Normal Methylation Patterns Research Articles

Quantification of 5-methyl-2'-deoxycytidine in the DNA.

Methylation at position 5 of cytosine (Cyt) at the CpG sequences leading to formation of 5-methyl-cytosine (m(5)Cyt) is an important element of epigenetic regulation of gene expression. Modification of the normal methylation pattern, unique to each organism, leads to the development of pathological processes and diseases, including cancer. Therefore, quantification of the DNA methylation and analysis of changes in the methylation pattern is very important from a practical point of view and can be used for diagnostic purposes, as well as monitoring of the treatment progress. In this paper we present a new method for quantification of 5-methyl-2'deoxycytidine (m(5)C) in the DNA. The technique is based on conversion of m(5)C into fluorescent 3,N(4)-etheno-5-methyl-2'deoxycytidine (εm(5)C) and its identification by reversed-phase high-performance liquid chromatography (RP-HPLC). The assay was used to evaluate m(5)C concentration in DNA of calf thymus and peripheral blood of cows bred under different conditions. This approach can be applied for measuring of 5-methylcytosine in cellular DNA from different cells and tissues.

Report and review of described associations of Mayer-Rokitansky-Küster-Hauser syndrome and Silver-Russell syndrome.

Silver-Russell syndrome (SRS) and Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome are described in isolation. However, their co-occurrence has only been rarely reported. Here, we present a case report of an adolescent with SRS who was diagnosed with MRKH during the evaluation of primary amenorrhoea. Multiplex ligation-dependent probe amplification analysis showed a normal methylation pattern and normal dosage at 11p15.5. A PubMed search for all peer-reviewed publications (original articles and reviews) using the key words Silver-Russell syndrome, Mayer-Rokitansky-Küster-Hauser syndrome, genetics, hypomethylation and reproductive anomalies identified three cases of SRS with MRKH, two of which were associated with significant hypomethylation of the H19 imprinting control region of the 11p15.5 locus. This report highlights the association between SRS and MRKH. The absence of hypomethylation and normal dosage at 11p15.5 suggests these two rare entities share alternative aetiopathogenic mechanisms.

DNA methylation patterns and karyotype analysis of off-type and normal phenotype somatic embryos of feijoa

Acca sellowiana is a native species to the southern region of Brazil, with secondary dispersion in Uruguay northeast. The potentially economic interest of this species motivated many fundamental and applied researches, such as micropropagation protocols development, including somatic embryogenesis (SE). Previous studies have reported the high frequency of off-type somatic embryos in A. sellowiana SE and low conversion efficiency, possibly due to this high off-type somatic embryos formation. In this sense, this study was aimed to evaluate the effect of 2,4-dichlorophenoxyacetic acid (2,4-D) in the induction and development of somatic embryos in direct and indirect SE through cytogenetic and DNA methylation analysis. The results of the present work reinforces the previous findings that 2,4-D plays a key role in the SE induction in A. sellowiana. However, the continuous presence of this auxin in the culture medium enhances the rate of off-type somatic embryos. This auxin also apparently affects the cells and chromosomes size in normal and off-type somatic embryos and DNA methylation patterns of the analyzed samples. These findings may indicate that the off-type somatic embryos formation may be related to genetic and/or epigenetic changes caused by long-term cultivation in A. sellowiana. The intermediate phase of callus also indicated increased formation of off-types, corroborating other studies that showed that indirect SE could cause increased formation of anomalies in regenerated plantlets.

Epigenetic germline mosaicism in infertile men.

Imprinted genes are expressed either from the paternal or the maternal allele, because the other allele has been silenced in the mother's or father's germline. Imprints are characterized by DNA methylation at cytosine phosphate guanine sites. Recently, abnormal sperm parameters and male infertility have been linked to aberrant methylation patterns of imprinted genes in sperm DNA. However, these studies did not account for possible epigenetic heterogeneity in sperm. We have investigated whether spermatozoa are a homogeneous cell population regarding DNA methylation of imprinted genes. Swim-up sperm was obtained from 45 men with normal (n = 19) and abnormal (n = 26) sperm parameters. DNA methylation of the imprinted gene KCNQ1OT1 was measured in multiple pools of 10 spermatozoa by a highly sensitive pyrosequencing-based oligo-sperm methylation assay (OSMA). DNA methylation of four imprinted genes (KCNQ1OT1, MEST, H19 and MEG3) was further analysed by deep bisulfite sequencing, which allows analysis at the single-cell level. Using OSMA, we found a significantly increased variation in the DNA methylation values of the maternally methylated gene KCNQ1OT1 in samples with abnormal sperm parameters. DBS showed that normozoospermic samples had a homogenous pattern of DNA methylation, whereas oligoasthenozoospermic samples contained discrete populations of spermatozoa with either normal or abnormal methylation patterns. Aberrant methylation of H19 appears to occur preferentially on the maternally inherited allele. Our results demonstrate the presence of epigenetic mosaicism in the semen of oligoasthenozoospermic men, which probably results from errors in imprint erasure.

