Methylation-sensitive Enzyme Research Articles

Evaluation of fetal hypermethylated RASSF1A in pre-eclampsia and its relationship with placental protein-13, pregnancy associated plasma protein-A and urine protein.

Cell free fetal DNA (cffDNA) and its hypermethylated RASSF1A gene signify a recent advancement in non-invasive prenatal diagnosis of feto-placental anomalies like pre-eclampsia. The study uses hypermethylated RASSF1A gene to quantify cffDNA and to assess its relationship with placental and urine proteins in pre-eclampsia cases. DNA was isolated from plasma samples of clinically diagnosed cases of pre-eclampsia (n=103) and normal pregnancy (n=616) from 21weeks of gestation. Through methylation sensitive enzyme (BstUI) digestion; followed by real time-polymerase chain reaction (RT-PCR), quantification of hypermethylated RASSF1A was done. Immunoassays determined: placental protein-13 (pp-13) and pregnancy associated plasma protein A (PAPP-A) and pyrogallol red molybdate assay for 24h urine protein. Highly significant differences between control and pre-eclampsia cases for hypermethylated RASSF1A concentrations were found; Group I: 33±7.35 vs 74.46±16.71, Group II: 53.75±16.65 vs 244.22±35.68, Group III: 93.25±19.08 vs 412.31±80.18, Group IV: 144.30±18.13 vs 1056.89±153.78, Group V: 307.55±40.76 vs 2763.76±259.76copies/ml. Multivariate Pearson's correlation analysis of hypermethylated RASSF1A with pp-13, PAPP-A and urine proteins showed positive and very highly significant (P<0.001) associations. Diagnostic potential of fetal specific, hypermethylated RASSF1A was evaluated. Its positive relationship with placental and urine proteins submit the case for considering it as a reliable marker for pre-eclampsia.

Relationship between methylation status of vitamin D-related genes, vitamin D levels, and methyl-donor biochemistry

Vitamin D is known for its role in the regulation of gene expression via the vitamin D receptor, a nuclear transcription factor. More recently, a role for vitamin D in regulating DNA methylation has been identified as an additional mechanism of modulation of gene expression. How methylation status influences vitamin D metabolism and response pathways is not yet clear. Therefore, we aimed to assess the relationship between plasma 25-hydroxycholecalciferol (25(OH)D) and the methylation status of vitamin D metabolism enzyme genes (CYP2R1, CYP27B1 and CYP24A1) and the vitamin D receptor gene (VDR). This analysis was conducted in the context of dietary vitamin D, and background methyl donor related biochemistry, with adjustment for several dietary and lifestyle variables. Percentage methylation at CpG sites was assessed in peripheral blood cells using methylation sensitive and dependent enzymes and qPCR. Standard analytical techniques were used to determine plasma 25(OH)D and homocysteine, and serum folate and B12, with the relationship to methylation status assessed using multi-variable regression analysis. CYP2R1 and VDR methylation were found to be independent predictors of plasma 25(OH)D, when adjusted for vitamin D intake and other lifestyle variables. CYP24A1 was related to plasma 25(OH)D directly, but not in the context of vitamin D intake. Methyl-group donor biochemistry was associated with the methylation status of some genes, but did not alter the relationship between methylation and plasma 25(OH)D. Modulation of methylation status of CYP2R1, CYP24A1 and VDR in response to plasma 25(OH)D may be part of feedback loops involved in maintaining vitamin D homeostasis, and may explain a portion of the variance in plasma 25(OH)D levels in response to intake and sun exposure. Methyl-group donor biochemistry, while a potential independent modulator, did not alter this effect.

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.

Presence of DNA methyltransferase activity and CpC methylation in Drosophila melanogaster.

