CpG Island Arrays Research Articles

Finasteride induces Epigenetic Modulation of LSP1: A Gene implicated in Neutrophil Actin Dysfunction disease

Lymphocyte Specific Protein 1 (LSP1) gene translates F‐actin protein expressed in leucocytes such as neutrophils, lymphocytes, macrophages, and endothelium. It controls neutrophil motility, adhesion to fibrinogen matrix proteins, and transendothelial migration. Overexpression or increased level of Lymphocyte Specific Protein 1 (LSP1) is implicated in a rare immunologic disorder Neutrophil Actin Dysfunction characterized by spontaneous onset of recurrent infections and inflammation of oral cavity, skin, and respiratory tract. In this experiment aimed at studying the methylation profile and genes expression following finasteride treatment, we cultured human Leydig cells in the presence of 0.5 μM finasteride for 14 days and performed whole‐genome DNA methylation analysis using the NimbleGen Human DNA Methylation 3×720K Promoter Plus CpG Island Array and Ingenuity Pathway Analysis. Results identified several genes that were differentially methylated following finasteride treatment. Lymphocyte Specific Protein 1 (LSP1) gene was downregulated through methylation. We therefore deduced that finasteride could be a gateway to the treatment of Neutrophil Actin Dysfunction disease through epigenetic modification and decreased expression of LSP1 gene.

DNA methylation changes in the hippocampus of learning and memory disorder offspring rats of lead exposure during pregnant and lactation period.

Pregnant female rats exposed to lead may give birth to offspring with learning and memory disorders. Many studies have shown that there are many mechanisms that cause learning and memory impairment. Epigenetic mechanisms may play an important function in the learning and memory impairment. We examined DNA methylation changes in the hippocampus of rats with learning and memory disorder that were the offsprings of rats exposed to lead during pregnant and lactation period. The Morris Water Maze was applied as a learning and memory test, and a Roche NimbleGen's rat DNA methylation 385K Promoter Plus CpG Island Array was used for array hybridization. The results of the integrated navigation and spacial exploration test showed that until 21 days after birth and the lactation period, the learning and memory abilities of offsprings with lead exposure during pregnant and lactation period were significantly lower than those of the control group. The hippocampus DNA methylation levels of the three types of promoters increased compared with those of the control group. According to the Gene Ontology (GO) terms, metal ion transport, cell connections, the lamellar body, the axon bulge, and methylation of various metal transporters were found to be significantly enriched. Pathway analysis showed that the hedgehog signaling pathway, neuroactive receptor-ligand interaction with the ligase pathway, and interaction between cytokines had high methylation. DNA methylation of the whole genome in the hippocampus of the rats with learning and memory impairment induced by perinatal lead contact showed a lot of changes compared with that in the group of control.

Epigenetic Effects of Finasteride on Human Leydig Cells

Finasteride is a synthetic 4‐azasteroid, used in the treatment of both androgenetic alopecia and benign prostatic hyperplasia. In some men, its use is associated with persistent erectile dysfunction, ejaculatory dysfunction, loss of libido, altered sperm counts and infertility ‐ even after cessation of the drug. This condition has been termed “Post‐finasteride Syndrome” (PFS). We want to understand the underlying biochemical mechanisms that govern the development of this condition. Therefore in this experiment we tested the effects of exposure of finasteride on human Leydig cells, the main hormone‐producing cell of the testis, to identify epigenetic changes in the cells that could be at least partially responsible for PFS. We cultured human Leydig cells in the presence of 0.5 μM finasteride for 14 days and performed whole‐genome DNA methylation analysis using the NimbleGen Human DNA Methylation 3×720K Promoter Plus CpG Island Array and Ingenuity Pathway Analysis. Here we describe initial identification of specific genes and pathways involved that may ultimately lead to treatments to reverse PFS.Support or Funding InformationNIH/NIA (IR25AG047843‐01) to ABC

MTSS1 hypermethylation is associated with prostate cancer progression.

