carlos; Urquiza, José M.; Tegnér, Jesper; Rodríguez-Gallego, Carlos; López-Granados, Eduardo; Ballestar, Esteban

    2015-01-01

    Common variable immunodeficiency (CVID), the most frequent primary immunodeficiency characterized by loss of B-cell function, depends partly on genetic defects, and epigenetic changes are thought to contribute to its aetiology. Here we perform a high-throughput DNA methylation analysis of this disorder using a pair of CVID-discordant MZ twins and show predominant gain of DNA methylation in CVID B cells with respect to those from the healthy sibling in critical B lymphocyte genes, such as PIK3CD, BCL2L1, RPS6KB2, TCF3 and KCNN4. Individual analysis confirms hypermethylation of these genes. Analysis in naive, unswitched and switched memory B cells in a CVID patient cohort shows impaired ability to demethylate and upregulate these genes in transitioning from naive to memory cells in CVID. Our results not only indicate a role for epigenetic alterations in CVID but also identify relevant DNA methylation changes in B cells that could explain the clinical manifestations of CVID individuals. PMID:26081581

  10. Kaempferol Modulates DNA Methylation and Downregulates DNMT3B in Bladder Cancer.

    PubMed

    Qiu, Wei; Lin, Jun; Zhu, Yichen; Zhang, Jian; Zeng, Liping; Su, Ming; Tian, Ye

    2017-01-01

    Genomic DNA methylation plays an important role in both the occurrence and development of bladder cancer. Kaempferol (Kae), a natural flavonoid that is present in many fruits and vegetables, exhibits potent anti-cancer effects in bladder cancer. Similar to other flavonoids, Kae possesses a flavan nucleus in its structure. This structure was reported to inhibit DNA methylation by suppressing DNA methyltransferases (DNMTs). However, whether Kae can inhibit DNA methylation remains unclear. Nude mice bearing bladder cancer were treated with Kae for 31 days. The genomic DNA was extracted from xenografts and the methylation changes was determined using an Illumina Infinium HumanMethylation 450 BeadChip Array. The ubiquitination was detected using immuno-precipitation assay. Our data indicated that Kae modulated DNA methylation in bladder cancer, inducing 103 differential DNA methylation positions (dDMPs) associated with genes (50 hyper-methylated and 53 hypo-methylated). DNA methylation is mostly relied on the levels of DNMTs. We observed that Kae specifically inhibited the protein levels of DNMT3B without altering the expression of DNMT1 or DNMT3A. However, Kae did not downregulate the transcription of DNMT3B. Interestingly, we observed that Kae induced a premature degradation of DNMT3B by inhibiting protein synthesis with cycloheximide (CHX). By blocking proteasome with MG132, we observed that Kae induced an increased ubiquitination of DNMT3B. These results suggested that Kae could induce the degradation of DNMT3B through ubiquitin-proteasome pathway. Our data indicated that Kae is a novel DNMT3B inhibitor, which may promote the degradation of DNMT3B in bladder cancer. © 2017 The Author(s)Published by S. Karger AG, Basel.

  11. Divergent cytosine DNA methylation patterns in single-cell, soybean root hairs.

    PubMed

    Hossain, Md Shakhawat; Kawakatsu, Taiji; Kim, Kyung Do; Zhang, Ning; Nguyen, Cuong T; Khan, Saad M; Batek, Josef M; Joshi, Trupti; Schmutz, Jeremy; Grimwood, Jane; Schmitz, Robert J; Xu, Dong; Jackson, Scott A; Ecker, Joseph R; Stacey, Gary

    2017-04-01

    Chromatin modifications, such as cytosine methylation of DNA, play a significant role in mediating gene expression in plants, which affects growth, development, and cell differentiation. As root hairs are single-cell extensions of the root epidermis and the primary organs for water uptake and nutrients, we sought to use root hairs as a single-cell model system to measure the impact of environmental stress. We measured changes in cytosine DNA methylation in single-cell root hairs as compared with multicellular stripped roots, as well as in response to heat stress. Differentially methylated regions (DMRs) in each methylation context showed very distinct methylation patterns between cell types and in response to heat stress. Intriguingly, at normal temperature, root hairs were more hypermethylated than were stripped roots. However, in response to heat stress, both root hairs and stripped roots showed hypomethylation in each context, especially in the CHH context. Moreover, expression analysis of mRNA from similar tissues and treatments identified some associations between DMRs, genes and transposons. Taken together, the data indicate that changes in DNA methylation are directly or indirectly associated with expression of genes and transposons within the context of either specific tissues/cells or stress (heat). © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  12. Dietary vitamin A impacts DNA methylation patterns of adipogenesis-related genes in suckling rats.

    PubMed

    Arreguín, A; Ribot, J; Mušinović, H; von Lintig, J; Palou, A; Bonet, M L

    2018-05-11

    We previously showed that vitamin A supplementation in early life impacts white adipose tissue (WAT) biology. We here studied the vitamin's effects on DNA methylation of genes crucial for WAT cell development, determination and metabolism. CpG promoter methylation and mRNA expression of Pparg, Zfp423, Pcna, and Rbp4 was compared in inguinal WAT of 21-day-old rats supplemented during the suckling period with vehicle (controls) or an emulsion of vitamin A as retinyl ester (RE) or β-carotene (BC). The methylation profile of promoters was affected by vitamin A supplementation with pronounced differences between the RE and BC groups. In the RE group, hypermethylation of the Rbp4 (at multiple CpGs) and the Pparg2 (at a specific CpG) promoters and hypomethylation of the Pcna promoter (at multiple CpGs) was observed, together with inverse changes in gene expression levels. In the BC group, hypomethylation of the Rbp4 and hypermethylation of the Pcna promoter at distinct CpGs was observed, with no effects on gene expression. In both supplemention groups, hypomethylation and increased expression was found for Zfp423. Thus, modest vitamin A supplementation in early postnatal life impacts methylation marks in developing WAT. Differential epigenetic effects of RE and BC in early life may affect adipose tissue programming activity. Copyright © 2018 Elsevier Inc. All rights reserved.

  13. Potential link between Fusobacterium enrichment and DNA methylation accumulation in the inflammatory colonic mucosa in ulcerative colitis

    PubMed Central

    Tahara, Tomomitsu; Hirata, Ichiro; Nakano, Naoko; Tahara, Sayumi; Horiguchi, Noriyuki; Kawamura, Tomohiko; Okubo, Masaaki; Ishizuka, Takamitsu; Yamada, Hyuga; Yoshida, Dai; Ohmori, Takafumi; Maeda, Kohei; Komura, Naruomi; Ikuno, Hirokazu; Jodai, Yasutaka; Kamano, Toshiaki; Nagasaka, Mitsuo; Nakagawa, Yoshihito; Tuskamoto, Tetsuya; Urano, Makoto; Shibata, Tomoyuki; Kuroda, Makoto; Ohmiya, Naoki

    2017-01-01

    BACKGROUND AND AIM Fusobacterium enrichment has been associated with colorectal cancer development. Ulcerative colitis (UC) associated tumorigenesis is characterized as high degree of methylation accumulation through continuous colonic inflammation. The aim of this study was to investigate a potential link between Fusobacterium enrichment and DNA methylation accumulation in the inflammatory colonic mucosa in UC. METHODS In the candidate analysis, inflamed colonic mucosa from 86 UC patients were characterized the methylation status of colorectal a panel of cancer related 24 genes. In the genome-wide analysis, an Infinium HumanMethylation450 BeadChip array was utilized to characterize the methylation status of >450,000 CpG sites for fourteen UC patients. Results were correlated with Fusobacterium status. RESULTS UC with Fusobacterium enrichment (FB-high) was characterized as high degree of type C (for cancer-specific) methylation compared to other (FB-low/neg) samples (P<0.01). Genes hypermethylated in FB-high samples included well-known type C genes in colorectal cancer, such as MINT2 and 31, P16 and NEUROG1. Multivariate analysis demonstrated that the FB high status held an increased likelihood for methylation high as an independent factor (odds ratio: 16.18, 95% confidence interval: 1.94-135.2, P=0.01). Genome-wide methylation analysis demonstrated a unique methylome signature of FB-high cases irrespective of promoter, outside promoter, CpG and non-CpG sites. Group of promoter CpG sites that were exclusively hypermethylated in FB-high cases significantly codified the genes related to the catalytic activity (P=0.039). CONCLUSION Our findings suggest that Fusobacterium accelerates DNA methylation in specific groups of genes in the inflammatory colonic mucosa in UC. PMID:28977914

  14. Epigenetic events underlie the pathogenesis of sinonasal papillomas.

    PubMed

    Stephen, Josena K; Vaught, Lori E; Chen, Kang M; Sethi, Seema; Shah, Veena; Benninger, Michael S; Gardner, Glendon M; Schweitzer, Vanessa G; Khan, Mumtaz; Worsham, Maria J

    2007-10-01

    Benign inverted papillomas have been reported as monoclonal but lacking common genetic alterations identified in squamous cell carcinoma of the head and neck. Epigenetic changes alter the heritable state of gene expression and chromatin organization without change in DNA sequence. We investigated whether epigenetic events of aberrant promoter hypermethylation in genes known to be involved in squamous head and neck cancer underlie the pathogenesis of sinonasal papillomas. Ten formalin-fixed paraffin DNA samples from three inverted papilloma cases, two exophytic (everted) papilloma cases, and two cases with inverted and exophytic components were studied. DNA was obtained from microdissected areas of normal and papilloma areas and examined using a panel of 41 gene probes, designed to interrogate 35 unique genes for aberrant methylation status (22 genes) using the methylation-specific multiplex-ligation-specific polymerase assay. Methylation-specific PCR was employed to confirm aberrant methylation detected by the methylation-specific multiplex-ligation-specific polymerase assay. All seven cases indicated at least one epigenetic event of aberrant promoter hypermethylation. The CDKN2B gene was a consistent target of aberrant methylation in six of seven cases. Methylation-specific PCR confirmed hypermethylation of CDKN2B. Recurrent biopsies from two inverted papilloma cases had common epigenetic events. Promoter hypermethylation of CDKN2B was a consistent epigenetic event. Common epigenetic alterations in recurrent biopsies underscore a monoclonal origin for these lesions. Epigenetic events contribute to the underlying pathogenesis of benign inverted and exophytic papillomas. As a consistent target of aberrant promoter hypermethylation, CDKN2B may serve as an important epigenetic biomarker for gene reactivation studies.

  15. Association of homocysteine with global DNA methylation in vegetarian Indian pregnant women and neonatal birth anthropometrics.

    PubMed

    Gadgil, Maithili S; Joshi, Kalpana S; Naik, Sadanand S; Pandit, Anand N; Otiv, Suhas R; Patwardhan, Bhushan K

    2014-11-01

    The present study was designed to evaluate if plasma maternal folate, vitamin B-12 and homocysteine levels had an effect on maternal global DNA methylation and neonatal anthropometrics in Indian pregnant women. A total of 49 participants having completed ≥36 weeks of pregnancy were enrolled in the study. Estimation of folate was by Ion capture assay, vitamin B-12 by microparticle enzyme immunoassay, total homocysteine by fluorescence polarization immunoassay and global DNA methylation using Cayman's DNA methylation enzyme immunoassay (EIA) kit. Folate and vitamin B-12 were inversely correlated to homocysteine in pregnant women consuming vegetarian and non-vegetarian diet. No difference in global DNA methylation was found between the vegetarian and non-vegetarian pregnant women. Folate and vitamin B-12 did not show association with global DNA methylation, however plasma total homocysteine of the vegetarian group showed significant correlation to global DNA methylation (r(2 )= 0.49, p = 0.011). Plasma total homocysteine was inversely related to tricep skinfold (r(2 )= -0.484, p = 0.01) and chest circumference (r(2 )= -0.104, p = 0.04) of neonates in vegetarian group. Moderate vitamin B-12 deficiency in vegetarian pregnant women might be the cause of hyperhomocystinemia, hypermethylation when compared to vitamin B-12 sufficient non-vegetarian group.

  16. Variation of DNA methylation patterns associated with gene expression in rice (Oryza sativa) exposed to cadmium.

    PubMed

    Feng, Sheng Jun; Liu, Xue Song; Tao, Hua; Tan, Shang Kun; Chu, Shan Shan; Oono, Youko; Zhang, Xian Duo; Chen, Jian; Yang, Zhi Min

    2016-12-01

    We report genome-wide single-base resolution maps of methylated cytosines and transcriptome change in Cd-exposed rice. Widespread differences were identified in CG and non-CG methylation marks between Cd-exposed and Cd-free rice genomes. There are 2320 non-redundant differentially methylated regions detected in the genome. RNA sequencing revealed 2092 DNA methylation-modified genes differentially expressed under Cd exposure. More genes were found hypermethylated than those hypomethylated in CG, CHH and CHG (where H is A, C or T) contexts in upstream, gene body and downstream regions. Many of the genes were involved in stress response, metal transport and transcription factors. Most of the DNA methylation-modified genes were transcriptionally altered under Cd stress. A subset of loss of function mutants defective in DNA methylation and histone modification activities was used to identify transcript abundance of selected genes. Compared with wide type, mutation of MET1 and DRM2 resulted in general lower transcript levels of the genes under Cd stress. Transcripts of OsIRO2, OsPR1b and Os09g02214 in drm2 were significantly reduced. A commonly used DNA methylation inhibitor 5-azacytidine was employed to investigate whether DNA demethylation affected physiological consequences. 5-azacytidine provision decreased general DNA methylation levels of selected genes, but promoted growth of rice seedlings and Cd accumulation in rice plant. © 2016 John Wiley & Sons Ltd.

  17. Prognostic relevance of aberrant DNA methylation in g1 and g2 pancreatic neuroendocrine tumors.

    PubMed

    Stefanoli, Michele; La Rosa, Stefano; Sahnane, Nora; Romualdi, Chiara; Pastorino, Roberta; Marando, Alessandro; Capella, Carlo; Sessa, Fausto; Furlan, Daniela

    2014-01-01

    The occurrence and clinical relevance of DNA hypermethylation and global hypomethylation in pancreatic neuroendocrine tumours (PanNETs) are still unknown. We evaluated the frequency of both epigenetic alterations in PanNETs to assess the relationship between methylation profiles and chromosomal instability, tumour phenotypes and prognosis. In a well-characterized series of 56 sporadic G1 and G2 PanNETs, methylation-sensitive multiple ligation-dependent probe amplification was performed to assess hypermethylayion of 33 genes and copy number alterations (CNAs) of 53 chromosomal regions. Long interspersed nucleotide element-1 (LINE-1) hypomethylation was quantified by pyrosequencing. Unsupervised hierarchical clustering allowed to identify a subset of 22 PanNETs (39%) exhibiting high frequency of gene-specific methylation and low CNA percentages. This tumour cluster was significantly associated with stage IV (p = 0.04) and with poor prognosis in univariable analysis (p = 0.004). LINE-1 methylation levels in PanNETs were significantly lower than in normal samples (p < 0.01) and were approximately normally distributed. 12 tumours (21%) were highly hypomethylated, showing variable levels of CNA. Interestingly, only 5 PanNETs (9%) were observed to show simultaneously LINE-1 hypomethylation and high frequency of gene-specific methylation. LINE-1 hypomethylation was strongly correlated with advanced stage (p = 0.002) and with poor prognosis (p < 0.0001). In the multivariable analysis, low LINE-1 methylation status and methylation clusters were the only independent significant predictors of outcome (p = 0.034 and p = 0.029, respectively). The combination of global DNA hypomethylation and gene hypermethylation analyses may be useful to define distinct subsets of PanNETs. Both alterations are common in PanNETs and could be directly correlated with tumour progression. © 2014 S. Karger AG, Basel.

  18. Supra-physiological folic acid concentrations induce aberrant DNA methylation in normal human cells in vitro.

    PubMed

    Charles, Michelle A; Johnson, Ian T; Belshaw, Nigel J

    2012-07-01

    The micronutrients folate and selenium may modulate DNA methylation patterns by affecting intracellular levels of the methyl donor S-adenosylmethionine (SAM) and/or the product of methylation reactions S-adenosylhomocysteine (SAH). WI-38 fibroblasts and FHC colon epithelial cells were cultured in the presence of two forms of folate or four forms of selenium at physiologically-relevant doses, and their effects on LINE-1 methylation, gene-specific CpG island (CGI) methylation and intracellular SAM:SAH were determined. At physiologically-relevant doses the forms of folate or selenium had no effect on LINE-1 or CGI methylation, nor on intracellular SAM:SAH. However the commercial cell culture media used for the selenium studies, containing supra-physiological concentrations of folic acid, induced LINE-1 hypomethylation, CGI hypermethylation and decreased intracellular SAM:SAH in both cell lines. We conclude that the exposure of normal human cells to supra-physiological folic acid concentrations present in commercial cell culture media perturbs the intracellular SAM:SAH ratio and induces aberrant DNA methylation.

  19. Whole Genome DNA Methylation Analysis of Obstructive Sleep Apnea: IL1R2, NPR2, AR, SP140 Methylation and Clinical Phenotype.

    PubMed

    Chen, Yung-Che; Chen, Ting-Wen; Su, Mao-Chang; Chen, Chung-Jen; Chen, Kuang-Den; Liou, Chia-Wei; Tang, Petrus; Wang, Ting-Ya; Chang, Jen-Chieh; Wang, Chin-Chou; Lin, Hsin-Ching; Chin, Chien-Hung; Huang, Kuo-Tung; Lin, Meng-Chih; Hsiao, Chang-Chun

    2016-04-01

    We hypothesized that DNA methylation patterns may contribute to disease severity or the development of hypertension and excessive daytime sleepiness (EDS) in patients with obstructive sleep apnea (OSA). Illumina's (San Diego, CA, USA) DNA methylation 27-K assay was used to identify differentially methylated loci (DML). DNA methylation levels were validated by pyrosequencing. A discovery cohort of 15 patients with OSA and 6 healthy subjects, and a validation cohort of 72 patients with sleep disordered breathing (SDB). Microarray analysis identified 636 DMLs in patients with OSA versus healthy subjects, and 327 DMLs in patients with OSA and hypertension versus those without hypertension. In the validation cohort, no significant difference in DNA methylation levels of six selected genes was found between the primary snoring subjects and OSA patients (primary outcome). However, a secondary outcome analysis showed that interleukin-1 receptor 2 (IL1R2) promoter methylation (-114 cytosine followed by guanine dinucleotide sequence [CpG] site) was decreased and IL1R2 protein levels were increased in the patients with SDB with an oxygen desaturation index > 30. Androgen receptor (AR) promoter methylation (-531 CpG site) and AR protein levels were both increased in the patients with SDB with an oxygen desaturation index > 30. Natriuretic peptide receptor 2 (NPR2) promoter methylation (-608/-618 CpG sites) were decreased, whereas levels of both NPR2 and serum C type natriuretic peptide protein were increased in the SDB patients with EDS. Speckled protein 140 (SP140) promoter methylation (-194 CpG site) was increased, and SP140 protein levels were decreased in the patients with SDB and EDS. IL1R2 hypomethylation and AR hypermethylation may constitute an important determinant of disease severity, whereas NPR2 hypomethylation and SP140 hypermethylation may provide a biomarker for vulnerability to EDS in OSA. A commentary on this article appears in this issue on page 723. © 2016

  20. Abnormal plasma DNA profiles in early ovarian cancer using a non-invasive prenatal testing platform: implications for cancer screening.

    PubMed

    Cohen, Paul A; Flowers, Nicola; Tong, Stephen; Hannan, Natalie; Pertile, Mark D; Hui, Lisa

    2016-08-24

    Non-invasive prenatal testing (NIPT) identifies fetal aneuploidy by sequencing cell-free DNA in the maternal plasma. Pre-symptomatic maternal malignancies have been incidentally detected during NIPT based on abnormal genomic profiles. This low coverage sequencing approach could have potential for ovarian cancer screening in the non-pregnant population. Our objective was to investigate whether plasma DNA sequencing with a clinical whole genome NIPT platform can detect early- and late-stage high-grade serous ovarian carcinomas (HGSOC). This is a case control study of prospectively-collected biobank samples comprising preoperative plasma from 32 women with HGSOC (16 'early cancer' (FIGO I-II) and 16 'advanced cancer' (FIGO III-IV)) and 32 benign controls. Plasma DNA from cases and controls were sequenced using a commercial NIPT platform and chromosome dosage measured. Sequencing data were blindly analyzed with two methods: (1) Subchromosomal changes were called using an open source algorithm WISECONDOR (WIthin-SamplE COpy Number aberration DetectOR). Genomic gains or losses ≥ 15 Mb were prespecified as "screen positive" calls, and mapped to recurrent copy number variations reported in an ovarian cancer genome atlas. (2) Selected whole chromosome gains or losses were reported using the routine NIPT pipeline for fetal aneuploidy. We detected 13/32 cancer cases using the subchromosomal analysis (sensitivity 40.6 %, 95 % CI, 23.7-59.4 %), including 6/16 early and 7/16 advanced HGSOC cases. Two of 32 benign controls had subchromosomal gains ≥ 15 Mb (specificity 93.8 %, 95 % CI, 79.2-99.2 %). Twelve of the 13 true positive cancer cases exhibited specific recurrent changes reported in HGSOC tumors. The NIPT pipeline resulted in one "monosomy 18" call from the cancer group, and two "monosomy X" calls in the controls. Low coverage plasma DNA sequencing used for prenatal testing detected 40.6 % of all HGSOC, including 38 % of early stage cases. Our

  1. Induced DNA demethylation by targeting Ten-Eleven Translocation 2 to the human ICAM-1 promoter

    PubMed Central

    Chen, Hui; Kazemier, Hinke G; de Groote, Marloes L.; Ruiters, Marcel H. J.; Xu, Guo-Liang; Rots, Marianne G.

    2014-01-01

    Increasing evidence indicates that active DNA demethylation is involved in several processes in mammals, resulting in developmental stage-specificity and cell lineage-specificity. The recently discovered Ten-Eleven Translocation (TET) dioxygenases are accepted to be involved in DNA demethylation by initiating 5-mC oxidation. Aberrant DNA methylation profiles are associated with many diseases. For example in cancer, hypermethylation results in silencing of tumor suppressor genes. Such silenced genes can be re-expressed by epigenetic drugs, but this approach has genome-wide effects. In this study, fusions of designer DNA binding domains to TET dioxygenase family members (TET1, -2 or -3) were engineered to target epigenetically silenced genes (ICAM-1, EpCAM). The effects on targeted CpGs’ methylation and on expression levels of the target genes were assessed. The results indicated demethylation of targeted CpG sites in both promoters for targeted TET2 and to a lesser extent for TET1, but not for TET3. Interestingly, we observed re-activation of transcription of ICAM-1. Thus, our work suggests that we provided a mechanism to induce targeted DNA demethylation, which facilitates re-activation of expression of the target genes. Furthermore, this Epigenetic Editing approach is a powerful tool to investigate functions of epigenetic writers and erasers and to elucidate consequences of epigenetic marks. PMID:24194590

  2. Genome-wide analysis of day/night DNA methylation differences in Populus nigra.

    PubMed

    Ding, Chang-Jun; Liang, Li-Xiong; Diao, Shu; Su, Xiao-Hua; Zhang, Bing-Yu

    2018-01-01

    DNA methylation is an important mechanism of epigenetic modification. Methylation changes during stress responses and developmental processes have been well studied; however, their role in plant adaptation to the day/night cycle is poorly understood. In this study, we detected global methylation patterns in leaves of the black poplar Populus nigra 'N46' at 8:00 and 24:00 by methylated DNA immunoprecipitation sequencing (MeDIP-seq). We found 10,027 and 10,242 genes to be methylated in the 8:00 and 24:00 samples, respectively. The methylated genes appeared to be involved in multiple biological processes, molecular functions, and cellular components, suggesting important roles for DNA methylation in poplar cells. Comparing the 8:00 and 24:00 samples, only 440 differentially methylated regions (DMRs) overlapped with genic regions, including 193 hyper- and 247 hypo-methylated DMRs, and may influence the expression of 137 downstream genes. Most hyper-methylated genes were associated with transferase activity, kinase activity, and phosphotransferase activity, whereas most hypo-methylated genes were associated with protein binding, ATP binding, and adenyl ribonucleotide binding, suggesting that different biological processes were activated during the day and night. Our results indicated that methylated genes were prevalent in the poplar genome, but that only a few of these participated in diurnal gene expression regulation.

  3. Evaluation of Genomic Instability in the Abnormal Prostate

    DTIC Science & Technology

    2006-12-01

    array CGH maps copy number aberrations relative to the genome sequence by using arrays of BAC or cDNA clones as the hybridization target instead of...data produced from these analyses complicate the interpretation of results . For these reasons, and as outlined by Davies et al., 22 it is desirable...There have been numerous studies of these abnormalities and several techniques, including 9 chromosome painting, array CGH and SNP arrays , have

  4. Initial analysis of sperm DNA methylome in Holstein bulls

    USDA-ARS?s Scientific Manuscript database

    Aberrant DNA methylation patterns have been associated with abnormal semen parameters, idiopathic male infertility and early embryonic loss in mammals. Using Holstein bulls with high (Bull1) or low (Bull2) fertility rates, we created two representative sperm DNA methylomes at a single-base resolutio...

  5. DNA methylation levels analysis in four tissues of sea cucumber Apostichopus japonicus based on fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) during aestivation.

    PubMed

    Zhao, Ye; Chen, Muyan; Storey, Kenneth B; Sun, Lina; Yang, Hongsheng

    2015-03-01

    DNA methylation plays an important role in regulating transcriptional change in response to environmental stimuli. In the present study, DNA methylation levels of tissues of the sea cucumber Apostichopus japonicus were analyzed by the fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) technique over three stages of the aestivation cycle. Overall, a total of 26,963 fragments were amplified including 9112 methylated fragments among four sea cucumber tissues using 18 pairs of selective primers. Results indicated an average DNA methylation level of 33.79% for A. japonicus. The incidence of DNA methylation was different across tissue types in the non-aestivation stage: intestine (30.16%), respiratory tree (27.61%), muscle (27.94%) and body wall (56.25%). Our results show that hypermethylation accompanied deep-aestivation in A. japonicus, which suggests that DNA methylation may have an important role in regulating global transcriptional suppression during aestivation. Further analysis indicated that the main DNA modification sites were focused on intestine and respiratory tree tissues and that full-methylation but not hemi-methylation levels exhibited significant increases in the deep-aestivation stage. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Altered intragenic DNA methylation of HOOK2 gene in adipose tissue from individuals with obesity and type 2 diabetes

    PubMed Central

    Fernández-Bayón, Gustavo; Morales-Sánchez, Paula; Sanz, Lourdes; Turienzo, Estrella; González, Juan José; Martinez-Faedo, Ceferino; Suarez-Gutiérrez, Lorena; Ares, Jessica; Díaz-Naya, Lucia; Martin-Nieto, Alicia; Fernández-Morera, Juan L.; Fraga, Mario F.

    2017-01-01

    Aims/Hypothesis Failure in glucose response to insulin is a common pathology associated with obesity. In this study, we analyzed the genome wide DNA methylation profile of visceral adipose tissue (VAT) samples in a population of individuals with obesity and assessed whether differential methylation profiles are associated with the presence of type 2 diabetes (T2D). Methods More than 485,000 CpG genome sites from VAT samples from women with obesity undergoing gastric bypass (n = 18), and classified as suffering from type 2 diabetes (T2D) or not (no type 2 diabetes, NT2D), were analyzed using DNA methylation arrays. Results We found significant differential methylation between T2D and NT2D samples in 24 CpGs that map with sixteen genes, one of which, HOOK2, demonstrated a significant correlation between differentially hypermethylated regions on the gene body and the presence of type 2 diabetes. This was validated by pyrosequencing in a population of 91 samples from both males and females with obesity. Furthermore, when these results were analyzed by gender, female T2D samples were found hypermethylated at the cg04657146-region and the cg 11738485-region of HOOK2 gene, whilst, interestingly, male samples were found hypomethylated in this latter region. Conclusion The differential methylation profile of the HOOK2 gene in individuals with T2D and obesity might be related to the attendant T2D, but further studies are required to identify the potential role of HOOK2 gene in T2D disease. The finding of gender differences in T2D methylation of HOOK2 also warrants further investigation. PMID:29228058

  7. Recognition and repair of chemically heterogeneous structures at DNA ends

    PubMed Central

    Andres, Sara N.; Schellenberg, Matthew J.; Wallace, Bret D.; Tumbale, Percy; Williams, R. Scott

    2014-01-01

    Exposure to environmental toxicants and stressors, radiation, pharmaceutical drugs, inflammation, cellular respiration, and routine DNA metabolism all lead to the production of cytotoxic DNA strand breaks. Akin to splintered wood, DNA breaks are not “clean”. Rather, DNA breaks typically lack DNA 5'-phosphate and 3'-hydroxyl moieties required for DNA synthesis and DNA ligation. Failure to resolve damage at DNA ends can lead to abnormal DNA replication and repair, and is associated with genomic instability, mutagenesis, neurological disease, ageing and carcinogenesis. An array of chemically heterogeneous DNA termini arises from spontaneously generated DNA single-strand and double-strand breaks (SSBs and DSBs), and also from normal and/or inappropriate DNA metabolism by DNA polymerases, DNA ligases and topoisomerases. As a front line of defense to these genotoxic insults, eukaryotic cells have accrued an arsenal of enzymatic first responders that bind and protect damaged DNA termini, and enzymatically tailor DNA ends for DNA repair synthesis and ligation. These nucleic acid transactions employ direct damage reversal enzymes including Aprataxin (APTX), Polynucleotide kinase phosphatase (PNK), the tyrosyl DNA phosphodiesterases (TDP1 and TDP2), the Ku70/80 complex and DNA polymerase β (POLβ). Nucleolytic processing enzymes such as the MRE11/RAD50/NBS1/CtIP complex, Flap endonuclease (FEN1) and the apurinic endonucleases (APE1 and APE2) also act in the chemical "cleansing" of DNA breaks to prevent genomic instability and disease, and promote progression of DNA- and RNA-DNA damage response (DDR and RDDR) pathways. Here, we provide an overview of cellular first responders dedicated to the detection and repair of abnormal DNA termini. PMID:25111769

  8. Hypermethylation of 28S ribosomal RNA in β-thalassemia trait carriers.

    PubMed

    Sornjai, Wannapa; Lithanatudom, Pathrapol; Erales, Jenny; Joly, Philippe; Francina, Alain; Hacot, Sabine; Fucharoen, Suthat; Svasti, Saovaros; Diaz, Jean Jacques; Mertani, Hichem C; Smith, Duncan R

    2017-01-01

    Ribosome biogenesis is the process of synthesis of the cellular ribosomes which mediate protein translation. Integral with the ribosomes are four cytoplasmic ribosomal RNAs (rRNAs) which show extensive post-transcriptional modifications including 2'-O-methylation and pseudouridylation. Several hereditary hematologic diseases including Diamond-Blackfan anemia have been shown to be associated with defects in ribosome biogenesis. Thalassemia is the most important hematologic inherited genetic disease worldwide, and this study examined the post-transcriptional ribose methylation status of three specific active sites of the 28S rRNA molecule at positions 1858, 4197 and 4506 of β-thalassemia trait carriers and normal controls. Samples from whole blood and cultured erythroid cells were examined. Results showed that site 4506 was hypermethylated in β-thalassemia trait carriers in both cohorts. Expression of fibrillarin, the ribosomal RNA methyltransferase as well as snoRNAs were additionally quantified by RT-qPCR and evidence of dysregulation was seen. Hemoglobin E trait carriers also showed evidence of dysregulation. These results provide the first evidence that ribosome biogenesis is dysregulated in β-thalassemia trait carriers. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Association of MTHFR polymorphisms and chromosomal abnormalities in leukemia.

    PubMed

    Sinthuwiwat, Thivaratana; Poowasanpetch, Phanasit; Wongngamrungroj, Angsana; Soonklang, Kamonwan; Promso, Somying; Auewarakul, Chirayu; Tocharoentanaphol, Chintana

    2012-01-01

    Genetic variation in MTHFR gene might explain the interindividual differences in the reduction of DNA repaired and the increase of chromosome breakage and damage. Nowadays, chromosomal rearrangement is recognized as a major cause of lymphoid malignancies. In addition, the association of MTHFR polymorphisms with aneuploidy was found in several studies, making the MTHFR gene as a good candidate for leukemia etiology. Therefore, in this study, we investigated the common sequence variation, 677C>T and 1298A>C in the MTHFR gene of 350 fixed cell specimens archived after chromosome analysis. The distribution of the MTHFR polymorphisms frequency was compared in leukemic patients with structural chromosome abnormality and chromosome aneuploidy, as well as in those with no evidence of chromosome abnormalities. We observed a significant decrease in the distribution of T allele in 677C>T polymorphisms among patients with chromosomal abnormalities including both structural aberration and aneuploidy. The same significance result also found in patients with structural aberration when compare with the normal karyotype patients. Suggesting that polymorphism in the MTHFR gene was involved in chromosome abnormalities of leukemia. However, further investigation on the correlation with the specific types of chromosomal aberrations is needed.