Drosophila melanogaster lacks DNMT1/DNMT3 based methylation machinery. Despite recent reports confirming the presence of low DNA methylation in Drosophila; little is known about the methyltransferase. Therefore, in this study, we have aimed to investigate the possible functioning of DNA methyltransferase in Drosophila. The 14K oligo microarray slide was incubated with native cell extract from adult Drosophila to check the presence of the methyltransferase activity. After incubation under appropriate conditions, the methylated oligo sequences were identified by the binding of anti 5-methylcytosine monoclonal antibody. The antibody bound to the methylated oligos was detected using Cy3 labeled secondary antibody. Methylation sensitive restriction enzyme mediated PCR was used to assess the methylation at a few selected loci identified on the array. It could be seen that a few of the total oligos got methylated under the assay conditions. Analysis of methylated oligo sequences provides evidence for the presence of de novo methyltransferase activity and allows identification of its sequence specificity in adult Drosophila. With the help of methylation sensitive enzymes we could detect presence of CpC methylation in the selected genomic regions. This study reports presence of an active DNA methyltransferase in adult Drosophila, which exhibits sequence specificity confirmed by presence of asymmetric methylation at corresponding sites in the genomic DNA. It also provides an innovative approach to investigate methylation specificity of a native methyltransferase.

On-Chip Sequence-Specific Immunochemical Epigenomic Analysis Utilizing Outward-Turned Cytosine in a DNA Bulge with Handheld Surface Plasmon Resonance Equipment.

This paper reports a sequence-specific immunoassay chip for DNA methylation assessment by microfluidic-based surface plasmon resonance (SPR) detection. This was achieved by utilizing an affinity measurement involving the target, (methyl)cytosine, in a single-base bulge region and an anti-methylcytosine antibody in a microchannel, following hybridization with a biotinylated bulge-inducing DNA probe. The probe alters the target cytosine in a looped-out state because of the π-π stacking between flanking bases of the target. The probe design is simple and consists of the elimination of guanine paired with the target cytosine from a fragmented full-match sequence. We obtained the single methylation status in 6 amol (48 fg) of synthesized oligo DNA in 45 min, which is the fastest DNA methylation assessment yet reported, without employing a conventional bisulfite reaction, PCR, or sequencing. We also succeeded in discrimination of the methylation status of single cytosine in genomic λ DNA and HCT116 human colon cancer cells. The advantages of the proposed method are its small equipment, simple microfluidics design, ease of handling (two injections of DNA and antibody), lack of need for a methylation-sensitive enzyme, and neutral buffer conditions.

Hypermethylation of promoter region of LATS1--a CDK interacting protein in oral squamous cell carcinomas--a pilot study in India.

Epigenetic silencing of tumor suppressor genes due to promoter hypermethylation is one of the frequent mechanisms observed in cancers. Hypermethylation of several tumor suppressor genes involved in cell cycle regulation has been reported in many types of tumors including oral squamous cell carcinomas. LATS1 (Large Tumor Suppressor, isoform 1) is a novel tumor suppressor gene that regulates cell cycle progression by forming complexes with the cyclin dependent kinase, CDK1. Promoter hypermethylation of the LATS1 gene has been observed in several carcinomas and also has been linked with prognosis. However, the methylation status of LATS1 in oral squamous cell carcinomas is not known. As oral cancer is one of the most prevalent forms of cancer in India, the present study was designed to investigate the methylation status of LATS1 promoter and associate it with histopathological findings in order to determine any associations of the genetic status with stage of differentiation. Tumor chromosomal DNA isolated from biopsy tissues of thirteen oral squamous cell carcinoma biopsy tissues were subjected to digestion with methylation sensitive HpaII enzyme followed by amplification with primers flanking CCGG motifs in promoter region of LATS1 gene. The PCR amplicons were subsequently subjected to agarose gel electrophoresis along with undigested amplification control. HpaII enzyme based methylation sensitive PCR identified LATS1 promoter hypermethylation in seven out of thirteen oral squamous cell carcinoma samples. The identification of LATS1 promoter hypermethylation in seven oral squamous cell carcinoma samples (54%), which included one sample with epithelial dysplasia, two early invasive and one moderately differentiated lesions indicates that the hypermethylation of this gene may be one of the early event during carcinogenesis. To the best of our knowledge, this is the first study to have explored and identified positive association between LATS1 promoter hypermethylation with histopathological features in oral squamous cell carcinomas.