Abstract 2308: Heterogeneous and low-level methylation of novel biomarker candidates for breast cancer clinical management

Abstract Breast cancer is a highly heterogeneous malignancy, which is best exemplified by the fact that despite similar clinical and pathological features large number breast cancer patients experience different outcome of the disease. Therefore there is an urgent need for identification of new biomarkers to stratify patients for personalized treatment. Methylation of gene promoters regulates genes transcription and provides normal cell function. At the same time, changes of the normal pattern of methylation are directly involved in malignant transformation, and have been shown to provide a good source of clinical biomarkers for cancer management. Nevertheless it is still challenging to firstly identify methylation changes suitable for clinical biomarkers and secondly evaluate the clinical specificity and sensitivity of those biomarkers. We used ultra high-resolution microarrays to compare genome wide methylation patterns of breast carcinomas (N=20) with non-malignant breast tissue (N=5) and identified 1000+ differentially methylated regions (DMR) in the genome, which undergo methylation changes during breast cancer development. The results of genome wide screening technologies (due to the high complexity of those methods) require validation with PCR based technology. Therefore, we validated 19 of the regions identified in the genome wide screening with the Methylation Sensitive High Resolution Melting (MS-HRM) technique in a panel of breast carcinomas (N=275) and non-malignant controls (N=74). The validation experiments corroborated the results, confirming that methylation changes within the identified regions can potentially be used as biomarkers. Interestingly, we have observed two types of methylation gain at the validated loci: 1) loci undergoing predominantly complete methylation, and 2) loci acquiring mainly heterogeneous methylation during carcinogenesis. It can be speculated that the different types of methylation gain have different physiological consequences for transcriptional activity of the affected genes. Consequently the involvement of the gene in carcinogenesis may depend on the type of methylation acquired. However more research is needed to elaborate these findings. Our validation experiments also showed that almost all tested DMRs (17 out of 19) displayed low-level methylation in non-malignant breast tissue. The physiological consequences of the low level methylation of non-malignant tissue are largely unknown. These changes does not seem to have immediate pathological consequences but may predispose to breast cancer and hence have significant impact on evaluation of the clinical sensitivity and specificity of methylation biomarkers. Summarizing in our study, we have identified two phenomena that have significant consequences for the locus specific methylation biomarker development and need to be further researched. Citation Format: Tomasz K. Wojdacz, Johanne A. Windeløv, Britta B. Thestrup, Tine E. Damsgaard, Jens Overgaard, Lise Lotte Hansen. Heterogeneous and low-level methylation of novel biomarker candidates for breast cancer clinical management. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2308. doi:10.1158/1538-7445.AM2014-2308

Flow-dependent epigenetic DNA methylation regulates endothelial gene expression and atherosclerosis.

In atherosclerosis, plaques preferentially develop in arterial regions of disturbed blood flow (d-flow), which alters endothelial gene expression and function. Here, we determined that d-flow regulates genome-wide DNA methylation patterns in a DNA methyltransferase-dependent (DNMT-dependent) manner. Induction of d-flow by partial carotid ligation surgery in a murine model induced DNMT1 in arterial endothelium. In cultured endothelial cells, DNMT1 was enhanced by oscillatory shear stress (OS), and reduction of DNMT with either the inhibitor 5-aza-2'-deoxycytidine (5Aza) or siRNA markedly reduced OS-induced endothelial inflammation. Moreover, administration of 5Aza reduced lesion formation in 2 mouse models of atherosclerosis. Using both reduced representation bisulfite sequencing (RRBS) and microarray, we determined that d-flow in the carotid artery resulted in hypermethylation within the promoters of 11 mechanosensitive genes and that 5Aza treatment restored normal methylation patterns. Of the identified genes, HoxA5 and Klf3 encode transcription factors that contain cAMP response elements, suggesting that the methylation status of these loci could serve as a mechanosensitive master switch in gene expression. Together, our results demonstrate that d-flow controls epigenomic DNA methylation patterns in a DNMT-dependent manner, which in turn alters endothelial gene expression and induces atherosclerosis.