  10. Prenatal Nutritional Deficiency Reprogrammed Postnatal Gene Expression in Mammal Brains: Implications for Schizophrenia

    PubMed Central

    Xu, Jiawei; He, Guang; Zhu, Jingde; Zhou, Xinyao; St Clair, David; Wang, Teng; Xiang, Yuqian; Zhao, Qingzhu; Xing, Qinghe; Liu, Yun; Wang, Lei; Li, Qiaoli

    2015-01-01

    Background: Epidemiological studies have identified prenatal exposure to famine as a risk factor for schizophrenia, and animal models of prenatal malnutrition display structural and functional brain abnormalities implicated in schizophrenia. Methods: The offspring of the RLP50 rat, a recently developed animal model of prenatal famine malnutrition exposure, was used to investigate the changes of gene expression and epigenetic modifications in the brain regions. Microarray gene expression analysis was carried out in the prefrontal cortex and the hippocampus from 8 RLP50 offspring rats and 8 controls. MBD-seq was used to test the changes in DNA methylation in hippocampus depending on prenatal malnutrition exposure. Results: In the prefrontal cortex, offspring of RLP50 exhibit differences in neurotransmitters and olfactory-associated gene expression. In the hippocampus, the differentially-expressed genes are related to synaptic function and transcription regulation. DNA methylome profiling of the hippocampus also shows widespread but systematic epigenetic changes; in most cases (87%) this involves hypermethylation. Remarkably, genes encoded for the plasma membrane are significantly enriched for changes in both gene expression and DNA methylome profiling screens (p = 2.37×10–9 and 5.36×10–9, respectively). Interestingly, Mecp2 and Slc2a1, two genes associated with cognitive impairment, show significant down-regulation, and Slc2a1 is hypermethylated in the hippocampus of the RLP50 offspring. Conclusions: Collectively, our results indicate that prenatal exposure to malnutrition leads to the reprogramming of postnatal brain gene expression and that the epigenetic modifications contribute to the reprogramming. The process may impair learning and memory ability and result in higher susceptibility to schizophrenia. PMID:25522397

  11. Immunohistochemical null-phenotype for mismatch repair proteins in colonic carcinoma associated with concurrent MLH1 hypermethylation and MSH2 somatic mutations.

    PubMed

    Wang, Tao; Stadler, Zsofia K; Zhang, Liying; Weiser, Martin R; Basturk, Olca; Hechtman, Jaclyn F; Vakiani, Efsevia; Saltz, Lenard B; Klimstra, David S; Shia, Jinru

    2018-04-01

    Microsatellite instability, a well-established driver pathway in colorectal carcinogenesis, can develop in both sporadic and hereditary conditions via different molecular alterations in the DNA mismatch repair (MMR) genes. MMR protein immunohistochemistry (IHC) is currently widely used for the detection of MMR deficiency in solid tumors. The IHC test, however, can show varied staining patterns, posing challenges in the interpretation of the staining results in some cases. Here we report a case of an 80-year-old female with a colonic adenocarcinoma that exhibited an unusual "null" IHC staining pattern with complete loss of all four MMR proteins (MLH1, MSH2, MSH6, and PMS2). This led to subsequent MLH1 methylation testing and next generation sequencing which demonstrated that the loss of all MMR proteins was associated with concurrent promoter hypermethylation of MLH1 and double somatic truncating mutations in MSH2. These molecular findings, in conjunction with the patient's age being 80 years and the fact that the patient had no personal or family cancer history, indicated that the MMR deficiency was highly likely sporadic in nature. Thus, the stringent Lynch syndrome type surveillance programs were not recommended to the patient and her family members. This case illustrates a rare but important scenario where a null IHC phenotype signifies complex underlying molecular alternations that bear clinical management implications, highlighting the need for recognition and awareness of such unusual IHC staining patterns.

  12. Abnormal brain aging as a radical-related disease: A new target for nuclear medicine

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Fujibayashi, Y.; Yamamoto, S.; Waki, A.

    DNA damages caused by endogenously produced radicals are closely correlated with aging. Among them, mitochondrial DNA (mtDNA) deletions have been reported as a memory of DNA damage by oxygen radicals. In fact, clinical as well as experimental studies indicated the accumulation of deleted mtDNA in the brain, myocardium and son on, in aged subjects. In our previous work, radioiodinated radical trapping agent, p-iodophenyl-N-t-butylnitrone, and hypoxia imaging agent, Cu-62 diacetyl-bis-N-4-methyl-thiosemicarbazone have been developed for the diagnosis of radical-related diseases, such as ischemic, inflammation, cancer or aging. The aim of the present work was to evaluate these agents for brain aging studies.more » In our university, an unique animal model, a senescence accelerated model mouse (SAM), has been established. Among the various substrains, SAMP8 showing memory deterioration in its young age ({approximately}3 month) was basically evaluated as an abnormal brain aging model with mtDNA deletion. As controls, SAMR1 showing normal aging and ddY mice were used. MtDNA deletion n the brain was analyzed with polymerase-chain reaction (PCR) method, and relationship between mtDNA deletion and brain uptake of IPBN or Cu-62-ATSM was studied. In 1-3 month old SAMP8 brain, multiple mtDNa deletions were already found and their content was significantly higher than that of SAMR1 or age-matched ddY control. Thus, it was cleared that SAMP8 brain has high tendency to be attacked by endogenously produced oxygen radicals, possibly from its birth. Both IPBN and Cu-ATSM showed significantly higher accumulation in the SAMP8 brain than in the SAMR1 brain, indicating that these agents have high possibility for the early detection of abnormal brain aging as a radical-related disease.« less

  13. Identification of DNA methylation changes associated with disease progression in subchondral bone with site-matched cartilage in knee osteoarthritis.

    PubMed

    Zhang, Yanfei; Fukui, Naoshi; Yahata, Mitsunori; Katsuragawa, Yozo; Tashiro, Toshiyuki; Ikegawa, Shiro; Lee, Ming Ta Michael

    2016-09-30

    Subchondral bone plays a key role in the development of osteoarthritis, however, epigenetics of subchondral bone has not been extensively studied. In this study, we examined the genome-wide DNA methylation profiles of subchondral bone from three regions on tibial plateau representing disease progression using HumanMethylation450 BeadChip to identify progression associated DNA methylation alterations. Significant differential methylated probes (DMPs) and differential methylated genes (DMGs) were identified in the intermediate and late stages and during the transition from intermediate to late stage of OA in the subchondral bone. Over half of the DMPs were hyper-methylated. Genes associated with OA and bone remodeling were identified. DMGs were enriched in morphogenesis and development of skeletal system, and HOX transcription factors. Comparison of DMGs identified in subchondral bone and site-matched cartilage indicated that DNA methylation changes occurred earlier in subchondral bone and identified different methylation patterns at the late stage of OA. However, shared DMPs, DMGs and common pathways that implicated the tissue reparation were also identified. Methylation is one key mechanism to regulate the crosstalk between cartilage and subchondral bone.

  14. Cervical cytology of atypical squamous cells, cannot exclude high-grade squamous intra-epithelial lesion: significance of age, human papillomavirus DNA detection and previous abnormal cytology on follow-up outcomes.

    PubMed

    Sung, Chang Ohk; Oh, Young Lyun; Song, Sang Yong

    2011-11-01

    Despite the usefulness of Pap tests for cancer screening, outcomes can be difficult to predict when atypical squamous cells (ASCs) are identified. According to the 2001 Bethesda system, ASCs can be subdivided into two groups: ASCs of undetermined significance (ASC-US); and ASCs, cannot exclude high-grade squamous intra-epithelial lesion (ASC-H). ASC-H interpretations are uncommon, and studies involving this type of lesion are based on small numbers of cases. Cross-sectional, retrospective study of 392 ASC-H cases. The follow-up outcomes of ASC-H cases that were diagnosed during routine primary screening between 2002 and 2008 were investigated, and relationships between clinicopathological parameters were assessed, particularly positive test for high-risk HPV (HPV) DNA, patient age at diagnosis and previous abnormal cytology. Of the 392 cases, high-grade squamous intra-epithelial lesion (HSIL) was detected in 111 (28.3%) cases, squamous cell carcinoma was detected in 15 (3.8%) cases, low-grade squamous intra-epithelial lesion was detected in 37 (9.4%) cases, reactive change was detected in 178 (45.4%) cases, atrophy was detected in 47 (12.0%) cases, and adenocarcinoma was detected in four (1.0%) cases. The prevalence of HSIL or greater was 27.8% for women aged ≥ 40 years, and 52.3% for women aged <40 years (p<0.001). HPV positivity in ASC-H smears was significantly associated with HSIL or greater, irrespective of age (<40 years, p=0.003; ≥ 40 years, p<0.001). ASC-H with previous abnormal cytology greater than ASC-US showed a significantly higher detection rate for HSIL or greater at follow-up (p<0.001). Patient age, positive HPV DNA test and previous abnormal cytology are useful predictors of underlying HSIL or greater in women with ASC-H. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  15. Maternal pre-pregnancy BMI and gestational weight gain, offspring DNA methylation and later offspring adiposity: findings from the Avon Longitudinal Study of Parents and Children

    PubMed Central

    Sharp, Gemma C; Lawlor, Debbie A; Richmond, Rebecca C; Fraser, Abigail; Simpkin, Andrew; Suderman, Matthew; Shihab, Hashem A; Lyttleton, Oliver; McArdle, Wendy; Ring, Susan M; Gaunt, Tom R; Davey Smith, George; Relton, Caroline L

    2015-01-01

    Background: Evidence suggests that in utero exposure to undernutrition and overnutrition might affect adiposity in later life. Epigenetic modification is suggested as a plausible mediating mechanism. Methods: We used multivariable linear regression and a negative control design to examine offspring epigenome-wide DNA methylation in relation to maternal and offspring adiposity in 1018 participants. Results: Compared with neonatal offspring of normal weight mothers, 28 and 1621 CpG sites were differentially methylated in offspring of obese and underweight mothers, respectively [false discovert rate (FDR)-corrected P-value < 0.05), with no overlap in the sites that maternal obesity and underweight relate to. A positive association, where higher methylation is associated with a body mass index (BMI) outside the normal range, was seen at 78.6% of the sites associated with obesity and 87.9% of the sites associated with underweight. Associations of maternal obesity with offspring methylation were stronger than associations of paternal obesity, supporting an intrauterine mechanism. There were no consistent associations of gestational weight gain with offspring DNA methylation. In general, sites that were hypermethylated in association with maternal obesity or hypomethylated in association with maternal underweight tended to be positively associated with offspring adiposity, and sites hypomethylated in association with maternal obesity or hypermethylated in association with maternal underweight tended to be inversely associated with offspring adiposity. Conclusions: Our data suggest that both maternal obesity and, to a larger degree, underweight affect the neonatal epigenome via an intrauterine mechanism, but weight gain during pregnancy has little effect. We found some evidence that associations of maternal underweight with lower offspring adiposity and maternal obesity with greater offspring adiposity may be mediated via increased DNA methylation. PMID:25855720

  16. Novel Methylated Biomarkers and a Robust Assay to Detect Circulating Tumor DNA in Metastatic Breast Cancer

    PubMed Central

    Fackler, Mary Jo; Bujanda, Zoila Lopez; Umbricht, Christopher; Teo, Wei Wen; Cho, Soonweng; Zhang, Zhe; Visvanathan, Kala; Jeter, Stacie; Argani, Pedram; Wang, Chenguang; Lyman, Jaclyn P.; de Brot, Marina; Ingle, James N.; Boughey, Judy; McGuire, Kandace; King, Tari A.; Carey, Lisa A.; Cope, Leslie; Wolff, Antonio C.; Sukumar, Saraswati

    2015-01-01

    The ability to consistently detect cell-free tumor-specific DNA in peripheral blood of patients with metastatic breast cancer provides the opportunity to detect changes in tumor burden and to monitor response to treatment. We developed cMethDNA, a quantitative multiplexed methylation-specific PCR assay for a panel of ten genes, consisting of novel and known breast cancer hypermethylated markers identified by mining our previously reported study of DNA methylation patterns in breast tissue (103 cancer, 21 normal on the Illumina HumanMethylation27 Beadchip) and then validating the 10-gene panel in a TCGA breast cancer methylome database. For cMethDNA, a fixed physiological level (50 copies) of artificially constructed, standard non-human reference DNA specific for each gene is introduced into in a constant volume of serum (300 μl) prior to purification of the DNA, facilitating a sensitive, specific, robust and quantitative assay of tumor DNA, with broad dynamic range. Cancer-specific methylated DNA was detected in Training (28 normal, 24 cancer) and Test (27 normal, 33 cancer) sets of recurrent Stage 4 patient sera with a sensitivity of 91% and a specificity of 96% in the test set. In a pilot study, cMethDNA assay faithfully reflected patient response to chemotherapy (N = 29). A core methylation signature present in the primary breast cancer was retained in serum and metastatic tissues collected at autopsy 2–11 years after diagnosis of the disease. Together, our data suggest that the cMethDNA assay can detect advanced breast cancer, and monitor tumor burden and treatment response in women with metastatic breast cancer. PMID:24737128

  17. A novel interplay between HOTAIR and DNA methylation in osteosarcoma cells indicates a new therapeutic strategy.

    PubMed

    Li, Xingang; Lu, Hongming; Fan, Guilian; He, Miao; Sun, Yu; Xu, Kai; Shi, Fengjun

    2017-11-01

    Osteosarcoma (OS) is one of the most prevalent primary malignant bone tumors in adolescent. HOTAIR is highly expressed and associated with the epigenetic modifications, especially DNA methylation, in cancer. However, the regulation mechanism between HOTAIR and DNA methylation and the biological effects of them in the pathogenesis of osteosarcoma remains elusive. Through RNA-sequencing and computational analysis, followed by a variety of experimental validations, we report a novel interplay between HOTAIR, miR-126, and DNA methylation in OS. We found that HOTAIR is highly expressed in OS cells and the knockdown of HOTAIR leads to the down-regulation of DNMT1, as well as the decrease of global DNA methylation level. RNA-sequencing analysis of HOTAIR-regulated gene shows that CDKN2A is significantly repressed by HOTAIR. A series of experiments show that HOTAIR represses the expression of CDKN2A through inhibiting the promoter activity of CDKN2A by DNA hypermethylation. Further evidence shows that HOTAIR activates the expression of DNMT1 through repressing miR-126, which is the negative regulator of DNMT1. Functionally, HOTAIR depletion increases the sensibility of OS cells to DNMT1 inhibitor through regulating the viability and apoptosis of OS cells via HOTAIR-miR126-DNMT1-CDKN2A axis. These results not only enrich our understanding of the regulation relationship between non-coding RNA, DNA methylation, and gene expression, however, also provide a novel direction in developing more sophisticated therapeutic strategies for OS patients.

  18. High expression of DNA methyltransferases in primary human medulloblastoma.

    PubMed

    Pócza, T; Krenács, T; Turányi, E; Csáthy, J; Jakab, Z; Hauser, P

    2016-01-01

    Epigenetic alterations have been implicated in cancer development. DNA methylation modulates gene expression, which is catalyzed by DNA methyltransferases (DNMTs). The objective of our study was to evaluate expression of DNMTs in medulloblastoma and analyze its correlation with clinical features. Nuclear expression of DNMT1, DNMT3A and DNMT3B was analyzed in human primary medulloblastoma of 44 patients using immunohistochemistry. Correlation of expression of DNMT levels with classical histological subtypes, novel molecular subgroups and survival of patients was analyzed. Elevated expression of DNMT1, DNMT3A and DNMT3B was observed in 63.64%, 68.18% and 72.73% of all cases, respectively. None of them showed a correlation with classical histology or survival. Concerning molecular subtypes, significantly higher expression of DNMT1 was observed in the SHH group compared to non-SHH samples (p = 0.02), but without significant difference in DNMT3A or DNMT3B levels between any subtypes. In conclusion, DNMT1, DNMT3A and DNMT3B are highly expressed in human medulloblastoma samples, suggesting that promoter hypermethylation may play a role in medulloblastoma development. Demethylation of tumor suppressor gene promoters may be considered as a possible future target in therapy of medulloblastoma.

  19. Analysis of DNA methylation and gene expression in radiation-resistant head and neck tumors.

    PubMed

    Chen, Xiaofei; Liu, Liang; Mims, Jade; Punska, Elizabeth C; Williams, Kristin E; Zhao, Weiling; Arcaro, Kathleen F; Tsang, Allen W; Zhou, Xiaobo; Furdui, Cristina M

    2015-01-01

    Resistance to radiation therapy constitutes a significant challenge in the treatment of head and neck squamous cell cancer (HNSCC). Alteration in DNA methylation is thought to play a role in this resistance. Here, we analyzed DNA methylation changes in a matched model of radiation resistance for HNSCC using the Illumina HumanMethylation450 BeadChip. Our results show that compared to radiation-sensitive cells (SCC-61), radiation-resistant cells (rSCC-61) had a significant increase in DNA methylation. After combining these results with microarray gene expression data, we identified 84 differentially methylated and expressed genes between these 2 cell lines. Ingenuity Pathway Analysis revealed ILK signaling, glucocorticoid receptor signaling, fatty acid α-oxidation, and cell cycle regulation as top canonical pathways associated with radiation resistance. Validation studies focused on CCND2, a protein involved in cell cycle regulation, which was identified as hypermethylated in the promoter region and downregulated in rSCC-61 relative to SCC-61 cells. Treatment of rSCC-61 and SCC-61 with the DNA hypomethylating agent 5-aza-2'deoxycitidine increased CCND2 levels only in rSCC-61 cells, while treatment with the control reagent cytosine arabinoside did not influence the expression of this gene. Further analysis of HNSCC data from The Cancer Genome Atlas found increased methylation in radiation-resistant tumors, consistent with the cell culture data. Our findings point to global DNA methylation status as a biomarker of radiation resistance in HNSCC, and suggest a need for targeted manipulation of DNA methylation to increase radiation response in HNSCC.

  20. Novel Insights into DNA Methylation Features in Spermatozoa: Stability and Peculiarities

    PubMed Central

    Sayols, Sergi; Chianese, Chiara; Giachini, Claudia; Heyn, Holger; Esteller, Manel

    2012-01-01

    Data about the entire sperm DNA methylome are limited to two sperm donors whereas studies dealing with a greater number of subjects focused only on a few genes or were based on low resolution arrays. This implies that information about what we can consider as a normal sperm DNA methylome and whether it is stable among different normozoospermic individuals is still missing. The definition of the DNA methylation profile of normozoospermic men, the entity of inter-individual variability and the epigenetic characterization of quality-fractioned sperm subpopulations in the same subject (intra-individual variability) are relevant for a better understanding of pathological conditions. We addressed these questions by using the high resolution Infinium 450K methylation array and compared normal sperm DNA methylomes against somatic and cancer cells. Our study, based on the largest number of subjects (n = 8) ever considered for such a large number of CpGs (n = 487,517), provided clear evidence for i) a highly conserved DNA methylation profile among normozoospermic subjects; ii) a stable sperm DNA methylation pattern in different quality-fractioned sperm populations of the same individual. The latter finding is particularly relevant if we consider that different quality fractioned sperm subpopulations show differences in their structural features, metabolic and genomic profiles. We demonstrate, for the first time, that DNA methylation in normozoospermic men remains highly uniform regardless the quality of sperm subpopulations. In addition, our analysis provided both confirmatory and novel data concerning the sperm DNA methylome, including its peculiar features in respect to somatic and cancer cells. Our description about a highly polarized sperm DNA methylation profile, the clearly distinct genomic and functional organization of hypo- versus hypermethylated loci as well as the association of histone-enriched hypomethylated loci with embryonic development, which we now

  1. DNA methylation and childhood asthma in the inner city.

    PubMed

    Yang, Ivana V; Pedersen, Brent S; Liu, Andrew; O'Connor, George T; Teach, Stephen J; Kattan, Meyer; Misiak, Rana Tawil; Gruchalla, Rebecca; Steinbach, Suzanne F; Szefler, Stanley J; Gill, Michelle A; Calatroni, Agustin; David, Gloria; Hennessy, Corinne E; Davidson, Elizabeth J; Zhang, Weiming; Gergen, Peter; Togias, Alkis; Busse, William W; Schwartz, David A

    2015-07-01

    Epigenetic marks are heritable, influenced by the environment, direct the maturation of T lymphocytes, and in mice enhance the development of allergic airway disease. Thus it is important to define epigenetic alterations in asthmatic populations. We hypothesize that epigenetic alterations in circulating PBMCs are associated with allergic asthma. We compared DNA methylation patterns and gene expression in inner-city children with persistent atopic asthma versus healthy control subjects by using DNA and RNA from PBMCs. Results were validated in an independent population of asthmatic patients. Comparing asthmatic patients (n = 97) with control subjects (n = 97), we identified 81 regions that were differentially methylated. Several immune genes were hypomethylated in asthma, including IL13, RUNX3, and specific genes relevant to T lymphocytes (TIGIT). Among asthmatic patients, 11 differentially methylated regions were associated with higher serum IgE concentrations, and 16 were associated with percent predicted FEV1. Hypomethylated and hypermethylated regions were associated with increased and decreased gene expression, respectively (P < 6 × 10(-12) for asthma and P < .01 for IgE). We further explored the relationship between DNA methylation and gene expression using an integrative analysis and identified additional candidates relevant to asthma (IL4 and ST2). Methylation marks involved in T-cell maturation (RUNX3), TH2 immunity (IL4), and oxidative stress (catalase) were validated in an independent asthmatic cohort of children living in the inner city. Our results demonstrate that DNA methylation marks in specific gene loci are associated with asthma and suggest that epigenetic changes might play a role in establishing the immune phenotype associated with asthma. Published by Elsevier Inc.

  2. Aflatoxin B₁-Induced Developmental and DNA Damage in Caenorhabditis elegans.

    PubMed

    Feng, Wei-Hong; Xue, Kathy S; Tang, Lili; Williams, Phillip L; Wang, Jia-Sheng

    2016-12-26

    Aflatoxin B₁ (AFB₁) is a ubiquitous mycotoxin produced by toxicogenic Aspergillus species. AFB₁ has been reported to cause serious adverse health effects, such as cancers and abnormal development and reproduction, in animals and humans. AFB₁ is also a potent genotoxic mutagen that causes DNA damage in vitro and in vivo. However, the link between DNA damage and abnormal development and reproduction is unclear. To address this issue, we examined the DNA damage, germline apoptosis, growth, and reproductive toxicity following exposure to AFB₁, using Caenorhabditis elegans as a study model. Results found that AFB₁ induced DNA damage and germline apoptosis, and significantly inhibited growth and reproduction of the nematodes in a concentration-dependent manner. Exposure to AFB₁ inhibited growth or reproduction more potently in the DNA repair-deficient xpa-1 nematodes than the wild-type N2 strain. According to the relative expression level of pathway-related genes measured by real-time PCR, the DNA damage response (DDR) pathway was found to be associated with AFB₁-induced germline apoptosis, which further played an essential role in the dysfunction of growth and reproduction in C. elegans .

  3. Genome-wide DNA methylation patterns of bovine blastocysts derived from in vivo embryos subjected to in vitro culture before, during or after embryonic genome activation.

    PubMed

    Salilew-Wondim, Dessie; Saeed-Zidane, Mohammed; Hoelker, Michael; Gebremedhn, Samuel; Poirier, Mikhaël; Pandey, Hari Om; Tholen, Ernst; Neuhoff, Christiane; Held, Eva; Besenfelder, Urban; Havlicek, Vita; Rings, Franca; Fournier, Eric; Gagné, Dominic; Sirard, Marc-André; Robert, Claude; Gad, Ahmed; Schellander, Karl; Tesfaye, Dawit

    2018-06-01

    Aberrant DNA methylation patterns of genes required for development are common in in vitro produced embryos. In this regard, we previously identified altered DNA methylation patterns of in vivo developed blastocysts from embryos which spent different stages of development in vitro, indicating carryover effects of suboptimal culture conditions on epigenetic signatures of preimplantation embryos. However, epigenetic responses of in vivo originated embryos to suboptimal culture conditions are not fully understood. Therefore, here we investigated DNA methylation patterns of in vivo derived bovine embryos subjected to in vitro culture condition before, during or after major embryonic genome activation (EGA). For this, in vivo produced 2-, 8- and 16-cell stage embryos were cultured in vitro until the blastocyst stage and blastocysts were used for genome-wide DNA methylation analysis. The 2- and 8-cell flushed embryo groups showed lower blastocyst rates compared to the 16-cell flush group. This was further accompanied by increased numbers of differentially methylated genomic regions (DMRs) in blastocysts of the 2- and 8-cell flush groups compared to the complete in vivo control ones. Moreover, 1623 genomic loci including imprinted genes were hypermethylated in blastocyst of 2-, 8- and 16-cell flushed groups, indicating the presence of genomic regions which are sensitive to the in vitro culture at any stage of embryonic development. Furthermore, hypermethylated genomic loci outnumbered hypomethylated ones in blastocysts of 2- and 16-cell flushed embryo groups, but the opposite occurred in the 8-cell group. Moreover, DMRs which were unique to blastocysts of the 2-cell flushed group and inversely correlated with corresponding mRNA expression levels were involved in plasma membrane lactate transport, amino acid transport and phosphorus metabolic processes, whereas DMRs which were specific to the 8-cell group and inversely correlated with corresponding mRNA expression levels

  4. Hepatitis B virus X protein (HBx)-induced abnormalities of nucleic acid metabolism revealed by (1)H-NMR-based metabonomics.

    PubMed

    Dan Yue; Zhang, Yuwei; Cheng, Liuliu; Ma, Jinhu; Xi, Yufeng; Yang, Liping; Su, Chao; Shao, Bin; Huang, Anliang; Xiang, Rong; Cheng, Ping

    2016-04-14

    Hepatitis B virus X protein (HBx) plays an important role in HBV-related hepatocarcinogenesis; however, mechanisms underlying HBx-mediated carcinogenesis remain unclear. In this study, an NMR-based metabolomics approach was applied to systematically investigate the effects of HBx on cell metabolism. EdU incorporation assay was conducted to examine the effects of HBx on DNA synthesis, an important feature of nucleic acid metabolism. The results revealed that HBx disrupted metabolism of glucose, lipids, and amino acids, especially nucleic acids. To understand the potential mechanism of HBx-induced abnormalities of nucleic acid metabolism, gene expression profiles of HepG2 cells expressing HBx were investigated. The results showed that 29 genes involved in DNA damage and DNA repair were differentially expressed in HBx-expressing HepG2 cells. HBx-induced DNA damage was further demonstrated by karyotyping, comet assay, Western blotting, immunofluorescence and immunohistochemistry analyses. Many studies have previously reported that DNA damage can induce abnormalities of nucleic acid metabolism. Thus, our results implied that HBx initially induces DNA damage, and then disrupts nucleic acid metabolism, which in turn blocks DNA repair and induces the occurrence of hepatocellular carcinoma (HCC). These findings further contribute to our understanding of the occurrence of HCC.

  5. Germline DNA methylation in reef corals: patterns and potential roles in response to environmental change.

    PubMed

    Dimond, James L; Roberts, Steven B

    2016-04-01

    DNA methylation is an epigenetic mark that plays an inadequately understood role in gene regulation, particularly in nonmodel species. Because it can be influenced by the environment, DNA methylation may contribute to the ability of organisms to acclimatize and adapt to environmental change. We evaluated the distribution of gene body methylation in reef-building corals, a group of organisms facing significant environmental threats. Gene body methylation in six species of corals was inferred from in silico transcriptome analysis of CpG O/E, an estimate of germline DNA methylation that is highly correlated with patterns of methylation enrichment. Consistent with what has been documented in most other invertebrates, all corals exhibited bimodal distributions of germline methylation suggestive of distinct fractions of genes with high and low levels of methylation. The hypermethylated fractions were enriched with genes with housekeeping functions, while genes with inducible functions were highly represented in the hypomethylated fractions. High transcript abundance was associated with intermediate levels of methylation. In three of the coral species, we found that genes differentially expressed in response to thermal stress and ocean acidification exhibited significantly lower levels of methylation. These results support a link between gene body hypomethylation and transcriptional plasticity that may point to a role of DNA methylation in the response of corals to environmental change. © 2015 John Wiley & Sons Ltd.

  6. Genome-wide identification of DNA methylation provides insights into the association of gene expression in rice exposed to pesticide atrazine

    PubMed Central

    Lu, Yi Chen; Feng, Sheng Jun; Zhang, Jing Jing; Luo, Fang; Zhang, Shuang; Yang, Hong

    2016-01-01

    Atrazine (ATR) is a pesticide widely used for controlling weeds for crop production. Crop contamination with ATR negatively affects crop growth and development. This study presents the first genome-wide single-base-resolution maps of DNA methylation in ATR-exposed rice. Widespread differences were identified in CG and non-CG methylation marks between the ATR-exposed and ATR-free (control) rice. Most of DNA methyltransferases, histone methyltransferases and DNA demethylase were differentially regulated by ATR. We found more genes hypermethylated than those hypomethylated in the regions of upstream, genebody and downstream under ATR exposure. A stringent group of 674 genes (p < 0.05, two-fold change) with a strong preference of differential expression in ATR-exposed rice was identified. Some of the genes were identified in a subset of loss of function mutants defective in DNA methylation/demethylation. Provision of 5-azacytidine (AZA, inhibitor of DNA methylation) promoted the rice growth and reduced ATR content. By UPLC/Q-TOF-MS/MS, 8 degraded products and 9 conjugates of ATR in AZA-treated rice were characterized. Two of them has been newly identified in this study. Our data show that ATR-induced changes in DNA methylation marks are possibly involved in an epigenetic mechanism associated with activation of specific genes responsible for ATR degradation and detoxification. PMID:26739616

  7. PITX2 and PANCR DNA methylation predicts overall survival in patients with head and neck squamous cell carcinoma.

    PubMed

    Sailer, Verena; Holmes, Emily Eva; Gevensleben, Heidrun; Goltz, Diane; Dröge, Freya; de Vos, Luka; Franzen, Alina; Schröck, Friederike; Bootz, Friedrich; Kristiansen, Glen; Schröck, Andreas; Dietrich, Dimo

    2016-11-15

    Squamous cell carcinoma of the head and neck region (HNSCC) is a common malignant disease accompanied by a high risk of local or distant recurrence after curative-intent treatment. Biomarkers that allow for the prediction of disease outcome can guide clinicians with respect to treatment and surveillance strategies. Here, the methylation status of PITX2 and an adjacent lncRNA (PANCR) were evaluated for their ability to predict overall survival in HNSCC patients. PITX2 hypermethylation was associated with a better overall survival (hazard ratio, HR = 0.51, 95%CI: 0.35-0.74, p<0.001), while PANCR hypermethylation was significantly associated with an increased risk of death (HR = 1.64, 95%CI: 1.12-2.39, p=0.010). Quantitative, methylation-specific real-time PCR assays for PITX2 and PANCR were employed to measure bisulfite-converted DNA from formalin-fixed, paraffin-embedded (FFPE) tissues in a cohort of 399 patients with localized or locally advanced HNSCC who received curative-intent treatment (surgery with optional adjuvant radiochemotherapy or definite radiochemotherapy). PITX2 and PANCR methylation status were shown to be independent predictors for overall survival in HNSCC patients. Tissue-based methylation testing could therefore potentially be employed to identify patients with a high risk for death who might benefit from a more radical or alternative treatment.

  8. Effects of arsenic exposure on DNA methylation in cord blood samples from newborn babies and in a human lymphoblast cell line

    PubMed Central

    2012-01-01

    hypermethylation (p < 0.05). However, a slight LINE-1 hypomethylation and transient p53 promoter hypermethylation were observed following long-term in vitro treatment. Conclusions This study provides an important finding that in utero arsenic exposure affects DNA methylation, particularly at the p53 promoter region, which may be linked to the mechanism of arsenic carcinogenesis and the observed increased incidence of cancer later in life. PMID:22551203

  9. Plasma genetic and genomic abnormalities predict treatment response and clinical outcome in advanced prostate cancer.

    PubMed

    Xia, Shu; Kohli, Manish; Du, Meijun; Dittmar, Rachel L; Lee, Adam; Nandy, Debashis; Yuan, Tiezheng; Guo, Yongchen; Wang, Yuan; Tschannen, Michael R; Worthey, Elizabeth; Jacob, Howard; See, William; Kilari, Deepak; Wang, Xuexia; Hovey, Raymond L; Huang, Chiang-Ching; Wang, Liang

    2015-06-30

    Liquid biopsies, examinations of tumor components in body fluids, have shown promise for predicting clinical outcomes. To evaluate tumor-associated genomic and genetic variations in plasma cell-free DNA (cfDNA) and their associations with treatment response and overall survival, we applied whole genome and targeted sequencing to examine the plasma cfDNAs derived from 20 patients with advanced prostate cancer. Sequencing-based genomic abnormality analysis revealed locus-specific gains or losses that were common in prostate cancer, such as 8q gains, AR amplifications, PTEN losses and TMPRSS2-ERG fusions. To estimate tumor burden in cfDNA, we developed a Plasma Genomic Abnormality (PGA) score by summing the most significant copy number variations. Cox regression analysis showed that PGA scores were significantly associated with overall survival (p < 0.04). After androgen deprivation therapy or chemotherapy, targeted sequencing showed significant mutational profile changes in genes involved in androgen biosynthesis, AR activation, DNA repair, and chemotherapy resistance. These changes may reflect the dynamic evolution of heterozygous tumor populations in response to these treatments. These results strongly support the feasibility of using non-invasive liquid biopsies as potential tools to study biological mechanisms underlying therapy-specific resistance and to predict disease progression in advanced prostate cancer.

