Significant Differences In Methylation Status Research Articles

Association of promoter methylation status of NRF2 and PNPLA3 genes in alcoholic liver disease.

Alcoholic liver disease (ALD) is the leading cause of chronic liver disease. In the liver, metabolism of alcohol occurs through multiple mechanisms and it results in the generation of various toxic products. Multiple genetic causes have been identified that are associated with the development and progression of ALD. The present study assessed the promoter site methylation status ofnuclear factor erythroid 2-related factor 2 (NRF2) and patatin-like phospholipase domain-containing protein-3 (PNPLA3) genes in different subgroups of ALD. The patients recruited were cases of alcohol dependence syndrome with hepatic dysfunction, compensated cirrhosis, decompensated cirrhosis, and acute-on-chronicliver failure due to alcohol as an etiology along with healthy control subjects. Routine biochemical investigations were performed along with methylation-specific polymerase chain reaction (MS-PCR) to qualitatively assess the promoter methylation status of NRF2 and PNPLA3 in all these cases. There was significant difference in methylation status of NRF2 gene in ALD when compared to healthy controls but there was no such difference in PNPLA3. All biochemical and clinical parameters studied were significantly different in subgroups of ALD except the serum aspartate aminotransferase (AST) level. Subgroups of ALD did not show any significant association with NRF2 or PNPLA3 methylation status. Gamma-glutamyl transferase (GGT) and creatinine levels in serum were significantly associated with the methylation status of NRF2 gene while no such association was seen with PNPLA3 gene. Model for end-stage liver disease (MELD) score varied differentially with NRF2 methylation and PNPLA3 methylation but there was no statistical significance. The present study showed that methylation status of NRF2 and PNPLA3 genes could not differentiate between subgroups of alcoholic liver diseases. However, the unmethylation of NRF2 promoter is associated with higher serum levels of GGT.

DNA Methylation Patterns in the HLA-DPB1 and PDCD1LG2 Gene Regions in Patients with Autoimmune Thyroiditis from Different Water Iodine Areas.

Background: Epigenetic disorders play an important role in the pathogenesis of autoimmune thyroiditis (AIT). Therefore, the study of the possible role of DNA methylation in AIT is of great significance to explore the pathogenesis of AIT. Methods: From May 2019 to June 2019, whole blood samples were collected from 176 AIT patients and 176 controls from different water iodine levels in Shandong Province, China. We used the Illumina Methylation 850K BeadChip to determine significant differences in methylation status of genes and used the MethylTarget™ assay to verify the methylation level in 176 cases and 176 controls. The relative mRNA levels of genes were detected by quantitative real-time-polymerase chain reaction. Results: There were multiple differential methylation sites in the HLA-DPB1 and PDCD1LG2 genes between the case and control population with different water iodine levels. Some target regions of HLA-DPB1 and PDCD1LG2 genes were negatively correlated with relative mRNA expression in the case and control populations and with different water iodine levels. Conclusions: There is differential methylation status in genomic DNA in patients with AIT. The methylation patterns of HLA-DPB1 and PDCD1LG2 genes related to cell adhesion molecule pathway may be different based on different water iodine levels. HLA-DPB1 and PDCD1LG2 genes related to the cell adhesion molecules pathway may play a role in the development of AIT. This study is registered with Chinese Clinical Trial Registry, www.chictr.org.cn, number ChiCTR2000039105.

Associations Between Physical Effort and DNA Methylation in the Promotor Region of the Dopamine Transporter Gene (DAT1).

The purpose of this study was to investigate the association between physical effort and DNA methylation in the promoter region of the dopamine transporter gene (DAT1). The research group included 100 athletes (mean age = 22.88, SD = 6.35), whereas the control group were 239 healthy male volunteers matched for age (mean age = 21.69, SD = 3.39). Both, the control and the research group, included individuals with Caucasian origin from the same region of Poland. DNA was extracted from peripheral blood leukocytes using a DNA isolation kit (A&A Biotechnology, Gdynia, Poland). Bisulfite modification of 250 ng DNA was performed using the EZ DNA Methylation Kit (Zymo Research, Orange, CA, USA), according to manufacturer's instructions. The methylation-specific PCR assay was carried out in a Mastercycler epgradient S (Eppendorf, Germany). We observed that the level of general methylation of the CpG island was similar for both groups. Further exploration of individual CpG sites allowed to notice that there were significant differences in methylation status in specific positions. Nonetheless, there was no rule that would indicate either higher or lower methylation of individual sites, four of them were methylated at a higher level (positions 1, 4, 5, 7, 8, 9, 10, 11, 12, 13, 16, 17, 18, 23, 25, 26, 27, 29 and 30), while one showed an inverse trend (position 3). More precise analysis with the usage of Bonferroni correction for multiple tests indicated that differences in CpG site methylation were mainly increased in several positions and decreased in position 3.

