Evaluation Of DNA Methylation Research Articles

EPIGENETIC AGE AND MUSCLE CHARACTERISTICS IN BALTIMORE LONGITUDINAL STUDY OF AGING PARTICIPANTS

Abstract Changes in muscle, including loss of muscle mass and fat infiltration, contribute to loss of muscle strength and subsequent declines in physical function in later life. Measures of body composition are consistently associated with DNA methylation (DNAm) in cross sectional studies. Whether differences in DNAm are informative with respect to longitudinal muscle characteristics has not been fully explored. Here we evaluate the relationship between epigenetic age metrics and computed tomography (CT) derived measures, including areas of muscle, subcutaneous fat, and intermuscular fat, as well as the mean and standard deviation of muscle intensity at mid-femur, in the Baltimore Longitudinal Study of Aging. Participants were at least 50 years old at the evaluation of DNAm (n = 471, 51% female), 61.8% of participants had three or more CT scans and mean follow up time was 6.0 years. Relationships between epigenetic age metrics including Horvath and Hannum DNAm age, PhenoAge, GrimAge and DunedinPACE at the index visit, and the trajectory of muscle composition were evaluated using multivariable mixed effects models adjusted for key demographic characteristics, health behaviors and body size. Epigenetic age metrics at the index visit were negatively correlated with the trajectory of muscle area over follow-up. Higher GrimAge at the index visit was associated with faster loss in muscle area and faster increase in fat areas (p < .05). Our analyses suggest that further exploration of DNAm with trajectories of muscle parameters may elucidate novel pathways of muscle dysfunction.

Proteomic Analysis Identifies Epigenetic Modifiers and Potential Negative Regulators of Leukemogenesis KMD6A and SETDB1 to be Induced Post In Vivo Decitabine Exposure in Pediatric AML

Deregulation of epigenetic mechanisms has been associated with AML pathogenesis. In an integrated transcriptomic and DNA methylation evaluation in pediatric AML, we demonstrated that reduced methylation and increased expression of DNMT3B were associated with greater genome-wide methylation and worse outcome in pediatric AML patients treated on St Jude AML02 and COG clinical trials. This epigenetic deregulation has been leveraged through use of DNA methylation inhibitors (DMTis) such as azacytidine and decitabine for treatment of MDS and AML. Given promising results in adults and relapsed pediatric AML, the multi-site AML16 clinical trial (NCT03164057) evaluated single agent DMTi "priming” followed by standard chemotherapy for the treatment of pediatric AML. Despite the use of DMTi to treat heme-malignancies, the underlying molecular mechanisms are still under investigation. Thus, we evaluated molecular in vivo response of pediatric AML cells to decitabine by evaluating paired specimens obtained at diagnosis and after five days of single agent decitabine therapy for global proteomic profiling. In a pilot study we previously showed modest correlation (10.7%) between transcript and protein levels, suggesting that the transcriptome may not be a reliable surrogate for proteomics in pediatric AML. Additionally, given that the majority of biological functions within cells are operated by proteins and most drugs target the proteome rather than the genome or transcriptome, understanding the proteomic signatures is critical. Thus, in this evaluation, we focused on decitabine-induced alterations in cellular proteomic profiles of paired diagnostic leukemic samples and 5 days post in vivo decitabine exposure from 9 patients treated on AML16. Completely automated high-resolution TMT (tandem-mass-tag) -LC/LC-MS/MS system was utilized to capture high resolution proteomic data and 10,471 proteins were detected in samples. At p<0.01, 109 proteins increased and 41 decreased post decitabine exposure. In vivoexposure to decitabine resulted in increased levels of several epigenetic regulators, including KMD6A and SETDB1. KMD6A, a histone3 lysine 27 demethylase, was recently shown to be mutated in relapsed AML and loss of KMD6A has been associated with resistance to cytarabine and daunorubicin (PMID: 31201358). SETDB1 is a methyltransferase that suppresses leukemogenesis and higher SETB1 expression has been shown to alter the promoter epigenome and reduce expression of AML associated genes such as DOCK1, HOXA9, SIX1. A recent study demonstrated SETDB1 to be negatively regulating pro-leukemic target genes thereby suppressing AML (PMID: 33054052). Among the top genes that were reduced at protein levels was DNMT3A, which is a well-established prognostic marker in adult AML. Combined, alterations in these protein levels suggest that decitabine exposure may increase sensitivity of AML blasts to chemotherapy and interfere with the leukemia program. Of particular interest were proteins that are targetable by drugs and demonstrated increased expression post decitabine exposure such as dihydrofolate reductase (DHFR), which is essential for cellular metabolism and growth. DHFR is inhibited by the antileukemic agent methotrexate and our data suggest that DNMTi priming might increase sensitivity of leukemia cells to methotrexate. Other targetable proteins are listed in Table 1. In summary, this pilot study provides significant insight into the DMTi-mediated proteomics changes that hold prognostic and therapeutic relevance in AML. Ongoing studies are focused on expanding the cohort size and evaluate association of proteomics with clinical outcome. Additionally, we plan to perform a multi-omics analysis by integrating proteomics with DNA methylation, RNAseq and metabolomics. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Evaluation of DNA methylation in BDNF, SLC6A4, NR3C1 and FKBP5 before and after treatment with selective serotonin-reuptake inhibitor in major depressive disorder.