This study was conducted to evaluate the influence of DNA methylation of metastasis suppressor 1 (MTSS1) on prostate cancer (PCa) progression. Forty-nine paired PCa tissue samples and normal tissue samples from The Cancer Genome Atlas were analyzed. Methylome analysis, CpG island arrays and Hierarchical clustering were used to analyze methylation profiles of PCa tissues. MTSS1 methylation level was detected by methylation-specific PCR. Relative messenger RNA and the expression level of MTSS1 protein were identified by quantitative real-time PCR (qRT-PCR) and western blot analysis. The migration, invasion, proliferation, and cell cycle were detected separately by wound-healing assay, transwell chamber assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and flow cytometry. The roles of MTSS1 in PCa progression were demonstrated in vivo by tumor formation assays in nude mice. MTSS1 expression was decreased in PCa tissues in comparison with paired adjacent normal prostate tissues. Compared to the methylation of MTSS1 in normal prostate tissues based on the MethHC website, the MTSS1 in PCa tissues was hypermethylated. The expression of MTSS1 detected by qRT-PCR and western blot analysis was found to be downregulated in PCa cells and tissues. The reduced expression of MTSS1 by small interfering RNA-MTSS1 was recovered by 5-aza-2'-deoxycytidine treatment. Besides, MTSS1 demethylation inhibited migration, invasion, and proliferation of PCa cells, and induced cell cycle to be arrested at G0/G1 phase. Furthermore, it was shown by tumor xenograft assay that MTSS1 inhibited the growth of tumor in vivo. Hypermethylated MTSS1 promoted PCa cells migration, invasion, and proliferation, and suppressed cell cycle arrest at the G0/G1 phase.

The Effect of Citalopram on Genome-Wide DNA Methylation of Human Cells.

Commonly used pharmaceutical drugs might alter the epigenetic state of cells, leading to varying degrees of long-term repercussions to human health. To test this hypothesis, we cultured HEK-293 cells in the presence of 50 μM citalopram, a common antidepressant, for 30 days and performed whole-genome DNA methylation analysis using the NimbleGen Human DNA Methylation 3x720K Promoter Plus CpG Island Array. A total of 626 gene promoters, out of a total of 25,437 queried genes on the array (2.46%), showed significant differential methylation (p < 0.01); among these, 272 were hypomethylated and 354 were hypermethylated in treated versus control. Using Ingenuity Pathway Analysis, we found that the chief gene networks and signaling pathways that are differentially regulated include those involved in nervous system development and function and cellular growth and proliferation. Genes implicated in depression, as well as genetic networks involving nucleic acid metabolism, small molecule biochemistry, and cell cycle regulation were significantly modified. Involvement of upstream regulators such as BDNF, FSH, and NFκB was predicted based on differential methylation of their downstream targets. The study validates our hypothesis that pharmaceutical drugs can have off-target epigenetic effects and reveals affected networks and pathways. We view this study as a first step towards understanding the long-term epigenetic consequences of prescription drugs on human health.

DNA methylation in lung tissues of mouse offspring exposed in utero to polycyclic aromatic hydrocarbons

Polycyclic aromatic hydrocarbons (PAHs) comprise an important class of environmental pollutants that are known to cause lung cancer in animals and are suspected lung carcinogens in humans. Moreover, evidence from cell-based studies points to PAHs as modulators of the epigenome. The objective of this work was to assess patterns of genome-wide DNA methylation in lung tissues of adult offspring initiated in utero with the transplacental PAH carcinogens dibenzo [def,p]chrysene (DBC) or benzo [a]pyrene (BaP). Genome-wide methylation patterns for normal (not exposed), normal adjacent and lung tumor tissues obtained from adult offspring were determined using methylated DNA immunoprecipitation (MeDIP) with the NimbleGen mouse DNA methylation CpG island array. Lung tumor incidence in 45-week old mice initiated with BaP was 32%, much lower than that of the DBC-exposed offspring at 96%. Also, male offspring appeared more susceptible to BaP as compared to females. Distinct patterns of DNA methylation were associated with non-exposed, normal adjacent and adenocarcinoma lung tissues, as determined by principal components, hierarchical clustering and gene ontology analyses. From these methylation profiles, a set of genes of interest was identified that includes potential important targets for epigenetic modification during the process of lung tumorigenesis in animals exposed to environmental PAHs.

DNA methylome changes by estradiol benzoate and bisphenol A links early-life environmental exposures to prostate cancer risk