Abstract 831: Hypermethylation in promoter regions of CpG islands of multiple genes is associated with disease progression and molecular response in chronic myelogenous leukemia

Abstract Chronic myelogenous leukemia (CML) has a typical progressive course with transition from a less aggressive chronic phase to a terminal more aggressive blast crisis. The molecular mechanisms leading to CML disease progression remain to be elucidated. However, it has been proposed in many studies that alterations in the methylation status of various genes may contribute to the progression of different malignancies. Hence, in this study we tried to understand the role of aberrant methylation of six tumor suppressor genes in the disease progression and molecular response in CML. We investigated 100 CML patients and 100 age and sex matched healthy controls for the methylation status of six tumor suppressor genes. The methylation status of these genes was evaluated by MS-PCR. In this study, 100 CML samples and 100 controls were analyzed for the DAPK1, RASSF1A, RIZ-1, p16, p14ARF and SOCS-1 promoter methylation status. These genes, except p14ARF and SOCS-1 showed significant differences in methylation status among CML patients and healthy controls. Also DAPK1, RASSF1A, RIZ-1, p16 and p14ARF genes were observed to be hypermethylated during progression of CML disease to advanced phases. The methylation frequencies of these five genes were seen to increase progressively during CML disease progression. This increase in hypermethylation of these five genes from was statistically significant with a p-value of <0.0001, 0.001, 0.009,0.002 and 0.01 respectively. Although, there was a progressive increase in the promoter methylation of SOCS1 gene during the CML disease progression (CP= 4%, AP=8% and BC=16%) but this did not reached a statistical significance (p-value 0.1). We also assessed the molecular response of these 100 CML patients after imatinib therapy and the patients were categorized into two groups; major molecular response group and loss of molecular response group. Out of 100 CML patients on imatinib therapy 52 achieved major molecular response while 48 patients showed loss of molecular response. Out of the above studied genes RASSF1 and RIZ1 promoter hypermethylation was significantly associated with loss of molecular response in CML patients receiving imatininb therapy. Out of 48 CML patients who were in loss of molecular response, 23.0% and 16.77% showed promoter hypermethylation of RASSF1 and RIZ1 genes respectively while out of 52 patients that were in major molecular response only 9.6% and 1.92% of patients showed hypermethylation in RASSF1 and RIZ1 genes respectively. These results suggest that hypermethylation of tumor suppressor genes plays a significant role in the progression and response in CML. Note: This abstract was not presented at the meeting. Citation Format: Mamta P. Sumi, Sameer A. Guru, Imtiyaz A Najar, Mariyam Zuberi, N. Gupta, Alpana Saxena. Hypermethylation in promoter regions of CpG islands of multiple genes is associated with disease progression and molecular response in chronic myelogenous leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 831.

Identification of genes and pathways, including the CXCL2 axis, altered by DNA methylation in hepatocellular carcinoma.

Recent genetic studies have suggested that tumor suppressor genes are often silenced during carcinogenesis via epigenetic modification caused by methylation of promoter CpG islands. Here, we characterized genes inactivated by DNA methylation in human hepatocellular carcinoma (HCC) to identify the genes and pathways involved in DNA methylation in hepatocellular carcinoma. Eight HCC-derived cell lines were treated with a DNA demethylating agent, 5-aza-2'-deoxycytidine. Additionally, 100 pairs of primary HCC and adjacent non-cancerous tissues as well as 15 normal liver tissues were analyzed by comprehensive gene expression analysis using microarrays. Moreover, gene set enrichment analysis identified the major molecular pathways associated with DNA methylation. Validation of gene expression and DNA methylation status was performed by real-time PCR after bisulfite modification. We showed that CXCL2, an immune-related chemokine, expression was significantly downregulated in tumor tissues and also significantly upregulated by DAC treatment in cell lines. Furthermore, we observed a statistically significant difference in methylation status between normal liver tissues and tumor tissues (P < 0.05). In addition, tumors with higher CXCL2 expression included significantly more numbers of multiple tumors than the lower expression group. We identified CXCL2, an immune-related chemokine, decreased in hepatocellular carcinoma and the regulation mechanism may be controlled by methylation. Further studies should be warranted to examine if and to what extent the gene is actually suppressed by methylation and if there is a possibility that the CXCL2 axis plays a role for diagnosis and treatment of hepatocellular carcinoma.