Physiological Oxygen Prevents Frequent Silencing of the DLK1-DIO3 Cluster during Human Embryonic Stem Cells Culture

Genetic and epigenetic alterations are observed in long-term culture (>30 passages) of human embryonic stem cells (hESCs); however, little information is available in early cultures. Through a large-scale gene expression analysis between initial-passage hESCs (ihESCs, <10 passages) and early-passage hESCs (ehESCs, 20-30 passages) of 12 hESC lines, we found that the DLK1-DIO3 gene cluster was normally expressed and showed normal methylation pattern in ihESC, but was frequently silenced after 20 passages. Both the DLK1-DIO3 active status in ihESCs and the inactive status in ehESCs were inheritable during differentiation. Silencing of the DLK1-DIO3 cluster did not seem to compromise the multilineage differentiation ability of hESCs, but was associated with reduced DNA damage-induced apoptosis in ehESCs and their differentiated hepatocyte-like cell derivatives, possibly through attenuation of the expression and phosphorylation of p53. Furthermore, we demonstrated that 5% oxygen, instead of the commonly used 20% oxygen, is required for preserving the expression of the DLK1-DIO3 cluster. Overall, the data suggest that active expression of the DLK1-DIO3 cluster represents a new biomarker for epigenetic stability of hESCs and indicates the importance of using a proper physiological oxygen level during the derivation and culture of hESCs.

Dnmt3b Has Few Specific Functions In Adult Hematopoietic Stem Cells But Shows Abnormal Activity In The Absence Of Dnmt3a

DNA methylation is one of the major epigenetic modifications in the vertebrate genome and is important for development, stem cell differentiation, and malignant transformation. DNA methylation is catalyzed by the DNA methyltransferase enzymes Dnmt1, Dnmt3a, and Dnmt3b. We have recently shown that Dnmt3a is essential for hematopoietic stem cell (HSC) differentiation. Ablation of Dnmt3a in hematopoietic cells (Mx1-CRE; Dnmt3a-KO) resulted in HSCs that could not sustain peripheral blood generation after serial transplantation, while phenotypically defined HSCs accumulated in the bone marrow. Recurrent somatic mutations in DNTM3A have been discovered in patients with a wide range of hematopoietic malignancies (AML, MDS, MPN, CML, T-ALL, T-cell lymphoma) suggesting a critical role for de novo DNA methylation in normal and leukemic hematopoiesis. As Dnmt3b is also highly expressed in HSCs and congenital mutations in DNMT3B can cause ICF (immunodeficiency, centromeric instability, and facial anomalies) syndrome, in this study we used a mouse model to investigate if Dnmt3b had distinct roles in HSCs.We conditionally inactivated Dnmt3b in HSCs using the Mx1-CRE system (Dnmt3b-KO) and performed serial competitive transplantation. Loss of Dnmt3b had minimal functional consequences for adult HSC function even after three rounds of transplantation. However, combinatorial deletion of both Dnmt3a and Dnmt3b (Dnmt3ab-dKO) exacerbated the differentiation defect seen in Dnmt3a-KO HSCs, leading to a dramatic accumulation of mutant HSCs in the bone marrow (>50-fold), suggesting a synergistic effect resulting from simultaneous ablation of both de novo DNA methyltransferases. The accumulation of Dnmt3ab-dKO HSCs cannot be attributed to altered proliferation or apoptosis, but is due to an imbalance between self-renewal and differentiation. RNA-SEQ of the mutant HSCs revealed loss of transcriptional integrity in Dnmt3ab-dKO HSCs including increased expression of repetitive elements, inappropriate mRNA splicing, and over-expression of HSC-specific genes.To examine the impact of loss of Dnmt3a and -3b on DNA methylation in HSCs, we performed Whole Genome Bisulfite Sequencing (WGBS). Ablation of both enzymes resulted in loss of DNA methylation that was much more extensive than that seen in the absence of Dnmt3a alone, while loss of Dnmt3b alone showed only minimal changes in DNA methylation compared to control HSCs. One puzzling finding was the observation that a subset of promoter CpG islands (CGIs) actually gained DNA methylation in Dnmt3a-KO HSCs. This CGI hypermethylation is a cancer methylome phenotype and was specific to Dnmt3a-KO HSCs (Figure 1A). The HSC transplant experiments suggest that Dnmt3a can compensate for Dnmt3b in HSCs, but Dnmt3b cannot reciprocate in the reverse situation. An explanation for increases in DNA methylation is that in the absence of Dnmt3a, abnormal function of Dnmt3b may lead to aberrant CGI hypermethylation as the hypermethylation was lost when both enzymes were conditionally inactivated. To confirm the mechanism, post-transplant Dnmt3ab-dKO HSCs were transduced with a retroviral vector encoding ectopic expression of Dnmt3b (MIG-Dnmt3b) or a control empty vector (MIG) and assessed for DNA methylation by bisulfite PCR. Using the promoter CGI of Praf2 as an example, enforced expression of Dnmt3b in Dnmt3ab-dKO HSCs resulted in increased DNA methylation at this loci compared to Dnmt3ab-dKO HSCs transduced with a control empty vector (MIG), control HSCs transduced with either MIG or MIG-Dnmt3b and untransduced HSCs (Figure 1B). It is possible that when Dnmt3b tries to compensate for Dnmt3a, the locus-specificity for targets is reduced, leading to aberrant DNA methylation patterns. Promoter CGI hypermethylation is a cancer phenotype observed in a wide range of tumors, including hematopoietic neoplasms driven by mutation in DNMT3A. Targeting DNMT3B in DNMT3A-mutation hematopoietic pathologies may be a therapeutic option for restoring normal DNA methylation and gene expression patterns. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Silver–Russell syndrome due to paternal H19/IGF2 hypomethylation in a twin girl born after in vitro fertilization