  10. Hypermethylation in the ZBTB20 gene is associated with major depressive disorder

    PubMed Central

    2014-01-01

    Background Although genetic variation is believed to contribute to an individual’s susceptibility to major depressive disorder, genome-wide association studies have not yet identified associations that could explain the full etiology of the disease. Epigenetics is increasingly believed to play a major role in the development of common clinical phenotypes, including major depressive disorder. Results Genome-wide MeDIP-Sequencing was carried out on a total of 50 monozygotic twin pairs from the UK and Australia that are discordant for depression. We show that major depressive disorder is associated with significant hypermethylation within the coding region of ZBTB20, and is replicated in an independent cohort of 356 unrelated case-control individuals. The twins with major depressive disorder also show increased global variation in methylation in comparison with their unaffected co-twins. ZBTB20 plays an essential role in the specification of the Cornu Ammonis-1 field identity in the developing hippocampus, a region previously implicated in the development of major depressive disorder. Conclusions Our results suggest that aberrant methylation profiles affecting the hippocampus are associated with major depressive disorder and show the potential of the epigenetic twin model in neuro-psychiatric disease. PMID:24694013

  11. pH-Controlled Assembly of DNA Tiles

    DOE PAGES

    Amodio, Alessia; Adedeji, Abimbola Feyisara; Castronovo, Matteo; ...

    2016-09-15

    We demonstrate a strategy to trigger and finely control the assembly of supramolecular DNA nanostructures with pH. Control is achieved via a rationally designed strand displacement circuit that responds to pH and activates a downstream DNA tile self-assembly process. We observe that the DNA structures form under neutral/basic conditions, while the self-assembly process is suppressed under acidic conditions. The strategy presented here demonstrates a modular approach toward building systems capable of processing biochemical inputs and finely controlling the assembly of DNA-based nanostructures under isothermal conditions. In particular, the presented architecture is relevant for the development of complex DNA devices ablemore » to sense and respond to molecular markers associated with abnormal metabolism.« less

  12. pH-Controlled Assembly of DNA Tiles

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Amodio, Alessia; Adedeji, Abimbola Feyisara; Castronovo, Matteo

    We demonstrate a strategy to trigger and finely control the assembly of supramolecular DNA nanostructures with pH. Control is achieved via a rationally designed strand displacement circuit that responds to pH and activates a downstream DNA tile self-assembly process. We observe that the DNA structures form under neutral/basic conditions, while the self-assembly process is suppressed under acidic conditions. The strategy presented here demonstrates a modular approach toward building systems capable of processing biochemical inputs and finely controlling the assembly of DNA-based nanostructures under isothermal conditions. In particular, the presented architecture is relevant for the development of complex DNA devices ablemore » to sense and respond to molecular markers associated with abnormal metabolism.« less

  13. Intensive hypermethylation of the CpG island of Ras association domain family 1A in hepatitis B virus-associated hepatocellular carcinomas.

    PubMed

    Zhong, Sheng; Yeo, Winnie; Tang, Mandy W; Wong, Nathalie; Lai, Paul B S; Johnson, Phillip J

    2003-08-15

    The human Ras association domain family 1A gene (RASSF1A) is a newly isolated tumor suppressor gene. In this study, we analyzed the methylation status of the promoter region of RASSF1A using bisulfite sequencing and PCR-RFLP in four liver cancer cell lines (Hep3B, HepG(2), SK-HEP-1, and Huh-7) and a cohort of 43 hepatitis B virus-associated hepatocellular carcinoma (HCC) tissues and their corresponding nontumor tissue specimens. The methylation of the CpG islands in the RASSF1A promoter was not detected in 4 samples of normal liver tissue or 10 samples of peripheral blood mononuclear cells from normal subjects. However, the CpG islands were completely methylated, and transcription of the RASSF1A was silenced in the four cell lines. Treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine reactivated the expression of RASSF1A in the Hep3B and HepG2 cells. In 41 of 43 (95%) HCC specimens studied, the promoter region of RASSF1A was intensively methylated at its CpG sites. Although heterogeneous methylation was also detected in 16 of the 23 (70%) corresponding nontumorous tissues analyzed, the level of methylation was significantly lower than in the corresponding tumor tissues. HCC has the highest incidence of promoter methylation of RASSF1A among all malignancies yet reported suggesting that hypermethylation of the CpG island promoter of RASSF1A may play an important pathological role in this tumor.

  14. Regulation of Active DNA Demethylation by a Methyl-CpG-Binding Domain Protein in Arabidopsis thaliana

    PubMed Central

    Sun, Han; Zeng, Jun; Cao, Zhendong; Li, Yan; Qian, Weiqiang

    2015-01-01

    Active DNA demethylation plays crucial roles in the regulation of gene expression in both plants and animals. In Arabidopsis thaliana, active DNA demethylation is initiated by the ROS1 subfamily of 5-methylcytosine-specific DNA glycosylases via a base excision repair mechanism. Recently, IDM1 and IDM2 were shown to be required for the recruitment of ROS1 to some of its target loci. However, the mechanism(s) by which IDM1 is targeted to specific genomic loci remains to be determined. Affinity purification of IDM1- and IDM2- associating proteins demonstrated that IDM1 and IDM2 copurify together with two novel components, methyl-CpG-binding domain protein 7 (MBD7) and IDM2-like protein 1 (IDL1). IDL1 encodes an α-crystallin domain protein that shows high sequence similarity with IDM2. MBD7 interacts with IDM2 and IDL1 in vitro and in vivo and they form a protein complex associating with IDM1 in vivo. MBD7 directly binds to the target loci and is required for the H3K18 and H3K23 acetylation in planta. MBD7 dysfunction causes DNA hypermethylation and silencing of reporter genes and a subset of endogenous genes. Our results suggest that a histone acetyltransferase complex functions in active DNA demethylation and in suppression of gene silencing at some loci in Arabidopsis. PMID:25933434

  15. Combined analysis of DNA methylome and transcriptome reveal novel candidate genes with susceptibility to bovine Staphylococcus aureus subclinical mastitis.

    PubMed

    Song, Minyan; He, Yanghua; Zhou, Huangkai; Zhang, Yi; Li, Xizhi; Yu, Ying

    2016-07-14

    Subclinical mastitis is a widely spread disease of lactating cows. Its major pathogen is Staphylococcus aureus (S. aureus). In this study, we performed genome-wide integrative analysis of DNA methylation and transcriptional expression to identify candidate genes and pathways relevant to bovine S. aureus subclinical mastitis. The genome-scale DNA methylation profiles of peripheral blood lymphocytes in cows with S. aureus subclinical mastitis (SA group) and healthy controls (CK) were generated by methylated DNA immunoprecipitation combined with microarrays. We identified 1078 differentially methylated genes in SA cows compared with the controls. By integrating DNA methylation and transcriptome data, 58 differentially methylated genes were shared with differently expressed genes, in which 20.7% distinctly hypermethylated genes showed down-regulated expression in SA versus CK, whereas 14.3% dramatically hypomethylated genes showed up-regulated expression. Integrated pathway analysis suggested that these genes were related to inflammation, ErbB signalling pathway and mismatch repair. Further functional analysis revealed that three genes, NRG1, MST1 and NAT9, were strongly correlated with the progression of S. aureus subclinical mastitis and could be used as powerful biomarkers for the improvement of bovine mastitis resistance. Our studies lay the groundwork for epigenetic modification and mechanistic studies on susceptibility of bovine mastitis.

  16. Combined analysis of DNA methylome and transcriptome reveal novel candidate genes with susceptibility to bovine Staphylococcus aureus subclinical mastitis

    PubMed Central

    Song, Minyan; He, Yanghua; Zhou, Huangkai; Zhang, Yi; Li, Xizhi; Yu, Ying

    2016-01-01

    Subclinical mastitis is a widely spread disease of lactating cows. Its major pathogen is Staphylococcus aureus (S. aureus). In this study, we performed genome-wide integrative analysis of DNA methylation and transcriptional expression to identify candidate genes and pathways relevant to bovine S. aureus subclinical mastitis. The genome-scale DNA methylation profiles of peripheral blood lymphocytes in cows with S. aureus subclinical mastitis (SA group) and healthy controls (CK) were generated by methylated DNA immunoprecipitation combined with microarrays. We identified 1078 differentially methylated genes in SA cows compared with the controls. By integrating DNA methylation and transcriptome data, 58 differentially methylated genes were shared with differently expressed genes, in which 20.7% distinctly hypermethylated genes showed down-regulated expression in SA versus CK, whereas 14.3% dramatically hypomethylated genes showed up-regulated expression. Integrated pathway analysis suggested that these genes were related to inflammation, ErbB signalling pathway and mismatch repair. Further functional analysis revealed that three genes, NRG1, MST1 and NAT9, were strongly correlated with the progression of S. aureus subclinical mastitis and could be used as powerful biomarkers for the improvement of bovine mastitis resistance. Our studies lay the groundwork for epigenetic modification and mechanistic studies on susceptibility of bovine mastitis. PMID:27411928

  17. Lack of Evidence for Green Tea Polyphenols as DNA Methylation Inhibitors in Murine Prostate

    PubMed Central

    Morey Kinney, Shannon R.; Zhang, Wa; Pascual, Marien; Greally, John M.; Gillard, Bryan M.; Karasik, Ellen; Foster, Barbara A.; Karpf, Adam R.

    2009-01-01

    Green tea polyphenols (GTPs) have been reported to inhibit DNA methylation in cultured cells. Here we tested whether oral consumption of GTPs affects normal or cancer specific DNA methylation in vivo, using mice. Wildtype (WT) and Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice were administered 0.3% GTPs in drinking water beginning at 4 weeks of age. To monitor DNA methylation, we measured 5-methyl-deoxycytidine (5mdC) levels, methylation of the B1 repetitive element, and methylation of the Mage-a8 gene. Each of these parameters were unchanged in prostate, gut, and liver from WT mice at both 12 and 24 weeks of age, with the single exception of a decrease of 5mdC in the liver at 12 weeks. In GTP-treated TRAMP mice, 5mdC levels and the methylation status of four loci hypermethylated during tumor progression were unaltered in TRAMP prostates at 12 or 24 weeks. Quite surprisingly, GTP treatment did not inhibit tumor progression in TRAMP mice, although known pharmacodynamic markers of GTPs were altered in both WT and TRAMP prostates. We also administered 0.1%, 0.3%, or 0.6% GTPs to TRAMP mice for 12 weeks and measured 5mdC levels and methylation of B1 and Mage-a8 in prostate, gut, and liver tissues. No dose-dependent alterations in DNA methylation status were observed. Genome-wide DNA methylation profiling using the HELP assay also revealed no significant hypomethylating effect of GTP. These data indicate that oral administration of GTPs does not affect normal or cancer-specific DNA methylation in the murine prostate. PMID:19934341

  18. Whole Genome DNA Methylation Analysis of Obstructive Sleep Apnea: IL1R2, NPR2, AR, SP140 Methylation and Clinical Phenotype

    PubMed Central

    Chen, Yung-Che; Chen, Ting-Wen; Su, Mao-Chang; Chen, Chung-Jen; Chen, Kuang-Den; Liou, Chia-Wei; Tang, Petrus; Wang, Ting-Ya; Chang, Jen-Chieh; Wang, Chin-Chou; Lin, Hsin-Ching; Chin, Chien-Hung; Huang, Kuo-Tung; Lin, Meng-Chih; Hsiao, Chang-Chun

    2016-01-01

    Study Objectives: We hypothesized that DNA methylation patterns may contribute to disease severity or the development of hypertension and excessive daytime sleepiness (EDS) in patients with obstructive sleep apnea (OSA). Methods: Illumina's (San Diego, CA, USA) DNA methylation 27-K assay was used to identify differentially methylated loci (DML). DNA methylation levels were validated by pyrosequencing. A discovery cohort of 15 patients with OSA and 6 healthy subjects, and a validation cohort of 72 patients with sleep disordered breathing (SDB). Results: Microarray analysis identified 636 DMLs in patients with OSA versus healthy subjects, and 327 DMLs in patients with OSA and hypertension versus those without hypertension. In the validation cohort, no significant difference in DNA methylation levels of six selected genes was found between the primary snoring subjects and OSA patients (primary outcome). However, a secondary outcome analysis showed that interleukin-1 receptor 2 (IL1R2) promoter methylation (−114 cytosine followed by guanine dinucleotide sequence [CpG] site) was decreased and IL1R2 protein levels were increased in the patients with SDB with an oxygen desaturation index > 30. Androgen receptor (AR) promoter methylation (−531 CpG site) and AR protein levels were both increased in the patients with SDB with an oxygen desaturation index > 30. Natriuretic peptide receptor 2 (NPR2) promoter methylation (−608/−618 CpG sites) were decreased, whereas levels of both NPR2 and serum C type natriuretic peptide protein were increased in the SDB patients with EDS. Speckled protein 140 (SP140) promoter methylation (−194 CpG site) was increased, and SP140 protein levels were decreased in the patients with SDB and EDS. Conclusions: IL1R2 hypomethylation and AR hypermethylation may constitute an important determinant of disease severity, whereas NPR2 hypomethylation and SP140 hypermethylation may provide a biomarker for vulnerability to EDS in OSA. Commentary: A

  19. Recent thymic emigrants and mature naive T cells exhibit differential DNA methylation at key cytokine loci.

    PubMed

    Berkley, Amy M; Hendricks, Deborah W; Simmons, Kalynn B; Fink, Pamela J

    2013-06-15

    Recent thymic emigrants (RTEs) are the youngest T cells in the lymphoid periphery and exhibit phenotypic and functional characteristics distinct from those of their more mature counterparts in the naive peripheral T cell pool. We show in this study that the Il2 and Il4 promoter regions of naive CD4(+) RTEs are characterized by site-specific hypermethylation compared with those of both mature naive (MN) T cells and the thymocyte precursors of RTEs. Thus, RTEs do not merely occupy a midpoint between the thymus and the mature T cell pool, but represent a distinct transitional T cell population. Furthermore, RTEs and MN T cells exhibit distinct CpG DNA methylation patterns both before and after activation. Compared with MN T cells, RTEs express higher levels of several enzymes that modify DNA methylation, and inhibiting methylation during culture allows RTEs to reach MN T cell levels of cytokine production. Collectively, these data suggest that the functional differences that distinguish RTEs from MN T cells are influenced by epigenetic mechanisms and provide clues to a mechanistic basis for postthymic maturation.

  20. DNA methylation of miRNA coding sequences putatively associated with childhood obesity.

    PubMed

    Mansego, M L; Garcia-Lacarte, M; Milagro, F I; Marti, A; Martinez, J A

    2017-02-01

    Epigenetic mechanisms may be involved in obesity onset and its consequences. The aim of the present study was to evaluate whether DNA methylation status in microRNA (miRNA) coding regions is associated with childhood obesity. DNA isolated from white blood cells of 24 children (identification sample: 12 obese and 12 non-obese) from the Grupo Navarro de Obesidad Infantil study was hybridized in a 450 K methylation microarray. Several CpGs whose DNA methylation levels were statistically different between obese and non-obese were validated by MassArray® in 95 children (validation sample) from the same study. Microarray analysis identified 16 differentially methylated CpGs between both groups (6 hypermethylated and 10 hypomethylated). DNA methylation levels in miR-1203, miR-412 and miR-216A coding regions significantly correlated with body mass index standard deviation score (BMI-SDS) and explained up to 40% of the variation of BMI-SDS. The network analysis identified 19 well-defined obesity-relevant biological pathways from the KEGG database. MassArray® validation identified three regions located in or near miR-1203, miR-412 and miR-216A coding regions differentially methylated between obese and non-obese children. The current work identified three CpG sites located in coding regions of three miRNAs (miR-1203, miR-412 and miR-216A) that were differentially methylated between obese and non-obese children, suggesting a role of miRNA epigenetic regulation in childhood obesity. © 2016 World Obesity Federation.

  1. Genome-wide screening identifies Plasmodium chabaudi-induced modifications of DNA methylation status of Tlr1 and Tlr6 gene promoters in liver, but not spleen, of female C57BL/6 mice.

    PubMed

    Al-Quraishy, Saleh; Dkhil, Mohamed A; Abdel-Baki, Abdel Azeem S; Delic, Denis; Santourlidis, Simeon; Wunderlich, Frank

    2013-11-01

    Epigenetic reprogramming of host genes via DNA methylation is increasingly recognized as critical for the outcome of diverse infectious diseases, but information for malaria is not yet available. Here, we investigate the effect of blood-stage malaria of Plasmodium chabaudi on the DNA methylation status of host gene promoters on a genome-wide scale using methylated DNA immunoprecipitation and Nimblegen microarrays containing 2,000 bp oligonucleotide features that were split into -1,500 to -500 bp Ups promoters and -500 to +500 bp Cor promoters, relative to the transcription site, for evaluation of differential DNA methylation. Gene expression was analyzed by Agilent and Affymetrix microarray technology. Challenging of female C57BL/6 mice with 10(6) P. chabaudi-infected erythrocytes resulted in a self-healing outcome of infections with peak parasitemia on day 8 p.i. These infections induced organ-specific modifications of DNA methylation of gene promoters. Among the 17,354 features on Nimblegen arrays, only seven gene promoters were identified to be hypermethylated in the spleen, whereas the liver exhibited 109 hyper- and 67 hypomethylated promoters at peak parasitemia in comparison with non-infected mice. Among the identified genes with differentially methylated Cor-promoters, only the 7 genes Pigr, Ncf1, Klkb1, Emr1, Ndufb11, and Tlr6 in the liver and Apol6 in the spleen were detected to have significantly changed their expression. Remarkably, the Cor promoter of the toll-like receptor Tlr6 became hypomethylated and Tlr6 expression increased by 3.4-fold during infection. Concomitantly, the Ups promoter of the Tlr1 was hypermethylated, but Tlr1 expression also increased by 11.3-fold. TLR6 and TLR1 are known as auxillary receptors to form heterodimers with TLR2 in plasma membranes of macrophages, which recognize different pathogen-associated molecular patterns (PAMPs), as, e.g., intact 3-acyl and sn-2-lyso-acyl glycosylphosphatidylinositols of P. falciparum

  2. DNA cytosine methylation in the bovine leukemia virus promoter is associated with latency in a lymphoma-derived B-cell line: potential involvement of direct inhibition of cAMP-responsive element (CRE)-binding protein/CRE modulator/activation transcription factor binding.

    PubMed

    Pierard, Valérie; Guiguen, Allan; Colin, Laurence; Wijmeersch, Gaëlle; Vanhulle, Caroline; Van Driessche, Benoît; Dekoninck, Ann; Blazkova, Jana; Cardona, Christelle; Merimi, Makram; Vierendeel, Valérie; Calomme, Claire; Nguyên, Thi Liên-Anh; Nuttinck, Michèle; Twizere, Jean-Claude; Kettmann, Richard; Portetelle, Daniel; Burny, Arsène; Hirsch, Ivan; Rohr, Olivier; Van Lint, Carine

    2010-06-18

    Bovine leukemia virus (BLV) proviral latency represents a viral strategy to escape the host immune system and allow tumor development. Besides the previously demonstrated role of histone deacetylation in the epigenetic repression of BLV expression, we showed here that BLV promoter activity was induced by several DNA methylation inhibitors (such as 5-aza-2'-deoxycytidine) and that overexpressed DNMT1 and DNMT3A, but not DNMT3B, down-regulated BLV promoter activity. Importantly, cytosine hypermethylation in the 5'-long terminal repeat (LTR) U3 and R regions was associated with true latency in the lymphoma-derived B-cell line L267 but not with defective latency in YR2 cells. Moreover, the virus-encoded transactivator Tax(BLV) decreased DNA methyltransferase expression levels, which could explain the lower level of cytosine methylation observed in the L267(LTaxSN) 5'-LTR compared with the L267 5'-LTR. Interestingly, DNA methylation inhibitors and Tax(BLV) synergistically activated BLV promoter transcriptional activity in a cAMP-responsive element (CRE)-dependent manner. Mechanistically, methylation at the -154 or -129 CpG position (relative to the transcription start site) impaired in vitro binding of CRE-binding protein (CREB) transcription factors to their respective CRE sites. Methylation at -129 CpG alone was sufficient to decrease BLV promoter-driven reporter gene expression by 2-fold. We demonstrated in vivo the recruitment of CREB/CRE modulator (CREM) and to a lesser extent activating transcription factor-1 (ATF-1) to the hypomethylated CRE region of the YR2 5'-LTR, whereas we detected no CREB/CREM/ATF recruitment to the hypermethylated corresponding region in the L267 cells. Altogether, these findings suggest that site-specific DNA methylation of the BLV promoter represses viral transcription by directly inhibiting transcription factor binding, thereby contributing to true proviral latency.

  3. Analysis of the machinery and intermediates of the 5hmC-mediated DNA demethylation pathway in aging on samples from the MARK-AGE Study.

    PubMed

    Valentini, Elisabetta; Zampieri, Michele; Malavolta, Marco; Bacalini, Maria Giulia; Calabrese, Roberta; Guastafierro, Tiziana; Reale, Anna; Franceschi, Claudio; Hervonen, Antti; Koller, Bernhard; Bernhardt, Jürgen; Slagboom, P Eline; Toussaint, Olivier; Sikora, Ewa; Gonos, Efstathios S; Breusing, Nicolle; Grune, Tilman; Jansen, Eugène; Dollé, Martijn E T; Moreno-Villanueva, María; Sindlinger, Thilo; Bürkle, Alexander; Ciccarone, Fabio; Caiafa, Paola

    2016-08-29

    Gradual changes in the DNA methylation landscape occur throughout aging virtually in all human tissues. A widespread reduction of 5-methylcytosine (5mC), associated with highly reproducible site-specific hypermethylation, characterizes the genome in aging. Therefore, an equilibrium seems to exist between general and directional deregulating events concerning DNA methylation controllers, which may underpin the age-related epigenetic changes. In this context, 5mC-hydroxylases (TET enzymes) are new potential players. In fact, TETs catalyze the stepwise oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), driving the DNA demethylation process based on thymine DNA glycosylase (TDG)-mediated DNA repair pathway. The present paper reports the expression of DNA hydroxymethylation components, the levels of 5hmC and of its derivatives in peripheral blood mononuclear cells of age-stratified donors recruited in several European countries in the context of the EU Project 'MARK-AGE'. The results provide evidence for an age-related decline of TET1 , TET3 and TDG gene expression along with a decrease of 5hmC and an accumulation of 5caC. These associations were independent of confounding variables, including recruitment center, gender and leukocyte composition. The observed impairment of 5hmC-mediated DNA demethylation pathway in blood cells may lead to aberrant transcriptional programs in the elderly.

  4. Identification of DNA methylation changes associated with disease progression in subchondral bone with site-matched cartilage in knee osteoarthritis

    PubMed Central

    Zhang, Yanfei; Fukui, Naoshi; Yahata, Mitsunori; Katsuragawa, Yozo; Tashiro, Toshiyuki; Ikegawa, Shiro; Lee, Ming Ta Michael

    2016-01-01

    Subchondral bone plays a key role in the development of osteoarthritis, however, epigenetics of subchondral bone has not been extensively studied. In this study, we examined the genome-wide DNA methylation profiles of subchondral bone from three regions on tibial plateau representing disease progression using HumanMethylation450 BeadChip to identify progression associated DNA methylation alterations. Significant differential methylated probes (DMPs) and differential methylated genes (DMGs) were identified in the intermediate and late stages and during the transition from intermediate to late stage of OA in the subchondral bone. Over half of the DMPs were hyper-methylated. Genes associated with OA and bone remodeling were identified. DMGs were enriched in morphogenesis and development of skeletal system, and HOX transcription factors. Comparison of DMGs identified in subchondral bone and site-matched cartilage indicated that DNA methylation changes occurred earlier in subchondral bone and identified different methylation patterns at the late stage of OA. However, shared DMPs, DMGs and common pathways that implicated the tissue reparation were also identified. Methylation is one key mechanism to regulate the crosstalk between cartilage and subchondral bone. PMID:27686527

  5. Abnormal Transmethylation/Transsulfuration Metabolism and DNA Hypomethylation among Parents of Children with Autism

    ERIC Educational Resources Information Center

    James, S. Jill; Melnyk, Stepan; Jernigan, Stefanie; Hubanks, Amanda; Rose, Shannon; Gaylor, David W.

    2008-01-01

    An integrated metabolic profile reflects the combined influence of genetic, epigenetic, and environmental factors that affect the candidate pathway of interest. Recent evidence suggests that some autistic children may have reduced detoxification capacity and may be under chronic oxidative stress. Based on reports of abnormal methionine and…

  6. Detection of chromosomal abnormalities, congenital abnormalities and transfusion syndrome in twins.

    PubMed

    Sperling, L; Kiil, C; Larsen, L U; Brocks, V; Wojdemann, K R; Qvist, I; Schwartz, M; Jørgensen, C; Espersen, G; Skajaa, K; Bang, J; Tabor, A

    2007-05-01

    To evaluate the outcome of screening for structural malformations in twins and the outcome of screening for twin-twin transfusion syndrome (TTTS) among monochorionic twins through a number of ultrasound scans from 12 weeks' gestation. Enrolled into this prospective multicenter observational study were women with twin pregnancies diagnosed before 14 + 6 gestational weeks. The monochorionic pregnancies were scanned every second week until 23 weeks in order to rule out early TTTS. All pregnancies had an anomaly scan in week 19 and fetal echocardiography in week 21 that was performed by specialists in fetal echocardiography. Zygosity was determined by DNA analysis in all twin pairs with the same sex. Among the 495 pregnancies the prenatal detection rate for severe structural abnormalities including chromosomal aneuploidies was 83% by the combination of a first-trimester nuchal translucency scan and the anomaly scan in week 19. The incidence of severe structural abnormalities was 2.6% and two-thirds of these anomalies were cardiac. There was no significant difference between the incidence in monozygotic and dizygotic twins, nor between twins conceived naturally or those conceived by assisted reproduction. The incidence of TTTS was 23% from 12 weeks until delivery, and all those monochorionic twin pregnancies that miscarried had signs of TTTS. Twin pregnancies have an increased risk of congenital malformations and one out of four monochorionic pregnancies develops TTTS. Ultrasound screening to assess chorionicity and follow-up of monochorionic pregnancies to detect signs of TTTS, as well as malformation screening, are therefore essential in the antenatal care of twin pregnancies. Copyright (c) 2007 ISUOG.

  7. Cross talk between poly(ADP-ribose) polymerase 1 methylation and oxidative stress involved in the toxic effect of anatase titanium dioxide nanoparticles.

    PubMed

    Bai, Wenlin; Chen, Yujiao; Gao, Ai

    2015-01-01

    Given the tremendous growth in the application of titanium dioxide nanoparticles (TNPs), concerns about the potential health hazards of TNPs to humans have been raised. Poly(ADP-ribose) polymerase 1 (PARP-1), a highly conserved DNA-binding protein, is involved in many molecular and cellular processes. Limited data demonstrated that certain nanomaterials induced the aberrant hypermethylation of PARP-1. However, the mechanism involved in TNP-induced PARP-1 abnormal methylation has not been studied. A549 cells were incubated with anatase TNPs (22.1 nm) for 24 hours pretreatment with or without methyltransferase inhibitor 5-aza-2'-deoxycytidine and the reactive oxygen species (ROS) scavenger α-lipoic acid to assess the possible role of methylation and ROS in the toxic effect of TNPs. After TNPs characterization, a battery of assays was performed to evaluate the toxic effect of TNPs, PARP-1 methylation status, and oxidative damage. Results showed that TNPs decreased the cell viability in a dose-dependent manner, in accordance with the increase of lactate dehydrogenase activity, which indicated membrane damage of cells. Similar to the high level of PARP-1 methylation, the generation of ROS was significantly increased after exposure to TNPs for 24 hours. Furthermore, α-lipoic acid decreased TNP-induced ROS generation and then attenuated TNP-triggered PARP-1 hypermethylation. Meanwhile, 5-aza-2'-deoxycytidine simultaneously decreased the ROS generation induced by TNPs, resulting in the decline of PARP-1 methylation. In summary, TNPs triggered the aberrant hypermethylation of the PARP-1 promoter and there was a cross talk between oxidative stress and PARP-1 methylation in the toxic effect of TNPs.

  8. Significance of phosphatase and tensin homologue (PTEN), O(6)-methylguanine-DNA methyltransferase (MGMT), and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) protein expression in gynaecomastia.

    PubMed

    Zhu, L; Liu, Z; Yang, J; Cai, J

    2009-01-01

    This study was designed to investigate the pathogenesis of gynaecomastia by measuring phosphatase and tensin homologue (PTEN), O(6)-methylguanine-DNA methyltransferase (MGMT) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) protein in breast tissue specimens from 68 patients with gynaecomastia and 24 normal male controls using immunohistochemical staining. The gynaecomastia cases were divided into three different histological types: florid, intermediate and fibrous. The PTEN, MGMT and DNA-PKcs proteins were detected in both gynaecomastia and normal breast tissue, but the levels of immunohistochemical staining of each protein were significantly lower in gynaecomastia breast tissue than in normal breast tissue. There were also significant differences in the levels of immunohistochemical staining for the three proteins according to gynaecomastia histological type. These results suggest that abnormally low levels of PTEN, MGMT and DNA-PKcs protein in gynaecomastia breast tissue may play a role in the development of gynaecomastia. Further research is required to elucidate fully their individual roles in the pathophysiology of gynaecomastia.

  9. Impairment of DNA Methylation Maintenance Is the Main Cause of Global Demethylation in Naive Embryonic Stem Cells.

    PubMed

    von Meyenn, Ferdinand; Iurlaro, Mario; Habibi, Ehsan; Liu, Ning Qing; Salehzadeh-Yazdi, Ali; Santos, Fátima; Petrini, Edoardo; Milagre, Inês; Yu, Miao; Xie, Zhenqing; Kroeze, Leonie I; Nesterova, Tatyana B; Jansen, Joop H; Xie, Hehuang; He, Chuan; Reik, Wolf; Stunnenberg, Hendrik G

    2016-06-16

    Global demethylation is part of a conserved program of epigenetic reprogramming to naive pluripotency. The transition from primed hypermethylated embryonic stem cells (ESCs) to naive hypomethylated ones (serum-to-2i) is a valuable model system for epigenetic reprogramming. We present a mathematical model, which accurately predicts global DNA demethylation kinetics. Experimentally, we show that the main drivers of global demethylation are neither active mechanisms (Aicda, Tdg, and Tet1-3) nor the reduction of de novo methylation. UHRF1 protein, the essential targeting factor for DNMT1, is reduced upon transition to 2i, and so is recruitment of the maintenance methylation machinery to replication foci. Concurrently, there is global loss of H3K9me2, which is needed for chromatin binding of UHRF1. These mechanisms synergistically enforce global DNA hypomethylation in a replication-coupled fashion. Our observations establish the molecular mechanism for global demethylation in naive ESCs, which has key parallels with those operating in primordial germ cells and early embryos. Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.

  10. Protein Affinity Chromatography with Purified Yeast DNA Polymerase α Detects Proteins that Bind to DNA Polymerase

    NASA Astrophysics Data System (ADS)

    Miles, Jeff; Formosa, Tim

    1992-02-01

    We have overexpressed the POL1 gene of the yeast Saccharomyces cerevisiae and purified the resulting DNA polymerase α polypeptide in an apparently intact form. We attached the purified DNA polymerase covalently to an agarose matrix and used this matrix to chromatograph extracts prepared from yeast cells. At least six proteins bound to the yeast DNA polymerase α matrix that did not bind to a control matrix. We speculate that these proteins might be DNA polymerase α accessory proteins. Consistent with this interpretation, one of the binding proteins, which we have named POB1 (polymerase one binding), is required for normal chromosome transmission. Mutations in this gene cause increased chromosome loss and an abnormal cell morphology, phenotypes that also occur in the presence of mutations in the yeast α or δ polymerase genes. These results suggest that the interactions detected by polymerase affinity chromatography are biologically relevant and may help to illuminate the architecture of the eukaryotic DNA replication machinery.

  11. miR-125b targets DNMT3b and mediates p53 DNA methylation involving in the vascular smooth muscle cells proliferation induced by homocysteine

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Cao, ChengJian; Zhang, HuiPing; Zhao, Li

    MicroRNAs (miRNAs) are short non-coding RNA and play crucial roles in a wide array of biological processes, including cell proliferation, differentiation and apoptosis. Our previous studies found that homocysteine(Hcy) can stimulate the proliferation of vascular smooth muscle cells (VSMCs), however, the underlying mechanisms were not fully elucidated. Here, we found proliferation of VSMCs induced by Hcy was of correspondence to the miR-125b expression reduced both in vitro and in the ApoE knockout mice, the hypermethylation of p53, its decreased expression, and DNA (cytosine-5)-methyltransferase 3b (DNMT3b) up-regulated. And, we found DNMT3b is a target of miR-125b, which was verified by themore » Dual-Luciferase reporter assay and western blotting. Besides, the siRNA interference for DNMT3b significantly decreased the methylation level of p53, which unveiled the causative role of DNMT3b in p53 hypermethylation. miR-125b transfection further confirmed its regulative roles on p53 gene methylation status and the VSMCs proliferation. Our data suggested that a miR-125b-DNMT3b-p53 signal pathway may exist in the VSMCs proliferation induced by Hcy.« less

  12. The impact of polyunsaturated fatty acids on DNA methylation and expression of DNMTs in human colorectal cancer cells.