Epigenetic regulation of inflammation in localized aggressive periodontitis

BackgroundWe have previously demonstrated a Toll-like receptor (TLR)-mediated hyper-responsive phenotype in our cohort of localized aggressive periodontitis (LAP) individuals. However, mechanisms related to this phenotype are still not clear in the literature. The objective of this cross-sectional study is to examine the role of epigenetic regulation, specifically DNA methylation status of genes in the TLR pathway in this cohort. Peripheral blood was collected from 20 LAP patients and 20 healthy unrelated controls. Whole blood was stimulated with 1 μl (100 ng/μl) of purified Escherichia coli lipopolysaccharide (LPS) for 24 h and cyto/chemokines in the supernatants analyzed by Luminex multiplex assays. Genomic DNA extracted from buffy coats prepared from a second tube of whole blood was used for DNA methylation analysis by pyrosequencing of seven TLR signaling genes (FADD, MAP3K7, MYD88, IL6R, PPARA, IRAK1BP1, RIPK2).ResultsSignificant differences in the methylation status were observed at specific CpG positions in LAP patients compared to healthy controls and interestingly also between severe and moderate LAP. Specifically, subjects with moderate LAP presented hypermethylation of both the upregulating (MAP3K7, MYD88, IL6R, and RIPK2) and downregulating (FADD, IRAK, and PPARA) genes, while severe LAP presented hypomethylation of these genes. Further analysis on CpG sites with significant differences in methylation status correlates with an increased pro-inflammatory cytokine profile for LAP patients.ConclusionsOur findings suggest that epigenetic modifications of genes in the TLR pathway may orchestrate the thresholds for balancing induction and prevention of tissue destruction during the course of disease, and thus differ significantly at different stages of the disease, where moderate LAP shows hypermethylation and severe LAP shows hypomethylation of several genes.Trial registrationhttps://clinicaltrials.gov, NCT01330719

Detection of aberrant methylated SEPT9 and NTRK3 genes in sporadic colorectal cancer patients as a potential diagnostic biomarker.

Colorectal cancer (CRC) is one of the most common malignancies, and the third leading cause of cancer mortality worldwide. Timely detection of CRC in patients with earlier stages provides the highest rate of survival. Epigenetic alterations are important in the occurrence and progression of CRC, and represent the primary modifications of cancer cells. Therefore, detection of these alterations in CRC cases are thought to hold great promise as diagnostic biomarkers. It has been shown that the SEPT9 and NTRK3 genes are aberrantly methylated and their detection can be used as biomarkers for early diagnosis of CRC. The present study analyzed promoter methylation status of these genes in CRC patients. Genomic DNA was extracted from 45 CRC and paired adjacent healthy tissues and undergone bisulfite conversion, and the methylation status of NTRK3 and SEPT9 were defined using the MS-HRM assay. Our results showed that there are statistically significant differences in methylation status of NTRK3 and specially SEPT9 between CRC and adjacent normal tissues (P<0.001). High sensitivity and specificity for a specific location in SEPT9 gene promoter as a diagnostic biomarker was observed. SEPT9 promoter hypermethylation may serve as a promising biomarker for the detection of CRC development. However, to validate the biomarker potential of NTRK3 there is a requirement for further investigation.

Type 3 iodothyronine deiodinase in neonatal goats: molecular cloning, expression, localization, and methylation signature.

Type 3 iodothyronine deiodinase (DIO3) is an important enzyme in the metabolism of thyroid hormones. It plays critical roles in fetal development and neonatal growth and is especially important for brain development in mammals. In the present study, we profiled the expression pattern and methylation signature of the DIO3 gene in goats. The complete coding sequence of caprine DIO3 encoded a protein of 301 amino acids and harbored an internal selenocysteine-encoding TGA codon. The DIO3 messenger RNA (mRNA) was predominantly expressed in the neonatal goat liver (P < 0.01), while expression in other tissues was quite low, with the lowest levels in the lung. In in situ hybridization, the DIO3 mRNA was predominantly localized in the liver and the lowest content was detected in the lung. The DIO3 transcript was widely localized in neurons and the neuropil. Methylation profiling of the DIO3 CpG island showed a significant difference between the 5' region (CpGs_1∼24) and the 3' region (CpG_25∼51) of the coding region. Furthermore, no significant difference in methylation status was observed among the six tested tissues with levels in the range of 29.11-33.12%. The CpG islands in the intergenic-differentially methylated region (IG-DMR) showed significantly different methylated levels among tissues, and the highest methylated level was observed in lung (CpG island 1, 69.34%) and longissimus dorsi (LD) (CpG island 2, 52.62%) tissues. Our study lays a foundation for understanding DIO3 function and the diseases caused by altered methylation profiles of the DIO3 gene.