Aim: To identify the DNA methylation status of related genes in major depressive disorder following selective serotonin-reuptake inhibitor treatment. Materials & methods: 45 patients with major depressive disorder and 45 healthy volunteers were considered experimental and control groups,respectively. High-resolution melting real-time PCR was implemented to evaluate DNA methylation. Results: After 100days of selective serotonin-reuptake inhibitor treatment, methylation of promoter CpG sites of BDNF, NR3C1, FKBP5 and SLC6A4 was significantly reduced. Compared with before treatment, patients' Hamilton Depression Rating Scale scores were significantly reduced after selective serotonin-reuptake inhibitor treatment (p≤0.0001). Conclusion: Based on the proven effect of antidepressants on DNA methylation and gene expression, these medications can improve the treatment process and reduce depression scores after treatment.

Evaluation of DNA methylation of MAP9 gene in breast cancer as epigenetic biomarker

Introduction and Aim: The DNA methylation is involved in the regulation of gene activity and abnormal DNA methylation is associated with various diseases, including cancer. MAP9 (Microtubule-Associated Protein 9) gene methylation was investigated as a potential epigenetic biomarker for cancer in this work. The results were published in Cancer Research. Materials and Methods: The present study was on 40 breast cancer samples and 20 healthy samples to identify diagnosis biomarkers for breast cancer. DNA was extracted from the whole blood of breast cancer patients and healthy samples and were converted to bisulfite by using EpiTect Fast DNA Bisulfite Kit –Part 1 from Qiagen company. Then used Qia gene methylation kit to identify methylated site an epigenetic marker (MAP9) using HRM software in RT-PCR. Results: The findings of the present investigation revealed that the methylation of the MAP9 gene in breast cancer patients were 28 (70%) compared to healthy patients 2 (10%) at a significant difference (P&lt;0.01). Conclusion: The MAP9 gene is hypermethylated in breast cancer patients, and it has the potential to be exploited as a molecular biomarker for the detection of breast cancer.

Genetic Polymorphisms and DNA Methylation Evaluation in a Rare Pediatric Case Carrying a Solid Pseudo Papillary Neoplasm of the Pancreas

Genetic Polymorphisms and DNA Methylation Evaluation in a Rare Pediatric Case Carrying a Solid Pseudo Papillary Neoplasm of the Pancreas

Association of Aberrant DNA Methylation Level in the CD4 and JAK-STAT-Pathway-Related Genes with Mastitis Indicator Traits in Chinese Holstein Dairy Cattle.