199 IN VITRO-DEVELOPED BOVINE BLASTOCYSTS ARE MARKED WITH ABERRANT HYPER- AND HYPO-METHYLATED GENOMIC REGIONS

Most often, in vitro produced embryos display poor quality and altered gene expression patterns compared to their in vivo counterparts. Aberrant DNA methylation occurring during in vitro embryo development is believed to be one of the multifaceted factors which may cause altered gene expression and poor embryo quality. Here, we investigated the genome-wide DNA methylation patterns of in vitro derived embryos using the recently developed Bovine EmbryoGENE Methylation Platform (BEGMP) array (Shojaei Saadi et al. BMC Genomics 2014 15, 451. doi: 10.1186/1471-2164-15-451) to unravel the aberrantly methylated genomic region in in vitro developed embryos. For this, in vitro and in vivo produced blastocysts were produced and used for genome-wide DNA methylation analysis. In vitro blastocysts were produced from oocytes retrieved from ovaries collected from the local abattoir and matured, fertilized, and cultured in vitro using SOF media. The in vivo blastocysts were produced by superovulation and AI of Simmental heifers followed by uterine flushing. Genomic DNA (gDNA) was then isolated from four replicates (each 10 blastocysts) of in vivo and in vitro derived blastocysts using Allprep DNA/RNA micro kit (Qiagen, Valencia, CA, USA) and the gDNA was then fragmented using the MseI enzyme. Following this, MseLig21 and MseLig were ligated to the MseI-digested genomic fragments in the presence of Ligase enzyme. Methyl-sensitive enzymes, HpaII, AciI, and Hinp1I, were used to cleave unmethlayted genomic regions within the MseI-MseI region of the fragmented DNA. The gDNA was subjected to two rounds of ligation-mediated polymerase chain reaction (LM-PCR) amplification. After removal of the adapters, the amplified gDNA samples from in vivo or in vitro groups were labelled either Cy-3 or Cy-5 dyes in dye-swap design using ULS Fluorescent gDNA labelling kit (Kreatech Biotechnology BV, Amsterdam, The Netherlands). Hybridization was performed for 40 h at 65°C. Slides were scanned using Agilent's High-Resolution C Scanner (Agilent Technologies Inc., Santa Clara, CA, USA) and features were extracted with Agilent's Feature Extraction software (Agilent Technologies Inc.). The results have shown that from a total of 414 566 probes harboured by the BEGMP array, 248 453 and 253 147 probes were detected in in vitro and in vivo derived blastocysts, respectively. Data analysis using the linear modelling for microarray (LIMMA) package and R software (The R Project for Statistical Computing, Vienna, Austria) revealed a total of 3434 differentially methylated regions (DMRs; Fold change ≥1.5, P-value &lt;0.05), of which 42 and 58% were hyper- and hypo-methylated, respectively, in in vitro derived blastocysts compared to their in vivo counterparts. The DMRs were found to be localised in the intronic, exonic, promoter, proximal promoter, and distal promoter, and some of the probes did not have nearby genes. In addition, 10.8% of the DMRs were found to be stretched in short, long, or intermediate CpG islands. Thus, this study demonstrated genome-wide dysregulation in the epigenome landscape of in vitro-derived embryos by the time they reach to the blastocysts stage.

B13 Can Dna Methylation Be A Factor In The Pathogenesis Of Huntington's Disease?

Background Age at onset (AO) of Huntington’s disease (HD) usually occurs in midlife and it is strongly associated with the length of CAG expansion. However, a variation in the AO has been observed between individuals carrying comparable numbers of (CAG)n repeats. It has led to the search of the modifying factors impacting the variation in the AO, with the genetic imprinting suggested as a potential factor. Interestingly, the epigenetic modifications have been found to play a role in the pathogenesis of other neurodegenerative disorders associated with expanded trinucleotide repeats, e.g. fragile X syndrome and Friedreich’s ataxia. Aim The aim of the study is to investigate whether DNA methylation status including genetic imprinting is involved in pathogenesis, in particular the AO of HD. This issue is addressed by examining what proportion of variation in the AO is explained by the parental sex in a population of HD patients with the dominant impact of (CAG)n repeats included in statistical analyses. Further, DNA methylation status of selected regions is analysed in both HD patients and healthy subjects. Methods/techniques Nested regression models are used to assess the proportion of variation in the AO explained by the parental sex. Gene methylation levels are determined in DNA extracted from the whole blood by using CpG methylation-sensitive enzyme digestion followed by qPCR. Results No differences in DNA methylation within the HTT promoter between HD patients and healthy subjects have been found to date. Further analyses to evaluate the hypothesis are conducted. Conclusions To our best knowledge this is the first study to examine a question as to whether DNA methylation status including genetic imprinting will add to an impact of (CAG)n repeat to the variation in the AO in HD. We believe that our research may contribute considerable insight into the molecular mechanism of HD. This project is funded by National Research Centre, Poland, No UMO-2011/03/B/NZ2/01422.