    PubMed

    Sarabi, Mostafa Moradi; Naghibalhossaini, Fakhraddin

    2018-05-01

    Growing evidence suggests a role of polyunsaturated fatty acids (PUFA) in the prevention of various types of malignancy, including colorectal cancer (CRC). No published studies have yet examined the direct effect of PUFA treatment on DNA methylation in CRC cells. In this study, 5 human CRC cells were treated with 100 μM DHA, EPA, and LA for 6 days and changes in their global- and gene-specific DNA methylation status as well as expression of DNA methyl transferases (DNMT) were investigated. Cell-type specific differences in DNA methylation and expression of DNMTs were observed in PUFA-treated cells. DHA and EPA treatment induced global hypermethylation in HT29/219 and HCT116 cells, but reduced methylation in Caco2 cells (p < 0.05). Among 10 tumor related genes tested in 5 CRC cell lines, DHA and EPA induced promoter demethylation of Cox2 in HT29/219, p14 and PPARγ in HCT116, and ECAD in SW742 cells. Cell-type specific differences in expression of DNMT1, DNMT3a, and 3b genes were also observed between PUFA-treated and control cells (p < 0.05). Overall, treatment of PUFAs coordinately induced the expression of DNMTs in HT29/219, but suppressed in other 4 cell lines investigated in this study. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  13. Hypermethylation of MDFI promoter with NSCLC is specific for females, non-smokers and people younger than 65.

    PubMed

    Ma, Hongying; Chen, Xiaoying; Hu, Haochang; Li, Bin; Ying, Xiuru; Zhou, Cong; Zhong, Jie; Zhao, Guofang; Duan, Shiwei

    2018-06-01

    Non-small cell lung carcinoma (NSCLC) is a major subtype of lung cancer. Aberrant DNA methylation has been frequently observed in NSCLC. The aim of the present study was to investigate the role of MyoD family inhibitor ( MDFI ) methylation in NSCLC. Formalin-fixed paraffin-embedded tumor tissues and adjacent non-cancerous tissues were collected from a total of 111 patients with NSCLC. A methylation assay was performed using the quantitative methylation-specific polymerase chain reaction method. The percentage of methylated reference was used to represent the methylation level of the MDFI promoter. Data mining of a dataset from The Cancer Genome Atlas (TCGA) demonstrated that MDFI promoter methylation levels were significantly increased in 830 tumor tissues compared with 75 non-tumor tissues (P=0.012). However, the results on tissues obtained in the present study indicated that the MDFI promoter methylation levels in tumor tissues were not significantly different compared with those in the adjacent non-tumor tissues (P=0.159). Subsequent breakdown analysis identified that higher MDFI promoter methylation levels were significantly associated with NSCLC in females (P=0.031), but not in males (P=0.832). Age-based subgroup analysis demonstrated that higher MDFI promoter methylation levels were significantly associated with NSCLC in younger patients (≤65 years; P=0.003), but not in older patients (P=0.327). In addition, the association of MDFI methylation with NSCLC was significant in non-smokers (P=0.014), but not in smokers (P=0.832). Similar results also have been determined from subgroup analysis of the TCGA datasets. The Gene Expression Omnibus database indicated MDFI expression restoration in partial lung cancer cell lines (H1299 and Hotz) following demethylation treatment. However, it was identified that MDFI promoter hypermethylation was not significantly associated with prognosis of NSCLC (P>0.05). In conclusion, the present study indicated that the

  14. Methylation of Notch3 modulates chemoresistance via P-glycoprotein.

    PubMed

    Gu, Xiaoting; Lu, Yangfan; He, Dongxu; Lu, Chunxiao; Jin, Jian; Lu, Xiaojie; Ma, Xin

    2016-12-05

    The global gene expression and DNA methylation of genes in adriamycin-resistant human breast cancer cells (MCF-7/ADM cells) are similar to those in paclitaxel-resistant MCF-7 cells (MCF-7/PTX) and are significantly different from those in wild-type MCF-7 cells. DNA methylation is associated with chemoresistance in breast cancer and changes the characteristics of chemoresistant and chemosensitive cells. Here, we showed that the tumor-suppressor gene Notch3 was inactivated due to epigenetic silencing DNA hypermethylation in MCF-7/ADM cells. In addition, the drug efflux pump P-glycoprotein was negatively regulated by Notch3 and highly expressed in MCF-7/ADM cells. Taken together, our findings demonstrated that hypermethylation of Notch3 causes activation of P-glycoprotein in adriamycin-resistant cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Integrative Analysis Reveals Relationships of Genetic and Epigenetic Alterations in Osteosarcoma

    PubMed Central

    Skårn, Magne; Namløs, Heidi M.; Barragan-Polania, Ana H.; Cleton-Jansen, Anne-Marie; Serra, Massimo; Liestøl, Knut; Hogendoorn, Pancras C. W.; Hovig, Eivind; Myklebost, Ola; Meza-Zepeda, Leonardo A.

    2012-01-01

    Background Osteosarcomas are the most common non-haematological primary malignant tumours of bone, and all conventional osteosarcomas are high-grade tumours showing complex genomic aberrations. We have integrated genome-wide genetic and epigenetic profiles from the EuroBoNeT panel of 19 human osteosarcoma cell lines based on microarray technologies. Principal Findings The cell lines showed complex patterns of DNA copy number changes, where genomic copy number gains were significantly associated with gene-rich regions and losses with gene-poor regions. By integrating the datasets, 350 genes were identified as having two types of aberrations (gain/over-expression, hypo-methylation/over-expression, loss/under-expression or hyper-methylation/under-expression) using a recurrence threshold of 6/19 (>30%) cell lines. The genes showed in general alterations in either DNA copy number or DNA methylation, both within individual samples and across the sample panel. These 350 genes are involved in embryonic skeletal system development and morphogenesis, as well as remodelling of extracellular matrix. The aberrations of three selected genes, CXCL5, DLX5 and RUNX2, were validated in five cell lines and five tumour samples using PCR techniques. Several genes were hyper-methylated and under-expressed compared to normal osteoblasts, and expression could be reactivated by demethylation using 5-Aza-2′-deoxycytidine treatment for four genes tested; AKAP12, CXCL5, EFEMP1 and IL11RA. Globally, there was as expected a significant positive association between gain and over-expression, loss and under-expression as well as hyper-methylation and under-expression, but gain was also associated with hyper-methylation and under-expression, suggesting that hyper-methylation may oppose the effects of increased copy number for detrimental genes. Conclusions Integrative analysis of genome-wide genetic and epigenetic alterations identified dependencies and relationships between DNA copy number, DNA

  16. Detection of changes in DNA methylation induced by different doses of ground-base ion radiation in rice(oryza sativa L.)

    NASA Astrophysics Data System (ADS)

    Zhao, Qian; Sun, Yeqing; Wang, Wei; Wen, Bin

    Spaceflight represents a very complex environmental condition with highly ionizing radiations (HZE). To further investigate the incentives of ion effects in space environment, we performed on-ground simulated HZE particle radiations to rice seeds with different cumulative doses (0Gy, 0.01Gy, 0.02Gy, 0.1Gy, 0.2Gy, 1Gy , 2Gy, 5Gy, 20Gy ). Using Methylation-Sensitive Amplification Polymorphism (MSAP) analysis technology, differential polymorphism sites of DNA methylation of seedlings were analysed and acquired. The results showed that changes of methylation and demethylation on CCGG sites had taken place after irradiated treatments in all doses. It was noted that there was a stimulating effect in low-dose radiation ≤1 Gy. The minimum proportion of DNA methylation polymorphism level was 3.15% in 0.1Gy, whereas the maximum proportion was 9.87% in 2Gy, interestingly the proportion reduced with radiation doses increased, suggesting the dosage effects of radiation. We further found that the CG site tended to have a higher proportion of cytosine methylation alterations than CNG site in six of the eight dose groups. The results also indicated that different dose treatment groups showed various frequencies of methylation variation patterns: The type of CG hypermethylation was higher than CG hypormethylation in four low-dose groups (<≤2 Gy) ,whereas the result presented the opposite trends in all high-dose groups(>≥1 Gy). In addition, the type of CNG hypormethylation was obviously higher than the CNG hypermethylation in seven dose groups. This result indicated that the methylation variation patterns caused by radiation had site preferences. To investigate the mechanisms of sequences underlying alterations in DNA methylation after ion irradiation, we isolated, cloned and sequenced a subset of bands which showed obvious mutational bias. BLAST analysis indicated that many sequences showed significant homology to known function genes, most of which were related to resistance

  17. Epigenetic repression of HOXB cluster in oral cancer cell lines.

    PubMed

    Xavier, Flávia Caló Aquino; Destro, Maria Fernanda de Souza Setubal; Duarte, Carina Magalhães Esteves; Nunes, Fabio Daumas

    2014-08-01

    Aberrant DNA methylation is a fundamental transcriptional control mechanism in carcinogenesis. The expression of homeobox genes is usually controlled by an epigenetic mechanism, such as the methylation of CpG islands in the promoter region. The aim of this study was to describe the differential methylation pattern of HOX genes in oral squamous cell carcinoma (OSCC) cell lines and transcript status in a group of hypermethylated and hypomethylated genes. Quantitative analysis of DNA methylation was performed on two OSCC cell lines (SCC4 and SCC9) using a method denominated Human Homeobox Genes EpiTect Methyl qPCR Arrays, which allowed fast, precise methylation detection of 24 HOX specific genes without bisulfite conversion. Methylation greater than 50% was detected in HOXA11, HOXA6, HOXA7, HOXA9, HOXB1, HOXB2, HOXB3, HOXB4, HOXB5, HOXB6, HOXC8 and HOXD10. Both cell lines demonstrated similar hypermethylation status for eight HOX genes. A similar pattern of promoter hypermethylation and hypomethylation was demonstrated for the HOXB cluster and HOXA cluster, respectively. Moreover, the hypermethylation profile of the HOXB cluster, especially HOXB4, was correlated with decreased transcript expression, which was restored following treatment with 5-aza-2'-deoxycytidine. The homeobox methylation profile in OSCC cell lines is consistent with an epigenetic biomarker. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Fanconi anemia: a disorder defective in the DNA damage response.

    PubMed

    Kitao, Hiroyuki; Takata, Minoru

    2011-04-01

    Fanconi anemia (FA) is a cancer predisposition disorder characterized by progressive bone marrow failure, congenital developmental defects, chromosomal abnormalities, and cellular hypersensitivity to DNA interstrand crosslink (ICL) agents. So far mutations in 14 FANC genes were identified in FA or FA-like patients. These gene products constitute a common ubiquitin-phosphorylation network called the "FA pathway" and cooperate with other proteins involved in DNA repair and cell cycle control to repair ICL lesions and to maintain genome stability. In this review, we summarize recent exciting discoveries that have expanded our view of the molecular mechanisms operating in DNA repair and DNA damage signaling.

  19. Perinatal depression and DNA methylation of oxytocin-related genes: a study of mothers and their children.

    PubMed

    King, Leonora; Robins, Stephanie; Chen, Gang; Yerko, Volodymyr; Zhou, Yi; Nagy, Corina; Feeley, Nancy; Gold, Ian; Hayton, Barbara; Turecki, Gustavo; Zelkowitz, Phyllis

    2017-11-01

    The present study investigated the association of perinatal depression (PD) with differential methylation of 3 genomic regions among mother and child dyads: exon 3 within the oxytocin receptor (OXTR) gene and 2 intergenic regions (IGR) between the oxytocin (OXT) and vasopressin (AVP) genes. Maternal PD was assessed at 5 time-points during pregnancy and postpartum. Four groups were established based on Edinburgh Postnatal Depression Scale (EPDS) cut-off scores: no PD, prenatal or postpartum depressive symptoms only and persistent PD (depressive symptoms both prenatally and postpartum). Salivary DNA was collected from mothers and children at the final time-point, 2.9years postpartum. Mothers with persistent PD had significantly higher overall OXTR methylation than the other groups and this pattern extended to 16/22 individual CpG sites. For the IGR, only the region closer to the AVP gene (AVP IGR) showed significant differential methylation, with the persistent PD group displaying the lowest levels of methylation overall, but not for individual CpG sites. These results suggest that transient episodes of depression may not be associated with OXTR hypermethylation. Validation studies need to confirm the downstream biological effects of AVP IGR hypomethylation as it relates to persistent PD. Differential methylation of the OXTR and IGR regions was not observed among children exposed to maternal PD. The consequences of OXTR hypermethylation and AVP IGR hypomethylation found in mothers with persistent PDS may not only impact the OXT system, but may also compromise maternal behavior, potentially resulting in negative outcomes for the developing child. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Effects of Replication and Transcription on DNA Structure-Related Genetic Instability.

    PubMed

    Wang, Guliang; Vasquez, Karen M

    2017-01-05

    Many repetitive sequences in the human genome can adopt conformations that differ from the canonical B-DNA double helix (i.e., non-B DNA), and can impact important biological processes such as DNA replication, transcription, recombination, telomere maintenance, viral integration, transposome activation, DNA damage and repair. Thus, non-B DNA-forming sequences have been implicated in genetic instability and disease development. In this article, we discuss the interactions of non-B DNA with the replication and/or transcription machinery, particularly in disease states (e.g., tumors) that can lead to an abnormal cellular environment, and how such interactions may alter DNA replication and transcription, leading to potential conflicts at non-B DNA regions, and eventually result in genetic stability and human disease.

  1. Effects of Replication and Transcription on DNA Structure-Related Genetic Instability

    PubMed Central

    Wang, Guliang; Vasquez, Karen M.

    2017-01-01

    Many repetitive sequences in the human genome can adopt conformations that differ from the canonical B-DNA double helix (i.e., non-B DNA), and can impact important biological processes such as DNA replication, transcription, recombination, telomere maintenance, viral integration, transposome activation, DNA damage and repair. Thus, non-B DNA-forming sequences have been implicated in genetic instability and disease development. In this article, we discuss the interactions of non-B DNA with the replication and/or transcription machinery, particularly in disease states (e.g., tumors) that can lead to an abnormal cellular environment, and how such interactions may alter DNA replication and transcription, leading to potential conflicts at non-B DNA regions, and eventually result in genetic stability and human disease. PMID:28067787

  2. Aberrant Hypermethylation of SALL3 with HPV Involvement Contributes to the Carcinogenesis of Cervical Cancer.

    PubMed

    Wei, Xing; Zhang, Shaohua; Cao, Di; Zhao, Minyi; Zhang, Qian; Zhao, Juan; Yang, Ting; Pei, Meili; Wang, Li; Li, Yang; Yang, Xiaofeng

    2015-01-01

    This study aimed to investigate the methylation status of the promoter region of spalt-like transcription factor 3 (SALL3) and the expression of SALL3 in cervical cancer to explore the function of this gene in cervical cancer carcinogenesis. The methylation status of SALL3 was detected by methylation-specific PCR, and SALL3 gene expression was assessed by real-time quantitative PCR in the cervical cancer cell lines, SiHa, HeLa and C33A, as well as in cervical cancer tissue samples (n = 23), matched pericarcinomatous tissue samples (n = 23) and normal cervix tissue samples (n = 17). MTT was used to measure the cell viability and proliferation capacity of SiHa and HeLa cells. The SALL3 promoter was completely methylated in SiHa cells, unmethylated in C33A cells and partially methylated in HeLa cells. After treatment of SiHa and HeLa cells with 5 μM and 10 μM of 5-Azacytidine (5-Aza), respectively, the methylation level of the SALL3 promoter decreased and observed increase in the degree of unmethylation in a dose-dependent manner. Moreover, the relative expression of SALL3 mRNA increased as the concentration of 5-Aza increased in SiHa (p<0.05) and HeLa (p<0.05) cells. This above-mentioned increase in SALL3 mRNA in SiHa cells was more remarkable than that observed in HeLa cells. Cell proliferation capacity also decreased after administration of 5-Aza to SiHa and HeLa cells (p<0.05). Methylation of the SALL3 promoter was observed in 15 of 23 (65.21%) cervical cancer tissue samples, 15 of 23 (65.21%) matched pericarcinomatous tissue samples and 5 of 17 (29.41%) normal cervical tissue samples (p<0.05). SALL3 mRNA expression was significantly lower in cervical cancer and pericarcinomatous tissues compared with normal cervical tissues (p<0.05). In all cervix tissue samples, HPV infection was positively associated with hypermethylation of the promoter region of SALL3 (p<0.05, r = 0.408), and the expression of SALL3 mRNA in HPV-positive tissues was lower than that in HPV

  3. Predicting DNA Methylation State of CpG Dinucleotide Using Genome Topological Features and Deep Networks

    NASA Astrophysics Data System (ADS)

    Wang, Yiheng; Liu, Tong; Xu, Dong; Shi, Huidong; Zhang, Chaoyang; Mo, Yin-Yuan; Wang, Zheng

    2016-01-01

    The hypo- or hyper-methylation of the human genome is one of the epigenetic features of leukemia. However, experimental approaches have only determined the methylation state of a small portion of the human genome. We developed deep learning based (stacked denoising autoencoders, or SdAs) software named “DeepMethyl” to predict the methylation state of DNA CpG dinucleotides using features inferred from three-dimensional genome topology (based on Hi-C) and DNA sequence patterns. We used the experimental data from immortalised myelogenous leukemia (K562) and healthy lymphoblastoid (GM12878) cell lines to train the learning models and assess prediction performance. We have tested various SdA architectures with different configurations of hidden layer(s) and amount of pre-training data and compared the performance of deep networks relative to support vector machines (SVMs). Using the methylation states of sequentially neighboring regions as one of the learning features, an SdA achieved a blind test accuracy of 89.7% for GM12878 and 88.6% for K562. When the methylation states of sequentially neighboring regions are unknown, the accuracies are 84.82% for GM12878 and 72.01% for K562. We also analyzed the contribution of genome topological features inferred from Hi-C. DeepMethyl can be accessed at http://dna.cs.usm.edu/deepmethyl/.

  4. Predicting DNA Methylation State of CpG Dinucleotide Using Genome Topological Features and Deep Networks.

    PubMed

    Wang, Yiheng; Liu, Tong; Xu, Dong; Shi, Huidong; Zhang, Chaoyang; Mo, Yin-Yuan; Wang, Zheng

    2016-01-22

    The hypo- or hyper-methylation of the human genome is one of the epigenetic features of leukemia. However, experimental approaches have only determined the methylation state of a small portion of the human genome. We developed deep learning based (stacked denoising autoencoders, or SdAs) software named "DeepMethyl" to predict the methylation state of DNA CpG dinucleotides using features inferred from three-dimensional genome topology (based on Hi-C) and DNA sequence patterns. We used the experimental data from immortalised myelogenous leukemia (K562) and healthy lymphoblastoid (GM12878) cell lines to train the learning models and assess prediction performance. We have tested various SdA architectures with different configurations of hidden layer(s) and amount of pre-training data and compared the performance of deep networks relative to support vector machines (SVMs). Using the methylation states of sequentially neighboring regions as one of the learning features, an SdA achieved a blind test accuracy of 89.7% for GM12878 and 88.6% for K562. When the methylation states of sequentially neighboring regions are unknown, the accuracies are 84.82% for GM12878 and 72.01% for K562. We also analyzed the contribution of genome topological features inferred from Hi-C. DeepMethyl can be accessed at http://dna.cs.usm.edu/deepmethyl/.

  5. Genome-wide screen of DNA methylation changes induced by low dose X-ray radiation in mice.

    PubMed

    Wang, Jingzi; Zhang, Youwei; Xu, Kai; Mao, Xiaobei; Xue, Lijun; Liu, Xiaobei; Yu, Hongjun; Chen, Longbang; Chu, Xiaoyuan

    2014-01-01

    Epigenetic mechanisms play a key role in non-targeted effects of radiation. The purpose of this study was to investigate global hypomethylation and promoter hypermethylation of particular genes induced by low dose radiation (LDR). Thirty male BALB/c mice were divided into 3 groups: control, acutely exposed (0.5 Gy X-rays), and chronic exposure for 10 days (0.05Gy/d×10d). High-performance liquid chromatography (HPLC) and MeDIP-quantitative polymerase chain reaction (qPCR) were used to study methylation profiles. DNMT1 and MBD2 expression was determined by qPCR and western blot assays. Methylation and expression of Rad23b and Ddit3 were determined by bisulfate sequencing primers (BSP) and qPCR, respectively. The results show that LDR induced genomic hypomethylation in blood 2 h postirraditaion, but was not retained at 1-month. DNMT1 and MBD2 were downregulated in a tissue-specific manner but did not persist. Specific hypermethylation was observed for 811 regions in the group receiving chronic exposure, which covered almost all key biological processes as indicated by GO and KEGG pathway analysis. Eight hypermethylated genes (Rad23b, Tdg, Ccnd1, Ddit3, Llgl1, Rasl11a, Tbx2, Scl6a15) were verified by MeDIP-qPCR. Among them, Rad23b and Ddit3 gene displayed tissue-specific methylation and downregulation, which persisted for 1-month postirradiation. Thus, LDR induced global hypomethylation and tissue-specific promoter hypermethylation of particular genes. Promoter hypermethylation, rather than global hypomethylation, was relatively stable. Dysregulation of methylation might be correlated with down-regulation of DNMT1 and MBD2, but much better understanding the molecular mechanisms involved in this process will require further study.

  6. Postmortem brain abnormalities of the glutamate neurotransmitter system in autism.

    PubMed

    Purcell, A E; Jeon, O H; Zimmerman, A W; Blue, M E; Pevsner, J

    2001-11-13

    Studies examining the brains of individuals with autism have identified anatomic and pathologic changes in regions such as the cerebellum and hippocampus. Little, if anything, is known, however, about the molecules that are involved in the pathogenesis of this disorder. To identify genes with abnormal expression levels in the cerebella of subjects with autism. Brain samples from a total of 10 individuals with autism and 23 matched controls were collected, mainly from the cerebellum. Two cDNA microarray technologies were used to identify genes that were significantly up- or downregulated in autism. The abnormal mRNA or protein levels of several genes identified by microarray analysis were investigated using PCR with reverse transcription and Western blotting. alpha-Amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA)- and NMDA-type glutamate receptor densities were examined with receptor autoradiography in the cerebellum, caudate-putamen, and prefrontal cortex. The mRNA levels of several genes were significantly increased in autism, including excitatory amino acid transporter 1 and glutamate receptor AMPA 1, two members of the glutamate system. Abnormalities in the protein or mRNA levels of several additional molecules in the glutamate system were identified on further analysis, including glutamate receptor binding proteins. AMPA-type glutamate receptor density was decreased in the cerebellum of individuals with autism (p < 0.05). Subjects with autism may have specific abnormalities in the AMPA-type glutamate receptors and glutamate transporters in the cerebellum. These abnormalities may be directly involved in the pathogenesis of the disorder.

  7. Methylation Landscape of Human Breast Cancer Cells in Response to Dietary Compound Resveratrol

    PubMed Central

    Medina-Aguilar, Rubiceli; Pérez-Plasencia, Carlos; Marchat, Laurence A.; Gariglio, Patricio; García Mena, Jaime; Rodríguez Cuevas, Sergio; Ruíz-García, Erika; Astudillo-de la Vega, Horacio; Hernández Juárez, Jennifer; Flores-Pérez, Ali; López-Camarillo, César

    2016-01-01

    Aberrant DNA methylation is a frequent epigenetic alteration in cancer cells that has emerged as a pivotal mechanism for tumorigenesis. Accordingly, novel therapies targeting the epigenome are being explored with the aim to restore normal DNA methylation patterns on oncogenes and tumor suppressor genes. A limited number of studies indicate that dietary compound resveratrol modulates DNA methylation of several cancer-related genes; however a complete view of changes in methylome by resveratrol has not been reported yet. In this study we performed a genome-wide survey of DNA methylation signatures in triple negative breast cancer cells exposed to resveratrol. Our data showed that resveratrol treatment for 24 h and 48 h decreased gene promoter hypermethylation and increased DNA hypomethylation. Of 2476 hypermethylated genes in control cells, 1,459 and 1,547 were differentially hypomethylated after 24 h and 48 h, respectively. Remarkably, resveratrol did not induce widespread non-specific DNA hyper- or hypomethylation as changes in methylation were found in only 12.5% of 27,728 CpG loci. Moreover, resveratrol restores the hypomethylated and hypermethylated status of key tumor suppressor genes and oncogenes, respectively. Importantly, the integrative analysis of methylome and transcriptome profiles in response to resveratrol showed that methylation alterations were concordant with changes in mRNA expression. Our findings reveal for the first time the impact of resveratrol on the methylome of breast cancer cells and identify novel potential targets for epigenetic therapy. We propose that resveratrol may be considered as a dietary epidrug as it may exert its anti-tumor activities by modifying the methylation status of cancer -related genes which deserves further in vivo characterization. PMID:27355345

  8. Comparison of cancer-associated genetic abnormalities in columnar-lined esophagus tissues with and without goblet cells.

    PubMed

    Bandla, Santhoshi; Peters, Jeffrey H; Ruff, David; Chen, Shiaw-Min; Li, Chieh-Yuan; Song, Kunchang; Thoms, Kimberly; Litle, Virginia R; Watson, Thomas; Chapurin, Nikita; Lada, Michal; Pennathur, Arjun; Luketich, James D; Peterson, Derick; Dulak, Austin; Lin, Lin; Bass, Adam; Beer, David G; Godfrey, Tony E; Zhou, Zhongren

    2014-07-01

    To determine and compare the frequency of cancer-associated genetic abnormalities in esophageal metaplasia biopsies with and without goblet cells. Barrett's esophagus is associated with increased risk of esophageal adenocarcinoma (EAC), but the appropriate histologic definition of Barrett's esophagus is debated. Intestinal metaplasia (IM) is defined by the presence of goblet cells whereas nongoblet cell metaplasia (NGM) lacks goblet cells. Both have been implicated in EAC risk but this is controversial. Although IM is known to harbor genetic changes associated with EAC, little is known about NGM. We hypothesized that if NGM and IM infer similar EAC risk, then they would harbor similar genetic aberrations in genes associated with EAC. Ninety frozen NGM, IM, and normal tissues from 45 subjects were studied. DNA copy number abnormalities were identified using microarrays and fluorescence in situ hybridization. Targeted sequencing of all exons from 20 EAC-associated genes was performed on metaplasia biopsies using Ion AmpliSeq DNA sequencing. Frequent copy number abnormalities targeting cancer-associated genes were found in IM whereas no such changes were observed in NGM. In 1 subject, fluorescence in situ hybridization confirmed loss of CDKN2A and amplification of chromosome 8 in IM but not in a nearby NGM biopsy. Targeted sequencing revealed 11 nonsynonymous mutations in 16 IM samples and 2 mutations in 19 NGM samples. This study reports the largest and most comprehensive comparison of DNA aberrations in IM and NGM genomes. Our results show that IM has a much higher frequency of cancer-associated mutations than NGM.

  9. CaMV-35S promoter sequence-specific DNA methylation in lettuce.

    PubMed

    Okumura, Azusa; Shimada, Asahi; Yamasaki, Satoshi; Horino, Takuya; Iwata, Yuji; Koizumi, Nozomu; Nishihara, Masahiro; Mishiba, Kei-ichiro

    2016-01-01

    We found 35S promoter sequence-specific DNA methylation in lettuce. Additionally, transgenic lettuce plants having a modified 35S promoter lost methylation, suggesting the modified sequence is subjected to the methylation machinery. We previously reported that cauliflower mosaic virus 35S promoter-specific DNA methylation in transgenic gentian (Gentiana triflora × G. scabra) plants occurs irrespective of the copy number and the genomic location of T-DNA, and causes strong gene silencing. To confirm whether 35S-specific methylation can occur in other plant species, transgenic lettuce (Lactuca sativa L.) plants with a single copy of the 35S promoter-driven sGFP gene were produced and analyzed. Among 10 lines of transgenic plants, 3, 4, and 3 lines showed strong, weak, and no expression of sGFP mRNA, respectively. Bisulfite genomic sequencing of the 35S promoter region showed hypermethylation at CpG and CpWpG (where W is A or T) sites in 9 of 10 lines. Gentian-type de novo methylation pattern, consisting of methylated cytosines at CpHpH (where H is A, C, or T) sites, was also observed in the transgenic lettuce lines, suggesting that lettuce and gentian share similar methylation machinery. Four of five transgenic lettuce lines having a single copy of a modified 35S promoter, which was modified in the proposed core target of de novo methylation in gentian, exhibited 35S hypomethylation, indicating that the modified sequence may be the target of the 35S-specific methylation machinery.

  10. Unique patterns of CpG island methylation in inflammatory bowel disease-associated colorectal cancers

    PubMed Central

    Olaru, Alexandru V.; Cheng, Yulan; Agarwal, Rachana; Yang, Jian; David, Stefan; Abraham, John M.; Yu, Wayne; Lazarev, Mark; Brant, Steven R.; Marohn, Michael R.; Hutcheon, David F.; Harpaz, Noam; Meltzer, Stephen J.; Mori, Yuriko

    2011-01-01

    Objective CpG island (CGI) hypermethylation at discrete loci is a prevalent cancer-promoting abnormality in sporadic colorectal carcinomas (S-CRCs). We investigated genome-wide CGI methylation in inflammatory bowel disease (IBD)-associated CRCs (IBD-CRCs). Design Methylation microarray analyses were conducted on 7 IBD-CRCs, 17 S-CRCs, and 8 normal control colonic tissues from patients without CRC or IBD. CGI methylator phenotype (CIMP), a surrogate marker for widespread cancer-specific CGI hypermethylation, was examined in 30 IBD-CRCs and 43 S-CRCs. Results The genome-wide CGI methylation pattern of IBD-CRCs was CIMP status-dependent. Based on methylation array data profiling of all autosomal loci, CIMP+ IBD-CRCs grouped together with S-CRCs, while CIMP− IBD-CRCs grouped together with control tissues. CIMP− IBD-CRCs demonstrated less methylation than did age-matched CIMP− S-CRCs at all autosomal CGIs (z-score −0.17 vs. 0.09, p=3×10−3) and CRC-associated hypermethylation target CGIs (z-score −0.43 vs. 0.68, p=1×10-4). Age-associated hypermethylation target CGIs were significantly overrepresented in CGIs that were hypermethylated in S-CRCs (p=1×10−192), but not in CGIs that were hypermethylated in IBD-CRCs (p=0.11). In contrast, KRAS mutation prevalence were similar between IBD-CRCs and S-CRCs. Notably, CIMP+ prevalence was significantly higher in older than in younger IBD-CRC cases (4.2% vs. 50.0%, p=0.02), but not in S-CRC cases (16.7% vs. 9.7%, p=0.92). Conclusions Cancer-specific CGI hypermethylation and age-associated CGI hypermethylation are diminished in IBD-CRCs relative to S-CRCs, while KRAS mutation rate is comparable between these cancers. CGI hypermethylation appears to play only a minor role in IBD-associated carcinogenesis. We speculate that aging, rather than inflammation per se, promotes CIMP+ CRCs in IBD patients. PMID:21830278

  11. Analysis of the machinery and intermediates of the 5hmC-mediated DNA demethylation pathway in aging on samples from the MARK-AGE Study

    PubMed Central

    Valentini, Elisabetta; Zampieri, Michele; Malavolta, Marco; Bacalini, Maria Giulia; Calabrese, Roberta; Guastafierro, Tiziana; Reale, Anna; Franceschi, Claudio; Hervonen, Antti; Koller, Bernhard; Bernhardt, Jürgen; Slagboom, P. Eline; Toussaint, Olivier; Sikora, Ewa; Gonos, Efstathios S.; Breusing, Nicolle; Grune, Tilman; Jansen, Eugène; Dollé, Martijn E.T.; Moreno-Villanueva, María; Sindlinger, Thilo; Bürkle, Alexander; Ciccarone, Fabio; Caiafa, Paola

    2016-01-01

    Gradual changes in the DNA methylation landscape occur throughout aging virtually in all human tissues. A widespread reduction of 5-methylcytosine (5mC), associated with highly reproducible site-specific hypermethylation, characterizes the genome in aging. Therefore, an equilibrium seems to exist between general and directional deregulating events concerning DNA methylation controllers, which may underpin the age-related epigenetic changes. In this context, 5mC-hydroxylases (TET enzymes) are new potential players. In fact, TETs catalyze the stepwise oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), driving the DNA demethylation process based on thymine DNA glycosylase (TDG)-mediated DNA repair pathway. The present paper reports the expression of DNA hydroxymethylation components, the levels of 5hmC and of its derivatives in peripheral blood mononuclear cells of age-stratified donors recruited in several European countries in the context of the EU Project ‘MARK-AGE’. The results provide evidence for an age-related decline of TET1, TET3 and TDG gene expression along with a decrease of 5hmC and an accumulation of 5caC. These associations were independent of confounding variables, including recruitment center, gender and leukocyte composition. The observed impairment of 5hmC-mediated DNA demethylation pathway in blood cells may lead to aberrant transcriptional programs in the elderly. PMID:27587280

  12. Functional analyses of PtRDM1 gene overexpression in poplars and evaluation of its effect on DNA methylation and response to salt stress.