ABSTRACTDevelopmental exposure to endocrine-disrupting chemicals (EDCs), 17β-estradiol-3-benzoate (EB) and bisphenol A (BPA), increases susceptibility to prostate cancer (PCa) in rodent models. Here, we used the methylated-CpG island recovery assay (MIRA)-assisted genomic tiling and CpG island arrays to identify treatment-associated methylome changes in the postnatal day (PND)90 dorsal prostate tissues of Sprague-Dawley rats neonatally (PND1, 3, and 5) treated with 25 µg/pup or 2,500 µg EB/kg body weight (BW) or 0.1 µg BPA/pup or 10 µg BPA/kg BW. We identified 111 EB-associated and 86 BPA-associated genes, with 20 in common, that have significant differentially methylated regions. Pathway analysis revealed cancer as the top common disease pathway. Bisulfite sequencing validated the differential methylation patterns observed by array analysis in 15 identified candidate genes. The methylation status of 7 (Pitx3, Wnt10b, Paqr4, Sox2, Chst14, Tpd52, Creb3l4) of these 15 genes exhibited an inverse correlation with gene expression in tissue samples. Cell-based assays, using 5-aza-cytidine-treated normal (NbE-1) and cancerous (AIT) rat prostate cells, added evidence of DNA methylation-mediated gene expression of 6 genes (exception: Paqr4). Functional connectivity of these genes was linked to embryonic stem cell pluripotency. Furthermore, clustering analyses using the dataset from The Cancer Genome Atlas revealed that expression of this set of 7 genes was associated with recurrence-free survival of PCa patients. In conclusion, our study reveals that gene-specific promoter methylation changes, resulting from early-life EDC exposure in the rat, may serve as predictive epigenetic biomarkers of PCa recurrence, and raises the possibility that such exposure may impact human disease.

Promoter DNA methylation is associated with KLF11 expression in epithelial ovarian cancer.

As a transforming growth factor-β (TGF-β)-inducible gene, the expression of Krüppel-like transcription factor 11 (KLF11) is altered in several types of cancer. In the current study, through using human 9K CpG island array, KLF11 was identified as one of hypermethylated genes in RAS-transformed ovarian T29H cells. Methylation of the KLF11 promoter was also observed in ovarian cancer tissue samples accompanied by significantly reduced KLF11 gene expression. Interestingly, the expression of SMAD2, SMAD3, and SMAD7 genes was reduced in the tumour, whilst no change was found in TGF-β expression. Our data suggest a relationship between promoter DNA methylation and KLF11 gene expression in ovarian cancer tumorigenesis.

Identification of a DNA methylation signature to predict disease-free survival in locally advanced rectal cancer.

In locally advanced rectal cancer a preoperative predictive biomarker is necessary to adjust treatment specifically for those patients expected to suffer relapse. We applied whole genome methylation CpG island array analyses to an initial set of patients (n=11) to identify differentially methylated regions (DMRs) that separate a good from a bad prognosis group. Using a quantitative high-resolution approach, candidate DMRs were first validated in a set of 61 patients (test set) and then confirmed DMRs were further validated in additional independent patient cohorts (n=71, n=42). We identified twenty highly discriminative DMRs and validated them in the test set using the MassARRAY technique. Ten DMRs could be confirmed which allowed separation into prognosis groups (p=0.0207, HR=4.09). The classifier was validated in two additional cohorts (n=71, p=0.0345, HR=3.57 and n=42, p=0.0113, HR=3.78). Interestingly, six of the ten DMRs represented regions close to the transcriptional start sites of genes which are also marked by the Polycomb Repressor Complex component EZH2. In conclusion we present a classifier comprising 10 DMRs which predicts patient prognosis with a high degree of accuracy. These data may now help to discriminate between patients that may respond better to standard treatments from those that may require alternative modalities.

Global alterations of DNA methylation in cholangiocarcinoma target the Wnt signaling pathway

The molecular mechanisms underlying the genesis of cholangiocarcinomas (CCs) are poorly understood. Epigenetic changes such as aberrant hypermethylation and subsequent atypical gene expression are characteristic features of most human cancers. In CC, data regarding global methylation changes are lacking so far. We performed a genome-wide analysis for aberrant promoter methylation in human CCs. We profiled 10 intrahepatic and 8 extrahepatic CCs in comparison to non-neoplastic biliary tissue specimens, using methyl-CpG immunoprecipitation (MCIp) combined with whole-genome CpG island arrays. DNA methylation was confirmed by quantitative mass spectrometric analysis and functional relevance of promoter hypermethylation was shown in demethylation experiments of two CC cell lines using 5-aza-2'deoxycytidine (DAC) treatment. Immunohistochemical staining of tissue microarrays (TMAs) from 223 biliary tract cancers (BTCs) was used to analyze candidate gene expression at the protein level. Differentially methylated, promoter-associated regions were nonrandomly distributed and enriched for genes involved in cancer-related pathways including Wnt, transforming growth factor beta (TGF-β), and PI3K signaling pathways. In CC cell lines, silencing of genes involved in Wnt signaling, such as SOX17, WNT3A, DKK2, SFRP1, SFRP2, and SFRP4 was reversed after DAC administration. Candidate protein SFRP2 was substantially down-regulated in neoplastic tissues of all BTC subtypes as compared to normal tissues. A significant inverse correlation of SFRP2 protein expression and pT status was found in BTC patients. We provide a comprehensive analysis to define the genome-wide methylation landscape of human CC. Several candidate genes of cancer-relevant signaling pathways were identified, and closer analysis of selected Wnt pathway genes confirmed the relevance of this pathway in CC. The presented global methylation data are the basis for future studies on epigenetic changes in cholangiocarcinogenesis.