Protocol: a beginner's guide to the analysis of RNA-directed DNA methylation in plants.

BackgroundDNA methylation is a conserved epigenetic mark that controls genome stability, development and environmental responses in many eukaryotes. DNA methylation can be guided by non-coding RNAs that include small interfering RNAs and scaffold RNAs. Although measurement of DNA methylation and regulatory non-coding RNAs is desirable for many biologists who are interested in exploring epigenetic regulation in their areas, conventional methods have limitations and are technically challenging. For instance, traditional siRNA detection through RNA hybridization requires relatively large amount of small RNAs and involves radioactive isotopes. An alternative approach is RT-qPCR that employs stem loop primers during reverse transcription; however, it requires a prerequisite that the exact sequences of siRNAs should be known.ResultsBy using the model organism Arabidopsis thaliana, we developed an easy-to-follow, integrative procedure for time-efficient, quantitative measurement of DNA methylation, small interfering RNAs, and scaffold RNAs. Starting with simplified nucleic acid manipulation, we examined DNA methylation levels by using Chop PCR (methylation-sensitive enzyme digestion followed by PCR), which allowed for fast screening for DNA methylation mutants without the need of transgenic reporters. We deployed a simple bioinformatics method for mining published small RNA databases, in order to obtain the nucleotide (nt) sequences of individual 24nt siRNAs within the regions of interest. The protocol of commercial TaqMan Small RNA Assay was subsequently optimized for reliable quantitative detection of individual siRNAs. We used nested qPCR to quantify scaffold RNAs that are of low abundance and without Poly-A tails. In addition, nuclei fraction enables separation of chromatin-associated scaffold RNAs from their cognate non-scaffold transcripts that have been released from chromatin.ConclusionsWe have developed a procedure for quantitative investigations on nucleic acids that are core components of RNA-directed DNA methylation. Our results not only demonstrated the efficacy of this procedure, but also provide lists of methylation-sensitive restriction enzymes, novel DNA methylation marker loci, and related siRNA sequences, all of which can be valuable for future epigenetic studies. Importantly, step-by-step protocols are provided in details such that the approaches can be easily followed by biologists with little experience in epigenetics.

Characterization of Non-CG Genomic Hypomethylation Associated with Gamma-Ray-Induced Suppression of CMT3 Transcription in Arabidopsis thaliana

Ionizing radiation causes various epigenetic changes, as well as a variety of DNA lesions such as strand breaks, cross-links, oxidative damages, etc., in genomes. However, radiation-induced epigenetic changes have rarely been substantiated in plant genomes. The current study investigates whether DNA methylation of Arabidopsis thaliana genome is altered by gamma rays. We found that genomic DNA methylation decreased in wild-type plants with increasing doses of gamma rays (5, 50 and 200 Gy). Irradiation with 200 Gy significantly increased the expression of transcriptionally inactive centromeric 180-bp (CEN) and transcriptionally silent information (TSI) repeats. This increase suggested that there was a substantial release of transcriptional gene silencing by gamma rays, probably by induction of DNA hypomethylation. High expression of the DNA demethylase ROS1 and low expression of the DNA methyltransferase CMT3 supported this hypothesis. Moreover, Southern blot analysis following digestion of genomic DNA with methylation-sensitive enzymes revealed that the DNA hypomethylation occured preferentially at CHG or CHH sites rather than CG sites, depending on the radiation dose. Unlike CEN and TSI repeats, the number of Ta3, AtSN1 and FWA repeats decreased in transcription but increased in non-CG methylation. In addition, the cmt3-11 mutant showed neither DNA hypomethylation nor transcriptional activation of silenced repeats upon gamma irradiation. Furthermore, profiles of genome-wide transcriptomes in response to gamma rays differed between the wild-type and cmt3-11 mutant. These results suggest that gamma irradiation induced DNA hypomethylation preferentially at non-CG sites of transcriptionally inactive repeats in a locus-specific manner, which depends on CMT3 activity.