    PubMed

    Movahedi, Ali; Zhang, Jiaxin; Sun, Weibo; Mohammadi, Kourosh; Almasi Zadeh Yaghuti, Amir; Wei, Hui; Wu, Xiaolong; Yin, Tongming; Zhuge, Qiang

    2018-06-01

    Epigenetic modification by DNA methylation is necessary for all cellular processes, including genetic expression events, DNA repair, genomic imprinting and regulation of tissue development. It occurs almost exclusively at the C5 position of symmetric CpG and asymmetric CpHpG and CpHpH sites in genomic DNA. The RNA-directed DNA methylation (RDM1) gene is crucial for heterochromatin and DNA methylation. We overexpressed PtRDM1 gene from Populus trichocarpa to amplify transcripts of orthologous RDM1 in 'Nanlin895' (P. deltoides × P. euramericana 'Nanlin895'). This overexpression resulted in increasing RDM1 transcript levels: by ∼150% at 0 mM NaCl treatment and by ∼300% at 60 mM NaCl treatment compared to WT (control) poplars. Genomic cytosine methylation was monitored within 5.8S rDNA and histone H3 loci by bisulfite sequencing. In total, transgenic poplars revealed more DNA methylation than WT plants. In our results, roots revealed more methylated CG contexts than stems and leaves whereas, histone H3 presented more DNA methylation than 5.8S rDNA in both WT and transgenic poplars. The NaCl stresses enhanced more DNA methylation in transgenic poplars than WT plants through histone H3 and 5.8 rDNA loci. Also, the overexpression of PtRDM1 resulted in hyper-methylation, which affected plant phenotype. Transgenic poplars revealed significantly more regeneration of roots than WT poplars via NaCl treatments. Our results proved that RDM1 protein enhanced the DNA methylation by chromatin remodeling (e.g. histone H3) more than repetitive DNA sequences (e.g. 5.8S rDNA). Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  13. Pretreatment dietary intake is associated with tumor suppressor DNA methylation in head and neck squamous cell carcinomas

    PubMed Central

    Colacino, Justin A.; Arthur, Anna E.; Dolinoy, Dana C.; Sartor, Maureen A.; Duffy, Sonia A.; Chepeha, Douglas B.; Bradford, Carol R.; Walline, Heather M.; McHugh, Jonathan B.; D'Silva, Nisha; Carey, Thomas E.; Wolf, Gregory T.; Taylor, Jeremy M.G.; Peterson, Karen E.; Rozek, Laura S.

    2012-01-01

    Diet is associated with cancer prognosis, including head and neck cancer (HNC), and has been hypothesized to influence epigenetic state by determining the availability of functional groups involved in the modification of DNA and histone proteins. The goal of this study was to describe the association between pretreatment diet and HNC tumor DNA methylation. Information on usual pretreatment food and nutrient intake was estimated via food frequency questionnaire (FFQ) on 49 HNC cases. Tumor DNA methylation patterns were assessed using the Illumina Goldengate Methylation Cancer Panel. First, a methylation score, the sum of individual hypermethylated tumor suppressor associated CpG sites, was calculated and associated with dietary intake of micronutrients involved in one-carbon metabolism and antioxidant activity, and food groups abundant in these nutrients. Second, gene specific analyses using linear modeling with empirical Bayesian variance estimation were conducted to identify if methylation at individual CpG sites was associated with diet. All models were controlled for age, sex, smoking, alcohol and HPV status. Individuals reporting in the highest quartile of folate, vitamin B12 and vitamin A intake, compared with those in the lowest quartile, showed significantly less tumor suppressor gene methylation, as did patients reporting the highest cruciferous vegetable intake. Gene specific analyses identified differential associations between DNA methylation and vitamin B12 and vitamin A intake when stratifying by HPV status. These preliminary results suggest that intake of folate, vitamin A and vitamin B12 may be associated with the tumor DNA methylation profile in HNC and enhance tumor suppression. PMID:22722388

  14. Feeling Abnormal: Simulation of Deviancy in Abnormal and Exceptionality Courses.

    ERIC Educational Resources Information Center

    Fernald, Charles D.

    1980-01-01

    Describes activity in which student in abnormal psychology and psychology of exceptional children classes personally experience being judged abnormal. The experience allows the students to remember relevant research, become sensitized to the feelings of individuals classified as deviant, and use caution in classifying individuals as abnormal.…

  15. Promoter hypermethylation of the RECK gene is associated with its low expression and poor survival of esophageal squamous cell carcinoma

    PubMed Central

    Zhu, Jing; Ling, Yang; Xu, Yun; Lu, Mingzhu; Liu, Yongping; Zhang, Changsong

    2017-01-01

    The present study aimed to investigate the association between the methylation status of the reversion-inducing cysteine-rich protein with kazal motifs (RECK) gene and its mRNA expression levels in patients with esophageal squamous cell carcinoma (ESCC). The methylation status of RECK was analyzed by methylation-specific polymerase chain reaction (PCR), and RECK mRNA expression levels were analyzed by quantitative PCR, in 310 paired ESCC tissues. The mean RECK methylation index (MI) was 0.65 in ESCCs and 0.49 in non-tumor samples. There was a significant association between RECK methylation and the American Joint Committee on Cancer stage and lymph node metastasis in ESCC (P<0.0001; P=0.001). The mRNA expression level of RECK was lower in ESCC tissues (mean-∆Cq=−4.66) compared with non-tumor tissues (mean-∆Cq=−2.79), and decreased RECK mRNA expression levels were associated with lymph node metastasis in ESCC. In addition, RECK mRNA levels were decreased in ESCC patients with hypermethylation of the RECK gene (∆MI >0.16; mean-∆∆Cq=−2.85) compared with those with hypomethylation of the RECK gene (∆MI ≤0.16; mean-∆∆Ct=−0.83), and there was a significant difference in the mRNA expression levels of RECK between those with N0–1 and N2–3 lymph node metastasis (P<0.0001). A significant correlation was observed between RECK mRNA expression levels, the MI of RECK and poor postoperative survival (P=0.0003; P<0.0001). The results of the present study suggested that promoter hypermethylation may be an important factor for loss of RECK mRNA expression and may be an indicator of poor survival in ESCC. PMID:28454343

  16. Longitudinal study of DNA methylation during the first 5 years of life.

    PubMed

    Urdinguio, Rocio G; Torró, María Isabel; Bayón, Gustavo F; Álvarez-Pitti, Julio; Fernández, Agustín F; Redon, Pau; Fraga, Mario F; Lurbe, Empar

    2016-06-03

    Early life epigenetic programming influences adult health outcomes. Moreover, DNA methylation levels have been found to change more rapidly during the first years of life. Our aim was the identification and characterization of the CpG sites that are modified with time during the first years of life. We hypothesize that these DNA methylation changes would lead to the detection of genes that might be epigenetically modulated by environmental factors during early childhood and which, if disturbed, might contribute to susceptibility to diseases later in life. The study of the DNA methylation pattern of 485577 CpG sites was performed on 30 blood samples from 15 subjects, collected both at birth and at 5 years old, using Illumina(®) Infinium 450 k array. To identify differentially methylated CpG (dmCpG) sites, the methylation status of each probe was examined using linear models and the Empirical Bayes Moderated t test implemented in the limma package of R/Bioconductor. Surogate variable analysis was used to account for batch effects. DNA methylation levels significantly changed from birth to 5 years of age in 6641 CpG sites. Of these, 36.79 % were hypermethylated and were associated with genes related mainly to developmental ontology terms, while 63.21 % were hypomethylated probes and associated with genes related to immune function. Our results suggest that DNA methylation alterations with age during the first years of life might play a significant role in development and the regulation of leukocyte-specific functions. This supports the idea that blood leukocytes experience genome remodeling related to their interaction with environmental factors, underlining the importance of environmental exposures during the first years of life and suggesting that new strategies should be take into consideration for disease prevention.

  17. A novel scoring system for gastric cancer risk assessment based on the expression of three CLIP4 DNA methylation-associated genes

    PubMed Central

    Hu, Chenggong; Zhou, Yongfang; Liu, Chang; Kang, Yan

    2018-01-01

    Gastric cancer (GC) is the fifth most common cancer and the third leading cause of cancer-associated mortality worldwide. In the current study, comprehensive bioinformatic analyses were performed to develop a novel scoring system for GC risk assessment based on CAP-Gly domain containing linker protein family member 4 (CLIP4) DNA methylation status. Two GC datasets with methylation sequencing information and mRNA expression profiling were downloaded from the The Cancer Genome Atlas and Gene Expression Omnibus databases. Differentially expressed genes (DEGs) between the CLIP4 hypermethylation and CLIP4 hypomethylation groups were screened using the limma package in R 3.3.1, and survival analysis of these DEGs was performed using the survival package. A risk scoring system was established via regression factor-weighted gene expression based on linear combination to screen the most important genes associated with CLIP4 methylation and prognosis. Genes associated with high/low-risk value were selected using the limma package. Functional enrichment analysis of the top 500 DEGs that positively and negatively associated with risk values was performed using DAVID 6.8 online and the gene set enrichment analysis (GSEA) software. In total, 35 genes were identified to be that significantly associated with prognosis and CLIP4 DNA methylation, and three prognostic signature genes, claudin-11 (CLDN11), apolipoprotein D (APOD), and chordin like 1 (CHRDL1), were used to establish a risk assessment system. The prognostic scoring system exhibited efficiency in classifying patients with different prognoses, where the low-risk groups had significantly longer overall survival times than those in the high-risk groups. CLDN11, APOD and CHRDL1 exhibited reduced expression in the hypermethylation and low-risk groups compare with the hypomethylation and high-risk groups, respectively. Multivariate Cox analysis indicated that risk value could be used as an independent prognostic factor. In

  18. Abnormal Uterine Bleeding

    MedlinePlus

    ... abnormal uterine bleeding? Abnormal uterine bleeding is any heavy or unusual bleeding from the uterus (through your ... one symptom of abnormal uterine bleeding. Having extremely heavy bleeding during your period can also be considered ...

  19. High frequency of genes' promoter methylation, but lack of BRAF V600E mutation among Iranian colorectal cancer patients.

    PubMed

    Naghibalhossaini, Fakhraddin; Hosseini, Hamideh Mahmoodzadeh; Mokarram, Pooneh; Zamani, Mozhdeh

    2011-12-01

    Gene silencing due to DNA hypermethylation is a major mechanism for loss of tumor suppressor genes function in colorectal cancer. Activating V600E mutation in BRAF gene has been linked with widespread methylation of CpG islands in sporadic colorectal cancers. The aim of the present study was to evaluate the methylation status of three cancer-related genes, APC2, p14ARF, and ECAD in colorectal carcinogenesis and their association with the mutational status of BRAF and KRAS among Iranian colorectal cancer patients. DNA from 110 unselected series of sporadic colorectal cancer patients was examined for BRAF V600E mutation by PCR-RFLP. Promoter methylation of genes in tumors was determined by methylation specific PCR. The frequency of APC2, E-CAD, and p14 methylation was 92.6%, 40.4% and 16.7%, respectively. But, no V600E mutation was identified in the BRAF gene in any sample. No association was found in cases showing epigenetic APC, ECAD, and p14 abnormality with the clinicopathological parameters under study. The association between KRAS mutations and the so called methylator phenotype was previously reported. Therefore, we also analyzed the association between the hot spot KRAS gene mutations in codons of 12 and 13 with genes' promoter hypermethylation in a subset of this group of patients. Out of 86 tumors, KRAS was mutated in 24 (28%) of tumors, the majority occurring in codon 12. KRAS mutations were not associated with genes' methylation in this tumor series. These findings suggest a distinct molecular pathway for methylation of APC2, p14, and ECAD genes from those previously described for colorectal cancers with BRAF or KRAS mutations.

  20. DNA Methylation and Methylation Polymorphism in Genetically Stable In vitro Regenerates of Jatropha curcas L. Using Methylation-Sensitive AFLP Markers.

    PubMed

    Rathore, Mangal S; Jha, Bhavanath

    2016-03-01

    The present investigation aimed to evaluate the degree and pattern of DNA methylation using methylation-sensitive AFLP (MS-AFLP) markers in genetically stable in vitro regenerates of Jatropha curcas L.. The genetically stable in vitro regenerates were raised through direct organogenesis via enhanced axillary shoot bud proliferation (Protocol-1) and in vitro-derived leaf regeneration (Protocol-2). Ten selective combinations of MS-AFLP primers produced 462 and 477 MS-AFLP bands in Protocol-1 (P-1) and Protocol-2 (P-2) regenerates, respectively. In P-1 regenerates, 15.8-31.17 % DNA was found methylated with an average of 25.24 %. In P-2 regenerates, 15.93-32.7 % DNA was found methylated with an average of 24.11 %. Using MS-AFLP in P-1 and P-2 regenerates, 11.52-25.53 % and 13.33-25.47 % polymorphism in methylated DNA was reported, respectively. Compared to the mother plant, P-1 regenerates showed hyper-methylation while P-2 showed hypo-methylation. The results clearly indicated alternation in degree and pattern of DNA methylation; hence, epigenetic instability in the genetically stable in vitro regenerates of J. curcas, developed so far using two different regeneration systems and explants of two different origins. The homologous nucleotide fragments in genomes of P-1 and P-2 regenerates showing methylation re-patterning might be involved in immediate adaptive responses and developmental processes through differential regulation of transcriptome under in vitro conditions.

  1. Lysosomal abnormalities in hereditary spastic paraplegia types SPG15 and SPG11

    PubMed Central

    Renvoisé, Benoît; Chang, Jaerak; Singh, Rajat; Yonekawa, Sayuri; FitzGibbon, Edmond J; Mankodi, Ami; Vanderver, Adeline; Schindler, Alice B; Toro, Camilo; Gahl, William A; Mahuran, Don J; Blackstone, Craig; Pierson, Tyler Mark

    2014-01-01

    Objective Hereditary spastic paraplegias (HSPs) are among the most genetically diverse inherited neurological disorders, with over 70 disease loci identified (SPG1-71) to date. SPG15 and SPG11 are clinically similar, autosomal recessive disorders characterized by progressive spastic paraplegia along with thin corpus callosum, white matter abnormalities, cognitive impairment, and ophthalmologic abnormalities. Furthermore, both have been linked to early-onset parkinsonism. Methods We describe two new cases of SPG15 and investigate cellular changes in SPG15 and SPG11 patient-derived fibroblasts, seeking to identify shared pathogenic themes. Cells were evaluated for any abnormalities in cell division, DNA repair, endoplasmic reticulum, endosomes, and lysosomes. Results Fibroblasts prepared from patients with SPG15 have selective enlargement of LAMP1-positive structures, and they consistently exhibited abnormal lysosomal storage by electron microscopy. A similar enlargement of LAMP1-positive structures was also observed in cells from multiple SPG11 patients, though prominent abnormal lysosomal storage was not evident. The stabilities of the SPG15 protein spastizin/ZFYVE26 and the SPG11 protein spatacsin were interdependent. Interpretation Emerging studies implicating these two proteins in interactions with the late endosomal/lysosomal adaptor protein complex AP-5 are consistent with shared abnormalities in lysosomes, supporting a converging mechanism for these two disorders. Recent work with Zfyve26−/− mice revealed a similar phenotype to human SPG15, and cells in these mice had endolysosomal abnormalities. SPG15 and SPG11 are particularly notable among HSPs because they can also present with juvenile parkinsonism, and this lysosomal trafficking or storage defect may be relevant for other forms of parkinsonism associated with lysosomal dysfunction. PMID:24999486

  2. Targeting of >1.5 Mb of Human DNA into the Mouse X Chromosome Reveals Presence of cis-Acting Regulators of Epigenetic Silencing

    PubMed Central

    Yang, Christine; McLeod, Andrea J.; Cotton, Allison M.; de Leeuw, Charles N.; Laprise, Stéphanie; Banks, Kathleen G.; Simpson, Elizabeth M.; Brown, Carolyn J.

    2012-01-01

    Regulatory sequences can influence the expression of flanking genes over long distances, and X chromosome inactivation is a classic example of cis-acting epigenetic gene regulation. Knock-ins directed to the Mus musculus Hprt locus offer a unique opportunity to analyze the spread of silencing into different human DNA sequences in the identical genomic environment. X chromosome inactivation of four knock-in constructs, including bacterial artificial chromosome (BAC) integrations of over 195 kb, was demonstrated by both the lack of expression from the inactive X chromosome in females with nonrandom X chromosome inactivation and promoter DNA methylation of the human transgene in females. We further utilized promoter DNA methylation to assess the inactivation status of 74 human reporter constructs comprising >1.5 Mb of DNA. Of the 47 genes examined, only the PHB gene showed female DNA hypomethylation approaching the level seen in males, and escape from X chromosome inactivation was verified by demonstration of expression from the inactive X chromosome. Integration of PHB resulted in lower DNA methylation of the flanking HPRT promoter in females, suggesting the action of a dominant cis-acting escape element. Female-specific DNA hypermethylation of CpG islands not associated with promoters implies a widespread imposition of DNA methylation during X chromosome inactivation; yet transgenes demonstrated differential capacities to accumulate DNA methylation when integrated into the identical location on the inactive X chromosome, suggesting additional cis-acting sequence effects. As only one of the human transgenes analyzed escaped X chromosome inactivation, we conclude that elements permitting ongoing expression from the inactive X are rare in the human genome. PMID:23023002

  3. The Real Culprit in Systemic Lupus Erythematosus: Abnormal Epigenetic Regulation

    PubMed Central

    Wu, Haijing; Zhao, Ming; Chang, Christopher; Lu, Qianjin

    2015-01-01

    Systemic lupus erythematosus (SLE) is an autoimmune disease involving multiple organs and the presence of anti-nuclear antibodies. The pathogenesis of SLE has been intensively studied but remains far from clear. B and T lymphocyte abnormalities, dysregulation of apoptosis, defects in the clearance of apoptotic materials, and various genetic and epigenetic factors are attributed to the development of SLE. The latest research findings point to the association between abnormal epigenetic regulation and SLE, which has attracted considerable interest worldwide. It is the purpose of this review to present and discuss the relationship between aberrant epigenetic regulation and SLE, including DNA methylation, histone modifications and microRNAs in patients with SLE, the possible mechanisms of immune dysfunction caused by epigenetic changes, and to better understand the roles of aberrant epigenetic regulation in the initiation and development of SLE and to provide an insight into the related therapeutic options in SLE. PMID:25988383

  4. Novel methylation panel for the early detection of colorectal tumors in stool DNA.

    PubMed

    Azuara, Daniel; Rodriguez-Moranta, Francisco; de Oca, Javier; Soriano-Izquierdo, Antonio; Mora, Josefina; Guardiola, Jordi; Biondo, Sebastiano; Blanco, Ignacio; Peinado, Miguel Angel; Moreno, Victor; Esteller, Manel; Capellá, Gabriel

    2010-07-01

    Previous studies showed that the assessment of promoter hypermethylation of a limited number of genes in tumor biopsies may identify the majority of colorectal tumors. This study aimed to assess the clinical usefulness of a panel of methylation biomarkers in stool DNA in the identification of colorectal tumors, using methylation-specific melting curve analysis (MS-MCA), a technique that simultaneously analyzes all cytosine-phosphate-guanine (CpG) residues within a promoter. The promoter methylation status of 4 tumor-related genes (RARB2, p16INK4a, MGMT, and APC) was analyzed in DNA stool samples and corresponding tissues in an initial set of 12 patients with newly diagnosed primary colorectal carcinomas and 20 patients with newly diagnosed colorectal adenomas, using methylation-specific polymerase chain reaction. Results were replicated in a set of 82 patients (20 healthy subjects, 16 patients with inflammatory bowel disease (IBD), 20 patients with adenomas, and 26 patients with carcinomas), using MS-MCA analyses. In the initial set, >or= 1 positive methylation marker was detected in the stools of 9 of 12 patients (75%) with carcinomas and 12 of 20 patients (60%) with adenomas, with no false-positive results. Stool analyses missed 7 methylated lesions (25%). In the replication set, stool DNA testing detected 16 of 26 carcinomas (62%) and 8 of 20 adenomas (40%). The MS-MCAs missed 14 methylated tumors (37%). No aberrant methylation was evident in healthy subjects, but the RARB2 marker was positive in 2 of 15 stool samples (13%) of patients with IBD. Analysis via MS-MCA of a panel of methylation markers in stool DNA may offer a good alternative in the early, noninvasive detection of colorectal tumors.

  5. DNA-dependent protein kinase (DNA-PK)-deficient human glioblastoma cells are preferentially sensitized by Zebularine

    PubMed Central

    Meador, Jarah A.; Su, Yanrong; Ravanat, Jean-Luc; Balajee, Adayabalam S.

    2010-01-01

    Brain tumor cells respond poorly to radiotherapy and chemotherapy due to inherently efficient anti-apoptotic and DNA repair mechanisms. This necessitates the development of new strategies for brain cancer therapy. Here, we report that the DNA-demethylating agent Zebularine preferentially sensitizes the killing of human glioblastomas deficient in DNA-dependent protein kinase (DNA-PK). In contrast to DNA-PK-proficient human glioblastoma cells (MO59K), cytotoxicity assay with increasing Zebularine concentrations up to 300 μM resulted in a specific elevation of cell killing in DNA-PK-deficient MO59J cells. Further, an elevated frequency of polyploid cells observed in MO59J cells after Zebularine treatment pointed out a deficiency in mitotic checkpoint control. Existence of mitotic checkpoint deficiency in MO59J cells was confirmed by the abnormal centrosome number observed in Zebularine-treated MO59J cells. Although depletion of DNA methyltransferase 1 by Zebularine occurred at similar levels in both cell lines, MO59J cells displayed increased extent of DNA demethylation detected both at the gene promoter-specific level and at the genome overall level. Consistent with increased sensitivity, deoxy-Zebularine adduct level in the genomic DNA was 3- to 6-fold higher in MO59J than in MO59K cells. Elevated micronuclei frequency observed after Zebularine treatment in MO59J cells indicates the impairment of DNA repair response in MO59J cells. Collectively, our study suggests that DNA-PK is the major determining factor for cellular response to Zebularine. PMID:19933707

  6. Gonadotrophin abnormalities in an infant with Lowe syndrome.

    PubMed

    Warner, Bronwen E; Inward, Carol D; Burren, Christine P

    2017-01-01

    This case, presenting with bilateral impalpable testes, illustrates the relevance of a broad differential disorders of sex development case management. It provides new insights on hypothalamic-pituitary-gonadal (HPG) axis and testicular function abnormalities in the multisystem disorder of Lowe syndrome. Lowe syndrome, also known as oculocerebrorenal syndrome, is a rare disorder characterised by eye abnormalities, central nervous system involvement and proximal renal tubular acidosis. There are a handful of reports of pubertal delay, infertility and cryptorchidism in Lowe syndrome. Biochemistry aged 72 h: testosterone 6.4 nmol/L, LH <0.5 IU/L and FSH <0.5 IU/L. Gonadotropin-releasing hormone stimulation test identified significantly raised baseline LH = 45.4 IU/L (contrasts with earlier undetectable LH), with a 20% increase on stimulation, while baseline FSH = 4.3 IU/L with no increase on stimulation. Day 14 HCG stimulation test produced an acceptable 50% increase in testosterone. The constellation of further abnormalities suggested Lowe syndrome: hypotonia, bilateral cataracts (surgical extraction and intraocular lens implantation) and renal tubular acidosis (microscopic haematuria, hypercalciuria, proteinuria, generalised aminoaciduria, hypophosphataemia and metabolic acidosis). DNA sequencing identified de novo hemizygous frameshift mutation OCRL c.2409_2410delCT in exon 22. Interpretation of initial and repeat GnRH and HCG testing indicates the likelihood of testicular failure. Partial testicular descent occurred but left orchidopexy was required. Improving long-term gonadal function in Lowe syndrome assumes increased importance for current cohorts as advances in renal replacement therapy have greatly improved life expectancy. Noting HPG axis abnormalities in Lowe syndrome in infancy can identify cases requiring increased surveillance of pubertal progress for earlier detection and management. Clinical endocrine problems in Lowe syndrome has

  7. Folate deprivation induces cell cycle arrest at G0/G1 phase and apoptosis in hippocampal neuron cells through down-regulation of IGF-1 signaling pathway.

    PubMed

    Yang, Yang; Li, Xi; Sun, Qinwei; He, Bin; Jia, Yimin; Cai, Demin; Zhao, Ruqian

    2016-10-01

    Folate deficiency contributes to impaired adult hippocampal neurogenesis, yet the mechanisms remain unclear. Here we use HT-22 hippocampal neuron cells as model to investigate the effect of folate deprivation (FD) on cell proliferation and apoptosis, and to elucidate the underlying mechanism. FD caused cell cycle arrest at G0/G1 phase and increased the rate of apoptosis, which was associated with disrupted expression of folate transport and methyl transfer genes. FOLR1 and SLC46A1 were (P<0.01) down-regulated, while SLC19A1 was up-regulated (P<0.01) in FD group. FD cells exhibited significantly (P<0.05) higher protein content of BHMT, MAT2b and DNMT3a, as well as increased SAM/SAH concentrations and global DNA hypermethylation. The expression of the total and all the 3 classes of IGF-1 mRNA variants was significantly (P<0.01) down-regulated and IGF-1 concentration was decreased (P<0.05) in the culture media. IGF-1 signaling pathway was also compromised with diminished activation (P<0.05) of STAT3, AKT and mTOR. CpG hypermethylation was detected in the promoter regions of IGF-1 and FOLR1 genes, while higher SLC19A1 mRNA corresponded to hypomethylation of its promoter. IGF-1 supplementation in FD media significantly abolished FD-induced decrease in cell viability. However, IGF-1 had limited effect in rescuing the cell phenotype when added 24h after FD. Taken together, down-regulation of IGF-1 expression and signaling is involved in FD-induced cell cycle arrest and apoptosis in HT-22 hippocampal neuron cells, which is associated with an abnormal activation of methyl transfer pathway and hypermethylation of IGF-1 gene promoter. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Epigenetic Modification of Hippocampal Bdnf DNA in Adult Rats in an Animal Model of Post-Traumatic Stress Disorder

    PubMed Central

    Roth, Tania L.; Zoladz, Phillip R.; Sweatt, J. David; Diamond, David M.

    2011-01-01

    Epigenetic alterations of the brain-derived neurotrophic factor (Bdnf) gene have been linked with memory, stress, and neuropsychiatric disorders. Here we examined whether there was a link between an established rat model of post-traumatic stress disorder (PTSD) and BdnfDNA methylation. Adult male Sprague-Dawley rats were given psychosocial stress composed of two acute cat exposures in conjunction with 31 days of daily social instability. These manipulations have been shown previously to produce physiological and behavioral sequelae in rats that are comparable to symptoms observed in traumatized people with PTSD. We then assessed BdnfDNA methylation patterns (at exon IV) and gene expression. We have found here that the psychosocial stress regimen significantly increased BdnfDNA methylation in the dorsal hippocampus, with the most robust hypermethylation detected in the dorsal CA1 subregion. Conversely, the psychosocial stress regimen significantly decreased methylation in the ventral hippocampus (CA3). No changes in BdnfDNA methylation were detected in the medial prefrontal cortex or basolateral amygdala. In addition, there were decreased levels of BdnfmRNA in both the dorsal and ventral CA1. These results provide evidence that traumatic stress occurring in adulthood can induce CNS gene methylation, and specifically, support the hypothesis that epigenetic marking of the Bdnfgene may underlie hippocampal dysfunction in response to traumatic stress. Furthermore, this work provides support for the speculative notion that altered hippocampal BdnfDNA methylation is a cellular mechanism underlying the persistent cognitive deficits which are prominent features of the pathophysiology of PTSD. PMID:21306736

  9. Fetal aneuploidy detection by maternal plasma DNA sequencing: a technology assessment.

    PubMed

    Walsh, Judith M E; Goldberg, James D

    2013-06-01

    The American College of Obstetricians and Gynecologists currently recommends that all pregnant women be offered screening for chromosomal abnormalities, regardless of maternal age. Traditional screening tests have detection rates ranging from 85% to 90% and false-positive rates of 3% to 5%. A woman with an abnormal noninvasive test is offered a diagnostic test, but diagnostic tests are associated with a risk of pregnancy loss. Recently, analysis of cell-free fetal DNA (cffDNA) in maternal blood has been shown to have potential for the accurate detection of some of the common fetal autosomal aneuploidies. As part of a technology assessment for the California Technology Assessment Forum, we critically reviewed the evidence for the use of cffDNA as a prenatal screening test. We evaluated the evidence for its use as either a 'primary' or an 'advanced' screening test and for its use in screening for three different trisomies: 21, 18, and 13. We evaluated whether the use of cffDNA met established technology assessment criteria and established conclusions about evidence-based use of this new technology. © 2013 John Wiley & Sons, Ltd.

  10. Stress induced gene expression drives transient DNA methylation changes at adjacent repetitive elements.

    PubMed

    Secco, David; Wang, Chuang; Shou, Huixia; Schultz, Matthew D; Chiarenza, Serge; Nussaume, Laurent; Ecker, Joseph R; Whelan, James; Lister, Ryan

    2015-07-21

    Cytosine DNA methylation (mC) is a genome modification that can regulate the expression of coding and non-coding genetic elements. However, little is known about the involvement of mC in response to environmental cues. Using whole genome bisulfite sequencing to assess the spatio-temporal dynamics of mC in rice grown under phosphate starvation and recovery conditions, we identified widespread phosphate starvation-induced changes in mC, preferentially localized in transposable elements (TEs) close to highly induced genes. These changes in mC occurred after changes in nearby gene transcription, were mostly DCL3a-independent, and could partially be propagated through mitosis, however no evidence of meiotic transmission was observed. Similar analyses performed in Arabidopsis revealed a very limited effect of phosphate starvation on mC, suggesting a species-specific mechanism. Overall, this suggests that TEs in proximity to environmentally induced genes are silenced via hypermethylation, and establishes the temporal hierarchy of transcriptional and epigenomic changes in response to stress.

  11. DNA methylation differences in exposed workers and nearby residents of the Ma Ta Phut industrial estate, Rayong, Thailand

    PubMed Central

    Peluso, Marco; Bollati, Valentina; Munnia, Armelle; Srivatanakul, Petcharin; Jedpiyawongse, Adisorn; Sangrajrang, Suleeporn; Piro, Sara; Ceppi, Marcello; Bertazzi, Pier Alberto; Boffetta, Paolo; Baccarelli, Andrea A

    2012-01-01

    Background Adverse biological effects from airborne pollutants are a primary environmental concern in highly industrialized areas. Recent studies linked air pollution exposures with altered blood Deoxyribo-nucleic acid (DNA) methylation, but effects from industrial sources and underlying biological mechanisms are still largely unexplored. Methods The Ma Ta Phut industrial estate (MIE) in Rayong, Thailand hosts one of the largest steel, oil refinery and petrochemical complexes in south-eastern Asia. We measured a panel of blood DNA methylation markers previously associated with air pollution exposures, including repeated elements [long interspersed nuclear element-1 (LINE-1) and Alu] and genes [p53, hypermethylated-in-cancer-1 (HIC1), p16 and interleukin-6 (IL-6)], in 67 MIE workers, 65 Ma Ta Phut residents and 45 rural controls. To evaluate the role of DNA damage and oxidation, we correlated DNA methylation measures with bulky DNA and 3-(2-deoxy-β-D-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine (M1dG) adducts. Results In covariate-adjusted models, MIE workers, compared with rural residents, showed lower LINE-1 (74.8% vs 78.0%; P < 0.001), p53 (8.0% vs 15.7%; P < 0.001) and IL-6 methylation (39.2% vs 45.0%; P = 0.027) and higher HIC1 methylation (22.2% vs 15.3%, P < 0.001). For all four markers, Ma Ta Phut residents exhibited methylation levels intermediate between MIE workers and rural controls (LINE-1, 75.7%, P < 0.001; p53, 9.0%, P < 0.001; IL-6, 39.8%, P = 0.041; HIC1, 17.8%, P = 0.05; all P-values vs rural controls). Bulky DNA adducts showed negative correlation with p53 methylation (P = 0.01). M1dG showed negative correlations with LINE-1 (P = 0.003) and IL-6 methylation (P = 0.05). Conclusions Our findings indicate that industrial exposures may induce alterations of DNA methylation patterns detectable in blood leucocyte DNA. Correlation of DNA adducts with DNA hypomethylation suggests potential mediation by DNA damage. PMID:23064502

  12. Inhibiting Mitochondrial DNA Ligase IIIα Activates Caspase 1-Dependent Apoptosis in Cancer Cells.

    PubMed

    Sallmyr, Annahita; Matsumoto, Yoshihiro; Roginskaya, Vera; Van Houten, Bennett; Tomkinson, Alan E

    2016-09-15

    Elevated levels of DNA ligase IIIα (LigIIIα) have been identified as a biomarker of an alteration in DNA repair in cancer cells that confers hypersensitivity to a LigIIIα inhibitor, L67, in combination with a poly (ADP-ribose) polymerase inhibitor. Because LigIIIα functions in the nucleus and mitochondria, we examined the effect of L67 on these organelles. Here, we show that, although the DNA ligase inhibitor selectively targets mitochondria, cancer and nonmalignant cells respond differently to disruption of mitochondrial DNA metabolism. Inhibition of mitochondrial LigIIIα in cancer cells resulted in abnormal mitochondrial morphology, reduced levels of mitochondrial DNA, and increased levels of mitochondrially generated reactive oxygen species that caused nuclear DNA damage. In contrast, these effects did not occur in nonmalignant cells. Furthermore, inhibition of mitochondrial LigIIIα activated a caspase 1-dependent apoptotic pathway, which is known to be part of inflammatory responses induced by pathogenic microorganisms in cancer, but not nonmalignant cells. These results demonstrate that the disruption of mitochondrial DNA metabolism elicits different responses in nonmalignant and cancer cells and suggests that the abnormal response in cancer cells may be exploited in the development of novel therapeutic strategies that selectively target cancer cells. Cancer Res; 76(18); 5431-41. ©2016 AACR. ©2016 American Association for Cancer Research.