Silver–Russell syndrome (SRS) is a clinically and genetically heterogeneous syndrome characterized by severe intrauterine and postnatal growth retardation, facial dysmorphism and body asymmetry. One of the main molecular mechanisms leading to the syndrome involves methylation abnormalities of chromosome 11p15. In the last decades, an increase of imprinting disorders have been reported in children born from assisted reproductive technology (ART); however there is currently little evidence linking SRS and ART. Only few infants with SRS born using ART, supported by molecular analysis, have been described. We report on a twin-girl conceived using intracytoplasmic sperm injection (ICSI) diagnosed with SRS. Molecular studies revealed a hypomethylation of the paternal H19/IGF2 Imprinting Control Region. Her twin sister had a normal prenatal and postnatal growth and a normal methylation pattern of the chromosome 11p15. This is the second reported case of a twin infant with SRS conceived using ART with hypomethylation of H19/IGF2; it provides additional evidence of a possible relationship between ART procedures and methylation defects observed in SRS. Given the clinical heterogeneity of SRS, and the increased risk of multiple and preterm births in the ART-conceived children, it is possible that a number of cases of SRS remains undiagnosed in this population. Future studies should investigate the possible link between ART and SRS, in order to better understand the causes of epimutations in ART pregnancies, and to help clinicians to adequately counsel parents who approach to ART and to assess the opportunity of a long-term follow-up of children conceived using ART. © 2013 Wiley Periodicals, Inc.

Abstract 4255: Imprintome-wide methylation alteration in human cancer: from occupation to clinic.

Abstract Background: Exposure to light at night through shiftwork is a widespread occupational exposure which has been associated with cancer risk. In this study, we sought to investigate whether this association may be mediated in part by disruption of genomic imprinting patterns. Methods: Using an Illumina Infinium Methylation Assay, we measured methylation differences at 397 CpG sites in the promoter regions of 56 imprinted genes in DNA isolated from 10 long-term shiftworkers as compared to 10 age- and folate intake-matched day workers. We subsequently measured methylation differences at these same CpG sites in a pooled sample of 12 pairs of prostate tumor as compared to adjacent normal tissue. Top differentially methylated loci were validated using the Sequenom EpiTYPER methylation assay. Results: Among long-term shiftworkers, statistically significant hypermethylation was observed at 20 CpG sites and statistically significant hypomethylation was observed at 30 CpG sites relative to matched day workers (P &amp;lt; 0.05). This differential methylation was observed across 26 unique genes, 15 of which contained CpG sites also statistically significantly differentially methylated between prostate tumor and adjacent normal tissue (P &amp;lt; 0.05). In total, statistically significant hypermethylation was observed at 52 CpG sites and hypomethylation was observed at 17 CpG sites in prostate tumor relative to adjacent normal tissue. Of particular interest was the disruption of methylation patterns at TP73, an important component of the cellular apoptotic response, among both shiftworkers and in prostate tumor tissue. Tumor suppressor genes SLC22A18 and WT1 also demonstrated altered methylation patterns in both comparison groups. A query of publicly available expression array data returned several studies demonstrating statistically significant differential expression levels of these three genes in prostate cancer tissue relative to tissue from healthy controls. Conclusion: In exploring the methylation patterns at normally imprinted genes among a set of shiftworkers and a sample of prostate tumor tissue, we have identified a set of 15 genes within which differential methylation is observed in comparison to day workers and adjacent normal prostate tissue, respectively. This altered methylation was observed at several important tumor suppressor genes, against a background of widespread changes in methylation at imprinted genes in both comparison groups. Taken together, these data suggest that loss of normal patterns of methylation at imprinted genes may be an important mechanism by which occupational exposure to light at night drives the increased risk of cancer development, and warrants further exploration. Citation Format: Daniel I. Jacobs, Johnni Hansen, Alan Fu, Richard G. Stevens, Anne Tjonneland, Ulla B. Vogel, Tongzhang Zheng, Yingying Mao, William K. Kelly, Yong Zhu. Imprintome-wide methylation alteration in human cancer: from occupation to clinic. [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 4255. doi:10.1158/1538-7445.AM2013-4255

Restoration of the methylation status of hypermethylated gene promoters by microRNA-29b in human breast cancer: A novel epigenetic therapeutic approach