RASSF1A in maternal plasma as a molecular marker of preeclampsia

This study aimed to quantitate cell free (cf) and cell free fetal (cff) DNA in maternal plasma by determining RASSF1A levels before and after enzyme digestion in women who subsequently developed preeclampsia (PE) and compare them with uncomplicated pregnancies. Twenty-four samples from pregnant women who developed PE and 48 samples from women with uncomplicated pregnancies were analysed. Blood samples were obtained at 11-13 weeks. cfDNA was determined by quantifying RASSF1A using qRT-PCR. A second qRT-PCR was performed following methylation-sensitive enzyme digestion by BstUI, to quantitate hypermethylated RASSF1A sequences of fetal origin. ACTB gene was used as control to confirm complete enzyme digestion. cfDNA and cffDNA levels were significantly increased in women who developed PE as compared with uncomplicated pregnancies (median cfDNA: 9402 vs 2698, median cffDNA: 934.5 vs 62, respectively). Following operating characteristic curve analysis, cut-off values of 7486 Εq/mL for cfDNA and 512 Εq/mL for cffDNA were chosen, which provided a sensitivity of 75% and 100% and specificity of 98% and 100%, respectively, to identify women at risk for PE. The study demonstrates potential use of cfDNA and cffDNA in maternal plasma as markers for the early prediction of women at risk for PE.

Revised Selection Criteria for Candidate Restriction Enzymes in Genome Walking

A new method to improve the efficiency of flanking sequence identification by genome walking was developed based on an expanded, sequential list of criteria for selecting candidate enzymes, plus several other optimization steps. These criteria include: step (1) initially choosing the most appropriate restriction enzyme according to the average fragment size produced by each enzyme determined using in silico digestion of genomic DNA, step (2) evaluating the in silico frequency of fragment size distribution between individual chromosomes, step (3) selecting those enzymes that generate fragments with the majority between 100 bp and 3,000 bp, step (4) weighing the advantages and disadvantages of blunt-end sites vs. cohesive-end sites, step (5) elimination of methylation sensitive enzymes with methylation-insensitive isoschizomers, and step (6) elimination of enzymes with recognition sites within the binary vector sequence (T-DNA and plasmid backbone). Step (7) includes the selection of a second restriction enzyme with highest number of recognition sites within regions not covered by the first restriction enzyme. Step (8) considers primer and adapter sequence optimization, selecting the best adapter-primer pairs according to their hairpin/dimers and secondary structure. In step (9), the efficiency of genomic library development was improved by column-filtration of digested DNA to remove restriction enzyme and phosphatase enzyme, and most important, to remove small genomic fragments (<100 bp) lacking the T-DNA insertion, hence improving the chance of ligation between adapters and fragments harbouring a T-DNA. Two enzymes, NsiI and NdeI, fit these criteria for the Arabidopsis thaliana genome. Their efficiency was assessed using 54 T3 lines from an Arabidopsis SK enhancer population. Over 70% success rate was achieved in amplifying the flanking sequences of these lines. This strategy was also tested with Brachypodium distachyon to demonstrate its applicability to other larger genomes.