Simple SummaryMastitis is the most common inflammatory disease of economic and animal welfare concern in dairy animals. The present study was designed to evaluate the gene expression and epigenetic modifications in cattle with mastitis and healthy cows. The CpG islands in the promoter regions of the JAK2 and the STAT5A revealed hypo-methylation levels and higher gene expression in cows with mastitis compared to the healthy control, and vice versa in those with the CD4 gene. DNA methylation was negatively correlated with gene expression in the JAK2 and CD4 genes. Findings of the current study showed that aberrant DNA methylation due to mastitis in the promoter region of the three genes under study could be used as potential epigenetic markers to predict the mastitis susceptibility in dairy cattle.The present study was designed to evaluate the gene expression and DNA methylation level in the promoter region of the CD4 and the JAK-STAT-pathway-related genes. A total of 24 samples were deployed in the gene expression and 118 samples were used in the DNA methylation study. Student’s t-tests were used to analyze the gene expression and DNA methylation. The evaluation of DNA methylation in promoter regions of JAK2 and STAT5A revealed hypo-methylation levels of CpG sites and higher gene expression in cows diagnosed with mastitis as compared to the healthy control, and vice versa in those with CD4. DNA methylation was negatively correlated with gene expression in JAK2, STAT5A, and CD4 genes. Six, two, and four active transcription factors were identified on the CpG sites in the promoter regions of JAK2, STAT5A, and CD4 genes, respectively. Regarding correlation analysis, the DNA methylation levels of CD4 showed significantly higher positive correlations with somatic cell counts (p < 0.05). Findings of the current study inferred that aberrant DNA methylation in the CpG sites at the 1 kb promoter region in JAK2, STAT5A, and CD4 genes due to mastitis in cows can be used as potential epigenetic markers to estimate bovine mastitis susceptibility in dairy cattle.

Evaluation of DNA methylation in promoter regions of hTERT, TWIST1, VIM and NID2 genes in Moroccan bladder cancer patients

Promoter hypermethylation have been reported to play a key role in bladder cancer development and progression. The aim of this study is to evaluate the methylation status of hTERT, TWIST1, VIM and NID2 genes in bladder cancer. The methylation status was evaluated using the Methylation-Specific PCR (MSP) approach on 70 tumour biopsies from Moroccan bladder cancer patients. Overall, methylation frequencies of hTERT, TWIST1, VIM and NID2 genes, were 90%, 85.71%, 67.14% and 67.14%, respectively. Hypermethylation of all studied genes was found in all pathological grades and stages of bladder cancer. Nevertheless, statistical analysis showed no significant association between promoter methylation of hTERT, TWIST1, VIM and NID2 genes and tumours stage/grade (p value >0.05). Moreover, we have investigated the association between the methylation pattern of selected genes and the treatment outcome in a sub-group of non-muscle-invasive bladder cancer cases (52/70). Hypermethylation of hTERT, TWIST1, VIM and NID2 was detected in 83.34%; 66.67%; 83.34% and 58.34% of recurrent cases, respectively, and in 80%; 80%; 80% and 60% of progressive cases, respectively. Statistical analysis highlighted a significant association between TWIST1 hypermethylation and tumour recurrence (p = 0.041<0.05).Our results indicate that hypermethylation of hTERT, TWIST1, VIM and NID2 genes is a frequent epigenetic event in bladder cancer and could be a promising therapeutic target to prevent bladder cancer progression and metastasis.

Evaluation of DNA Methylation at Birth in Monozygotic Twin Pairs Discordant for Acute Lymphoblastic Leukemia