Abstract A32: NISCH promoter hypermethylation, smoking and lung cancer: A case control study

Abstract Lung cancer is the most common cancer worldwide, both in terms of incidence and mortality. The high mortality of lung cancer is attributable to the presence of metastatic disease in nearly two thirds of patients at diagnosis. Detection of early stage lung cancer amenable to curative resection could potentially result in a manifold increase in survival rates. Epigenetic alterations seemingly contribute to cancer initiation and progression. Cancer specific methylation patterns of tumor suppressor genes, which precede precursor lesions, could possibly herald earlier diagnosis of lung cancer and may even have important preventive or therapeutic implications. Our aim was to evaluate the promoter hypermethylation of tumor suppressor gene NISCH in cfDNA from plasma of lung cancer patients and its possible correlation with smoking status and various clinicopathological parameters. NISCH codes for a nonadrenergic imidazoline-1 receptor protein nischarin that anchors to the inner layer of the plasma membrane. The orthologous mouse protein has been shown to bind alpha-5 beta-1 integrin and influence cytoskeletal organization. Forty histopathologically confirmed lung cancer cases were enrolled along with thirty smoker and thirty nonsmoker controls. Plasma cfDNA was extracted and subjected to bisulfite treatment followed by conventional methylation specific PCR. DNA methylated using CpG methylase by the whole genome methylation protocol, was used as positive control and Nuclease free PCR grade water was used as negative control. The serum nischarin levels were estimated using commercially available ELISA kits. Statistical analysis was performed using SPSS 22.0 software. We observed that frequency of promoter hypermethylation of tumor suppressor NISCH was significantly higher in lung cancer patients and in non-cancerous smokers as compared to lifelong nonsmoker controls. This difference in methylation status of NISCH between lung cancer cases, smoker and nonsmoker controls was found to be significant (P&amp;lt;0.05). There was no significant association between methylation status of tumor suppressor gene NISCH and clinicopathologic variables- staging, tumor size, lymph node status, metastasis and histopathological grading. We observed a higher frequency of NISCH methylation in adenocarcinoma as compared to Squamous Cell Carcinoma. Methylation status of tumor suppressor gene NISCH was found to be significantly higher among smokers as compared to nonsmokers. But there was no significant difference in the frequency of NISCH methylation between smoker lung cancer patients and nonsmoker lung cancer patients. There was no significant difference in methylation status of NISCH with type or duration of smoking. The pack years and packs per day were significantly higher in those with methylated NISCH as compared to the unmethylated group. There was no significant association of serum nischarin levels with methylation status of NISCH, demographic variables or any clinicopathological parameters. Our findings suggest that NISCH methylation occurs in high frequencies in cfDNA from plasma of lung cancer patients. Though it was not found to correlate with stage, tumor size, lymph node status, metastases or histological grade, it was found to be significantly associated with adenocarcinomas in our study. Hence, NISCH methylation could serve as a part of blood based biomarker panel for early diagnosis of lung cancer. In addition, since NISCH is found to be highly methylated in both high risk heavy smoker controls as well as lung cancer cases irrespective of smoking status, it can be hypothesized that NISCH methylation may be the common primogenitor at which varied etiologies for lung cancer converge. Hence, in the future, it may be investigated as a universal therapeutic target for lung cancers regardless of clinicopathological heterogeneity. Citation Format: Kritika Krishnamurthy, TK Mishra, Alpana Saxena, MK Daga, Nita Khurana, Masroor Mirza. NISCH promoter hypermethylation, smoking and lung cancer: A case control study. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A32.