Genome‐wide screening of aberrant DNA methylation which associated with gene expression in mouse skin cancers

Epigenetic alteration of genomic DNA is a common and key process in carcinogenesis. There is considerable evidence indicating that some of the somatic alterations occurring during carcinogenesis in humans also involve the same processes as those observed in mice. Therefore, we analyzed mouse skin cancer tissues induced by the 2-stage carcinogenesis model to identify skin tumor-specific differentially methylated regions (ST-DMRs) during the multistep carcinogenesis process. We have previously identified ST-DMRs using the restriction landmark genomic scanning (RLGS) technique and reported that some of the mouse ST-DMRs were also epigenetically modified in human cancers, such as melanoma, neuroblastoma, and brain tumor. These results encouraged us to pursue global methylation screening in mouse skin carcinogenesis. Using the methylated DNA immunoprecipitation (MeDIP) method combined with the NimbleGen promoter plus CpG island (CpGi) array, we identified 615 ST-DMRs. In combination with global gene expression analysis, 91 of these ST-DMRs were shown to be located on or around the genes differentially expressed between normal skin and tumor tissues, including a candidate human tumor suppressor gene Tfap2e. As observed in human colorectal cancers, Tfap2e was methylated at a CpGi located in intron 3 and downregulated in skin tumors. Our results identified aberrant methylated regions that were associated with gene expression regulation during carcinogenesis, which may indicate critical genetic regions also involved in human carcinogenesis. © 2013 Wiley Periodicals, Inc.

Abstract 4257: Genome-wide CpG island methylation analysis identifies tumor specifically methylated genes in non-small cell lung cancer patients.

Abstract DNA methylation is part of the epigenetic gene regulation complex and it has been shown that methylation of certain genes occurs frequently in non-small cell lung cancers (NSCLC). We performed a genome-wide search for methylated CpG islands in primary tumors and corresponding non-malignant lung tissue samples of 101 stage I-III NSCLC patients by combining methylated DNA immunoprecipitation and microarray analysis using NimbleGen's 385K Human CpG Island plus Promoter arrays (MeDIP-chip). To test for differences in methylation between tumors and corresponding non-malignant lung tissues, we calculated paired t-statistics with permutation adjusted p-values for step down multiple testing. Overall, we identified 2.414 genomic positions differentially methylated between tumor and corresponding non-malignant lung tissue samples by MeDIP-chip analyses. Ninety-seven % of them were found to be tumor-specifically methylated. Annotation of these genomic positions resulted in the identification of 477 tumor-specifically methylated genes. These genes were classified according to Gene Ontology (GO) categories and over-representation of certain GO terms was calculated. Interestingly, we found that a large number of tumor-specifically methylated genes act as regulators of gene expression or mediate homophilic cell adhesion. Tumor-specific methylation of selected genes was confirmed by methylation-sensitive high-resolution melting analysis and ROC curve analyses revealed that primary tumors may be distinguished from non-malignant lung tissue samples by methylation of certain genes. In addition, in the majority of tumors methylation of certain genes was associated with loss of their protein expression determined by immunohistochemistry. Moreover, treatment of NSCLC cells with epigenetically active drugs resulted in upregulated expression of many tumor-specifically methylated genes analysed by gene expression microarrays. In conclusion, we identified a large number of tumor-specifically methylated genes in NSCLC patients. Expression of many of them is regulated by methylation. Overall, our findings emphasize the impact of methylation on the pathogenesis of NSCLCs. Citation Format: Gerwin Heller, Valerie Babinsky, Barbara Ziegler, Marlene Weinzierl, Christian Noll, Corinna Altenberger, Leonhard Muellauer, Gerhard Dekan, Yuliya Grin, Gyoergy Lang, Adelheid End-Pfützenreuter, Irene Steiner, Sonja Zehetmayer, Balazs Doeme, Madeleine Arns, Kwun M. Fong, Casey M. Wright, Ian A. Yang, Walter Klepetko, Martin Posch, Christoph C. Zielinski, Sabine Zoechbauer-Mueller. Genome-wide CpG island methylation analysis identifies tumor specifically methylated genes in non-small cell lung cancer patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4257. doi:10.1158/1538-7445.AM2013-4257

Abstract 2: Genome-wide DNA methylation profiling identifies novel diagnostic and prognostic biomarkers in early stage prostate cancer.