Background: Aberrant patterns of DNA methylation constitute a key feature of pediatric acute lymphoblastic leukemia (ALL) at diagnosis, however its role as a predisposing or early contributor to the development of ALL remains unknown. We employed a discordant monozygotic twin model to identify epigenetic variation associated with future development of pediatric ALL through evaluation of DNA methylation at birth.Methods: Twin pairs discordant for the development of pediatric ALL were identified using linked data from the California Cancer Registry and California Birth Statistical Master File spanning from 1989 to 2015. Archived dried neonatal blood spots were obtained from 86 same-gender twin pairs with available materials from the California Biobank Program. Following isolation of genomic DNA from DBS samples, monozygosity was confirmed in 43 of 86 twin pairs through an identity-by-descent analysis from a genome-wide SNP-array. Epigenome-wide DNA methylation assessment of the 43 discordant monozygotic twin pairs was conducted using the Illumina Infinium MethylationEPIC BeadChip kit (Illumina, San Diego, CA, USA). Data preprocessing and quality control measures were conducted in R, using SeSAMe for data normalization. Two twin pairs were omitted due to failure to pass quality control measures. Within-pair analysis was conducted through identification of array probes with absolute differences in methylation beta values greater than 15% between case and control siblings of a twin pair unit. Differentially methylated probes (DMP) were identified using a conditional logistic regression model accounting for array-specific variation, nucleated cell proportions, and appropriate control for the paired nature of the dataset. Differentially methylated regions (DMR) were defined by regional correlation of p-values from the conditional logistic regression model. Gene set enrichment analysis was conducted on significant probes identified through the within pair and regression analysis.Results: The discordant twin cohort (n = 41 pairs) included 24 female and 17 male pairs. Median gestational age was 258 days, ranging from 184 to 306 days. Age of diagnosis in the case twin ranged from <1 to 23 years (median = 5). There was no significant association between birthweight and case status (paired Wilcoxon signed rank test p = 0.22). No significant differences in nucleated cell proportions were identified in deconvolution analysis. Within-pair analysis identified a total of 18,001 probes with absolute methylation variation greater than 15% across the 41 twin pairs, with 3,984 recurrently variable across more than one pair. Gene ontology analysis of these recurrently variable sites revealed an enrichment of immune-related processes in 7 of the top 15 terms with nominal p-value <0.05, though no terms were significant after correction for multiple comparisons. Conditional logistic regression was conducted on 37 twin pairs, with T-cell cases (n=4) omitted to improve data resolution. This resulted in 240 significant DMPs with p-values below an FDR threshold of 0.05. Of these significant probes, 20 associate with genes previously reported to have altered DNA methylation in ALL at diagnosis. Regional analysis identified 10 significant DMRs with adjusted p-values below 0.05, with the top association encompassing a 454bp region on chromosome 6 located near TRIM39-RPP21 (adjusted p-value 2.39e-05). Notably, conditional regression analysis revealed a significant negative bias in coefficients (409,812 of 710,010 probes, binomial exact test p <2.2e-16), indicating a global tendency toward hypomethylation in cases compared to unaffected siblings (Figure 1). The strength of this bias was greater in probes associated with open sea regions compared to those in island regions, as well as promoter-associated probes.Conclusions: This novel analysis of DNA methylation at birth in ALL-discordant monozygotic twins identified sites of differential methylation associated with immune regulation. In addition, these results provide evidence of an association between global DNA hypomethylation and future development of ALL in one member of a genetically identical twin pair. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Cell-Free DNA-Methylation-Based Methods and Applications in Oncology.

Liquid biopsy based on cell-free DNA (cfDNA) enables non-invasive dynamic assessment of disease status in patients with cancer, both in the early and advanced settings. The analysis of DNA-methylation (DNAm) from cfDNA samples holds great promise due to the intrinsic characteristics of DNAm being more prevalent, pervasive, and cell- and tumor-type specific than genomics, for which established cfDNA assays already exist. Herein, we report on recent advances on experimental strategies for the analysis of DNAm in cfDNA samples. We describe the main steps of DNAm-based analysis workflows, including pre-analytics of cfDNA samples, DNA treatment, assays for DNAm evaluation, and methods for data analysis. We report on protocols, biomolecular techniques, and computational strategies enabling DNAm evaluation in the context of cfDNA analysis, along with practical considerations on input sample requirements and costs. We provide an overview on existing studies exploiting cell-free DNAm biomarkers for the detection and monitoring of cancer in early and advanced settings, for the evaluation of drug resistance, and for the identification of the cell-of-origin of tumors. Finally, we report on DNAm-based tests approved for clinical use and summarize their performance in the context of liquid biopsy.

DNA Methylation Clusters and Their Relation to Cytogenetic Features in Pediatric AML.

Simple SummaryAcute myeloid leukemia is a heterogenous disease with dismal outcome. In order to improve currently used therapeutic strategies it is important to get a in depth understanding of the molecular and genomic landscape of AML. Adult AML studies have established the significant of mutational profile of epigenetic genes as well as epigenetic deregulation DNA methylation signatures. In the current study we focused on establishing the DNA methylation profile in pediatric AML. Our result show that in pediatric AML patients the risk group and cytogenetic features have distinctive epigenetic signatures. Additionally, we observed that distinctive epigenetic hotspots co-occur complementary to the known genomic lesions and contribute towards leukemogenesis.Acute Myeloid Leukemia (AML) is characterized by recurrent genetic and cytogenetic lesions that are utilized for risk stratification and for making treatment decisions. In recent years, methylation dysregulation has been extensively studied and associated with risk groups and prognosis in adult AML, however, such studies in pediatric AML are limited. Moreover, the mutations in epigenetic genes such as DNMT3A, IDH1 or IDH2 are almost absent or rare in pediatric patients as compared to their abundance in adult AML. In the current study, we evaluated methylation patterns that occur with or independent of the well-defined cytogenetic features in pediatric AML patients enrolled on multi-site AML02 clinical trial (NCT00136084). Our results demonstrate that unlike adult AML, cytosine DNA methylation does not result in significant unique clusters in pediatric AML, however, DNA methylation signatures correlated significantly with the most common and recurrent cytogenetic features. Paired evaluation of DNA methylation and expression identified genes and pathways of biological relevance that hold promise for novel therapeutic strategies. Our results further demonstrate that epigenetic signatures occur complimentary to the well-established chromosomal/mutational landscape, implying that dysregulation of oncogenes or tumor suppressors might be leveraging both genetic and epigenetic mechanisms to impact biological pathways critical for leukemogenesis.