Association between DNA hypomethylation at IL13 gene and allergic rhinitis in house dust mite‐sensitized subjects

Allergic rhinitis (AR) is a complex disease, in which gene-environment interactions contribute to its pathogenesis. Epigenetic modifications such as DNA methylation play an important role in the regulation of gene function. As IL13, a pleiotropic cytokine, may be important in conferring susceptibility to AR, the aim of the present work was to assess the relationship between a CpG island methylation status at the upstream of IL13 gene and house dust mite (HDM)-sensitized AR in Han Chinese subjects. A total of 60 patients with HDM-sensitized AR and 65 control subjects were enrolled as two independent cohorts from Beijing and Liaoning. MassARRAY EpiTYPER and pyrosequencing was used to systematically screen the status of DNA methylation in peripheral blood leucocytes. IL13 mRNA expression was measured by real-time quantitative PCR. Electrophoretic mobility shift assay was used to assess the function of methylation site. The mean level of methylation was decreased in the AR patient group compared with the control group (P=0.01). Two of a total of 33 IL13CpG units analysed (CpG units 24:25:26 and 38:39) showed significant differences in methylation status between the AR patient group and the control group, with DNA hypomethylation at CpG38 significantly associated with higher risk of HDM-sensitized AR in both independent cohorts and a combined cohort (Beijing: OR=1.24, 95%CI=1.01-1.52, P=0.036; Liaoning: OR=1.62, 95%CI=1.11-2.38, P=0.013; Combined: OR=1.31, 95%CI=1.10-1.56, P=0.002). Methylation level of CpG38 correlated negatively with both IL13 mRNA expression and serum total IgE level and affected the binding affinity of SP1. DNA hypomethylation of IL13 gene may be associated with increased risk of AR from HDM sensitization.

Patterns of DNA Methylation and Gene Expression in Higher-Risk Myelodysplastic Syndromes: Predictors of Demethylating Therapy Response

Background:Standard therapies for patients with higher-risk myelodysplastic syndromes (MDS) include the hypomethylating agents as azacitidine (AZA) and decitabine, of which they benefit significantly. However, the therapy is effective only in 60% of patients. The aim of the project was to identify patients in whom demethylating therapy might be appropriate.Methods:We performed gene expression and methylation profiling in parallel in CD34+ cells from 33 MDS patients before AZA treatment. Control group included 10 healthy individuals. HumanMethylation27 BeadChips and HumanHT-12 v4 Expression BeadChips (Illumina) were used to generate the profiles. DNA and RNA were isolated from the same CD34+ cells separated from bone marrow by magnetic beads. The expression data were preprocessed in lumi package. T-test was applied for differential expression testing. The method of Holm was employed to adjust p-values for multiple comparisons. COHCAP package was used to study differential methylation of the individual genome sites. Beta-values of statistics were considered.Results:We used gene expression and methylation profiling methods to identify predictors of therapy response and prognosis. Overall, 10 patients (30.3%) showed progression (PD), 9 (27.3%) had stable disease (SD), and 14 (42.4%) achieved complete (CR) or partial remission (PR). There were no differences between clinical parameters of these groups before administration of the treatment. The main efficacy indicator of the therapy was duration of overall survival, CR was not the main criterion. Therefore, we divided the patients into two groups for further analysis: patients with and without disease progression. Median survival after initiation of AZA treatment in PD was 11.1 months and in other groups was 25.9 months (Fig. 1). Comparison of gene expression levels between the groups revealed 110 differentially expressed probes (FC >1.5; p<0.05) related to following biological processes (according to the DAVID database): immune response (14 genes), cellular response to stress (12), DNA metabolic process (10), and response to DNA damage stimulus (9).We found affected Mismatch repair pathways (p<0.05). Among the most deregulated genes were F13A1, AR, TM4SF1 and BIRC3 withincreased expression and CXCR4, IRF8, LY86 and FABP4 genes with decreased expression (Fig. 2). Between the patient groups, we found significant differences in methylation status only in 5 genes (p<0.05), namely CLP24, LILRA4, KCNS2, DAAM2 and DBC1 genes. None of the genes showed a negative correlation between the expression and methylation status. Most of these deregulated genes are associated with a worse prognosis in AML.Conclusions:Although the data in our study still require a more comprehensive examination in a larger confirmative cohort of patients, the genomic signatures we detected before hypomethylation therapy may help to stratify patients regarding their response to treatment and thus optimize use of hypomethylating agents in order to achieve maximal benefit. Based on the altered expression and methylation status of AML-related genes, we assume that if a certain degree of the progression process has already started in the patient with MDS, azacitidine therapy is no longer effective.Supported by grant (NT/13899, NT/14377, NT/14539, NT/13847), the project for conceptual development of research organization (00023736) MHCZ and CZ.2.16/3.1.00/28007. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

MALDI-TOF Mass Array Analysis of Nell-1 Promoter Methylation Patterns in Human Gastric Cancer.