Abstract Prostate cancer (PCa) is the most prevailing cancer in the male population and the second-leading cause of cancer-related death in the Western world. Although genomic alterations during PCa development have been recently characterized [1], substantial evidence shows that genome-wide epigenetic insults complement or even precede genetic alterations [2]. Promoter CpG island methylation frequently correlates with gene silencing of functional genes. Therefore we hypothesized that scanning for early-silenced genes by profiling hypermethylation sites of promoter CpG islands would facilitate identification of novel methylation biomarkers with potential functions during prostate carcinogenesis. DNA from 9 primary pT2a tumors and matched normal tissue was subjected to enrichment of methylated sequences using Methyl CpG Immunoprecipitation (MCIp) [3], followed by array co-hybridization using Agilent CpG island arrays. Candidate genes were selected according to 1) hypermethylation in gene promoter regions in tumor tissue and 2) reduced expression in PCa in comparison to normal prostate tissue based on published gene expression datasets. DNA methylation was quantified by mass spectrometry-based EpiTYPER technology [4]. We identified 245 promoter CpG islands corresponding to 207 genes that were hypermethylated in at least 6/9 early stage PCa. Gene ontology enrichment analyses indicated enrichment of homeobox genes, and of gene functions such as regulation of transcription, cellular biosynthesis and morphogenesis, and neuron development. Quantitative methylation analysis of 9 selected candidate genes confirmed significant hypermethylation in the range of 20 -50% in 6/9 tumors of the initial discovery set in comparison with normal prostate tissue. Further validation in 37 sample triplets of matched normal tissue - prostate intraepithelial neoplasia (PIN) - tumor tissue indicated significant hypermethylation of SPG20 (spastic paraplegia 20, p=0.023, Wilcoxon signed-rank test), SPATA6 (spermatogenesis associated 6, p=0.017), and DUOXA1 (dual oxidase maturation factor 1 alpha, p=0.017) already at the PIN stage. Moreover, methylation analysis of 60 prostate tumor samples with increasing malignancy revealed a significant correlation between DNA hypermethylation patterns and biochemical recurrence of prostate cancer (p=0.008, log-rank test). From this genome-wide study we identified relevant biomarkers for early-detection and prognosis of early stage prostate cancer. The identified genes might represent interesting candidates to further investigate their function in the molecular pathogenesis and development of prostate cancer. Citation Format: Po-Hsien J. Huang, Wolfgang Göring, Thomas Hielscher, Dieter Weichenhan, Wolfgang A. Schulz, Christoph Plass, Clarissa Gerhäuser. Genome-wide DNA methylation profiling identifies novel diagnostic and prognostic biomarkers in early stage prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2. doi:10.1158/1538-7445.AM2013-2

Genome-wide analysis of DNA methylation in tongue squamous cell carcinoma

Tongue squamous cell carcinoma (TSCC) is one of the most common types of oral cancer; however, its molecular mechanisms remain unclear. In this study, methylated DNA immunoprecipitation (MeDIP) coupled with methylation microarray analysis was performed to screen for aberrantly methylated genes in adjacent normal control and TSCC tissues from 9 patients. Roche NimbleGen Human DNA Methylation 385K Promoter Plus CpG Island Arrays were used to detect 28,226 CpG sites. A total of 1,269 hypermethylated CpG sites covering 330 genes and 1,385 hypomethylated CpG sites covering 321 genes were found in TSCC tissue, compared to the adjacent normal tissue. Furthermore, we chose three candidate genes (FBLN1, ITIH5 and RUNX3) and validated the DNA methylation status by methylation-specific PCR (MS-PCR) and the mRNA expression levels by reverse transcription PCR (RT-PCR). In TSCC tissue, FBLN1 and ITIH5 were shown to be hypermethylated and their expression was found to be decreased, and RUNX3 was shown to be hypomethylated, however, its mRNA expression was found to be increased. In addition, another three genes (BCL2L14, CDCP1 and DIRAS3) were tested by RT-PCR. In TSCC tissue, BCL2L14 and CDCP1 expressions were markedly upregulated, and DIRAS3 expression was significantly downregulated. Our data demonstrated that aberrant DNA methylation is observed in TSCC tissue and plays an important role in the tumorigenesis, development and progression of TSCC.