It is well established that transcriptional silencing of critical tumor-suppressor genes by DNA methylation is a fundamental component in the initiation of breast cancer. However, the involvement of microRNAs (miRNAs) in restoring abnormal DNA methylation patterns in breast cancer is not well understood. Therefore, we investigated whether miRNA-29b, due to its complimentarity to the 3’- untranslated region of DNA methyltransferase 3A (DNMT3A) and DNMT3B, could restore normal DNA methylation patterns in human breast cancers and breast cancer cell lines. We demonstrated that transfection of pre-miRNA-29b into less aggressive MCF-7 cells, but not MDA-MB-231 mesenchymal cells, inhibited cell proliferation, decreased DNMT3A and DNMT3B messenger RNA (mRNA), and decreased promoter methylation status of ADAM23 , CCNA1, CCND2, CDH1, CDKN1C, CDKN2A, HIC1, RASSF1, SLIT2, TNFRSF10D, and TP73 tumor-suppressor genes. Using methylation polymerase chain reaction (PCR) arrays and real-time PCR, we also demonstrated that the methylation status of several critical tumor-suppressor genes increased as stage of breast disease increased, while miRNA-29b mRNA levels were significantly decreased in breast cancers versus normal breast. This increase in methylation status was accompanied by an increase in DNMT1 and DNMT3B mRNA in advanced stage of human breast cancers and in MCF-7, MDA-MB-361, HCC70, Hs-578T, and MDA-MB-231 breast cancer cells as compared to normal breast specimens and MCF-10-2A, a non-tumorigenic breast cell line, respectively. Our findings highlight the potential for a new epigenetic approach in improving breast cancer therapy by targeting DNMT3A and DNMT3B through miRNA-29b in non-invasive epithelial breast cancer cells.

Therapeutic Delivery of MicroRNA-29b by Cationic Lipoplexes for Lung Cancer

MicroRNA-29b (miR-29b) expression has been shown to be reduced in non-small–cell lung cancer (NSCLC) tissues. Here, we have identified the oncogene cyclin-dependent protein kinase 6 (CDK6) as a direct target of miR-29b in lung cancer. We hypothesized that in vivo restoration of miR-29b and thus targeting of genes important to tumor initiation and progression may represent an option for lung cancer treatment. We developed a cationic lipoplexes (LPs)-based carrier that efficiently delivered miR-29b both in vitro and in vivo. LPs containing miR-29b (LP-miR-29b) efficiently delivered miR-29b to NSCLC A549 cells, reduced the expression of key targets CDK6, DNMT3B, and myeloid cell leukemia sequence 1 (MCL1), as well as cell growth and clonogenicity of A549 cells. In addition, the IC50 for cisplatin in the miR-29b–treated cells was effectively reduced. In a xenograft murine model, LPs efficiently accumulated at tumor sites. Systemic delivery of LP-miR-29b increased the tumor miR-29b expression by approximately fivefold, downregulated the tumor mRNA expression of CDK6, DNMT3B, and MCL1 by ~57.4, ~40.5, and ~52.4%, respectively, and significantly inhibited tumor growth by ~60% compared with LP-miR-NC (negative control). Our results demonstrate that cationic LPs represent an efficient delivery system that holds great potential in the development of miRNA-based therapeutics for lung cancer treatment.

Dnmt3a Protects Active Chromosome Domains against Cancer-Associated Hypomethylation

Changes in genomic DNA methylation patterns are generally assumed to play an important role in the etiology of human cancers. The Dnmt3a enzyme is required for the establishment of normal methylation patterns, and mutations in Dnmt3a have been described in leukemias. Deletion of Dnmt3a in a K-ras–dependent mouse lung cancer model has been shown to promote tumor progression, which suggested that the enzyme might suppress tumor development by stabilizing DNA methylation patterns. We have used whole-genome bisulfite sequencing to comprehensively characterize the methylomes from Dnmt3a wildtype and Dnmt3a-deficient mouse lung tumors. Our results show that profound global methylation changes can occur in K-ras–induced lung cancer. Dnmt3a wild-type tumors were characterized by large hypomethylated domains that correspond to nuclear lamina-associated domains. In contrast, Dnmt3a-deficient tumors showed a uniformly hypomethylated genome. Further data analysis revealed that Dnmt3a is required for efficient maintenance methylation of active chromosome domains and that Dnmt3a-deficient tumors show moderate levels of gene deregulation in these domains. In summary, our results uncover conserved features of cancer methylomes and define the role of Dnmt3a in maintaining DNA methylation patterns in cancer.