Mass spectrometry (MS) enables rapid and sensitive qualitative and quantitative analyses of biomolecules (proteins, peptides, oligosaccharides, lipids, DNA, and RNA), drugs, and metabolites. MS has become an essential tool in modern biomedical research, including the analysis of DNA methylation. DNA methylation has been reported in many cancers, suggesting that it can be utilized as an early biomarker to improve the early diagnosis rate. Using matrix-assisted laser desorption/ionization time-of-flight MS and MassCLEAVE reagent, we compared Nell-1 hypermethylation levels among tumor tissues, paracarcinoma tissues, and normal tissues from gastric cancer patients. Almost 80% of the CpG sites in the amplicons produced were covered by the analysis. Our results indicate a significant difference in methylation status between gastric cancer tissue (a higher level) and normal tissue. The same trend was identified in gastric cancer tissue versus paracarcinoma tissue. We also detected lower relative expression of Nell-1 by real-time PCR. Furthermore, immunohistochemical analyses revealed that Nell-1 staining was less intense in cancer tissue relative to normal tissue and that the tumor cells had spread to the muscle layer. These findings may serve as a guide for the early diagnosis of gastric cancer.

Association of promoter methylation statuses of congenital heart defect candidate genes with Tetralogy of Fallot

BackgroundAlthough a lower methylation level of whole genome has been demonstrated in Tetralogy of Fallot (TOF) patients, little is known regarding changes in specific gene DNA methylation profiles and the possible associations with TOF. In current study, the promoter methylation statuses of congenital heart defect (CHD) candidate genes were measured in order to further understand epigenetic mechanisms that may play a role in the development of TOF.MethodsThe methylation levels of CHD candidate genes were measured using the Sequenom MassARRAY platform. QRT-PCR was used to analyze the mRNA levels of CHD candidate genes in the right ventricular myocardium of TOF cases and normal controls.ResultsMethylation status analysis was performed on the promoter regions of 71 CHD candidate genes (113 amplicons). We found significant differences in methylation status, between TOF cases and controls, in 26 amplicons (26 genes) (p < 0.05). Of the 26 amplicons, 17 were up regulated and 9 were down regulated. Additionally, 14 of them were located in the CpG islands, 7 were located in the CpG island shores, and 5 were covering the regions near the transcription start site (TSS). The methylation status was subsequently confirmed and mRNA levels were measured for 7 represented candidate genes, including EGFR, EVC2, NFATC2, NR2F2, TBX5, CFC1B and GJA5. The methylation values of EGFR, EVC2, TBX5 and CFC1B were significantly correlated with their mRNA levels (p < 0.05).ConclusionsAberrant promoter methylation statuses of CHD candidate genes presented in TOF cases may contribute to the TOF development and have potential prognostic and therapeutic significance for TOF disease.

Comparison of DNA Methylation and Expression Status Prior Azacytidine Treatment and their Relationship to Overall Survival and Clinical Response of MDS Patients

BackgroundMyelodysplastic syndromes (MDS) are clonal disorders of hematopoietic stem cells characterized by ineffective hematopoiesis. High-risk MDS patients are treated by hypomethylating agents, of which they benefit significantly. However, only half of the patients respond positively to the treatment. Aberrant DNA methylation and mRNA expression in MDS were documented in several studies, but their prognostic impact in response to hypomethylating therapy is still unclear. The aim of the project was to find a relationship between methylation and expression status prior to azacytidine (AZA) treatment and the overall survival and clinical response of MDS patients. MethodsWe performed methylation and expression profiling in CD34+ cells from 30 samples from MDS patients before AZA treatment and after 4-8 treatment cycles. HumanMethylation27 BeadChips and HumanHT-12 v4 Expression BeadChips (Illumina) were used to generate profiles. DNA and RNA were isolated from same CD34+ cells separated from bone marrow by magnetic beads. The β-values represent quantitative measurements of DNA methylation levels of specific CpGs, and range from 0 for completely unmethylated to 1 for completely methylated DNA. The nonparametric Mann-Whitney test was used for comparison of β-values and expression levels between responders and nonresponders. ResultsTo determine whether DNA methylation and expression might predict a response to AZA treatment, we compared methylation and expression status at baseline with clinical responses in 30 MDS patients. Twelve patients of 30 (40%) achieved complete remission or partial remission, 10 had stable disease (33.3%), and 8 showed progression (26.7%). Median survival after initiation of AZA treatment in progression patient group was 8.7 months, stable group 21.2 months, and group with complete or partial remission 24.5 months. We found significant differences in methylation status in 20 genes (p<0.05) between groups of responders and nonresponders and the largest methylation differences showed CALCA (0.61 vs. 0.16, p<0.05), MAGEE2 (0.71 vs. 0.30, p<0.05), HMP19 (0.62 vs. 0.23, p<0.05), MEOX1 (0.36 vs. 0.84, p<0.05), and KCNQ1DN genes (0.33 vs. 0.84, p<0.05). The aberrant expression status did not correlate with the response to AZA. We also measured methylation changes caused by AZA treatment. In the group of patients with progression, we did not find any change in the methylation profile after treatment. On the contrary, we found significant methylation changes after AZA treatment in the group of patients responding to treatment (e.g. AMT, NOTCH, and WT1genes). ConclusionsOur finding of different DNA methylation levels at baseline between groups of responders and nonresponders as well as detection of decreased methylation after AZA treatment in the group of patients with clinical response may represent useful prediction markers of treatment success. However, the data require detailed examination along with confirmative cohort of patients.Supported by grant (NT/13899, NT/14377, NT/14539, NT/13847) and the project for conceptual development of research organization (00023736) from the Ministry of Health of the Czech Republic. Disclosures:No relevant conflicts of interest to declare.