Pediatric Acute Myeloid Leukemia with t(8;16)(p11;p13): A Distinct Clinical and Biological Entity. Results of a Collaborative Study by the International Berlin-Frankfurt-Münster AML Study Group.

AbstractAbstract 2516▪▪This icon denotes a clinically relevant abstract Introduction:In pediatric acute myeloid leukemia (AML) cytogenetic abnormalities are important for prognosis and treatment stratification. Some recurring cytogenetic abnormalities occur so rarely that large collaborative studies are required to define their prognostic impact. t(8;16)(p11;p13) is an aberration reported in <1% of pediatric AML patients. Recently, Haferlach et al. (Leukemia, 2009) reported that t(8;16)(p11;p13) represents a distinct clinical and biological subgroup in adult AML, with dismal clinical outcome (median overall survival (OS) 4.7 months). In pediatric AML case reports are available, but studies of larger series are lacking. Methods:We collected data on patients with t(8;16)(p11;p13) (n=39) from 18 collaborative study groups belonging to the International Berlin-Frankfurt-Münster AML-Committee. Data collection included clinical characteristics, karyotype, morphology and immunophenotype, which were centrally reviewed by the co-authors. In addition, the literature was screened for reports on pediatric t(8;16)(p11;p13), which found an additional 23 cases. Results:A total of 62 pediatric AML patients with t(8;16)(p11;p13) were identified, diagnosed between 1978–2010, including 2 secondary AML cases. They had a median age of 1.2 years (range 0–17 years) and 56% of the patients were younger than 2 years old. Median white blood cell count was 21.3 × 109/l (n= 59, range 1.1–173 × 109/l). FAB type was M4 or M5 in 95% of the cases and immunophenotypes showed monocytic differentiation. Erythrophagocytosis (23/33, 70%), leukemia cutis (21/36, 58%) and disseminated intravascular coagulation (DIC, 15/38, 39%) occurred frequently. At initial diagnosis, 35/57 (61%) of the patients showed t(8;16)(p11;p13) as the sole aberration (5 incomplete karyotypes were excluded). In the remaining 22 patients, no recurrent additional cytogenetic aberrations were found. In patients diagnosed after January 1st1993, and treated with chemotherapy at initial diagnosis (n= 34), the CR rate was 85% (29/34), 5-year OS was 59% (±9%), event free survival (EFS) was 57% (±9%) and the cumulative incidence of relapse (CIR) was 28% (±8%). The median follow-up time of survivors was 4 years. Outcome was is comparable to a reference cohort of AML-BFM patients (n= 543) treated in the same era, with OS of 62% (±2%), EFS of 50% (±2%) and CIR of 42% (±2%). Most striking was that in 7 neonates (<1 month of age) diagnosed with AML and t(8;16)(p11;p13), spontaneous complete remission was achieved. Three of these infants survived without recurrence of disease (follow-up 0.9–4.0 years). For those who relapsed (0.7–4 years after initial diagnosis), chemotherapy treatment was successful in 2/4 patients.Gene expression data, using the Affymetrix Human Genome U133 plus 2.0 array, were available from 297 pediatric AML samples, of which 8 were t(8;16)(p11;p13). The gene expression signature of t(8;16)(p11;p13) AML clustered close to but distinct from MLL-rearranged AML using unsupervised analysis. Highly expressed genes included HOXA11, HOXA10, RET, PERP and GGA2, indicating a pathway in common with MLL-rearranged AML with distinct features. PERP is a direct target gene of p53 and acts as an effector of apoptosis, GGA2 codes for a protein that regulates protein trafficking within the cell. PERP and GGA2 knockdown in high expressing cell lines did not result in significant changes in cell proliferation or apoptosis, thus their function in leukemic blasts remains unknown.DNA methylation profiling was performed using Agilent 244K Human CpG Island arrays on 167 pediatric AML samples of which 12 had t(8;16)(p11;p13). Differential methylation of the TRIM59 promoter, an oncogene described in prostate cancer located on chromosome 3, was confirmed by pyrosequencing, with subsequent mRNA overexpression, validated by RT-qPCR. Conclusion:This study shows the distinct nature of t(8;16)(p11;p13) pediatric AML with remarkable clinical features such as high percentage of leukemia cutis and DIC. The 5-year OS of this pediatric cohort was intermediate (59%), and therefore different from the dismal prognosis reported in adult cases. In neonatal t(8;16)(p11;p13) AML, spontaneous remissions were reported. Profiling studies have shed light on potential oncogenic mechanisms involved in AML with t(8;16)(p11;p13), including HOX-gene upregulation similar to MLL-rearranged AML. Disclosures:No relevant conflicts of interest to declare.