84: First trimester free fetal DNA in the maternal circulation as a predictor of preeclampsia

circulation as a predictor of preeclampsia Bob Silver University of Utah Health Sciences Center and Intermountain Healthcare, Obstetrics and Gynecology, Salt Lake City, UT OBJECTIVE: To assess the relationship between cell-free fetal (CFF) DNA in maternal plasma in the first trimester and the subsequent development of preeclampsia. STUDY DESIGN: Nested case-control study of patients enrolled in the CAPPS prediction study (RCT of antioxidants for the prevention of preeclampsia) of 175 women who did and 175 women who did not develop preeclampsia. DNA was extracted from 600 L of maternal plasma obtained between 9 13 weeks gestation that had been frozen at 700C. Quantitative real time PCR using Taqman chemistry was used to amplify genes specific for the RASSF1A (methylated in the fetus) and B-actin (control) genes with and without digestion with the methylation sensitive enzymes Hinp1I and HhaI. Standard curves were generated using serial dilutions of a known DNA standard and samples were normalized. RESULTS: Women with and without preeclampsia were similar with regard to age, gestational age at blood-draw, education, smoking and race. As expected, cases had higher systolic and diastolic BP and BMI at the time of enrollment compared to controls. Total free DNA was higher in African American (median; 25 75%; 6.15; 0.14 28.7; p 0.024) and Hispanic (4.95; 0.20 26.82; p 0.037) compared to white women (2.33; 0.03 13.10). Levels of total free DNA also increased with increasing maternal BMI (p 0.016). After adjustment for BMI and race, total and fetal free DNA levels were similar between women with preeclampsia and controls. Levels of total free DNA were more predictive of preeclampsia in African Americans (ROC-AUC 0.69; 95% CI: 0.60 0.79; p 0.064). In African Americans, at levels 70, there was a 60.7% sensitivity and 80.4% specificity for the prediction of preeclampsia. CONCLUSION: Levels of total free DNA in the first trimester are increased in African American and Hispanic compared to white women, and levels increase with increasing BMI. There is no significant difference in levels of total or free fetal DNA in the first trimester in women who subsequently develop preeclampsia.

Phyloepigenomic comparison of great apes reveals a correlation between somatic and germline methylation states.

We have determined methylation state differences in the epigenomes of uncultured cells purified from human, chimpanzee, and orangutan, using digestion with a methylation-sensitive enzyme, deep sequencing, and computational analysis of the sequence data. The methylomes show a high degree of conservation, but the methylation states of ~10% of CpG island-like regions differ significantly between human and chimp. The differences are not associated with changes in CG content and recapitulate the known phylogenetic relationship of the three species, indicating that they are stably maintained within each species. Inferences about the relationship between somatic and germline methylation states can be made by an analysis of CG decay, derived from methylation and sequence data. This indicates that somatic methylation states are highly related to germline states and that the methylation differences between human and chimp have occurred in the germline. These results provide evidence for epigenetic changes that occur in the germline and distinguish closely related species and suggest that germline epigenetic states might constrain somatic states.

High-resolution methylome analysis of renal cell carcinoma and identification of aberrant epigenetic changes in gene bodies.

e15000 Background: Metastatic renal cell carcinoma (RCC) has a very poor prognosis with limited therapeutic options. Despite this, there have been very few global genetic and epigenetic studies of RCC. DNA methylation is an epigenetic change that has been extensively studied in cancer, but most studies have focused on changes in promoters and CpG islands. We utilized a novel high resolution, high throughput assay, the HELP (HpaII tiny fragment enrichment by ligation-mediated PCR) assay, to determine DNA methylation in RCC. The HELP assay examines CpG methylation at 1.3 million loci in the genome, including promoters, gene bodies and intergenic regions, making it the most comprehensive study of the RCC methylome available. Methods: We performed the HELP assay on 13 clear cell RCC and 13 control samples. The HELP assay compares restriction enzyme digestion with a methylation-sensitive enzyme (HpaII) to a methylation-insensitive enzyme (MspI) at CCGG sequences in the genome. These digestion fragments are amplified by PCR, fluorescently labeled, and co-hybridized onto a custom microarray. Methylation can be determined at each loci by comparison of the HpaII to MspI signal. Results: Unsupervised clustering showed that the RCC samples were epigenetically distinct and clustered completely separately from controls. Next, we determined common epigenetic alterations in all samples of RCC and observed that these tumors are characterized by a dramatic hypermethylation of a large number of loci in the gene bodies when compared to controls. Ingenuity pathway analysis of genes that were methylated and underexpressed in RCC revealed the TGFβ1 pathway to be the dominant pathway affected by epigenetic silencing, while cell death and cancer were two major biological functions influenced by epigenetic methylation. Conclusions: The HELP assay revealed widespread novel epigenetic changes and identified differentially regulated genes in RCC. This is the first study of DNA methylation changes in gene bodies in RCC. We hope to correlate methylation profiles with clinical response to uncover novel signatures of prognosis in RCC and identify new therapeutic targets within its epigenome.