Generation of Corneal Epithelial Cells from Induced Pluripotent Stem Cells Derived from Human Dermal Fibroblast and Corneal Limbal Epithelium

Induced pluripotent stem (iPS) cells can be established from somatic cells. However, there is currently no established strategy to generate corneal epithelial cells from iPS cells. In this study, we investigated whether corneal epithelial cells could be differentiated from iPS cells. We tested 2 distinct sources: human adult dermal fibroblast (HDF)-derived iPS cells (253G1) and human adult corneal limbal epithelial cells (HLEC)-derived iPS cells (L1B41). We first established iPS cells from HLEC by introducing the Yamanaka 4 factors. Corneal epithelial cells were successfully induced from the iPS cells by the stromal cell-derived inducing activity (SDIA) differentiation method, as Pax6+/K12+ corneal epithelial colonies were observed after prolonged differentiation culture (12 weeks or later) in both the L1B41 and 253G1 iPS cells following retinal pigment epithelial and lens cell induction. Interestingly, the corneal epithelial differentiation efficiency was higher in L1B41 than in 253G1. DNA methylation analysis revealed that a small proportion of differentially methylated regions still existed between L1B41 and 253G1 iPS cells even though no significant difference in methylation status was detected in the specific corneal epithelium-related genes such as K12, K3, and Pax6. The present study is the first to demonstrate a strategy for corneal epithelial cell differentiation from human iPS cells, and further suggests that the epigenomic status is associated with the propensity of iPS cells to differentiate into corneal epithelial cells.

Abstract 4924: Gene-specific hyper and hypomethylation in follicular lymphoma progression

Abstract Epigenetic mechanisms involved in the origin and transformation of indolent lymphomas to aggressive forms are poorly understood. Based on the comparison of methylation states of follicular lymphoma (FL) pre and post- transformation, it has been suggested that DNA methylation changes occur during the early stages of lymphomagenesis. To obtain a more complete understanding of methylation aberrations responsible for progression and transformation of FL, we analyzed 20 untreated FL biopsies (10 low grade, 10 high grade), 5 diffuse large B cell lymphoma (DLBCL) biopsies, and 5 benign reactive lymph node (RLN) biopsies by using methyl-DNA immunoprecipitation coupled with analysis with Agilent 244K human promoter ChIP-on-chip microarrays. Our analysis revealed statistically significant differences in methylation status for 120 genes between FL samples and RLN samples and for 23 genes between low and high grade FL samples. To investigate the biological significance of these findings, we used DAVID functional annotation clustering algorithms and Gominer software. We found that the most methylation-enriched functional groups were comprised of genes with roles in cell cycle control, adhesion and motility, transcription regulation, homeobox, and developmental genes. In order to validate the DNA methylation differences of the significant gene regions, we utilized the MassARRAY EpiTYPER assay. Our data identified 13 genes (CYP26B1, GRM7, HS3ST2, DOCK5, GRB10, SST, TAC1, PALM2-AKAP, FLJ40125, ST6GAL2, WDR35, NCAM1, SATB2) with increased levels of DNA methylation in FL samples compared to controls; 3 of these genes (ST6GAL2, WDR35, NCAM1) exhibited increased methylation in high grades FLs and 1 gene (SATB2) in low grades FLs. We are currently investigating gene specific DNA hypomethylation in low versus high grade FL and FL versus DLBCL. Our data are the first to characterize the global changes in methylation that occur in FL progression. Our findings suggest that high grade FL are associated with hypermethylation of a specific subset of genes compared to low grade subtypes. Ongoing efforts include investigating the functional relevance of these methylation changes by treating, in vitro, FL cell lines with demethylating agents to evaluate changes in gene expression levels and correlating with phenotype alterations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4924.