Evaluation of DNA methylation in matched oral swab and tissue specimens from Chinese patients with oral squamous cell carcinoma

The DNA methylation statuses of the paired box 1 (PAX1) and zinc finger protein 582 (ZNF582) genes have shown promise in the detection of oral squamous cell carcinoma (OSCC). The aim of this study was to investigate the ability of PAX1 and ZNF582 methylation to distinguish OSCC and the adjacent normal tissue among cancer patients. This study included 67 patients with OSCC. The methylation levels of these two genes were analysed in tissue specimens (lesion site and adjacent normal site) and in oral swabs (lesion site and contralateral normal site). Levels of DNA methylation were higher at lesion sites than at the corresponding normal sites. According to receiver operating characteristics curve analysis, the area under the curve for PAX1 and ZNF582 methylation ranged from 0.73 to 0.82. No significant difference was observed between tissue specimens and oral swabs (PAX1, P= 0.41; ZNF582, P=0.28). For the oral swab, PAX1 methylation was more pronounced in bone invasion (Z=1.988, P= 0.047), and ZNF582 methylation was more pronounced in early-stage (Z=2.354, P= 0.02) and well-differentiated tumours (Z=3.731, P= 0.0002). Hypermethylated PAX1 and ZNF582 are effective biomarkers to distinguish lesion sites and corresponding normal sites in tissue specimens and oral swabs from OSCC patients.

Abstract 817: Assessment and manipulation of DNA methylation in cancer cells using terahertz radiation

Abstract Carcinogenesis is closely associated with abnormal regulation of gene expression, among which DNA methylation in one of the key epigenetic modifications. Therefore, several studies have been directed towards the evaluation of DNA methylation. Here, we aimed to experimentally observe the specific signal of methylated DNA in various blood cancer cell lines using terahertz (THz) radiation, which is presented as a resonance in the spectrum. THz radiations are electromagnetic waves having frequencies of 0.1−10 THz, which falls between those of infrared radiation and microwaves. Molecular low-energy motions in THz region, such as collective vibrations, torsion of molecular structures, and hydrogen bonds, is correlated with biological characteristics, i.e., vibrational modes of DNA, structural modes in proteins, cell membrane vibrations, or enzyme activity. These features are usually presented as a characteristic resonance of the modes in the THz spectrum. In our study, the degree of global DNA methylation was obtained by the quantification of the resonance around 1.7 THz, and the results were in good agreement with those obtained by enzyme-linked immunosorbent assay (ELISA). Further, DNA demethylation was performed using high-power THz radiation around the resonance frequency of 1.7 THz, which lead to a 10% to 72% decrease in the degree of DNA methylation as shown in Table 1. Thus, our data shows the feasibility of using THz radiation for the assessment and manipulation of DNA methylation. This is the first study wherein the sensing and manipulation of DNA methylation could be achieved using a non-contact and non-invasive optical technique. Use of THz radiation for the assessment of DNA methylation might be helpful for its rapid processing, while THz manipulation of DNA methylation can avoid the toxicity of chemical demethylating agents. We propose that THz radiation may be useful for the diagnosis of cancer and we expect that THz demethylation might find potential application in cancer therapy. Change of methylation status of blood cancer DNA using high-power THz radiation.SamplesNo ExposureHigh-power THz exxpsoureChange (%) THzChange (%) ELISATHzELISARatio1THzELISARatio2119.621.8510.6111.661.0611.0040.5742.70211.341.388.2210.201.198.5710.0513.77315.151.4310.596.240.738.5558.8148.95417.731.4712.063.510.447.9880.2070.07511.271.437.8810.001.367.3511.274.90STDEV1.58STDEV1.24THz, the amplitude of terahertz resonance; ELISA, optical density of Enzyme linked immunosorbent assay; STDEV, standard deviation1 THz/ELISA of no exposure2 THz/ELISA of high-power THz exposure Citation Format: Hee-Jin Yang, Hwayeong Cheon, Jin Ho Paik, Joo-Hiuk Son. Assessment and manipulation of DNA methylation in cancer cells using terahertz radiation [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 817.