Genome-wide analysis of DNA methylation changes in the rat ovary after prenatal exposure to di-(2-ethylhexyl)-phthalate

To study the effects of prenatal exposure to Di-(2-ethylhexyl)-phthalate (DEHP) on genome-wide epigenetic alterations in ovary of adult offspring rat. Pregnant Wistar rats were randomly treated with DEHP (1000 mg/kg) or con oil at 12 - 17 days upon pregnance. DNA methylation changes in the ovary for the adult offsprings which were 70 days old were detected by Rat DNA methylation promoter plus CpG island arrays CpG island chip. Gene ontology (GO) method was performed to analyze the function of genes which were significantly different between exposed group and control group. Gene Igfbp1 (insulin-like growth factor binding protein 1) and Itga3 (integrin alpha 3) were randomly selected and the methylation status were verified by bisulfite genomic sequencing (BSP). The methylation status were significantly different between exposed and control group in 406 genes (71 genes as hypermethylation and 335 genes as hypomethylation) (P < 0.05). GO analysis revealed that molecular transducer activity, cell part, cell, cellular process, multicellular organismal process, response to stimulus, biological regulation, regulation of biological process, reproduction, reproductive process, and rhythmic process were involved. The sequencing results were consistent with the data obtained by chips. This study provides evidence that prenatal exposure of DEHP may be associated with methylation changes on the genes in the rat ovary. Genes related to reproductive process have highly significant methylation changes, which may shed new light on mechanisms of reproductive and developmental toxicity after prenatal exposure to DEHP.

Genome‐wide methylation screen in low‐grade breast cancer identifies novel epigenetically altered genes as potential biomarkers for tumor diagnosis

Aberrant DNA methylation constitutes a well-established epigenetic marker for breast cancer. Changes in methylation early in cancer development may be clinically relevant for cancer detection and prognosis-based therapeutic decisions. In the present study, a combination of methyl-CpG immunoprecipitation (MCIp) and human CpG island (CGI) arrays was applied to compare genome-wide DNA methylation profiles in 10 low-grade in situ and invasive breast cancers against 10 normal breast samples. In total, 214 CGIs were found to be hypermethylated in ≥6 of 10 tumors. Functional term enrichment analyses revealed an overrepresentation of homeobox genes and genes involved in transcription and regulation of transcription. Significant hypermethylation of 11 selected genes in tumor vs. normal tissue was validated in two independent sample sets (45 tumors and 11 controls, 43 tumors and 8 controls) using quantitative EpiTyper technology. In tumors, median methylation levels of BCAN, HOXD1, KCTD8, KLF11, NXPH1, POU4F1, SIM1, and TCF7L1 were ≥30% higher than in normal samples, representing potential biomarkers for tumor diagnosis. Using the 90th percentile of methylation levels in normal tissue as cutoff value, 62-92% of in situ samples (n=13), 72-97% of invasive samples from the first validation set (n=32), and 86-100% of invasive samples from the second validation set (n=43) were classified as hypermethylated. Hypermethylation of KLF11 and SIM1 might also be associated with increased risk of developing metastases. In summary, early methylation changes are frequent in the low-grade pathway of breast cancer and may be useful in the development of differential diagnostic and possibly also prognostic markers.

Screening significantly hypermethylated genes in fetal tissues compared with maternal blood using a methylated-CpG island recovery assay-based microarray