Effect of IVF and laser zona dissection on DNA methylation pattern of mouse zygotes

In vitro fertilization (IVF) and zona pellucida laser microdissection-facilitated IVF (Laser-IVF) are presently routine procedures in human assisted reproduction. The safety of these methods at the epigenetic level is not fully understood. Studies on mouse Laser-IVF embryos provide evidence that the use of Laser-IVF leads to reduced birth rate, indicating a potential harm of this technique for the embryo. Hence, the aim of this study was to examine the difference in DNA methylation pattern between IVF- and Laser-IVF-derived mouse zygotes. We examined two experimental groups of C3HeB/FeJ oocytes: (1) zona-intact and (2) laser-microdissected oocytes that were fertilized in vitro with freshly collected spermatozoa. Zygotes were fixed 5, 8, and 12 h after fertilization, and indirect immunofluorescence staining was studied using an anti-5-methylcytidine (5-MeC) antibody. The fluorescence intensities of paternal and maternal pronuclei were evaluated using the computer-assisted analysis of digital images. In addition, we performed a semiquantitative RT-PCR analysis of the presence of transcripts of three developmental marker genes, Oct4, Dab2, and Dnmt3b, in IVF- and Laser-IVF-derived blastocysts. We observed no significant differences in methylation status of the paternal genome and in the transcripts of the developmental marker genes after IVF and Laser-IVF. In conclusion, epigenetic patterns and early embryonic development are not altered by laser-assisted IVF techniques and another explanation must be sought for the poor implantation rates observed in mice.

Methylation‐Associated Silencing of Death‐Associated Protein Kinase Gene in Laryngeal Squamous Cell Cancer

Death-associated protein kinase (DAPK) is a Ca/calmodulin-regulated Ser/Thr kinase that functions as a positive mediator of programmed cell death. It has been found that DAPK gene is frequently inactivated by its promoter hypermethylation in some cancers and tumor cell lines. However, it is not clear whether promoter hypermethylation of DAPK gene exists in laryngeal squamous cell cancer (LSCC). The aim of this study was to investigate the promoter methylation status of the DAPK gene in LSCC and the effect of 5-Aza-2'-deoxycytidine (5-Aza-CdR), a demethylating agent, on Hep-2 cells, a human laryngeal cancer cell line, and on xenografts of Hep-2. Methylation-specific polymerase chain reaction (PCR) and reverse-transcription PCR techniques were used to determine the promoter methylation status and mRNA expression of DAPK gene in LSCC. Furthermore, Hep-2 cells in vitro and in vivo were treated by 5-Aza-CdR to explore the effect of demethylating agents on DAPK mRNA expression and tumor growth. Hypermethylation of DAPK gene promoter was found in 39 (67.2%) of 58 LSCC samples. There was no significant difference in the promoter hypermethylation rate among the samples of different histologic grades or samples from patients with different T stages. However, there was significant difference in methylation status of DAPK gene between the samples from patients in N0 stages and those from patients in N1 stages. No promoter hypermethylation of DAPK gene was found in any of the five normal laryngeal tissue samples. DAPK mRNA expression was not detected in tumor specimens with promoter hypermethylation. On the contrary, DAPK mRNA expression was observed in the unmethylated tumor specimens, specimens from tissues adjacent to the tumor, and normal laryngeal tissues samples. Promoter hypermethylation of DAPK gene was found, and no DAPK mRNA expression was detected in Hep-2 cells. DAPK mRNA expression in Hep-2 cells and xenografts could be restored by treating cells and xenografts with 5-Aza-CdR. The tumors' xenografts, induced by way of Hep-2 cell injection in nude mice treated with 5-Aza-CdR, were obviously smaller than those in nude mice treated with phosphate-buffered saline. Abnormal loss of DAPK expression could be associated with aberrant promoter region methylation in the LSCC. 5-Aza-CdR may slow the growth of Hep-2 cells in vitro and in vivo by reactivating tumor suppressor gene DAPK silenced by de novo methylation.