Evaluation of DNA methylation and mRNA expression of heat shock proteins in thermal manipulated chicken.

Thermal manipulation during embryogenesis has been demonstrated to enhance the thermotolerance capacity of broilers through epigenetic modifications. Heat shock proteins (HSPs) are induced in response to stress for guarding cells against damage. The present study investigates the effect of thermal conditioning during embryogenesis and thermal challenge at 42days of age on HSP gene and protein expression, DNA methylation and in vitro luciferase assay in brain tissue of Naked Neck (NN) and Punjab Broiler-2 (PB-2) chicken. On the 15th day of incubation, fertile eggs from two breeds, NN and PB-2, were randomly divided in to two groups: control (C)-eggs were incubated under standard incubation conditions, and thermal conditioning (TC)-eggs were exposed to higher incubation temperature (40.5°C) for 3h on the 15th, 16th, and 17th days of incubation. The chicks obtained from each group were further subdivided and reared under different environmental conditions from the 15th to the 42nd day as normal [N; 25±1°C, 70% relative humidity (RH)] and heat exposed (HE; 35±1°C, 50% RH) resulting in four treatment groups (CN, CHE, TCN, and TCHE). The results revealed that HSP promoter activity was stronger in CHE, which had lesser methylation and higher gene expression. The activity of promoter region was lesser in TCHE birds that were thermally manipulated at the embryonic stage, thus reflecting their stress-free condition. This was confirmed by the lower level of mRNA expression of all the HSP genes. In conclusion, thermal conditioning during embryogenesis has a positive impact and improves chicken thermotolerance capacity in postnatal life.

Evaluation of DNA methylation in promoter regions of SFRP4 and ZAR1 in urine and plasma of women with cervical lesions

Background: Aberrant DNA hypermethylation in tumor suppressor genes is a common feature in cervical cancer (CC). This abnormal hypermethylation could be used as potential biomarkers for detecting CC in non-invasive samples such as urine and plasma.

Determination of DNA Methylation and Hydroxymethylation Levels in Biological Samples by Field-Amplified Sample Injection-Capillary Zone Electrophoresis with UV Detection

In this study, a method of field-amplified sample injection coupled with capillary zone electrophoresis with ultraviolet detection was established for evaluation of DNA methylation and hydroxymethylation levels in biological materials. By modifying an existing method, the separation of cytosine (C), 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) was performed on an uncoated capillary column (40 cm × 75 μm I.D.) using 300 mmol L−1 tris solution (pH 2.90) as running buffer and detected at 280 nm. The detection limits (S/N = 3) of the method were 0.004 ng mL−1 for cytosine (C), 0.01 ng mL−1 for 5-methylcytosine (5-mC), and 0.02 ng mL−1 for 5-hydroxymethylcytosine (5-hmC). The proposed method has been successfully applied to the evaluation of DNA methylation and hydroxymethylation levels of blood samples from 15 hepatocellular carcinoma patients and 5 liver cirrhosis patients and liver tissues from 50 pairs of tumor and matched tumor-adjacent samples.

Evaluation of DNA methylation at imprinted DMRs in the spermatozoa of oligozoospermic men in association with MTHFR C677T genotype.