BackgroundThe noninvasive prenatal diagnosis procedures that are currently used to detect genetic diseases do not achieve desirable levels of sensitivity and specificity. Recently, fetal methylated DNA biomarkers in maternal peripheral blood have been explored for the noninvasive prenatal detection of genetic disorders. However, such efforts have covered only chromosomal aneuploidy, and fetal methylated DNA biomarkers in maternal whole blood for detecting single-gene diseases remain to be discovered.MethodsTo address this issue, we systematically screened significantly hypermethylated genes in fetal tissues and compared them with maternal peripheral blood potential in an attempt to detect fetal genes in maternal peripheral blood. First, the methylated-CpG island recovery assay combined with a CpG island array was performed for four fetus-toward placental tissues and the corresponding maternal peripheral bloods. Subsequently, direct bisulfite sequencing and combined bisulfite restriction analysis (COBRA) were carried out to validate the methylation status of the hypermethylated genes that were identified by the microarray analysis.ResultsThree hundred and ten significantly hypermethylated genes in the placental tissues were detected by microarray. From the top 15 hypermethylated genes detected by microarray, two were selected for sequencing validation in placental tissue and chorionic villus samples and four were selected for COBRA validation in four placental tissues, ten amniotic fluids and five chorionic villus samples. The six selected genes were confirmed to be hypermethylated in placental tissue and chorionic villus samples, but methylation of the genes could not be detected in the amniotic fluids.ConclusionsOf the many hypermethylated genes and methylation sites that were found in the fetal tissues, some have great potential to be developed into molecular markers for noninvasive prenatal diagnosis of monogenic disorders. Further clinical studies are warranted to confirm these findings.

Screening of significantly hypermethylated genes in breast cancer using microarray-based methylated-CpG island recovery assay and identification of their expression levels

To screen candidate methylation markers for early detection of breast cancer and to explore the relationship between methylation and gene expression, we performed methylated-CpG island recovery assay (MIRA) combined with CpG island array on 61982 CpG sites across 4162 genes in 10 cancerous and 10 non-cancerous breast tissues. Direct bisulfite sequencing and combined bisulfite restriction analysis (COBRA) were carried out in independent cancerous and non-cancerous samples. Gene expression was analyzed by microarrays and validated using RT-PCR. We detected 70 significantly hypermethylated genes in breast cancer tissues, including many novel hypermethylated genes such as ITGA4, NFIX, OTX2 and FGF12. Direct bisulfite sequencing showed widespread methylation occurring in intragenic regions of the WT1, PAX6 and ITGA4 genes and in the promoter region of the OTX2 gene in breast cancer tissues. COBRA assay confirmed that the WT1, OTX2 and PAX6 genes were hypermethylated in breast cancer tissues. Clustering analysis of the gene expression of 70 significantly hypermethylated genes revealed that most hypermethylated genes in breast cancer were not expressed in breast tissues. RT-PCR assay confirmed that WT1 and PITX2 were only weakly expressed in the breast cancer tissues and were not expressed in most non-cancerous breast tissues. OTX2 and PAX6 were not expressed in either breast cancer or non-cancerous tissues. In conclusion, these results will expand our knowledge of hypermethylated genes and methylation sites for early detection of breast cancer and deepen our understanding of the relationship between methylation and gene expression. The MIRA approach can screen candidate methylated genes for further clinical validation more effectively than gene expression microarray-based strategy.

Genome-wide survey reveals dynamic widespread tissue-specific changes in DNA methylation during development

BackgroundChanges in DNA methylation in the mammalian genome during development are frequent events and play major roles regulating gene expression and other developmental processes. It is necessary to identify these events so that we may understand how these changes affect normal development and how aberrant changes may impact disease.ResultsIn this study Methylated DNA ImmunoPrecipitation (MeDIP) was used in conjunction with a NimbleGen promoter plus CpG island (CpGi) array to identify Tissue and Developmental Stage specific Differentially Methylated DNA Regions (T-DMRs and DS-DMRs) on a genome-wide basis. Four tissues (brain, heart, liver, and testis) from C57BL/6J mice were analyzed at three developmental stages (15 day embryo, E15; new born, NB; 12 week adult, AD). Almost 5,000 adult T-DMRs and 10,000 DS-DMRs were identified. Surprisingly, almost all DS-DMRs were tissue specific (i.e. methylated in at least one tissue and unmethylated in one or more tissues). In addition our results indicate that many DS-DMRs are methylated at early development stages (E15 and NB) but are unmethylated in adult. There is a very strong bias for testis specific methylation in non-CpGi promoter regions (94%). Although the majority of T-DMRs and DS-DMRs tended to be in non-CpGi promoter regions, a relatively large number were also located in CpGi in promoter, intragenic and intergenic regions (>15% of the 15,979 CpGi on the array).ConclusionsOur data suggests the vast majority of unique sequence DNA methylation has tissue specificity, that demethylation has a prominent role in tissue differentiation, and that DNA methylation has regulatory roles in alternative promoter selection and in non-promoter regions. Overall, our studies indicate changes in DNA methylation during development are a dynamic, widespread, and tissue-specific process involving both DNA methylation and demethylation.