Overall Hydroxymethylation Levels As an Independent Prognostic Marker in Intermediate-Cytogenetic Risk Acute Myeloid Leukemia

AbstractAbstract 1375 Introduction:Acute myeloid leukemia (AML) is a highly heterogeneous disease, with diverse genetic and epigenetic variables determining sensitivity to current standard therapy. A disruption of the normal DNA methylation pattern, which can result in altered gene and microRNA (miRNAs) expression, has been observed in different AML subtypes. Hydroxymethylation of 5-methylcytosine (5-mC) has recently been described as an intermediate key step in the process of DNA demethylation. Nonetheless, the correlation of DNA methylation and hydroxymethylation levels with clinical and biological characteristics and clinical outcome in AML is mostly unknown. Aim:To investigate the prognostic impact of overall methylation and hydroxymethylation levels in patients with intermediate-cytogenetic risk AML (IR-AML) and to identify miRNAs correlated with methylation and hydroxymethylation in these patients. Patients and Methods:We have analyzed 86 IR-AML patients (median age, 53 [range, 17–74]; 52% males) who received intensive therapy from 1994 to 2009 in a single institution. The level of overall methylation and hydroxymethylation in total DNA was estimated after determining the percentage of 5-mC and hydroxymethylcytosine (5-hmC), using anti-5-mC and anti-5-hmC monoclonal antibodies (MethylFlash Methylated or Hydroxymethylated DNA Quantification Kit, Epigentek). The expression of 670 mature miRNAs was analyzed using TaqMan Human MicroRNA Arrays (Applied Biosystems). The statistical analysis was performed with SPSS version 15.0.1 and R software version 2.9.0. MaxStat package of R was used to determine the optimal cutoffs. Results:The univariate analysis for overall methylation showed that patients with lower levels of methylation (cutoff < percentile 75) had shorter overall survival (OS) than those with higher 5-mC levels (5-year OS: 30±6% vs 52±11%; p=0.03) and a trend for shorter leukemia-free survival (LFS)(p=0.06). Overall methylation levels did not show any correlation with clinical features at diagnosis or with gene mutations, including DNMT3A. Concerning hydroxymethylation, patients with lower 5-hmC levels had a worse prognosis than those with higher 5-hmC levels, with a lower complete response rate (79% vs. 96%; p=0.04), shorter OS (5-yr OS: 21± 6% vs. 55± 6%; p=0.008), and shorter LFS (5-yr LFS: 24±8% vs. 52 ±10%; p=0.03). Interestingly, when analyzed as a continuous variable, 5-hmC levels retained their prognostic value as a marker of response rate (T-test, p=0.007), OS (Cox, p=0.015), and LFS (Cox, p=0.041). Moreover, 5-hmC levels were inversely correlated with FLT3-ITD (p=0.001) and the FLT3-ITD/FLT3wild-type ratio (Pearson correlation:-0.6; p=0.01). The multivariate analyses, including the main clinical and biological variables, identified older age, wild-type NPM1, FLT3-ITD, and lower 5-hmC levels (HR=3.072; 95% CI: 1.096–3.917; p=0.025) as independent prognostic markers of shorter OS and wild-type NPM1, FLT3-ITD, and lower 5-hmC levels (HR=2.002, 95% CI: 1.032–3.881, p=0.040) as independent prognostic markers of shorter LFS. Of note, lower 5-hmC levels retained their value as a marker of worse prognosis in the subgroup of IR-AML patients with unfavorable molecular markers (wild-type NPM1 and CEBPA and/or FLT3-ITD; p= 0.037).Finally, we have identified a 3-miRNA signature (miR-378*, p=0.02; miR-493, p=0.02; and miR-181, p=0.02) associated with global methylation levels, and a 12-miRNA signature associated with hydroxymethylation, including miR-183* (p=0.001), miR-125a-3p (p= 0.01), miR-586 (p=0.02), and miR-513–3p (p=0.02). Conclusions:Hydroxymethylation levels appear as an independent prognostic factor in IR-AML and maintain their prognostic value in the subset of patients with unfavorable molecular markers. Moreover, methylation and hydroxymethylation are associated with specific miRNA profiles. Further studies are warranted to confirm the clinical impact of these findings and to clarify the underlying molecular mechanisms. Disclosures:No relevant conflicts of interest to declare.