  13. Patients with xeroderma pigmentosum complementation groups C, E and V do not have abnormal sunburn reactions.

    PubMed

    Sethi, M; Lehmann, A R; Fawcett, H; Stefanini, M; Jaspers, N; Mullard, K; Turner, S; Robson, A; McGibbon, D; Sarkany, R; Fassihi, H

    2013-12-01

    Xeroderma pigmentosum (XP) is a rare autosomal recessive disorder of DNA repair. It is divided into eight complementation groups: XP-A to XP-G (classical XP) and XP variant (XP-V). Severe and prolonged sunburn reactions on minimal sun exposure have been considered a cardinal feature of classical XP. However, it has recently become clear that not all patients have abnormal sunburn reactions. To examine sunburn reactions in a cohort of patients with XP and correlate this to the complementation group. Sixty patients with XP attending the U.K. National XP Service from 2010 to 2012 were studied. Their history of burning after minimal sun exposure was assessed using a newly developed sunburn severity score. The age at which the first skin cancer was histologically diagnosed in each patient, and the presence of any neurological abnormality, was also recorded. Sunburn severity scores were abnormally high in patients with XP-A, XP-D, XP-F and XP-G compared with non-XP controls. There was no significant difference in sunburn score of patients with XP-C, XP-E and XP-V compared with controls (P > 0·05). Patients with XP-C, XP-E and XP-V were more likely to have skin cancer diagnosed at an earlier age than those with severe sunburn on minimal sun exposure. In addition, patients with XP with severe sunburn had an increased frequency of neurological abnormalities. Not all patients with XP have a history of severe and prolonged sunburn on minimal sun exposure. The normal sunburn response of patients with XP-C, XP-E and XP-V may relate to the preservation of transcription-coupled DNA repair in these groups. Those with a history of severe sunburn on minimal sun exposure developed their first skin cancer at an older age compared with patients with XP-C, XP-E and XP-V, but they had an increased frequency of neurological abnormalities. Physicians need to be aware that about half of all patients with XP will present without a history of abnormal sunburn. © 2013 British Association of

  14. [Verification of a decrease in the rigidity of the phage lambda DNA polymeric chain in low ionic strength aqueous solutions by testing the polymer-polymer interlink interactions].

    PubMed

    Arutiunian, A V; Ivanova, M A; Kurliand, D I; Kapshin, Iu S; Landa, S B; Poshekhonov, S T; Drobchenko, E A; Shevelev, I V

    2011-01-01

    Changes in the rigidity of the polymetric chain of phage lambda double-strand DNA have been studied by laser correlation spectroscopy. It was shown that, as the ionic strength increases, the effect of the screening of the hydrodynamic interaction of the links of the polymeric chain specific for polymeric coils arises in a DNA solution. It is assumed that the screening occurs when the threshold of the overlapping of DNA coils is achieved. The overlapping of coils is the result of a previously observed significant rise of DNA coil size from abnormally small DNA coils in low ionic strength buffers (about 10(-2) M Na+ or less) to maximum possible large coils in the 5SSC and 5SSC-like buffers. Further analysis of the far interlink interactions in linear lambda phage DNA coils in similar buffers at pH 7 and 4 confirms the earlier proposal about the role of H+ ions in the appearance of abnormally small DNA coils. The abnormal decrease in the DNA coil size in low ionic strength buffers is not a specific feature of lambda phage DNA only.

  15. Epigenetic markers in circulating cell-free DNA as prognostic markers for survival of castration-resistant prostate cancer patients.

    PubMed

    Hendriks, Rianne J; Dijkstra, Siebren; Smit, Frank P; Vandersmissen, Johan; Van de Voorde, Hendrik; Mulders, Peter F A; van Oort, Inge M; Van Criekinge, Wim; Schalken, Jack A

    2018-04-01

    Noninvasive biomarkers to guide personalized treatment for castration-resistant prostate cancer (CRPC) are needed. In this study, we analyzed hypermethylation patterns of two genes (GSTP1 and APC) in plasma cell-free DNA (cfDNA) of CRPC patients. The aim of this study was to analyze the cfDNA concentrations and levels of the epigenetic markers and to assess the value of these biomarkers for prognosis. In this prospective study, patients were included before starting new treatment after developing CRPC. The blood samples were collected prior to start of the treatment and at three time points thereafter. cfDNA was extracted from 1.5 mL of plasma and before performing a methylation-specific PCR, bisulfate modification was carried out. The median levels of cfDNA, GSTP1, and APC copies in the baseline samples of CRPC patients (n = 47) were higher than in controls (n = 30). In the survival analysis, the group with baseline marker levels below median had significant less PCa-related deaths (P-values <0.02) and did not reach the median survival point. The survival distributions for the groups were statistically significant for the cfDNA concentration, GSTP1 and APC copies, as well as PSA combined with GSTP1 + APC (P-values <0.03). Furthermore, there were strong positive correlations between PSA and marker response after starting treatment (P-values <0.04). In conclusion, this study showed the kinetics of methylated cfDNA (GSTP1 and APC) in plasma of CRPC patients after starting treatment. Furthermore, the value of the markers before treatment is prognostic for overall survival. These results are promising for developing a test to guide treatment-decision-making for CRPC patients. © 2018 The Authors. The Prostate Published by Wiley Periodicals, Inc.

  16. Occult Hepatitis B Virus Among the Patients With Abnormal Alanine Transaminase

    PubMed Central

    Makvandi, Manoochehr; Neisi, Niloofar; Khalafkhany, Davod; Makvandi, Kamyar; Hajiani, Eskandar; Shayesteh, Ali Akbar; Masjedi Zadeh, Abdolrahim; Sina, Amir Hosein; Hamidifard, Mojtaba; Rasti, Mojtaba; Aryan, Ehsan; Ahmadi, Kambiz; Yad Yad, Mohammad Jafar

    2014-01-01

    Background: The occult hepatitis B infection (OBI) is defined as the presence of hepatitis B virus (HBV) DNA in the sera or in the liver biopsy and the absence of hepatitis B surface antigen (HBsAg) by serological test. Objectives: The current study aimed to evaluate the occult HBV infection by polymerase chain reaction (PCR) and determine HBV genotyping among the patients with abnormal alanine transaminase (ALT) in Ahvaz city, Iran. Patients and Methods: The sera of 120 patients, 54 (45%) females and 66 (55%) males, with abnormal ALT 40-152 IU were collected. All the patients were negative for HBsAg for more than one year. The patients` sera were tested by PCR using primers specified for the S region of HBV. Then the positive PCR products were sequenced to determine HBV genotyping and phylogenic tree. Results: Of these 120 subjects, 12 (10%) patients including 6 (5%) males and 6 (5%) females were found positive for HBV DNA by PCR, which indicated the presence of occult HBV infection among these patients. The sequencing results revealed that genotype D was predominant with sub-genotyping D1 among OBI patients. Conclusions: Occult hepatitis B infection is remarkably prevalent in Ahvaz, Iran, and should be considered as a potential risk factor for the transmission of Hepatitis B Virus throughout the community by the carriers. PMID:25485052

  17. Ribosomal DNA status inferred from DNA cloud assays and mass spectrometry identification of agarose-squeezed proteins interacting with chromatin (ASPIC-MS).

    PubMed

    Krol, Kamil; Jendrysek, Justyna; Debski, Janusz; Skoneczny, Marek; Kurlandzka, Anna; Kaminska, Joanna; Dadlez, Michal; Skoneczna, Adrianna

    2017-04-11

    Ribosomal RNA-encoding genes (rDNA) are the most abundant genes in eukaryotic genomes. To meet the high demand for rRNA, rDNA genes are present in multiple tandem repeats clustered on a single or several chromosomes and are vastly transcribed. To facilitate intensive transcription and prevent rDNA destabilization, the rDNA-encoding portion of the chromosome is confined in the nucleolus. However, the rDNA region is susceptible to recombination and DNA damage, accumulating mutations, rearrangements and atypical DNA structures. Various sophisticated techniques have been applied to detect these abnormalities. Here, we present a simple method for the evaluation of the activity and integrity of an rDNA region called a "DNA cloud assay". We verified the efficacy of this method using yeast mutants lacking genes important for nucleolus function and maintenance (RAD52, SGS1, RRM3, PIF1, FOB1 and RPA12). The DNA cloud assay permits the evaluation of nucleolus status and is compatible with downstream analyses, such as the chromosome comet assay to identify DNA structures present in the cloud and mass spectrometry of agarose squeezed proteins (ASPIC-MS) to detect nucleolar DNA-bound proteins, including Las17, the homolog of human Wiskott-Aldrich Syndrome Protein (WASP).

  18. Ribosomal DNA status inferred from DNA cloud assays and mass spectrometry identification of agarose-squeezed proteins interacting with chromatin (ASPIC-MS)

    PubMed Central

    Krol, Kamil; Jendrysek, Justyna; Debski, Janusz; Skoneczny, Marek; Kurlandzka, Anna; Kaminska, Joanna; Dadlez, Michal; Skoneczna, Adrianna

    2017-01-01

    Ribosomal RNA-encoding genes (rDNA) are the most abundant genes in eukaryotic genomes. To meet the high demand for rRNA, rDNA genes are present in multiple tandem repeats clustered on a single or several chromosomes and are vastly transcribed. To facilitate intensive transcription and prevent rDNA destabilization, the rDNA-encoding portion of the chromosome is confined in the nucleolus. However, the rDNA region is susceptible to recombination and DNA damage, accumulating mutations, rearrangements and atypical DNA structures. Various sophisticated techniques have been applied to detect these abnormalities. Here, we present a simple method for the evaluation of the activity and integrity of an rDNA region called a “DNA cloud assay”. We verified the efficacy of this method using yeast mutants lacking genes important for nucleolus function and maintenance (RAD52, SGS1, RRM3, PIF1, FOB1 and RPA12). The DNA cloud assay permits the evaluation of nucleolus status and is compatible with downstream analyses, such as the chromosome comet assay to identify DNA structures present in the cloud and mass spectrometry of agarose squeezed proteins (ASPIC-MS) to detect nucleolar DNA-bound proteins, including Las17, the homolog of human Wiskott-Aldrich Syndrome Protein (WASP). PMID:28212567

  19. Optical biosensing strategies for DNA methylation analysis.

    PubMed

    Nazmul Islam, Md; Yadav, Sharda; Hakimul Haque, Md; Munaz, Ahmed; Islam, Farhadul; Al Hossain, Md Shahriar; Gopalan, Vinod; Lam, Alfred K; Nguyen, Nam-Trung; Shiddiky, Muhammad J A

    2017-06-15

    DNA methylation is an epigenetic modification of DNA, where a methyl group is added at the fifth carbon of the cytosine base to form 5 methyl cytosine (5mC) without altering the DNA sequences. It plays important roles in regulating many cellular processes by modulating key genes expression. Alteration in DNA methylation patterns becomes particularly important in the aetiology of different diseases including cancers. Abnormal methylation pattern could contribute to the pathogenesis of cancer either by silencing key tumor suppressor genes or by activating oncogenes. Thus, DNA methylation biosensing can help in the better understanding of cancer prognosis and diagnosis and aid the development of therapies. Over the last few decades, a plethora of optical detection techniques have been developed for analyzing DNA methylation using fluorescence, Raman spectroscopy, surface plasmon resonance (SPR), electrochemiluminescence and colorimetric readouts. This paper aims to comprehensively review the optical strategies for DNA methylation detection. We also present an overview of the remaining challenges of optical strategies that still need to be focused along with the lesson learnt while working with these techniques. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Sperm DNA fragmentation: mechanisms of origin, impact on reproductive outcome, and analysis.

    PubMed

    Sakkas, Denny; Alvarez, Juan G

    2010-03-01

    To review the mechanisms responsible for DNA fragmentation in human sperm, including those occurring during spermatogenesis and transport through the reproductive tract. The mechanisms examined include: apoptosis in the seminiferous tubule epithelium, defects in chromatin remodeling during the process of spermiogenesis, oxygen radical-induced DNA damage during sperm migration from the seminiferous tubules to the epididymis, the activation of sperm caspases and endonucleases, damage induced by chemotherapy and radiotherapy, and the effect of environmental toxicants. The different tests currently used for sperm DNA fragmentation analysis and the factors that determine the predictive value of sperm DNA fragmentation testing and their implications in the diagnosis and treatment of infertility are also discussed. Finally, we also scrutinize how the presence in the embryonic genome of DNA strand breaks or modifications of DNA nucleotides inherited from the paternal genome could impact the embryo and offspring. In particular we discuss how abnormal sperm could be dealt with by the oocyte and how sperm DNA abnormalities, which have not been satisfactorily repaired by the oocyte after fertilization, may interfere with normal embryo and fetal development. Sperm DNA can be modified through various mechanisms. The integrity of the paternal genome is therefore of paramount importance in the initiation and maintenance of a viable pregnancy both in a natural conception and in assisted reproduction. The need to diagnose sperm at a nuclear level is an area that needs further understanding so that we can improve treatment of the infertile couple. Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  1. The Mechanism and Function of Epigenetics in Uterine Leiomyoma Development

    PubMed Central

    Yang, Qiwei; Mas, Aymara; Diamond, Michael P.; Al-Hendy, Ayman

    2015-01-01

    Uterine leiomyomas, also known as uterine fibroids, are the most common pelvic tumors, occurring in nearly 70% of all reproductive-aged women and are the leading indication for hysterectomy worldwide. The development of uterine leiomyomas involve a complex and heterogeneous constellation of hormones, growth factors, stem cells, genetic, and epigenetic abnormalities. An increasing body of evidence emphasizes the important contribution of epigenetics in the pathogenesis of leiomyomas. Genome-wide methylation analysis demonstrates that a subset of estrogen receptor (ER) response genes exhibit abnormal hypermethylation levels that are inversely correlated with their RNA expression. Several tumor suppressor genes, including Kruppel-like factor 11 (KLF11), deleted in lung and esophageal cancer 1 (DLEC1), keratin 19 (KRT19), and death-associated protein kinase 1 (DAPK1) also display higher hypermethylation levels in leiomyomas when compared to adjacent normal tissues. The important role of active DNA demethylation was recently identified with regard to the ten-eleven translocation protein 1 and ten-eleven translocation protein 3-mediated elevated levels of 5-hydroxymethylcytosine in leiomyoma. In addition, both histone deacetylase and histone methyltransferase are reported to be involved in the biology of leiomyomas. A number of deregulated microRNAs have been identified in leiomyomas, leading to an altered expression of their targets. More recently, the existence of side population (SP) cells with characteristics of tumor-initiating cells have been characterized in leiomyomas. These SP cells exhibit a tumorigenic capacity in immunodeficient mice when exposed to 17β-estradiol and progesterone, giving rise to fibroid-like tissue in vivo. These new findings will likely enhance our understanding of the crucial role epigenetics plays in the pathogenesis of uterine leiomyomas as well as point the way to novel therapeutic options. PMID:25922306

  2. Methylated Glutathione S-transferase 1 (mGSTP1) is a potential plasma free DNA epigenetic marker of prognosis and response to chemotherapy in castrate-resistant prostate cancer.

    PubMed

    Mahon, K L; Qu, W; Devaney, J; Paul, C; Castillo, L; Wykes, R J; Chatfield, M D; Boyer, M J; Stockler, M R; Marx, G; Gurney, H; Mallesara, G; Molloy, P L; Horvath, L G; Clark, S J

    2014-10-28

    Glutathione S-transferase 1 (GSTP1) inactivation is associated with CpG island promoter hypermethylation in the majority of prostate cancers (PCs). This study assessed whether the level of circulating methylated GSTP1 (mGSTP1) in plasma DNA is associated with chemotherapy response and overall survival (OS). Plasma samples were collected prospectively from a Phase I exploratory cohort of 75 men with castrate-resistant PC (CRPC) and a Phase II independent validation cohort (n=51). mGSTP1 levels in free DNA were measured using a sensitive methylation-specific PCR assay. The Phase I cohort identified that detectable baseline mGSTP1 DNA was associated with poorer OS (HR, 4.2 95% CI 2.1-8.2; P<0.0001). A decrease in mGSTP1 DNA levels after cycle 1 was associated with a PSA response (P=0.008). In the Phase II cohort, baseline mGSTP1 DNA was a stronger predictor of OS than PSA change after 3 months (P=0.02). Undetectable plasma mGSTP1 after one cycle of chemotherapy was associated with PSA response (P=0.007). We identified plasma mGSTP1 DNA as a potential prognostic marker in men with CRPC as well as a potential surrogate therapeutic efficacy marker for chemotherapy and corroborated these findings in an independent Phase II cohort. Prospective Phase III assessment of mGSTP1 levels in plasma DNA is now warranted.

  3. Genetic predisposition to high anxiety- and depression-like behavior coincides with diminished DNA methylation in the adult rat amygdala

    PubMed Central

    McCoy, Chelsea R.; Jackson, Nateka L.; Day, Jeremy; Clinton, Sarah M.

    2016-01-01

    Understanding biological mechanisms that shape vulnerability to emotional dysfunction is critical for elucidating the neurobiology of psychiatric illnesses like anxiety and depression. To elucidate molecular and epigenetic alterations in the brain that contribute to individual differences in emotionality, our laboratory utilized a rodent model of temperamental differences. Rats bred for low response to novelty (Low Responders, LRs) are inhibited in novel situations and display high anxiety, helplessness, and diminished sociability compared to High Novelty Responder (HR) rats. Our current transcriptome profiling experiment identified widespread gene expression differences in the amygdala of adult HR/LR rats; we hypothesize that HR/LR gene expression and downstream behavioral differences stem from distinct epigenetic (specifically DNA methylation) patterning in the HR/LR brain. Although we found similar levels of DNA methyltransferase proteins in the adult HR/LR amygdala, next-generation sequencing analysis of the methylome revealed 793 differentially methylated genomic sites between the groups. Most of the differentially methylated sites were hypermethylated in HR versus LR, so we next tested the hypothesis that enhancing DNA methylation in LRs would improve their anxiety/depression-like phenotype. We found that increasing DNA methylation in LRs (via increased dietary methyl donor content) improved their anxiety-like behavior and decreased their typically high levels of Forced Swim Test (FST) immobility; however, dietary methyl donor depletion exacerbated LRs’ high FST immobility. These data are generally consistent with findings in depressed patients showing that treatment with DNA methylation-promoting agents improves depressive symptoms, and highlight epigenetic mechanisms that may contribute to individual differences in risk for emotional dysfunction. PMID:27965039

  4. Use of DNA from human stools to detect aberrant CpG island methylation of genes implicated in colorectal cancer.

    PubMed

    Belshaw, Nigel J; Elliott, Giles O; Williams, Elizabeth A; Bradburn, David M; Mills, Sarah J; Mathers, John C; Johnson, Ian T

    2004-09-01

    Hypermethylation of cytosine residues in the CpG islands of tumor suppressor genes is a key mechanism of colorectal carcinogenesis. Detection and quantification of CpG island methylation in human DNA isolated from stools might provide a novel strategy for the detection and investigation of colorectal neoplasia. To explore the feasibility of this approach, colorectal biopsies and fecal samples were obtained from 32 patients attending for colonoscopy or surgery, who were found to have adenomatous polyps, colorectal cancer, or no evidence of neoplasia. A further 18 fecal samples were obtained from healthy volunteers, with no bowel symptoms. Isolated DNA was modified with sodium bisulfite and analyzed by methylation-specific PCR and combined bisulfite restriction analysis for CpG island methylation of ESR1, MGMT, HPP1, p16(INK4a), APC, and MLH1. CpG island methylation was readily detectable in both mucosal and fecal DNA with methylation-specific PCR. Using combined bisulfite restriction analysis, it was established that, in volunteers from whom biopsies were available, the levels of methylation at two CpG sites within ESR1 assayed using fecal DNA were significantly correlated with methylation in DNA from colorectal mucosa. Thus, noninvasive techniques can be used to obtain quantitative information about the level of CpG island methylation in human colorectal mucosa. The methods described here could be applied to a much expanded range of genes and may be valuable both for screening purposes and to provide greater insight into the functional consequences of epigenetic changes in the colorectal mucosa of free-living individuals.

  5. Meiotic abnormalities

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NONE

    1993-12-31

    Chapter 19, describes meiotic abnormalities. These include nondisjunction of autosomes and sex chromosomes, genetic and environmental causes of nondisjunction, misdivision of the centromere, chromosomally abnormal human sperm, male infertility, parental age, and origin of diploid gametes. 57 refs., 2 figs., 1 tab.

  6. Commentary: 'Unhealthy diet,' nutrient status, and ADHD symptoms: a confounding role for environmental nitrous oxide exposure - reflections on Rijlaarsdam et al. (2016).

    PubMed

    Fluegge, Keith

    2017-01-01

    Rijlaarsdam et al. (2016) recently published their findings utilizing a longitudinal design showing that prenatal 'unhealthy diet' was positively associated with IGF2 DNA methylation at birth across both youth cohorts. However, only in the EOP youth was prenatal 'unhealthy diet' positively associated with ADHD symptoms presumably through IGF2 DNA hypermethylation. Rijlaarsdam et al.'s () choice to assess high fat and sugar diet with the Food Frequency Questionnaire (FFQ) may offer some indication as to prenatal nutrient status, as the foods identified by the FFQ in their study are relatively low in free choline. It has been shown that gestational choline deficiency in rats leads to hypermethylation of IGF2. Consistent with the literature describing an association between air pollution and cognitive neurodevelopmental impairment, the author of this commentary has previously proposed through empirical investigation that chronic environmental exposure to the trace levels of the pervasive air pollutant, nitrous oxide (N 2 O), may facilitate core features of neurodevelopmental disorders, like ADHD. Impaired acetylcholine synthesis in rats exposed to N 2 O has been shown, with a 53% reduction in [1-2H2,2-2H2] choline. Low-dose N 2 O exposure is also thought to stimulate central release of opioid peptides, like dynorphin, which play a role in significantly increasing food intake behavior and/or modulating sucrose intake. Taken altogether, these studies present a strong confounder to the interpretation made by Rijlaarsdam et al. () that prenatal 'unhealthy diet' may play a role in the onset of ADHD symptoms in youth with EOP conduct problems through induction of IGF2 DNA hypermethylation. While the 'unhealthy diet' may represent possible maternal nutrient deficiencies during gestation, it is also possible that exposure to air pollutants, particularly N 2 O, may not only directly reduce fetal cholinergic status thereby enhancing IGF2 DNA hypermethylation but may also

  7. BOTH HYPOMETHYLATION AND HYPERMETHYLATION OF DNA ASSOCIATED WITH ARSENITE EXPOSURE IN CULTURES OF HUMAN CELLS IDENTIFIED BY METHYLATION-SENSITIVE ARBITRARILY-PRIMED PCR

    EPA Science Inventory

    Differentially Methylated DNA Sequences Associated with Exposure to Arsenite in Cultures of Human Cells Identified by Methylation-Sensitive-Primed PCR

    Arsenic, a known human carcinogen, is converted to methylated derivatives by a methyltransferase (Mtase) and its biotra...

  8. Genome-wide analysis of salinity-stress induced DNA methylation alterations in cotton (Gossypium hirsutum L.) using the Me-DIP sequencing technology.

    PubMed

    Lu, X K; Shu, N; Wang, J J; Chen, X G; Wang, D L; Wang, S; Fan, W L; Guo, X N; Guo, L X; Ye, W W

    2017-06-29

    Cytosine DNA methylation is a significant form of DNA modification closely associated with gene expression in eukaryotes, fungi, animals, and plants. Although the reference genomes of cotton (Gossypium hirsutum L.) have been publically available, the salinity-stress-induced DNA methylome alterations in cotton are not well understood. Here, we constructed a map of genome-wide DNA methylation characteristics of cotton leaves under salt stress using the methylated DNA immunoprecipitation sequencing method. The results showed that the methylation reads on chromosome 9 were most comparable with those on the other chromosomes, but the greatest changes occurred on chromosome 8 under salt stress. The DNA methylation pattern analysis indicated that a relatively higher methylation density was found in the upstream2k and downstream2k elements of the CDS region and CG-islands. Almost 94% of the reads belonged to LTR-gspsy and LTR-copia, and the number of methylation reads in LTR-gypsy was four times greater than that in LTR-copia in both control and stressed samples. The analysis of differentially methylated regions (DMRs) showed that the gene elements upstream2k, intron, and downstream2k were hypomethylated, but the CDS regions were hypermethylated. The GO (Gene Ontology) analysis suggested that the methylated genes were most enriched in cellular processes, metabolic processes, cell parts and catalytic activities, which might be closely correlated with response to NaCl stress. In this study, we completed a genomic DNA methylation profile and conducted a DMR analysis under salt stress, which provided valuable information for the better understanding of epigenetics in response to salt stress in cotton.

  9. Dietary black raspberries modulate DNA methylation in dextran sodium sulfate (DSS)-induced ulcerative colitis

    PubMed Central

    Wang, Li-Shu

    2013-01-01

    Ulcerative colitis (UC) is characterized by chronic inflammation of the colon. During inflammation, NF-κB is increased in colonic epithelial cells and in immune cells, leading to increases in proinflammatory cytokines. These events then increase DNA methyltransferases (DNMTs), which silence a subset of tumor suppressor genes by promoter methylation. Negative regulators of the Wnt pathway are frequently methylated in UC, leading to dysregulation of the pathway and, potentially, to colorectal cancer. We determined if black raspberries (BRBs) influence promoter methylation of suppressors in the Wnt pathway in dextran sodium sulfate (DSS)-induced UC. C57BL/6J mice received 1% DSS and were fed either control or 5% BRB diets. Mice were euthanized on days 7, 14 and 28, and their colons, spleen and bone marrow were collected. Berries reduced ulceration at day 28. This was accompanied by decreased staining of macrophages and neutrophils and decreased NF-κB p65 nuclear localization in the colon at all time points. At day 7, BRBs demethylated the promoter of dkk3, leading to its increased messenger RNA (mRNA) expression in colon, spleen and bone marrow. β-Catenin nuclear localization, c-Myc staining as well as protein expression of DNMT3B, histone deacetylases 1 and 2 (HDAC1 and HDAC2) and methyl-binding domain 2 (MBD2) were all decreased in colon; mRNA expression of these four proteins was decreased in bone marrow cells by BRBs. These results suggest that BRBs suppress colonic ulceration by correcting promoter hypermethylation of suppressor genes in the colon, as well as in the spleen and bone marrow that systematically regulate inflammation. Summary: Our results suggest that dietary BRBs suppress colonic ulceration by correcting promoter hypermethylation of suppressor genes in the colon, as well as in the spleen and bone marrow that systematically regulate inflammation in DSS-induced UC. PMID:24067901

  10. Clinical experience of laboratory follow-up with noninvasive prenatal testing using cell-free DNA and positive microdeletion results in 349 cases.

    PubMed

    Schwartz, S; Kohan, M; Pasion, R; Papenhausen, P R; Platt, L D

    2018-02-01

    Screening via noninvasive prenatal testing (NIPT) involving the analysis of cell-free DNA (cfDNA) from plasma has become readily available to screen for chromosomal and DNA aberrations through maternal blood. This report reviews a laboratory's experience with follow-up of positive NIPT screens for microdeletions. Patients that were screened positive by NIPT for a microdeletion involving 1p, 4p, 5p, 15q, or 22q who underwent diagnostic studies by either chorionic villus sampling or amniocentesis were evaluated. The overall positive predictive value for 349 patients was 9.2%. When a microdeletion was confirmed, 39.3% of the cases had additional abnormal microarray findings. Unrelated abnormal microarray findings were detected in 11.8% of the patients in whom the screen positive microdeletion was not confirmed. Stretches of homozygosity in the microdeletion were frequently associated with a false positive cfDNA microdeletion result. Overall, this report reveals that while cfDNA analysis will screen for microdeletions, the positive predictive value is low; in our series it is 9.2%. Therefore, the patient should be counseled accordingly. Confirmatory diagnostic microarray studies are imperative because of the high percentage of false positives and the frequent additional abnormalities not delineated by cfDNA analysis. © 2018 John Wiley & Sons, Ltd.

  11. Central nervous system abnormalities in Fanconi anaemia: patterns and frequency on magnetic resonance imaging

    PubMed Central

    Alston, Robert; Wright, Neville B; Chandler, Kate; Bonney, Denise; Wynn, Robert F; Will, Andrew M; Punekar, Maqsood; Loughran, Sean; Kilday, John-Paul; Schindler, Detlev; Patel, Leena; Meyer, Stefan

    2015-01-01

    Objective: Fanconi anaemia (FA) is an inherited disease associated with congenital and developmental abnormalities resulting from the disruption of a multigenic DNA damage response pathway. This study aimed to define the MRI appearances of the brain in patients with FA in correlation with their genetic and clinical features. Methods: A review of the brain MRI in 20 patients with FA was performed. Pituitary size and frequencies of the radiological findings of individuals with FA and age-matched controls were determined. Results: Abnormalities were identified in 18 (90%) patients with FA, the commonest being a small pituitary (68%, p < 0.01 females and p < 0.001 males). In five cases (25%, p = 0.02), the pituitary morphology was also abnormal. Posterior fossa abnormalities were seen in six cases (30%, p = 0.01) including Chiari I malformation (n = 3), Dandy–Walker variant (n = 2) and cerebellar atrophy (n = 2). Six patients (30%, p = 0.01) had morphological structural variation of the corpus callosum (CC). Conclusion: The incidence of central nervous system (CNS) abnormalities in FA is higher than previously reported, with a midline predominance that points to impact in the early stages of CNS development. MRI brain imaging is important for endocrine assessment and pre-transplant evaluation and can make an important contribution to clinical decision-making. Advances in knowledge: The incidence of brain structural abnormalities in FA is higher than previously reported, with abnormalities of the posterior fossa, CC and pituitary being common. There is an association with gender and reduction in pituitary size which does not strongly correlate with biochemically evident endocrine abnormality. PMID:26369989

  12. Molecular analysis and conventional cytology: association between HPV and bacterial vaginosis in the cervical abnormalities of a Brazilian population.

    PubMed

    Peres, A L; Camarotti, J R S L; Cartaxo, M; Alencar, N; Stocco, R C; Beçak, W; Pontes-Filho, N T; Araújo, R F F; Lima-Filho, J L; Martins, D B G

    2015-08-14

    We investigated the association between bacterial vaginosis (BV) and human papillomavirus (HPV) infection in Papanicolaou smears in a Brazilian population. Cross-sectional analysis was performed on 673 samples collected from women attending public health centers in Olinda (PE, Brazil) by conventional cytology methodology and molecular analysis, PCR tests (GP5+/6+ and MY09/11). Cytological abnormalities, BV, and HPV-DNA were detected in 23 (3.4%) samples, 189 samples (28.1%), and 210 samples (31.2%), respectively. GP5+/6+ primers resulted in higher detection performance than MY09/11 primers, with 81% concordance between both primers (P < 0.0001). The occurrence of HPV-DNA and BV had ORs of 8.59 (P < 0.0001) and 2.91 (P = 0.0089) for abnormal cytology, respectively, whereas the concomitant presence of both infections showed an OR equal to 3.82 (P = 0.0054). Therefore, we observed an association between abnormal cervical cytology and HPV infection, BV, or both HPV infection and BV. These results highlight the necessity of monitoring patients presenting not only HPV, but also BV, as risk factors for cervical lesion development.

  13. Abnormal branching and regression of the notochord and its relationship to foregut abnormalities.

    PubMed

    Vleesch Dubois, V N; Quan Qi, B; Beasley, S W; Williams, A

    2002-04-01

    An abnormally positioned notochord has been reported in embryos that develop foregut abnormalities, vertebral defects and other abnormalities of the VATER association. This study examines the patterns of regression of the abnormal notochord in the rat model of the VATER association and investigates the relationship between developmental abnormalities of the notochord and those of the vertebra and foregut. Timed-pregnant Sprague-Dawley rats were given daily intraperitoneal injections of 1.75 mg/kg adriamycin on gestational days 6 - 9 inclusive. Rats were sacrificed between days 14 and 20 and their embryos harvested, histologically sectioned and stained and examined serially. The location and appearance of the degenerating notochord and its relationship to regional structural defects were analysed. All 26 embryos exposed to adriamycin developed foregut abnormalities and had an abnormal notochord. The notochord disappeared by a process of apoptotic degeneration that lagged behind that of the normal embryo: the notochord persisted in the abnormal embryo beyond day 17, whereas in the normal rat it had already disappeared. Similarly, formation of the nucleus pulposus was delayed. Vertebral abnormalities occurred when the notochord was ventrally-positioned. The notochord disappears during day 16 in the normal embryo whereas abnormal branches of the notochord persist until day 19 in the adriamycin-treated embryo. Degeneration of the notochord is dominated by apoptosis. An excessively ventrally-placed notochord is closely associated with abnormalities of the vertebral column, especially hemivertebrae.

  14. Prenatal fine particulate exposure associated with reduced childhood lung function and nasal epithelia GSTP1 hypermethylation: Sex-specific effects.