Altered DNA methylation has been previously identified in the spermatozoa of infertile men; however, the origins of these errors are poorly understood. DNA methylation is an epigenetic modification which is thought to play a fundamental role in male germline development. DNA methylation reactions rely on the cellular availability of methyl donors, which are primarily products of folate metabolism, where a key enzyme is methylenetetrahydrofolate reductase (MTHFR). The MTHFR C677T single nucleotide polymorphism (SNP) reduces enzyme activity and may potentially alter DNA methylation processes during germline development. The objective of this study was to determine whether altered DNA methylation in spermatozoa is associated with the MTHFR C677T SNP. DNA methylation was evaluated at the H19, IG-GTL2, and MEST imprinted differentially methylated regions in the spermatozoa of 53 men - 44 oligozoospermic men and nine fertile men with normal sperm parameters via bisulfite sequencing of sperm clones. The 44 infertile men were stratified by severity of oligozoospermia - three normal (>15million spermatozoa/mL), eight moderate (5-15million spermatozoa/mL), 23 severe (1-5million spermatozoa/mL), and 10 very severe (<1million spermatozoa/mL). MTHFR C677T SNP genotyping was conducted in a subset of 44 peripheral blood samples via restriction fragment length polymorphism. A total of three men - severe oligozoospermic and CT genotype - were found to be altered, which is defined as having ≥50% of their clones altered, where an altered clone was in turn defined as ≥50% of CpGs with incorrect DNA methylation patterns. The incidence of three altered men within the CT subgroup, however, was not significantly higher than the incidence in the CC subgroup. Taken together, altered DNA methylation in spermatozoa was not significantly associated with the MTHFR C677T SNP; however, there was a trend for higher incidence of alterations among severe oligozoospermic infertile men with CT genotypes.

Evaluation of DNA Methylation of MAP9 Gene in Breast Cancer as Epigenetic Biomarker

Background: Recent findings indicate epigenetic modifications as key factors in breast carcinogenesis. The abnormal methylation patterns of genes are among the consequences of epigenetic changes. The DNA methylation is involved in the regulation of gene activity and abnormal DNA methylation is associated with various diseases, including cancer. Due to the importance of epigenetics in cancer, particularly breast cancer, it seems to perform the most effective methods of prediction, detection and tracking of recurrence, and the availability of suitable biomarkers. In this study, the MAP9 (Microtubule-Associated Protein 9) gene methylation was examined as an epigenetic biomarker of cancer. Methods: We evaluated 30 breast cancer samples and 30 normal samples to identify diagnosis biomarkers for breast cancer. Sample of breast cancers were identified, with different Clinical and pathological data, which might be related with changes in MAP9 gene methylation. DNA was extracted from whole blood of breast cancer patients and healthy samples. The methylation-sensitive restriction enzymes were used to identify methylated site in epigenetic marker. Methylation-sensitive enzyme cannot cut hypermethylated sequences. Methylation-sensitive enzyme is not capable of cutting sequences of MAP9 gene, thus replication occurs. Results: MAP9 gene is significantly hypermethylated in breast cancer (P 0.05). Conclusion: In this study indicated MAP9 gene methylation changes in breast cancer and it can be used as molecular biomarker for breast cancer diagnosis.

CXCL12 and ADAM23 hypermethylation are associated with advanced breast cancers

More than 25% of the patients with breast cancer (BC) develop metastatic disease. In the present study, we investigated the relationship between DNA methylation levels in genes regulating cell growth, invasiveness, and metastasis and advanced BCs and evaluated the clinical utility of methylation profiles for detecting metastatic potential. Pyrosequencing was used to quantify methylation levels in 11 cancer-associated genes in primary tumors (PTs), lymph node metastases (LNMs), plasma (PL), and blood cells from 206 patients with invasive BC. Protein expression was evaluated using immunohistochemistry. PTs showed hypermethylation of A isoform of the RAS-association domain family 1 (RASSF1A), adenomatous polyposis coli (APC), chemokine C-X-C motif ligand 12 (CXCL12), and disintegrin and metalloprotease domain 23 (ADAM23) (means 38.98%, 24.84%, 12.04%, and 10.01%, respectively). Positive correlations were identified between methylations in PTs and LNMs, but not between PL and PTs. The cumulative methylation of PTs and LNMs manifested similar spectrums of methylated genes that indicate the maintaining of aberrant methylation during breast tumorigenesis. Significantly increased methylation levels in RASSF1A, APC, CXCL12, and ADAM23 were found in estrogen receptor (ER) positive BCs in comparison with ER negative cases. Regarding these results, the evaluation of DNA methylation could be more informative in testing of patients with ER positive BC. The risk for LNMs development and higher proliferation of cancer cells measured through Ki-67 expression was increased by hypermethylation of CXCL12 and ADAM23, respectively. Therefore, the quantification of CXCL12 and ADAM23 methylation could be useful for the prediction of advanced stage of BC.