8. Understanding cryoinjuries: Effect of cryopreservation on expression of imprinted genes related to birth defects

Assisted reproductive technologies (ARTs) such as in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and embryo cryopreservation became an indispensible part of infertility treatments. Currently, ARTs account for up to 3% of annual births in industrialized countries and are considered safe. However, recent studies reported an increased incidence of epigenetic birth defects among children conceived by ARTs. Since the timing of ARTs coincides with re-establishment of epigenetic marks (genomic imprinting) during gametogenesis and embryogenesis, it is likely that suboptimal ARTs may alter normal methylation patterns, and thus expression of imprinted genes, leading to epigenetic birth defects. The objective of this study was to investigate the effect of oocyte cryopreservation on expression of the imprinted genes involved in epigenetic birth defects. To this end, mouse metaphase II (M II) oocytes were cryopreserved by using either standard slow freezing (i.e., cooling to −35 °C at 0.3 °C/min in 1.5 M Me 2 SO + 0.1 M sucrose, and then plunging into liquid nitrogen [LN2]) or vitrification technique (i.e., plunging of oocytes within a ⩽ 1-ml volume of 15% Me 2 SO + 15% ethylene glycol + 0.5 M Sucrose into LN2). After warming at 37 °C and removal of cryoprotectants by stepwise dilution, cryopreserved oocytes along with untreated controls were inseminated and cultured to the blastocyst stage to determine their gene expression levels. To do so, blastocysts derived from cryopreserved and control oocytes were subjected first to reverse transcription and subsequently to real-time PCR. Gene expression levels in three replicates of experiments were analyzed by the comparative CT method using Rest Software. So far, our results suggest that both cryopreservation techniques downregulate the expression level of two imprinted genes (i.e., H19, and Ube3a). These findings warrant further studies to address the effect of oocyte cryopreservation on imprinted genes.

Semen samples showing an increased rate of spermatozoa with imprinting errors have a negligible effect in the outcome of assisted reproduction techniques

The topic of imprinting defects present in the sperm of infertile patients has been addressed by several reports in the last few years. However, whether methylation abnormalities at one or few CpGs within an imprinted locus are pathological is a matter of debate. Moreover, whether imprinting anomalies in sperm could interfere with fertility treatment outcomes is still unknown. In this report we analyze the sperm DNA methylation profile of H19-ICR, KvDMR, SNRPN-ICR, IG-DMR and MEG3-DMR by pyrosequencing in 107 infertile men series and a control population of 30 proven fertile males. DNA methylation was statistically evaluated from two points of view: first, the methylation of each CpG was analyzed in the control population and the mean, standard deviation and range were determined and compared with infertile population data; second, in order to define altered methylation patterns for each region, a hierarchical cluster analysis was performed by which individuals were grouped in different clusters according to the degree of similarity of their methylation pattern. Two pieces of data supported the results obtained in the multi-variate analysis: the classification of the vast majority of control individuals in clusters with normal methylation patterns and the significant differences in methylation levels found between individuals within the normal and abnormal clusters. Individuals included in normal and abnormal methylation clusters were compared according to seminal parameters as well as to the outcome of assisted reproduction.

Genetic variants in metabolizing genes NQO1, NQO2, MTHFR and risk of prostate cancer: a study from North India

Quinone oxidoreductases (NAD(P)H): quinone oxidoreductase 1 (NQO1) and NRH: quinone oxidoreductase 2 (NQO2) are an antioxidant enzyme, important in the detoxification of environmental carcinogens. Methylene-tetra-hydrofolate reductase (MTHFR), plays a role in folate metabolism and may have oncogenic role through disruption of normal DNA methylation pattern, synthesis, and impaired DNA repair. In a case-control study, genotyping was done in 195 PCa and 250 age matched unrelated healthy controls of similar ethnicity to determine variants in NQO1 exon 4 (C > T, rs4986998), exon 6 (C > T, rs1800566), NQO2 -3423 (G > A, rs2070999) and MTHFR exon 4 (C > T, rs1801133) by PCR-RFLP methods. Heterozygous genotype CT and variant allele career genotype (CT + TT) of NQO1 exon 4 showed increased risk of PCa (OR = 2.06, p = 0.033; OR = 2.02, p = 0.027). Variant allele T also revealed increased risk (OR = 1.87, p = 0.029). Similarly variant genotype TT (OR = 2.71, p = 0.009), combined genotype (CT + TT) (OR = 1.59, p = 0.019) and T allele (OR = 1.63, p = 0.002) of NQO1 exon 6 demonstrated significant risk for PCa. Diplotypes of NQO1 (exon 4 and 6), C-T (OR = 1.56, Pc = 0.007) and T-T (OR = 0.011, Pc = 3.86) was associated with an increased risk for PCa. NQO2 and MTHFR did not show any risk with PCa. Our results strongly support that common sequence variants and diplotypes of NQO1 exon 4 and 6 genes may have role in PCa risk in the North Indian population, indicating the importance of genes involved in metabolism with respect to PCa risk. Additional studies on larger populations are needed to clarify the role of variation in these genes in PCa carcinogenesis.

Effects of chronic exposure to arsenic and estrogen on epigenetic regulatory genes expression and epigenetic code in human prostate epithelial cells.