    PubMed

    Lee, Alison G; Le Grand, Blake; Hsu, Hsiao-Hsien Leon; Chiu, Yueh-Hsiu Mathilda; Brennan, Kasey J; Bose, Sonali; Rosa, Maria José; Brunst, Kelly J; Kloog, Itai; Wilson, Ander; Schwartz, Joel; Morgan, Wayne; Coull, Brent A; Wright, Robert O; Baccarelli, Andrea A; Wright, Rosalind J

    2018-04-27

    In utero exposure to particulate matter with an aerodynamic diameter of less than 2.5 μm (PM 2.5 ) has been linked to child lung function. Overlapping evidence suggests that child sex and exposure timing may modify effects and associations may be mediated through glutathione S-transferase P1 (GSTP1) methylation. We prospectively examined associations among prenatal PM 2.5 exposure and child lung function and GSTP1 methylation in an urban pregnancy cohort study. We employed a validated satellite-based spatiotemporally resolved prediction model to estimate daily prenatal PM 2.5 exposure over gestation. We used Baysian distributed lag interaction models (BDLIMs) to identify sensitive windows for prenatal PM 2.5 exposure on child lung function and nasal epithelia GSTP1 methylation at age 7 years, and to examine effect modification by child sex. BDLIMs identified a sensitive window for prenatal PM 2.5 exposure at 35-40 weeks gestation [cumulative effect estimate (CEE) = - 0.10, 95%CI = - 0.19 to - 0.01, per μg/m 3 increase in PM 2.5 ] and at 36-40 weeks (CEE = - 0.12, 95%CI = - 0.20 to - 0.01) on FEV 1 and FVC, respectively, in boys. BDLIMs also identified a sensitive window of exposure at 37-40 weeks gestation between higher prenatal PM 2.5 exposure and increased GSTP1 percent methylation. The association between higher GSTP1 percent methylation and decreased FEV1 was borderline significant in the sample as a whole (β = - 0.37, SE = 0.20, p = 0.06) and in boys in stratified analyses (β = - 0.56, SE = 0.29, p = 0.05). Prenatal PM 2.5 exposure in late pregnancy was associated with impaired early childhood lung function and hypermethylation of GSTPI in DNA isolated from nasal epithelial cells. There was a trend towards higher GSTP1 percent methylation being associated with reduced FEV1. All findings were most evident among boys.

  15. Prognostic role of APC and RASSF1A promoter methylation status in cell free circulating DNA of operable gastric cancer patients.

    PubMed

    Balgkouranidou, I; Matthaios, D; Karayiannakis, A; Bolanaki, H; Michailidis, P; Xenidis, N; Amarantidis, K; Chelis, L; Trypsianis, G; Chatzaki, E; Lianidou, E S; Kakolyris, S

    2015-08-01

    Gastric carcinogenesis is a multistep process including not only genetic mutations but also epigenetic alterations. The best known and more frequent epigenetic alteration is DNA methylation affecting tumor suppressor genes that may be involved in various carcinogenetic pathways. The aim of the present study was to investigate the methylation status of APC promoter 1A and RASSF1A promoter in cell free DNA of operable gastric cancer patients. Using methylation specific PCR, we examined the methylation status of APC promoter 1A and RASSF1A promoter in 73 blood samples obtained from patients with gastric cancer. APC and RASSF1A promoters were found to be methylated in 61 (83.6%) and 50 (68.5%) of the 73 gastric cancer samples examined, but in none of the healthy control samples (p < 0.001). A significant association between methylated RASSF1A promoter status and lymph node positivity was observed (p = 0.005). Additionally, a significant correlation between a methylated APC promoter and elevated CEA (p = 0.033) as well as CA-19.9 (p = 0.032) levels, was noticed. The Kaplan-Meier estimates of survival, significantly favored patients with a non-methylated APC promoter status (p = 0.008). No other significant correlations between APC and RASSF1A methylation status and different tumor variables examined was observed. Serum RASSF1A and APC promoter hypermethylation is a frequent epigenetic event in patients with early operable gastric cancer. The observed correlations between APC promoter methylation status and survival as well as between a hypermethylated RASSF1A promoter and nodal positivity may be indicative of a prognostic role for those genes in early operable gastric cancer. Additional studies, in a larger cohort of patients are required to further explore whether these findings could serve as potential molecular biomarkers of survival and/or response to specific treatments. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Recent thymic emigrants and mature naïve T cells exhibit differential DNA methylation at key cytokine loci

    PubMed Central

    Berkley, Amy M.; Hendricks, Deborah W.; Simmons, Kalynn B.; Fink, Pamela J.

    2013-01-01

    Recent thymic emigrants (RTEs) are the youngest T cells in the lymphoid periphery, and exhibit phenotypic and functional characteristics distinct from those of their more mature counterparts in the naïve peripheral T cell pool. We show here that the Il2 and Il4 promoter regions of naïve CD4+ RTEs are characterized by site-specific hypermethylation compared to those of both mature naïve (MN) T cells and the thymocyte precursors of RTEs. Thus, RTEs do not merely occupy a midpoint between the thymus and the mature T cell pool, but represent a distinct transitional T cell population. Furthermore, RTEs and MN T cells exhibit distinct CpG DNA methylation patterns both before and after activation. Compared to MN T cells, RTEs express higher levels of several enzymes that modify DNA methylation, and inhibiting methylation during culture allows RTEs to reach MN T cell levels of cytokine production. Collectively, these data suggest that the functional differences that distinguish RTEs from MN T cells are influenced by epigenetic mechanisms and provide clues to a mechanistic basis for post-thymic maturation. PMID:23686491

  17. Genome-Wide DNA Methylation Analysis and Epigenetic Variations Associated with Congenital Aortic Valve Stenosis (AVS).

    PubMed

    Radhakrishna, Uppala; Albayrak, Samet; Alpay-Savasan, Zeynep; Zeb, Amna; Turkoglu, Onur; Sobolewski, Paul; Bahado-Singh, Ray O

    2016-01-01

    Congenital heart defect (CHD) is the most common cause of death from congenital anomaly. Among several candidate epigenetic mechanisms, DNA methylation may play an important role in the etiology of CHDs. We conducted a genome-wide DNA methylation analysis using an Illumina Infinium 450k human methylation assay in a cohort of 24 newborns who had aortic valve stenosis (AVS), with gestational-age matched controls. The study identified significantly-altered CpG methylation at 59 sites in 52 genes in AVS subjects as compared to controls (either hypermethylated or demethylated). Gene Ontology analysis identified biological processes and functions for these genes including positive regulation of receptor-mediated endocytosis. Consistent with prior clinical data, the molecular function categories as determined using DAVID identified low-density lipoprotein receptor binding, lipoprotein receptor binding and identical protein binding to be over-represented in the AVS group. A significant epigenetic change in the APOA5 and PCSK9 genes known to be involved in AVS was also observed. A large number CpG methylation sites individually demonstrated good to excellent diagnostic accuracy for the prediction of AVS status, thus raising possibility of molecular screening markers for this disorder. Using epigenetic analysis we were able to identify genes significantly involved in the pathogenesis of AVS.

  18. Genome-Wide DNA Methylation Analysis and Epigenetic Variations Associated with Congenital Aortic Valve Stenosis (AVS)

    PubMed Central

    Radhakrishna, Uppala; Albayrak, Samet; Alpay-Savasan, Zeynep; Zeb, Amna; Turkoglu, Onur; Sobolewski, Paul; Bahado-Singh, Ray O.

    2016-01-01

    Congenital heart defect (CHD) is the most common cause of death from congenital anomaly. Among several candidate epigenetic mechanisms, DNA methylation may play an important role in the etiology of CHDs. We conducted a genome-wide DNA methylation analysis using an Illumina Infinium 450k human methylation assay in a cohort of 24 newborns who had aortic valve stenosis (AVS), with gestational-age matched controls. The study identified significantly-altered CpG methylation at 59 sites in 52 genes in AVS subjects as compared to controls (either hypermethylated or demethylated). Gene Ontology analysis identified biological processes and functions for these genes including positive regulation of receptor-mediated endocytosis. Consistent with prior clinical data, the molecular function categories as determined using DAVID identified low-density lipoprotein receptor binding, lipoprotein receptor binding and identical protein binding to be over-represented in the AVS group. A significant epigenetic change in the APOA5 and PCSK9 genes known to be involved in AVS was also observed. A large number CpG methylation sites individually demonstrated good to excellent diagnostic accuracy for the prediction of AVS status, thus raising possibility of molecular screening markers for this disorder. Using epigenetic analysis we were able to identify genes significantly involved in the pathogenesis of AVS. PMID:27152866

  19. DNA Damage and Repair: Relevance to Mechanisms of Neurodegeneration

    PubMed Central

    Martin, Lee J.

    2008-01-01

    DNA damage is a form of cell stress and injury that has been implicated in the pathogenesis of many neurologic disorders, including amyotrophic lateral sclerosis, Alzheimer disease, Down syndrome, Parkinson disease, cerebral ischemia, and head trauma. However, most data reveal only associations, and the role for DNA damage in direct mechanisms of neurodegeneration is vague with respect to being a definitive upstream cause of neuron cell death, rather than a consequence of the degeneration. Although neurons seem inclined to develop DNA damage during oxidative stress, most of the existing work on DNA damage and repair mechanisms has been done in the context of cancer biology using cycling non-neuronal cells but not nondividing (i.e. postmitotic) neurons. Nevertheless, the identification of mutations in genes that encode proteins that function in DNA repair and DNA damage response in human hereditary DNA repair deficiency syndromes and ataxic disorders is establishing a mechanistic precedent that clearly links DNA damage and DNA repair abnormalities with progressive neurodegeneration. This review summarizes DNA damage and repair mechanisms and their potential relevance to the evolution of degeneration in postmitotic neurons. PMID:18431258

  20. Relationship between sperm aneuploidy, sperm DNA integrity, chromatin packaging, traditional semen parameters, and recurrent pregnancy loss.

    PubMed

    Zidi-Jrah, Ines; Hajlaoui, Amani; Mougou-Zerelli, Soumaya; Kammoun, Molka; Meniaoui, Imene; Sallem, Amira; Brahem, Sonia; Fekih, Meriem; Bibi, Mohammed; Saad, Ali; Ibala-Romdhane, Samira

    2016-01-01

    To study the possible relationship between sperm aneuploidy, sperm DNA integrity, chromatin packaging, traditional semen parameters, and recurrent pregnancy loss (RPL). Descriptive study. University-affiliated tertiary teaching. A total of 22 couples with history of RPL and 20 fertile men. Semen samples from case and control men were examined for differences in semen parameters, DNA fragmentation, chromatin condensation, and sperm aneuploidy. Sperm DNA and chromatin integrity and sperm aneuploidy. Sperm progressive motility (30.2% vs. 51.5%) was significantly lower and abnormal morphology (74.8% vs. 54.2%) was significantly higher in the RPL group versus the control group, respectively. The percentage of fragmented DNA was significantly increased in the RPL group (17.1% vs. 10.2%) as well as the rate of spermatozoa with nuclear chromatin decondensation (23.6% vs. 11.8%). There was a significantly higher sperm aneuploidy rate among the RPL group as well. The increase in abnormal sperm parameters, sperm DNA fragmentation, nuclear chromatin decondensation, and sperm aneuploidy suggest possible causes of unexplained RPL. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  1. Analysis of DNA methylation of perennial ryegrass under drought using the methylation-sensitive amplification polymorphism (MSAP) technique.

    PubMed

    Tang, Xiao-Mei; Tao, Xiang; Wang, Yan; Ma, Dong-Wei; Li, Dan; Yang, Hong; Ma, Xin-Rong

    2014-12-01

    Perennial ryegrass (Lolium perenne), an excellent grass for forage and turf, is widespread in temperate regions. Drought is an important factor that limits its growth, distribution, and yield. DNA methylation affects gene expression and plays an important role in adaptation to adverse environments. In this study, the DNA methylation changes in perennial ryegrass under drought stress were assessed using methylation-sensitive amplified polymorphism (MSAP). After 15 days of drought stress treatment, the plant height was less than half of the control, and the leaves were smaller and darker. Genome-wide, a total of 652 CCGG sites were detected by MSAP. The total methylation level was 57.67 and 47.39 % in the control and drought treatment, respectively, indicating a decrease of 10.28 % due to drought exposure. Fifteen differentially displayed DNA fragments in MSAP profiles were cloned for sequencing analysis. The results showed that most of the genes involved in stress responses. The relative expression levels revealed that three demethylated fragments were up-regulated. The expression of a predicted retrotransposon increased significantly, changing from hypermethylation to non-methylation. Although the extent of methylation in two other genes decreased, the sites of methylation remained, and the expression increased only slightly. All of these results suggested that drought stress decreased the total DNA methylation level in perennial ryegrass and demethylation up-regulated related gene expressions and that the extent of methylation was negatively correlated with expression. Overall, the induced epigenetic changes in genome probably are an important regulatory mechanism for acclimating perennial ryegrass to drought and possibly other environmental stresses.

  2. Treatment of PCR products with exonuclease I and heat-labile alkaline phosphatase improves the visibility of combined bisulfite restriction analysis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Watanabe, Kousuke; Emoto, Noriko; Sunohara, Mitsuhiro

    2010-08-27

    Research highlights: {yields} Incubating PCR products at a high temperature causes smears in gel electrophoresis. {yields} Smears interfere with the interpretation of methylation analysis using COBRA. {yields} Treatment with exonuclease I and heat-labile alkaline phosphatase eliminates smears. {yields} The elimination of smears improves the visibility of COBRA. -- Abstract: DNA methylation plays a vital role in the regulation of gene expression. Abnormal promoter hypermethylation is an important mechanism of inactivating tumor suppressor genes in human cancers. Combined bisulfite restriction analysis (COBRA) is a widely used method for identifying the DNA methylation of specific CpG sites. Here, we report that exonucleasemore » I and heat-labile alkaline phosphatase can be used for PCR purification for COBRA, improving the visibility of gel electrophoresis after restriction digestion. This improvement is observed when restriction digestion is performed at a high temperature, such as 60 {sup o}C or 65 {sup o}C, with BstUI and TaqI, respectively. This simple method can be applied instead of DNA purification using spin columns or phenol/chloroform extraction. It can also be applied to other situations when PCR products are digested by thermophile-derived restriction enzymes, such as PCR restriction fragment length polymorphism (RFLP) analysis.« less

  3. Asbestos-associated genome-wide DNA methylation changes in lung cancer.

    PubMed

    Kettunen, Eeva; Hernandez-Vargas, Hector; Cros, Marie-Pierre; Durand, Geoffroy; Le Calvez-Kelm, Florence; Stuopelyte, Kristina; Jarmalaite, Sonata; Salmenkivi, Kaisa; Anttila, Sisko; Wolff, Henrik; Herceg, Zdenko; Husgafvel-Pursiainen, Kirsti

    2017-11-15

    Previous studies have revealed a robust association between exposure to asbestos and human lung cancer. Accumulating evidence has highlighted the role of epigenome deregulation in the mechanism of carcinogen-induced malignancies. We examined the impact of asbestos on DNA methylation. Our genome-wide studies (using Illumina HumanMethylation450K BeadChip) of lung cancer tissue and paired normal lung from 28 asbestos-exposed or non-exposed patients, mostly smokers, revealed distinctive DNA methylation changes. We identified a number of differentially methylated regions (DMR) and differentially variable, differentially methylated CpGs (DVMC), with individual CpGs further validated by pyrosequencing in an independent series of 91 non-small cell lung cancer and paired normal lung. We discovered and validated BEND4, ZSCAN31 and GPR135 as significantly hypermethylated in lung cancer. DMRs in genes such as RARB (FDR 1.1 × 10 -19 , mean change in beta [Δ] -0.09), GPR135 (FDR 1.87 × 10 -8 , mean Δ -0.09) and TPO (FDR 8.58 × 10 -5 , mean Δ -0.11), and DVMCs in NPTN, NRG2, GLT25D2 and TRPC3 (all with p <0.05, t-test) were significantly associated with asbestos exposure status in exposed versus non-exposed lung tumors. Hypomethylation was characteristic to DVMCs in lung cancer tissue from asbestos-exposed subjects. When DVMCs related to asbestos or smoking were analyzed, 96% of the elements were unique to either of the exposures, consistent with the concept that the methylation changes in tumors may be specific for risk factors. In conclusion, we identified novel DNA methylation changes associated with lung tumors and asbestos exposure, suggesting that changes may be present in causal pathway from asbestos exposure to lung cancer. © 2017 UICC.

  4. CANCER BIOMARKERS IN HUMAN ATHEROSCLEROTIC LESIONS: DETECTION OF DNA ADDUCTS

    EPA Science Inventory

    Since somatic mutations are suspected to contribute to the pathogenesis not only of cancer but also of atherosclerotic plaques, we measured DNA adducts in the smooth muscle layer of atherosclerotic lesions in abnormal aorta specimens taken at surgery from seven patients. NA adduc...

  5. CAFE: an R package for the detection of gross chromosomal abnormalities from gene expression microarray data.

    PubMed

    Bollen, Sander; Leddin, Mathias; Andrade-Navarro, Miguel A; Mah, Nancy

    2014-05-15

    The current methods available to detect chromosomal abnormalities from DNA microarray expression data are cumbersome and inflexible. CAFE has been developed to alleviate these issues. It is implemented as an R package that analyzes Affymetrix *.CEL files and comes with flexible plotting functions, easing visualization of chromosomal abnormalities. CAFE is available from https://bitbucket.org/cob87icW6z/cafe/ as both source and compiled packages for Linux and Windows. It is released under the GPL version 3 license. CAFE will also be freely available from Bioconductor. sander.h.bollen@gmail.com or nancy.mah@mdc-berlin.de Supplementary data are available at Bioinformatics online.

  6. Molecular andrology as related to sperm DNA fragmentation/sperm chromatin biotechnology.

    PubMed

    Shafik, A; Shafik, A A; Shafik, I; El Sibai, O

    2006-01-01

    Genetic male infertility occurs throughout the life cycle from genetic traits carried by the sperm, to fertilization and post-fertilization genome alterations, and subsequent developmental changes in the blastocyst and fetus as well as errors in meiosis and abnormalities in spermatogenesis/spermatogenesis. Genes encoding proteins for normal development include SRY, SOX9, INSL3 and LGR8. Genetic abnormalities affect spermatogenesis whereas polymorphisms affect receptor affinity and hormone bioactivity. Transgenic animal models, the human genome project, and other techniques have identified numerous genes related to male fertility. Several techniques have been developed to measure the amount of sperm DNA damage in an effort to identify more objective parameters for evaluation of infertile men. The integrity of sperm DNA influences a couple's fertility and helps predict the chances of pregnancy and its successful outcome. The available tests of sperm DNA damage require additional large-scale clinical trials before their integration into routine clinical practice. The physiological/molecular integrity of sperm DNA is a novel parameter of semen quality and a potential fertility predictor. Although DNA integrity assessment appears to be a logical biomarker of sperm quality, it is not being assessed as a routine part of semen analysis by clinical andrologists. Extensive investigation has been conducted for the comparative evaluation of these techniques. However, some of these techniques require expensive instrumentation for optimal and unbiased analysis, are labor intensive, or require the use of enzymes whose activity and accessibility to DNA breaks may be irregular. Thus, these techniques are recommended for basic research rather than for routine andrology laboratories.

  7. The epigenome as a therapeutic target in prostate cancer.

    PubMed

    Perry, Antoinette S; Watson, R William G; Lawler, Mark; Hollywood, Donal

    2010-12-01

    During cancer development and progression, tumor cells undergo abnormal epigenetic modifications, including DNA methylation, histone deacetylation and nucleosome remodeling. Collectively, these aberrations promote genomic instability and lead to silencing of tumor-suppressor genes and reactivation of oncogenic retroviruses. Epigenetic modifications, therefore, provide exciting new avenues for prostate cancer research. Promoter hypermethylation is widespread during neoplastic transformation of prostate cells, which suggests that restoration of a 'normal' epigenome through treatment with inhibitors of the enzymes involved could be clinically beneficial. Global patterns of histone modifications are also being defined and have been associated with clinical and pathologic predictors of prostate cancer outcome. Although treatment for localized prostate cancer can be curative, the development of successful therapies for the management of castration-resistant metastatic disease is urgently needed. Reactivation of tumor-suppressor genes by demethylating agents and histone deacetylase inhibitors could be a potential treatment option for patients with advanced disease.

  8. Exporting DNA - striking a balance between preventing exploitation and promoting innovation.

    PubMed

    Pepper, M S

    2017-01-30

    DNA contains the blueprint of life. Variations in the script determine the great diversity that characterises our planet. As the analysis of large datasets derived from DNA reveals the hidden secrets of normal and abnormal structure and function as well as our ancestry, the movement of DNA between research laboratories is becoming commonplace. DNA is a resource that can be used for the benefit or to the detriment of the individuals and communities from which it is derived. But can DNA be treated as a simple commodity? How do we deal with questions such as sovereignty, discrimination and commercialisation? What underlies the current trends in attempting to regulate the movement of DNA? And how can we achieve a balance between preventing exploitation and promoting innovation? This brief overview attempts to contextualise the current landscape in South Africa with regard to the DNA that is destined to leave our shores.

  9. Protection against oxidative DNA damage and stress in human prostate by glutathione S-transferase P1

    PubMed Central

    Kanwal, Rajnee; Pandey, Mitali; Bhaskaran, Natarajan; MacLennan, Gregory T; Fu, Pingfu; Ponsky, Lee E; Gupta, Sanjay

    2014-01-01

    The pi-class glutathione S-transferase (GSTP1) actively protect cells from carcinogens and electrophilic compounds. Loss of GSTP1 expression via promoter hypermethylation is the most common epigenetic alteration observed in human prostate cancer. Silencing of GSTP1 can increase generation of reactive oxygen species (ROS) and DNA damage in cells. In this study we investigated whether loss of GSTP1 contributes to increased DNA damage that may predispose men to a higher risk of prostate cancer. We found significantly elevated (103%; P<0.0001) levels of 8-oxo-2′-deoxogunosine (8-OHdG), an oxidative DNA damage marker, in adenocarcinomas, compared to benign counterparts, which positively correlated (r=0.2) with loss of GSTP1 activity (34%; P<0.0001). Silencing of GSTP1 using siRNA approach in normal human prostate epithelial RWPE1 cells caused increased intracellular production of ROS and higher susceptibility of cells to H2O2-mediated oxidative stress. Additionally, human prostate carcinoma LNCaP cells, which contain a silenced GSTP1 gene, were genetically modified to constitutively express high levels of GSTP1. Induction of GSTP1 activity lowered endogenous ROS levels in LNCaP-pLPCX-GSTP1 cells, and when exposed to H2O2, these cells exhibited significantly reduced production of ROS and 8-OHdG levels, compared to vector control LNCaP-pLPCX cells. Furthermore, exposure of LNCaP cells to green tea polyphenols caused re-expression of GSTP1, which protected the cells from H2O2-mediated DNA damage through decreased ROS production compared to non-exposed cells. These results suggest that loss of GSTP1 expression in human prostate cells, a process that increases their susceptibility to oxidative stress-induced DNA damage, may be an important target for primary prevention of prostate cancer. PMID:22833520

  10. Combined SOM-portrayal of gene expression and DNA methylation landscapes disentangles modes of epigenetic regulation in glioblastoma.

    PubMed

    Hopp, Lydia; Löffler-Wirth, Henry; Galle, Jörg; Binder, Hans

    2018-06-11

    We present here a novel method that enables unraveling the interplay between gene expression and DNA methylation in complex diseases such as cancer. The method is based on self-organizing maps and allows for analysis of data landscapes from 'governed by methylation' to 'governed by expression'. We identified regulatory modules of coexpressed and comethylated genes in high-grade gliomas: two modes are governed by genes hypermethylated and underexpressed in IDH-mutated cases, while two other modes reflect immune and stromal signatures in the classical and mesenchymal subtypes. A fifth mode with proneural characteristics comprises genes of repressed and poised chromatin states active in healthy brain. Two additional modes enrich genes either in active or repressed chromatin states. The method disentangles the interplay between gene expression and methylation. It has the potential to integrate also mutation and copy number data and to apply to large sample cohorts.

  11. Environmental exposure and HPV infection may act synergistically to induce lung tumorigenesis in nonsmokers

    PubMed Central

    Cheng, Ya-Wen; Lin, Frank Cheau-Feng; Chen, Chih-Yi; Hsu, Nan-Yung

    2016-01-01

    Most studies of lung tumorigenesis have focused on smokers rather than nonsmokers. In this study, we used human papillomavirus (HPV)-positive and HPV-negative lung cancer cells to test the hypothesis that HPV infection synergistically increases DNA damage induced by exposure to the carcinogen benzo[a]pyrene (B[a]P), and contributes to lung tumorigenesis in nonsmokers. DNA adduct levels induced by B[a]P in HPV-positive cells were significantly higher than in HPV-negative cells. The DNA adduct formation was dependent on HPV E6 oncoprotein expression. Gene and protein expression of two DNA repair genes, XRCC3 and XRCC5, were lower in B[a]P-treated E6-positive cells than in E6-negative lung cancer cells. The reduced expression was also detected immunohistochemically and was caused by increased promoter hypermethylation. Moreover, mutations of p53 and epidermal growth factor receptor (EGFR) genes in lung cancer patients were associated with XRCC5 inactivation. In sum, our study indicates that HPV E6-induced promoter hypermethylation of the XRCC3 and XRCC5 DNA repair genes and the resultant decrease in their expression increases B[a]P-induced DNA adducts and contributes to lung tumorigenesis in nonsmokers. PMID:26918347

  12. Bioinformatics Approaches for Fetal DNA Fraction Estimation in Noninvasive Prenatal Testing

    PubMed Central

    Peng, Xianlu Laura; Jiang, Peiyong

    2017-01-01

    The discovery of cell-free fetal DNA molecules in plasma of pregnant women has created a paradigm shift in noninvasive prenatal testing (NIPT). Circulating cell-free DNA in maternal plasma has been increasingly recognized as an important proxy to detect fetal abnormalities in a noninvasive manner. A variety of approaches for NIPT using next-generation sequencing have been developed, which have been rapidly transforming clinical practices nowadays. In such approaches, the fetal DNA fraction is a pivotal parameter governing the overall performance and guaranteeing the proper clinical interpretation of testing results. In this review, we describe the current bioinformatics approaches developed for estimating the fetal DNA fraction and discuss their pros and cons. PMID:28230760

  13. Bioinformatics Approaches for Fetal DNA Fraction Estimation in Noninvasive Prenatal Testing.

    PubMed

    Peng, Xianlu Laura; Jiang, Peiyong

    2017-02-20

    The discovery of cell-free fetal DNA molecules in plasma of pregnant women has created a paradigm shift in noninvasive prenatal testing (NIPT). Circulating cell-free DNA in maternal plasma has been increasingly recognized as an important proxy to detect fetal abnormalities in a noninvasive manner. A variety of approaches for NIPT using next-generation sequencing have been developed, which have been rapidly transforming clinical practices nowadays. In such approaches, the fetal DNA fraction is a pivotal parameter governing the overall performance and guaranteeing the proper clinical interpretation of testing results. In this review, we describe the current bioinformatics approaches developed for estimating the fetal DNA fraction and discuss their pros and cons.

  14. Alteration of gene expression and DNA methylation in drug-resistant gastric cancer.

    PubMed

    Maeda, Osamu; Ando, Takafumi; Ohmiya, Naoki; Ishiguro, Kazuhiro; Watanabe, Osamu; Miyahara, Ryoji; Hibi, Yoko; Nagai, Taku; Yamada, Kiyofumi; Goto, Hidemi

    2014-04-01

    The mechanisms of drug resistance in cancer are not fully elucidated. To study the drug resistance of gastric cancer, we analyzed gene expression and DNA methylation profiles of 5-fluorouracil (5-FU)- and cisplatin (CDDP)-resistant gastric cancer cells and biopsy specimens. Drug-resistant gastric cancer cells were established with culture for >10 months in a medium containing 5-FU or CDDP. Endoscopic biopsy specimens were obtained from gastric cancer patients who underwent chemotherapy with oral fluoropyrimidine S-1 and CDDP. Gene expression and DNA methylation analyses were performed using microarray, and validated using real-time PCR and pyrosequencing, respectively. Out of 17,933 genes, 541 genes commonly increased and 569 genes decreased in both 5-FU- and CDDP-resistant AGS cells. Genes with expression changed by drugs were related to GO term 'extracellular region' and 'p53 signaling pathway' in both 5-FU- and CDDP-treated cells. Expression of 15 genes including KLK13 increased and 12 genes including ETV7 decreased, in both drug-resistant cells and biopsy specimens of two patients after chemotherapy. Out of 10,365 genes evaluated with both expression microarray and methylation microarray, 74 genes were hypermethylated and downregulated, or hypomethylated and upregulated in either 5-FU-resistant or CDDP-resistant cells. Of these genes, expression of 21 genes including FSCN1, CPT1C and NOTCH3, increased from treatment with a demethylating agent. There are alterations of gene expression and DNA methylation in drug-resistant gastric cancer; they may be related to mechanisms of drug resistance and may be useful as biomarkers of gastric cancer drug sensitivity.

  15. Abnormal Uterine Bleeding FAQ

    MedlinePlus

    ... Abnormal Uterine Bleeding • What is a normal menstrual cycle? • When is bleeding abnormal? • At what ages is ... abnormal bleeding? •Glossary What is a normal menstrual cycle? The normal length of the menstrual cycle is ...

  16. Large Sex Differences in Chicken Behavior and Brain Gene Expression Coincide with Few Differences in Promoter DNA-Methylation

    PubMed Central

    Nätt, Daniel; Agnvall, Beatrix; Jensen, Per

    2014-01-01

    While behavioral sex differences have repeatedly been reported across taxa, the underlying epigenetic mechanisms in the brain are mostly lacking. Birds have previously shown to have only limited dosage compensation, leading to high sex bias of Z-chromosome gene expression. In chickens, a male hyper-methylated region (MHM) on the Z-chromosome has been associated with a local type of dosage compensation, but a more detailed characterization of the avian methylome is limiting our interpretations. Here we report an analysis of genome wide sex differences in promoter DNA-methylation and gene expression in the brain of three weeks old chickens, and associated sex differences in behavior of Red Junglefowl (ancestor of domestic chickens). Combining DNA-methylation tiling arrays with gene expression microarrays we show that a specific locus of the MHM region, together with the promoter for the zinc finger RNA binding protein (ZFR) gene on chromosome 1, is strongly associated with sex dimorphism in gene expression. Except for this, we found few differences in promoter DNA-methylation, even though hundreds of genes were robustly differentially expressed across distantly related breeds. Several of the differentially expressed genes are known to affect behavior, and as suggested from their functional annotation, we found that female Red Junglefowl are more explorative and fearful in a range of tests performed throughout their lives. This paper identifies new sites and, with increased resolution, confirms known sites where DNA-methylation seems to affect sexually dimorphic gene expression, but the general lack of this association is noticeable and strengthens the view that birds do not have dosage compensation. PMID:24782041

  17. Aprataxin resolves adenylated RNA–DNA junctions to maintain genome integrity

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tumbale, Percy; Williams, Jessica S.; Schellenberg, Matthew J.

    2013-12-22

    Faithful maintenance and propagation of eukaryotic genomes is ensured by three-step DNA ligation reactions used by ATP-dependent DNA ligases. Paradoxically, when DNA ligases encounter nicked DNA structures with abnormal DNA termini, DNA ligase catalytic activity can generate and/or exacerbate DNA damage through abortive ligation that produces chemically adducted, toxic 5'-adenylated (5'-AMP) DNA lesions. Aprataxin (APTX) reverses DNA adenylation but the context for deadenylation repair is unclear. Here we examine the importance of APTX to RNase-H2-dependent excision repair (RER) of a lesion that is very frequently introduced into DNA, a ribonucleotide. We show that ligases generate adenylated 5' ends containing amore » ribose characteristic of RNase H2 incision. APTX efficiently repairs adenylated RNA–DNA, and acting in an RNA–DNA damage response (RDDR), promotes cellular survival and prevents S-phase checkpoint activation in budding yeast undergoing RER. Structure–function studies of human APTX–RNA–DNA–AMP–Zn complexes define a mechanism for detecting and reversing adenylation at RNA–DNA junctions. This involves A-form RNA binding, proper protein folding and conformational changes, all of which are affected by heritable APTX mutations in ataxia with oculomotor apraxia 1. Together, these results indicate that accumulation of adenylated RNA–DNA may contribute to neurological disease.« less

  18. A homozygote splice site PMS2 mutation as cause of Turcot syndrome gives rise to two different abnormal transcripts.

    PubMed

    Sjursen, Wenche; Bjørnevoll, Inga; Engebretsen, Lars F; Fjelland, Kristine; Halvorsen, Tore; Myrvold, Helge E

    2009-01-01

    Turcot syndrome is a rare, inherited disease predisposing of tumours in the central nerve system and in the colorectal system. This report describes a Turcot patient with an extraordinary clinical history. The patient is still alive at the age of 43. She was operated at the age of 10 by brain tumour and at the age of 16 by colorectal cancer. She has since then been treated for multiple cancers (gastrointestinal, endometrial, basal cell carcinomas), and removal of adenomatous polyps at several occasions. The aim of this work was to investigate if there was any specific genotype that explains her remarkable clinical history. Microsatellite instability and immunohistochemistry analysis for four DNA mismatch repair proteins were performed. DNA mutation analysis was done for genes involved in polyposis and mismatch repair by denaturing high performance liquid chromatography and sequencing. cDNA analysis was carried out for the mismatch repair gene PMS2. The patients genotype was found to be a homozygous splice site mutation in the PMS2 gene, c.989-1Gabnormal transcripts, not one as expected. The patient's long time survival may in part be explained by meticulous follow up by health care professionals. The other importing factor is probably the nature of here genotype. cDNA analysis showed that the homozygous mutation led to two abnormal transcripts, of which one is perhaps less detrimental. Thus cDNA analysis is of prime importance for the full evaluation of the effect of putative splicing mutations.