Evaluation of DNA methylation using methylation-sensitive amplification polymorphism in plant tissues grown in vivo and in vitro

In present study, methylation-sensitive AFLP (MSAP) markers were employed to assess DNA methylation, degree of alterations in DNA methylation and methylation polymorphism in plant tissues growing in vivo and in vitro. The leaf tissues of six plants growing in vivo and in vitro were subjected to MSAP profiling. A total of 717 MSAP markers in Salvadora persica, 801 in Commiphora wightii, 874 in male (M) and 845 in female (F) genotype of Simmondsia chinensis, 719 in Jatropha curcas and 880 in Withania coagulans were obtained with seventeen MSAP primer combinations. Percentage methylation in genome obtained was higher in in vivo-grown tissues of S. persica (39.47 %), S. chinensis—M (61.71 %) and W. coagulans (71.59 %); and in in vitro-grown tissues of C. wightii (65.17 %), S. chinensis—F (60.83 %) and J. curcas (68.29 %). The percentage polymorphism in methylated DNA obtained was 8.71 % in S. persica, 9.81 % in J. curcas, 10.10 % in S. chinensis—F, 10.26 % in W. coagulans, 10.66 % in S. chinensis—M and 13.98 % in C. wightii. The difference in DNA methylation and polymorphism in genomes reflect the plasticity in genomes of the plants growing under two different environments. Different pattern of DNA methylation of the homologous nucleotide sequences and polymorphism in the methylated DNA in tissues under in vitro and in vivo conditions suggest possibility of involvement of these fragments in the dynamic processes regulating plant growth and development under prevailing growth conditions.

100 ASSESSMENT OF LOCUS-SPECIFIC DNA METHYLATION: OPTIMIZATION FOR BOVINE EMBRYOS

Assessment of chromatin remodeling in early embryos is a major focus of studies today, and evaluation of DNA methylation at specific loci is one approach to study these epigenetic modifications. Our objective was to optimize the bisulfite sequencing methodology for use with very small cell numbers originating from pre-implantation embryos, making the process more time- and cost-efficient. The optimized steps include bisulfite conversion of small samples, bisulfite primer design, high-throughput plasmid DNA amplification, and preparation for sequencing. Methylation at 2 loci, Satellite I and Oct4, was investigated in bovine in vitro-produced (IVP) embryos collected at the 2-cell, 8-cell, and blastocyst stages. Bovine skin fibroblasts were first used to optimize the particular steps of the process. All reactions were run in duplicate and no-template negative and somatic cell-positive controls were treated alongside samples. Incorporating the use of Methyl Primer Express (Applied Biosystems, Foster City, CA), MacVector (Oxford Molecular Ltd., Campbell, CA), and Mfold software (Mathews DH et al. 1999 J. Mol. Biol. 288, 911–940; Zuker M 2003 Nucleic Acids Res. 31, 3406–3415) improved the specificity of bisulfite primers by exclusion of secondary or tertiary structures. The DNA from bisulfite treatment for 15 to 16 h was of better quality than DNA treated for 18 h. After initial PCR optimization, different cell concentrations were used to establish that detectable PCR products and subsequent methylation data could be obtained from DNA isolated from as few as 8 cells. Treating single blastocysts and pools of ten 8-cell and forty 2-cell embryos was sufficient for the entire scope of the experiment, allowing use of the same samples across all loci. After molecular cloning, plasmid DNA was amplified by 3 different methods and evaluated for efficiency: miniprep, TempliPhi (GE Healthcare, Piscataway, NJ), or 96-well glycerol stocks and automated TempliPhi format. Although TempliPhi alone was better than miniprep for small-scale experiments, it was the 96-well format that saved weeks of time and was most cost-effective. Sequencing was performed on a minimum of 8 clones/sample using ABI Prism sequencers (Applied Biosystems), and results were analyzed using Chromas Pro software (Technelysium Pty. Ltd., Helensvale, Australia). Percentage methylation of bovine IVP 2-cell, 8-cell, and blastocyst stage embryos for Satellite I was 25, 10, and 22%, respectively, and for Oct4 was 88, 88, and 79%, respectively. However, somatic cell methylation was 74% for Satellite I and 88% for Oct4, implying that Satellite I is demethylated during early embryo development, whereas Oct4 remains hypermethylated. In conclusion, these improved methods will benefit further studies of chromatin remodeling in early bovine pre-implantation embryos. This project was supported by National Research Initiative Competitive Grant no. 2006-35203-16620 from the USDA Cooperative State Research, Education, and Extension Service.