Abstract 2728: Clonal nature of benign and malignant salivary gland neoplasms

Abstract The pathogenesis of salivary gland neoplasms (SGN) has not been established. There is a real debate on clonality issues and all the definitive evidence is on the side of polyclonal tumor origin. Evidence that many tumor types may be polyclonal in origin has been published. Despite all this body of evidence, scientists have incorporated the clonal theory proposed by Knudson (1985) and only strengthen these results (and do not discuss contrary findings), because they think it is expected. There are few clonality studies on benign salivary neoplasms and almost no data on malignant neoplasms. The HUMARA assay was used in the present study to investigate the clonality of 27 samples of SGN (17 benign and ten malignant). Briefly, samples were digested with HhaI and HpaII methylation-sensitive enzymes and a PCR carried out. Eighteen out of 27 cases revealed monotypy, while three were non-informative and six showed heterotypy, indicating a true polyclonality. In summary, our results show evidence that a subset of SGN might be clonal in origin, while some samples are polyclonal. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2728. doi:10.1158/1538-7445.AM2011-2728

Abstract 3023: Rapid methylation profiling of multiple genes at the same time using the methylation ligation-dependent macroarray (MLM)

Abstract Background: Aberrant methylation of multiple promoter CpG islands is a common event in human cancers. Methylation profile of cancer-related genes has extraordinary potential in basic research as well as translational medicine. The aim of this study was to replace the tedious and inefficient bisulfite-based methods with a simple technology which could permit a rapid and robust analysis of large numbers of genes in large numbers of tumor samples simultaneously. Materials and methods: Genomic DNA was first sonicated, then couples of primers for each specific gene promoter were annealed to this DNA. In a one-step reaction, the CfoI methylation-sensitive enzyme digested unmethylated DNA, whereas the Taq ligase joined primers annealed to the methylated sequence. Tagged ligation products were amplified by PCR and finally hybridized to a nylon membrane macroarray. Methylation Ligation-dependent Macroarray (MLM) allows the establishment of DNA methylation profiles on many samples (up to 21) and on many gene promoters (up to 45) in a simple reaction in 2-3 days. To validate this novel method, we used it to detect aberrant methylation in 14 cell lines and in DNA samples of 40 patients with a colorectal cancer. Results: In tumor cell lines, among the 42 genes investigated 23.7 (11-32) were found methylated whereas only 9 have been observed methylated in a normal fibroblast cell line (BJ). In normal colorectal tissues, about 12/40 gene promoters were found methylated; in colorectal cancers 16 to 36 genes were methylated. Methylated gene promoters identified in colorectal cancers were ADAMTS18 (78%), APC (28%), CDKN2A (20%), DKK2 (50%), GPx3 (48%), Mal (72%), MLH1 (30%), NELL1 (72%), SFRP4 (35%), SFRP5 (52%) and WRN (20%): Conclusion: The MLM is a fast, non expensive and reliable technology that profiles the DNA methylation status of &amp;gt;40 promoter CpG islands in various human samples. This technology can be used in research for rapid methylation profiling of numerous samples and for identification of new epigenetic biomarkers, but also in clinic for rapid cancer diagnosis, for predictive response to cancer therapy and for distinction between progressive and non-progressive diseases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3023. doi:10.1158/1538-7445.AM2011-3023

Differential methylation of the gene encoding myo -inositol 3-phosphate synthase ( Isyna1 ) in rat tissues