  19. Longitudinal Analysis of Carcinogenic Human Papillomavirus Infection and Associated Cytologic Abnormalities in the Guanacaste Natural History Study: Looking Ahead to Cotesting

    PubMed Central

    Rodriguez, Ana C.; Burk, Robert D.; Hildesheim, Allan; Herrero, Rolando; Wacholder, Sholom; Hutchinson, Martha; Schiffman, Mark

    2012-01-01

    Background. Few studies have addressed the timing of cervical cytologic abnormalities and human papillomavirus (HPV) positivity during the course of an infection. It remains largely unknown how infections detected by HPV and cytology wax and wane relative to each other. The aim of this analysis was to assess the longitudinal relationship of abnormal cytology and HPV positivity in a 7-year prospective study of 2500 women in Guanacaste, Costa Rica. Methods. At each semiannual or annual visit, cervical specimens were screened using liquid-based cytology and tested for >40 HPV types with use of MY09/MY11 L1 degenerate primer polymerase chain reaction–based methods. On the basis of previous work, we separated prevalent and newly detected infections in younger and older women. Results. Among newly detected HPV- and/or cytology-positive events, HPV and cytology appeared together ∼60% of the time; when discordant, HPV tended to appear before cytology in younger and older women. Combining newly and prevalently detected events, HPV and cytology disappeared at the same time >70% of the time. When discordant, HPV tended to disappear after cytology in younger and older women. Conclusions. Detection of HPV DNA and associated cytological abnormalities tend to come and leave together; however, when discordant, detection of HPV DNA tends to precede and/or last longer than associated cytologic abnormalities. PMID:22147792

  20. Circulating DNA and its methylation level in inflammatory bowel disease and related colon cancer.

    PubMed

    Bai, Xuming; Zhu, Yaqun; Pu, Wangyang; Xiao, Li; Li, Kai; Xing, Chungen; Jin, Yong

    2015-01-01

    Both of chronic inflammation and abnormal immune in inflammatory bowel disease can induce colon cancer. Previous research showed that cell apoptosis and necrosis become the main source of circulating DNA in the peripheral blood during tumorigenesis that reduced along with methylation degree. However, its role in the process of colitis transforming to colon cancer is not clarified. Drinking 3% DSS was used to establish colitis model, while 3% dextran sodium sulfate (DSS) combined with azo oxidation methane (AOM) intraperitoneal injection was applied to establish colitis related colon cancer model. Circulating DNA and its methylation level in peripheral blood were tested. Morphology observation, HE staining, and p53 and β-catenin expression detection confirmed that drinking 3% DSS and 3% DSS combined with AOM intraperitoneal injection can successfully establish colitis and colitis associated colorectal cancer models. Circulating DNA level in colitis and colon cancer mice increased by gradient compared with control, while significant difference was observed between each other. Circulating DNA methylation level decreased obviously in colitis and colon cancer, and significant difference was observed between each other. Abnormal protein expression, circulating DNA and its methylation level in ulcerative colitis associated colorectal tissues change in gradient, suggesting that circulating DNA and its methylation level can be treated as new markers for colitis cancer transformation that has certain significance to explore the mechanism of human ulcerative colitis canceration.

  1. Whole Gene Capture Analysis of 15 CRC Susceptibility Genes in Suspected Lynch Syndrome Patients.

    PubMed

    Jansen, Anne M L; Geilenkirchen, Marije A; van Wezel, Tom; Jagmohan-Changur, Shantie C; Ruano, Dina; van der Klift, Heleen M; van den Akker, Brendy E W M; Laros, Jeroen F J; van Galen, Michiel; Wagner, Anja; Letteboer, Tom G W; Gómez-García, Encarna B; Tops, Carli M J; Vasen, Hans F; Devilee, Peter; Hes, Frederik J; Morreau, Hans; Wijnen, Juul T

    2016-01-01

    Lynch Syndrome (LS) is caused by pathogenic germline variants in one of the mismatch repair (MMR) genes. However, up to 60% of MMR-deficient colorectal cancer cases are categorized as suspected Lynch Syndrome (sLS) because no pathogenic MMR germline variant can be identified, which leads to difficulties in clinical management. We therefore analyzed the genomic regions of 15 CRC susceptibility genes in leukocyte DNA of 34 unrelated sLS patients and 11 patients with MLH1 hypermethylated tumors with a clear family history. Using targeted next-generation sequencing, we analyzed the entire non-repetitive genomic sequence, including intronic and regulatory sequences, of 15 CRC susceptibility genes. In addition, tumor DNA from 28 sLS patients was analyzed for somatic MMR variants. Of 1979 germline variants found in the leukocyte DNA of 34 sLS patients, one was a pathogenic variant (MLH1 c.1667+1delG). Leukocyte DNA of 11 patients with MLH1 hypermethylated tumors was negative for pathogenic germline variants in the tested CRC susceptibility genes and for germline MLH1 hypermethylation. Somatic DNA analysis of 28 sLS tumors identified eight (29%) cases with two pathogenic somatic variants, one with a VUS predicted to pathogenic and LOH, and nine cases (32%) with one pathogenic somatic variant (n = 8) or one VUS predicted to be pathogenic (n = 1). This is the first study in sLS patients to include the entire genomic sequence of CRC susceptibility genes. An underlying somatic or germline MMR gene defect was identified in ten of 34 sLS patients (29%). In the remaining sLS patients, the underlying genetic defect explaining the MMRdeficiency in their tumors might be found outside the genomic regions harboring the MMR and other known CRC susceptibility genes.

  2. Succinate Dehydrogenase Mutation Underlies Global Epigenomic Divergence in Gastrointestinal Stromal Tumor

    PubMed Central

    Killian, J. Keith; Kim, Su Young; Miettinen, Markku; Smith, Carly; Merino, Maria; Tsokos, Maria; Quezado, Martha; Smith, William I.; Jahromi, Mona S.; Xekouki, Paraskevi; Szarek, Eva; Walker, Robert L.; Lasota, Jerzy; Raffeld, Mark; Klotzle, Brandy; Wang, Zengfeng; Jones, Laura; Zhu, Yuelin; Wang, Yonghong; Waterfall, Joshua J.; O’Sullivan, Maureen J.; Bibikova, Marina; Pacak, Karel; Stratakis, Constantine; Janeway, Katherine A.; Schiffman, Joshua D.; Fan, Jian-Bing; Helman, Lee; Meltzer, Paul S.

    2014-01-01

    Gastrointestinal stromal tumors (GIST) harbor driver mutations of signal transduction kinases such as KIT, or, alternatively, manifest loss-of-function defects in the mitochondrial succinate dehydrogenase (SDH) complex, a component of the Krebs cycle and electron transport chain. We have uncovered a striking divergence between the DNA methylation profiles of SDH-deficient GIST (n = 24) versus KIT tyrosine kinase pathway–mutated GIST (n = 39). Infinium 450K methylation array analysis of formalin-fixed paraffin-embedded tissues disclosed an order of magnitude greater genomic hypermethylation relative to SDH-deficient GIST versus the KIT-mutant group (84.9 K vs. 8.4 K targets). Epigenomic divergence was further found among SDH-mutant paraganglioma/pheochromocytoma (n = 29), a developmentally distinct SDH-deficient tumor system. Comparison of SDH -mutant GIST with isocitrate dehydrogenase -mutant glioma, another Krebs cycle–defective tumor type, revealed comparable measures of global hypo- and hypermethylation. These data expose a vital connection between succinate metabolism and genomic DNA methylation during tumorigenesis, and generally implicate the mitochondrial Krebs cycle in nuclear epigenomic maintenance. SIGNIFICANCE This study shows that SDH deficiency underlies pervasive DNA hypermethylation in multiple tumor lineages, generally defining the Krebs cycle as mitochondrial custodian of the methylome. We propose that this phenomenon may result from a failure of maintenance CpG demethylation, secondary to inhibition of the TET 5-methylcytosine dioxgenase demethylation pathway, by inhibitory metabolites that accumulate in tumors with Krebs cycle dysfunction. PMID:23550148

  3. Urine - abnormal color

    MedlinePlus

    ... medlineplus.gov/ency/article/003139.htm Urine - abnormal color To use the sharing features on this page, please enable JavaScript. The usual color of urine is straw-yellow. Abnormally colored urine ...

  4. [Genetic diagnostics of pathogenic splicing abnormalities in the clinical laboratory--pitfalls and screening approaches].

    PubMed

    Niimi, Hideki; Ogawa, Tomomi; Note, Rhougou; Hayashi, Shirou; Ueno, Tomohiro; Harada, Kenu; Uji, Yoshinori; Kitajima, Isao

    2010-12-01

    In recent years, genetic diagnostics of pathogenic splicing abnormalities are increasingly recognized as critically important in the clinical genetic diagnostics. It is reported that approximately 10% of pathogenic mutations causing human inherited diseases are splicing mutations. Nonetheless, it is still difficult to identify splicing abnormalities in routine genetic diagnostic settings. Here, we studied two different kinds of cases with splicing abnormalities. The first case is a protein S deficiency. Nucleotide analyses revealed that the proband had a previously reported G to C substitution in the invariant AG dinucleotide at the splicing acceptor site of intronl/exon2, which produces multiple splicing abnormalities resulting in protein S deficiency. The second case is an antithrombin (AT) deficiency. This proband had a previously reported G to A substitution, at nucleotide position 9788 in intron 4, 14 bp in front of exon 5, which created a de novo exon 5 splice site and resulted in AT deficiency. From a practical standpoint, we discussed the pitfalls, attentions, and screening approaches in genetic diagnostics of pathogenic splicing abnormalities. Due to the difficulty with full-length sequence analysis of introns, and the lack of RNA samples, splicing mutations may escape identification. Although current genetic testing remains to be improved, to screen for splicing abnormalities more efficiently, it is significant to use an appropriate combination of various approaches such as DNA and/or RNA samples, splicing mutation databases, bioinformatic tools to detect splice sites and cis-regulatory elements, and in vitro and/or in vivo experimentally methods as needed.

  5. Genes Downregulated in Endometriosis Are Located Near the Known Imprinting Genes

    PubMed Central

    Higashiura, Yumi; Koike, Natsuki; Akasaka, Juria; Uekuri, Chiharu; Iwai, Kana; Niiro, Emiko; Morioka, Sachiko; Yamada, Yuki

    2014-01-01

    There is now accumulating evidence that endometriosis is a disease associated with an epigenetic disorder. Genomic imprinting is an epigenetic phenomenon known to regulate DNA methylation of either maternal or paternal alleles. We hypothesize that hypermethylated endometriosis-associated genes may be enriched at imprinted gene loci. We sought to determine whether downregulated genes associated with endometriosis susceptibility are associated with chromosomal location of the known paternally and maternally expressed imprinting genes. Gene information has been gathered from National Center for Biotechnology Information database geneimprint.com. Several researchers have identified specific loci with strong DNA methylation in eutopic endometrium and ectopic lesion with endometriosis. Of the 29 hypermethylated genes in endometriosis, 19 genes were located near 45 known imprinted foci. There may be an association of the genomic location between genes specifically downregulated in endometriosis and epigenetically imprinted genes. PMID:24615936

  6. Polε Instability Drives Replication Stress, Abnormal Development, and Tumorigenesis.

    PubMed

    Bellelli, Roberto; Borel, Valerie; Logan, Clare; Svendsen, Jennifer; Cox, Danielle E; Nye, Emma; Metcalfe, Kay; O'Connell, Susan M; Stamp, Gordon; Flynn, Helen R; Snijders, Ambrosius P; Lassailly, François; Jackson, Andrew; Boulton, Simon J

    2018-05-17

    DNA polymerase ε (POLE) is a four-subunit complex and the major leading strand polymerase in eukaryotes. Budding yeast orthologs of POLE3 and POLE4 promote Polε processivity in vitro but are dispensable for viability in vivo. Here, we report that POLE4 deficiency in mice destabilizes the entire Polε complex, leading to embryonic lethality in inbred strains and extensive developmental abnormalities, leukopenia, and tumor predisposition in outbred strains. Comparable phenotypes of growth retardation and immunodeficiency are also observed in human patients harboring destabilizing mutations in POLE1. In both Pole4 -/- mouse and POLE1 mutant human cells, Polε hypomorphy is associated with replication stress and p53 activation, which we attribute to inefficient replication origin firing. Strikingly, removing p53 is sufficient to rescue embryonic lethality and all developmental abnormalities in Pole4 null mice. However, Pole4 -/- p53 +/- mice exhibit accelerated tumorigenesis, revealing an important role for controlled CMG and origin activation in normal development and tumor prevention. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  7. Topological events in single molecules of E. coli DNA confined in nanochannels

    PubMed Central

    Reifenberger, Jeffrey G.; Dorfman, Kevin D.; Cao, Han

    2015-01-01

    We present experimental data concerning potential topological events such as folds, internal backfolds, and/or knots within long molecules of double-stranded DNA when they are stretched by confinement in a nanochannel. Genomic DNA from E. coli was labeled near the ‘GCTCTTC’ sequence with a fluorescently labeled dUTP analog and stained with the DNA intercalator YOYO. Individual long molecules of DNA were then linearized and imaged using methods based on the NanoChannel Array technology (Irys® System) available from BioNano Genomics. Data were collected on 189,153 molecules of length greater than 50 kilobases. A custom code was developed to search for abnormal intensity spikes in the YOYO backbone profile along the length of individual molecules. By correlating the YOYO intensity spikes with the aligned barcode pattern to the reference, we were able to correlate the bright intensity regions of YOYO with abnormal stretching in the molecule, which suggests these events were either a knot or a region of internal backfolding within the DNA. We interpret the results of our experiments involving molecules exceeding 50 kilobases in the context of existing simulation data for relatively short DNA, typically several kilobases. The frequency of these events is lower than the predictions from simulations, while the size of the events is larger than simulation predictions and often exceeds the molecular weight of the simulated molecules. We also identified DNA molecules that exhibit large, single folds as they enter the nanochannels. Overall, topological events occur at a low frequency (~7% of all molecules) and pose an easily surmountable obstacle for the practice of genome mapping in nanochannels. PMID:25991508

  8. The role and diagnostic value of cell-free DNA in systemic lupus erythematosus.

    PubMed

    Truszewska, Anna; Foroncewicz, Bartosz; Pączek, Leszek

    2017-01-01

    Cell-free DNA (cfDNA) represents a small fraction of total DNA pool that circulates freely in the blood both in normal and pathological conditions. Data indicate that cfDNA plays an important role in the pathogenesis of systemic lupus erythematosus (SLE) and hypomethylation may be crucial for its immunogenic properties. Although differences in quantification methodology hinder the comparison of results between the studies, it appears that levels of cfDNA are abnormally elevated in SLE patients and correlate with various antibody titers, but not with disease activity. Increased cfDNA concentration, however, may be associated with active lupus nephritis. Most of the studies confirmed apoptosis as the major cfDNA release mechanism in various conditions, but formation of neutrophil extracellular traps may significantly contribute to the cfDNA generation in SLE patients. In this review, we summarise current knowledge about the role and possible origin of cfDNA in SLE patients, and discuss why cfDNA testing for diagnostic and prognosis of SLE remains questionable.

  9. DNA methylation is differentially associated with environmental cadmium exposure based on sex and smoking status

    PubMed Central

    Virani, Shama; Rentschler, Katie M.; Nishijo, Muneko; Ruangyuttikarn, Werawan; Swaddiwudhipong, Witaya; Basu, Niladri; Rozek, Laura S.

    2016-01-01

    The adverse health effects of cadmium (Cd) are well known in human populations; however, much of what is known about biological mechanisms of Cd comes from in vitro and animal studies. The adverse health outcomes due to high levels of Cd exposure in the population of Mae Sot, Thailand have been extensively characterized. Here, for the first time, this population is being studied in an epigenetic context. The objective of this study was to characterize the association between DNA methylation markers and Cd exposure, taking into account sex and smoking differences, in an adult population at an increased risk of experiencing adverse health outcomes from high body burden of Cd. One hundred and sixty-nine residents from known exposure areas of Mae Sot, Thailand and one hundred residents from non-exposed areas nearby were surveyed in 2012. Urine and blood samples were collected for measurement of urinary Cd (UCd) and DNA methylation of Cd-related markers (DNMT3B, MGMT, LINE-1, MT2A). UCd levels were 7 times higher in the exposed compared to the unexposed populations (exposed median: 7.4 μg/L, unexposed median: 1.0 μg/L, p <0.001). MGMT hypomethylation was associated with increasing levels of UCd in the total population. Sex-specific associations included MT2A and DNMT3B hypomethylation in women and LINE-1 hypermethylation in men with increasing UCd. Upon subanalysis, these associations separated by smoking status. In summary, environmental Cd exposure is associated with gene-specific DNA methylation in a sex and smoking dependent manner. PMID:26688266

  10. Stress induced gene expression drives transient DNA methylation changes at adjacent repetitive elements

    PubMed Central

    Secco, David; Wang, Chuang; Shou, Huixia; Schultz, Matthew D; Chiarenza, Serge; Nussaume, Laurent; Ecker, Joseph R; Whelan, James; Lister, Ryan

    2015-01-01

    Cytosine DNA methylation (mC) is a genome modification that can regulate the expression of coding and non-coding genetic elements. However, little is known about the involvement of mC in response to environmental cues. Using whole genome bisulfite sequencing to assess the spatio-temporal dynamics of mC in rice grown under phosphate starvation and recovery conditions, we identified widespread phosphate starvation-induced changes in mC, preferentially localized in transposable elements (TEs) close to highly induced genes. These changes in mC occurred after changes in nearby gene transcription, were mostly DCL3a-independent, and could partially be propagated through mitosis, however no evidence of meiotic transmission was observed. Similar analyses performed in Arabidopsis revealed a very limited effect of phosphate starvation on mC, suggesting a species-specific mechanism. Overall, this suggests that TEs in proximity to environmentally induced genes are silenced via hypermethylation, and establishes the temporal hierarchy of transcriptional and epigenomic changes in response to stress. DOI: http://dx.doi.org/10.7554/eLife.09343.001 PMID:26196146

  11. Genome-wide screen of ovary-specific DNA methylation in polycystic ovary syndrome.

    PubMed

    Yu, Ying-Ying; Sun, Cui-Xiang; Liu, Yin-Kun; Li, Yan; Wang, Li; Zhang, Wei

    2015-07-01

    To compare genome-wide DNA methylation profiles in ovary tissue from women with polycystic ovary syndrome (PCOS) and healthy controls. Case-control study matched for age and body mass index. University-affiliated hospital. Ten women with PCOS who underwent ovarian drilling to induce ovulation and 10 healthy women who were undergoing laparoscopic sterilization, hysterectomy for benign conditions, diagnostic laparoscopy for pelvic pain, or oophorectomy for nonovarian indications. None. Genome-wide DNA methylation patterns determined by immunoprecipitation and microarray (MeDIP-chip) analysis. The methylation levels were statistically significantly higher in CpG island shores (CGI shores), which lie outside of core promoter regions, and lower within gene bodies in women with PCOS relative to the controls. In addition, high CpG content promoters were the most frequently hypermethylated promoters in PCOS ovaries but were more often hypomethylated in controls. Second, 872 CGIs, specifically methylated in PCOS, represented 342 genes that could be associated with various molecular functions, including protein binding, hormone activity, and transcription regulator activity. Finally, methylation differences were validated in seven genes by methylation-specific polymerase chain reaction. These genes correlated to several functional families related to the pathogenesis of PCOS and may be potential biomarkers for this disease. Our results demonstrated that epigenetic modification differs between PCOS and normal ovaries, which may help to further understand the pathophysiology of this disease. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  12. DNA Methylation Analysis of the Angiotensin Converting Enzyme (ACE) Gene in Major Depression

    PubMed Central

    Zill, Peter; Baghai, Thomas C.; Schüle, Cornelius; Born, Christoph; Früstück, Clemens; Büttner, Andreas; Eisenmenger, Wolfgang; Varallo-Bedarida, Gabriella; Rupprecht, Rainer; Möller, Hans-Jürgen; Bondy, Brigitta

    2012-01-01

    Background The angiotensin converting enzyme (ACE) has been repeatedly discussed as susceptibility factor for major depression (MD) and the bi-directional relation between MD and cardiovascular disorders (CVD). In this context, functional polymorphisms of the ACE gene have been linked to depression, to antidepressant treatment response, to ACE serum concentrations, as well as to hypertension, myocardial infarction and CVD risk markers. The mostly investigated ACE Ins/Del polymorphism accounts for ∼40%–50% of the ACE serum concentration variance, the remaining half is probably determined by other genetic, environmental or epigenetic factors, but these are poorly understood. Materials and Methods The main aim of the present study was the analysis of the DNA methylation pattern in the regulatory region of the ACE gene in peripheral leukocytes of 81 MD patients and 81 healthy controls. Results We detected intensive DNA methylation within a recently described, functional important region of the ACE gene promoter including hypermethylation in depressed patients (p = 0.008) and a significant inverse correlation between the ACE serum concentration and ACE promoter methylation frequency in the total sample (p = 0.02). Furthermore, a significant inverse correlation between the concentrations of the inflammatory CVD risk markers ICAM-1, E-selectin and P-selectin and the degree of ACE promoter methylation in MD patients could be demonstrated (p = 0.01 - 0.04). Conclusion The results of the present study suggest that aberrations in ACE promoter DNA methylation may be an underlying cause of MD and probably a common pathogenic factor for the bi-directional relationship between MD and cardiovascular disorders. PMID:22808171

  13. Association of abnormal morphology and altered gene expression in human preimplantation embryos.

    PubMed

    Wells, Dagan; Bermúdez, Mercedes G; Steuerwald, Nury; Malter, Henry E; Thornhill, Alan R; Cohen, Jacques

    2005-08-01

    We set out to characterize the expression of nine genes in human preimplantation embryos and determine whether abnormal morphology is associated with altered gene activity. Reverse transcription and real-time polymerase chain reaction were used to quantify the expression of multiple genes in each embryo. The genes studied have various important cellular roles (e.g., cell cycle regulation, DNA repair, and apoptosis). Research laboratory working closely with a clinical IVF practice. Over 50 embryos were donated by infertile patients (various etiologies). Among these, all major stages of preimplantation development and a variety of common morphologic abnormalities were represented. None. Quantification of mRNA transcripts. We detected an association between certain forms of abnormal morphology and disturbances of gene activity. Cellular fragmentation was associated with altered expression of several genes, including TP53, suggesting that fragmenting blastomeres are suffering stress of a type monitored by p53, possibly as a consequence of suboptimal culture conditions. Appropriate gene expression is vital for the regulation of metabolic pathways and key developmental events. Our data indicates a possible causal relationship between changes in gene expression and the formation of clinically relevant abnormal embryo morphologies. We hypothesize that embryos with expression profiles characteristic of good morphology and appropriate for their developmental stage have the greatest potential for implantation. If confirmed, this could lead to a new generation of preimplantation genetic diagnosis (PGD) tests for assessing embryo viability and predicting implantation potential.

  14. pitx2 Deficiency Results in Abnormal Ocular and Craniofacial Development in Zebrafish

    PubMed Central

    Liu, Yi; Semina, Elena V.

    2012-01-01

    Human PITX2 mutations are associated with Axenfeld-Rieger syndrome, an autosomal-dominant developmental disorder that involves ocular anterior segment defects, dental hypoplasia, craniofacial dysmorphism and umbilical abnormalities. Characterization of the PITX2 pathway and identification of the mechanisms underlying the anomalies associated with PITX2 deficiency is important for better understanding of normal development and disease; studies of pitx2 function in animal models can facilitate these analyses. A knockdown of pitx2 in zebrafish was generated using a morpholino that targeted all known alternative transcripts of the pitx2 gene; morphant embryos generated with the pitx2ex4/5 splicing-blocking oligomer produced abnormal transcripts predicted to encode truncated pitx2 proteins lacking the third (recognition) helix of the DNA-binding homeodomain. The morphological phenotype of pitx2ex4/5 morphants included small head and eyes, jaw abnormalities and pericardial edema; lethality was observed at ∼6–8-dpf. Cartilage staining revealed a reduction in size and an abnormal shape/position of the elements of the mandibular and hyoid pharyngeal arches; the ceratobranchial arches were also decreased in size. Histological and marker analyses of the misshapen eyes of the pitx2ex4/5 morphants identified anterior segment dysgenesis and disordered hyaloid vasculature. In summary, we demonstrate that pitx2 is essential for proper eye and craniofacial development in zebrafish and, therefore, that PITX2/pitx2 function is conserved in vertebrates. PMID:22303467

  15. Reduced MUC4 expression is a late event in breast carcinogenesis and is correlated with increased infiltration of immune cells as well as promoter hypermethylation in invasive breast carcinoma.

    PubMed

    Cho, Jin Seong; Park, Min Ho; Lee, Ji Shin; Yoon, Jung Han

    2015-01-01

    Altered expression of MUC4 is associated with tumor progression and immune surveillance, but the potential involvement of MUC4 in breast carcinogenesis has not been rigorously assessed. Immunohistochemical staining with anti-MUC4 antibody was performed in a total of 324 patients with 26 normal breasts, 25 usual ductal hyperplasia, 76 ductal carcinoma in situ, and 198 invasive breast carcinoma (IBC) using tissue microarray. Immunohistochemical staining for CD8, CD57, and CD1a and methylation-specific polymerase chain reaction were also performed in IBC. Reduced MUC4 expression in IBC was significantly higher than in usual ductal hyperplasia and ductal carcinoma in situ (P<0.001 and P<0.01, respectively). Reduced MUC4 expression in IBC was significantly correlated with promoter hypermethylation (P<0.05). No association between MUC4 expression and patient outcomes was identified. Intratumoral CD8 T cells and stromal CD57 natural killer cells were significantly increased in the reduced MUC4 expression group compared with those in the normal expression group (P<0.01 and P<0.05, respectively). Our results suggest that tumor progression in breast epithelium is accompanied by reduced MUC4 protein expression. Reduced MUC4 expression correlates with increased tumor-infiltrating CD8 T and NK cells as well as promoter hypermethylation in IBC.

  16. Phase 1 Study of Preoperative Chemoradiation Therapy With Temozolomide and Capecitabine in Patients With Locally Advanced Rectal Cancer

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jeong, Jae Ho; Hong, Yong Sang; Park, Yangsoon

    Purpose: Preoperative chemoradiation therapy (CRT) with capecitabine is a standard treatment strategy in patients with locally advanced rectal cancer (LARC). Temozolomide improves the survival of patients with glioblastoma with hypermethylated O{sup 6}-methylguanine DNA methyltransferase (MGMT); MGMT hypermethylation is one of the colorectal carcinogenesis pathways. We aimed to determine the dose-limiting toxicity (DLT) and recommended dose (RD) of temolozomide in combination with capecitabine-based preoperative CRT for LARC. Methods and Materials: Radiation therapy was delivered with 45 Gy/25 daily fractions with coned-down boost of 5.4 Gy/3 fractions. Concurrent chemotherapy comprised fixed and escalated doses of capecitabine and temozolomide, respectively. The MGMT hypermethylation was evaluatedmore » in pretreatment tumor samples. This trial is registered with (ClinicalTrials.gov) with the number (NCT01781403). Results: Twenty-two patients with LARC of cT3-4N0 or cT{sub any}N1-2 were accrued. Dose level 3 was chosen as the RD because DLT was noticeably absent in 10 patients treated up to dose level 3. An additional 12 patients were recruited in this group. Grade III adverse events were noted, and pathologic complete response (pCR) was observed in 7 patients (31.8%); MGMT hypermethylation was detected in 16. The pCR rate was 37.5% and 16.7% in the hypermethylated and unmethylated MGMT groups, respectively (P=.616). Conclusions: There was a tendency toward higher pCR rates in patients with hypermethylated MGMT. Future randomized studies are therefore warranted.« less

  17. Abnormal pressures as hydrodynamic phenomena

    USGS Publications Warehouse

    Neuzil, C.E.

    1995-01-01

    So-called abnormal pressures, subsurface fluid pressures significantly higher or lower than hydrostatic, have excited speculation about their origin since subsurface exploration first encountered them. Two distinct conceptual models for abnormal pressures have gained currency among earth scientists. The static model sees abnormal pressures generally as relict features preserved by a virtual absence of fluid flow over geologic time. The hydrodynamic model instead envisions abnormal pressures as phenomena in which flow usually plays an important role. This paper develops the theoretical framework for abnormal pressures as hydrodynamic phenomena, shows that it explains the manifold occurrences of abnormal pressures, and examines the implications of this approach. -from Author

  18. Amyloid protein-mediated differential DNA methylation status regulates gene expression in Alzheimer's disease model cell line

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sung, Hye Youn; Choi, Eun Nam; Ahn Jo, Sangmee

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Genome-wide DNA methylation pattern in Alzheimer's disease model cell line. Black-Right-Pointing-Pointer Integrated analysis of CpG methylation and mRNA expression profiles. Black-Right-Pointing-Pointer Identify three Swedish mutant target genes; CTIF, NXT2 and DDR2 gene. Black-Right-Pointing-Pointer The effect of Swedish mutation on alteration of DNA methylation and gene expression. -- Abstract: The Swedish mutation of amyloid precursor protein (APP-sw) has been reported to dramatically increase beta amyloid production through aberrant cleavage at the beta secretase site, causing early-onset Alzheimer's disease (AD). DNA methylation has been reported to be associated with AD pathogenesis, but the underlying molecular mechanism of APP-sw-mediated epigenetic alterationsmore » in AD pathogenesis remains largely unknown. We analyzed genome-wide interplay between promoter CpG DNA methylation and gene expression in an APP-sw-expressing AD model cell line. To identify genes whose expression was regulated by DNA methylation status, we performed integrated analysis of CpG methylation and mRNA expression profiles, and identified three target genes of the APP-sw mutant; hypomethylated CTIF (CBP80/CBP20-dependent translation initiation factor) and NXT2 (nuclear exporting factor 2), and hypermethylated DDR2 (discoidin domain receptor 2). Treatment with the demethylating agent 5-aza-2 Prime -deoxycytidine restored mRNA expression of these three genes, implying methylation-dependent transcriptional regulation. The profound alteration in the methylation status was detected at the -435, -295, and -271 CpG sites of CTIF, and at the -505 to -341 region in the promoter of DDR2. In the promoter region of NXT2, only one CpG site located at -432 was differentially unmethylated in APP-sw cells. Thus, we demonstrated the effect of the APP-sw mutation on alteration of DNA methylation and subsequent gene expression. This epigenetic regulatory

  19. Suppression of the heterotrimeric G protein causes abnormal morphology, including dwarfism, in rice

    PubMed Central

    Fujisawa, Yukiko; Kato, Teruhisa; Ohki, Shizuka; Ishikawa, Atsushi; Kitano, Hidemi; Sasaki, Takuji; Asahi, Tadashi; Iwasaki, Yukimoto

    1999-01-01

    Transgenic rice containing an antisense cDNA for the α subunit of rice heterotrimeric G protein produced little or no mRNA for the subunit and exhibited abnormal morphology, including dwarf traits and the setting of small seeds. In normal rice, the mRNA for the α subunit was abundant in the internodes and florets, the tissues closely related to abnormality in the dwarf transformants. The position of the α-subunit gene was mapped on rice chromosome 5 by mapping with the restriction fragment length polymorphism. The position was closely linked to the locus of a rice dwarf mutant, Daikoku dwarf (d-1), which is known to exhibit abnormal phenotypes similar to those of the transformants that suppressed the endogenous mRNA for the α subunit by antisense technology. Analysis of the cDNAs for the α subunits of five alleles of Daikoku dwarf (d-1), ID-1, DK22, DKT-1, DKT-2, and CM1361–1, showed that these dwarf mutants had mutated in the coding region of the α-subunit gene. These results show that the G protein functions in the formation of normal internodes and seeds in rice. PMID:10377457

  20. Maternal Gametic Transmission of Translocations or Inversions of Human Chromosome 11p15.5 Results in Regional DNA Hypermethylation and Downregulation of CDKN1C Expression

    PubMed Central

    Smith, Adam C.; Suzuki, Masako; Thompson, Reid; Choufani, Sanaa; Higgins, Michael J.; Chiu, Idy W.; Squire, Jeremy A.; Greally, John M.; Weksberg, Rosanna

    2015-01-01

    Beckwith-Wiedemann syndrome (BWS) is an overgrowth syndrome associated with genetic or epigenetic alterations in one of two imprinted domains on chromosome 11p15.5. Rarely, chromosomal translocations or inversions of chromosome 11p15.5 are associated with BWS but the molecular pathophysiology in such cases is not understood. In our series of 3 translocation and 2 inversion patients with BWS, the chromosome 11p15.5 breakpoints map within the centromeric imprinted domain, 2. We hypothesized that either microdeletions/microduplications adjacent to the breakpoints could disrupt genomic sequences important for imprinted gene regulation. An alternate hypothesis was that epigenetic alterations of as yet unknown regulatory DNA sequences, result in the BWS phenotype. A high resolution Nimblegen custom microarray was designed representing all non-repetitive sequences in the telomeric 33 MB of the short arm of human chromosome 11. For the BWS-associated chromosome 11p15.5 translocations and inversions, we found no evidence of microdeletions/microduplications. DNA methylation was also tested on this microarray using the HpaII tiny fragment enrichment by ligation-mediated PCR (HELP) assay. This high-resolution DNA methylation microarray analysis revealed a gain of DNA methylation in the translocation/inversion patients affecting the p-ter segment of chromosome 11p15, including both imprinted domains. BWS patients that inherited a maternal translocation or inversion also demonstrated reduced expression of the growth suppressing imprinted gene, CDKN1C in Domain 2. In summary, our data demonstrate that translocations and inversions involving imprinted domain 2 on chromosome 11p15.5, alter regional DNA methylation patterns and imprinted gene expression in cis, suggesting that these epigenetic alterations are generated by an alteration in “chromatin context”. PMID:22079941