Myo-inositol levels are frequently altered in several brain disorders. Myo-inositol 3-phosphate synthase, encoded by the Isyna1 gene, catalyzes the synthesis of myo-inositol in cells. Very little is known about the mechanisms regulating Isyna1 expression in brain and other tissues. In this study, we have examined the role of DNA methylation in regulating Isyna1 expression in rat tissues. Transfection analysis using in vitro methylated promoter constructs, Southern blot analysis of genomic DNA from various tissues digested with a methylation-sensitive enzyme and CpG methylation profiling of genomic DNA from different tissues were used to determine differential methylation of Isyna1 in tissues. Transfection analysis using plasmids harboring mutated CpG residues in the 5'-upstream region of Isyna1 was used to identify critical residues mediating promoter activity. The -700 bp to -500 bp region (region 1) of Isyna1 exhibited increased methylation in brain cortex compared with other tissues; it also exhibited sex-specific methylation differences between matched male and female brain cortices. Mutation analysis identified one CpG residue in region 1 necessary for promoter activity in neuronal cells. A tissue-specific differentially methylated region (T-DMR) was found to be localized between +450 bp and +650 bp (region 3). This DMR was comparatively highly methylated in spleen, moderately methylated in brain cortex and poorly methylated in testis, consistent with mRNA levels observed in these tissues. Rat Isyna1 exhibits tissue-specific DNA methylation. Brain DNA was uniquely methylated in the 5'-upstream region and displayed gender specificity. A T-DMR was identified within the gene body of Isyna1. These findings suggest that Isyna1 is regulated, in part, by DNA methylation and that significant alterations in methylation patterns during development could have a major impact on inositol phosphate synthase expression in later life.

Systematic evaluation of genome-wide methylated DNA enrichment using a CpG island array

BackgroundRecent progress in high-throughput technologies has greatly contributed to the development of DNA methylation profiling. Although there are several reports that describe methylome detection of whole genome bisulfite sequencing, the high cost and heavy demand on bioinformatics analysis prevents its extensive application. Thus, current strategies for the study of mammalian DNA methylomes is still based primarily on genome-wide methylated DNA enrichment combined with DNA microarray detection or sequencing. Methylated DNA enrichment is a key step in a microarray based genome-wide methylation profiling study, and even for future high-throughput sequencing based methylome analysis.ResultsIn order to evaluate the sensitivity and accuracy of methylated DNA enrichment, we investigated and optimized a number of important parameters to improve the performance of several enrichment assays, including differential methylation hybridization (DMH), microarray-based methylation assessment of single samples (MMASS), and methylated DNA immunoprecipitation (MeDIP). With advantages and disadvantages unique to each approach, we found that assays based on methylation-sensitive enzyme digestion and those based on immunoprecipitation detected different methylated DNA fragments, indicating that they are complementary in their relative ability to detect methylation differences.ConclusionsOur study provides the first comprehensive evaluation for widely used methodologies for methylated DNA enrichment, and could be helpful for developing a cost effective approach for DNA methylation profiling.

Differential DNA Methylation as a Tool for Noninvasive Prenatal Diagnosis (NIPD) of X Chromosome Aneuploidies

The demographic tendency in industrial countries to delay childbearing, coupled with the maternal age effect in common chromosomal aneuploidies and the risk to the fetus of invasive prenatal diagnosis, are potent drivers for the development of strategies for noninvasive prenatal diagnosis. One breakthrough has been the discovery of differentially methylated cell-free fetal DNA in the maternal circulation. We describe novel bisulfite conversion- and methylation-sensitive enzyme digestion DNA methylation-related approaches that we used to diagnose Turner syndrome from first trimester samples. We used an X-linked marker, EF3, and an autosomal marker, RASSF1A, to discriminate between placental and maternal blood cell DNA using real-time methylation-specific PCR after bisulfite conversion and real-time PCR after methylation-sensitive restriction digestion. By normalizing EF3 amplifications versus RASSF1A outputs, we were able to calculate sex chromosome/autosome ratios in chorionic villus samples, thus permitting us to correctly diagnose Turner syndrome. The identification of this new marker coupled with the strategy outlined here may be instrumental in the development of an efficient, noninvasive method of diagnosis of sex chromosome aneuploidies in plasma samples.