Chronic exposures to arsenic and estrogen are known risk factors for prostate cancer. Though the evidence suggests that exposure to arsenic or estrogens can disrupt normal DNA methylation patterns and histone modifications, the mechanisms by which these chemicals induce epigenetic changes are not fully understood. Moreover, the epigenetic effects of co-exposure to these two chemicals are not known. Therefore, the objective of this study was to evaluate the effects of chronic exposure to arsenic and estrogen, both alone and in combination, on the expression of epigenetic regulatory genes, their consequences on DNA methylation, and histone modifications. Human prostate epithelial cells, RWPE-1, chronically exposed to arsenic and estrogen alone and in combination were used for analysis of epigenetic regulatory genes expression, global DNA methylation changes, and histone modifications at protein level. The result of this study revealed that exposure to arsenic, estrogen, and their combination alters the expression of epigenetic regulatory genes and changes global DNA methylation and histone modification patterns in RWPE-1 cells. These changes were significantly greater in arsenic and estrogen combination treated group than individually treated group. The findings of this study will help explain the epigenetic mechanism of arsenic- and/or estrogen-induced prostate carcinogenesis.

From hepatitis to hepatocellular carcinoma: a proposed model for cross-talk between inflammation and epigenetic mechanisms.

Inflammation represents the body's natural response to tissue damage; however, chronic inflammation may activate cell proliferation and induce deregulation of cell death in affected tissues. Chronic inflammation is an important factor in the development of hepatocellular carcinoma (HCC), although the precise underlying mechanism remains unknown. Epigenetic events, which are considered key mechanisms in the regulation of gene activity states, are also commonly deregulated in HCC. Here, we review the evidence that chronic inflammation might deregulate epigenetic processes, thus promoting oncogenic transformation, and we propose a working hypothesis that epigenetic deregulation is an underlying mechanism by which inflammation might promote HCC development. In this scenario, different components of the inflammatory response might directly and indirectly induce changes in epigenetic machineries ('epigenetic switch'), including those involved in setting and propagating normal patterns of DNA methylation, histone modifications and non-coding RNAs in hepatocytes. We discuss the possibility that self-reinforcing cross-talk between inflammation and epigenetic mechanisms might amplify inflammatory signals and maintain a chronic state of inflammation culminating in cancer development. The potential role of inflammation-epigenome interactions in the emergence and maintenance of cancer stem cells is also discussed.

Epigenetic Profiling of Primary DLBCLs Reveals Novel DNA Methylation-Based Clusters and New Underlying Mechanisms of Lymphomagenesis

Abstract 556Epigenetic profiling of primary DLBCLs reveals novel DNA methylation-based clusters and new underlying mechanisms of lymphomagenesis. Nyasha Chambwe, Matthias Kormaksson, Subhajyoti De, Franziska Michor, Nathalie Johnson, David W. Scott, Randy D. Gascoyne, Ari Melnick, Fabien Campagne and Rita Shaknovich. Diffuse Large B Cell Lymphomas (DLBCLs) are a heterogeneous group of diseases from the clinical, molecular and genetic standpoints. While gene expression profiling has identified clinically and biologically relevant DLBCL subtypes, there is still considerable heterogeneity beyond what has been resolved through transcriptional and genetic profiling. It is increasingly clear that lesions in epigenetic regulatory proteins and transcription factors are a hallmark of DLBCL, which suggests that aberrant epigenetic programming is likely to be a significant factor in these tumors. Our recent preliminary data suggests that aberrant DNA methylation is also widespread in DLBCL and contributes to the abnormal expression patterns of GCB and ABC DLBCLs. Moreover recent data in the setting of AML show that DNA methylation profiles delineate disease subtypes not captured through transcriptional or genetic profiling. We hypothesized that DNA methylation profiles would allow us to identify new, biologically significant DLBCL subtypes. We therefore examined the DNA methylation of over 140 patients with DLBCL using the HELP assay covering multiple CpGs at over 14,000 gene loci. We next performed an unbiased (unsupervised) analysis of probesets that display significant variability (n=3,005), using K-means consensus clustering. This procedure identified four robust DLBCL subtypes based on epigenetic profiles. To identify the genes that define these four clusters we next performed supervised analysis of the DLBCL subtypes including the normal counterpart germinal center B-cells as a normal control, using three independent statistical methods. 46 genes defined cluster A, 236 genes defined cluster B, 376 genes defined cluster C and 1271 genes defined cluster D (selected genes displayed change in methylation of at least 30% at BH corrected p-value < 0.05). Each of these epigenetically defined DLBCL subtypes featured aberrant DNA methylation of genes and pathways with potential relevance to pathogenesis. For example cluster A was notable for aberrant DNA methylation of REL, STAT3, CD30; cluster B for aberrant methylation of the TNFa and IFN1 networks; cluster C of IDH2, FOXG1 genes; and cluster D of CDKN2A, ATF3, FOXL3 genes. Other defining characteristics of Cluster D was enrichment for ABC DLBCLs (Fisher exact test, p=0.007) and most remarkably, marked intra-tumor and inter-individual heterogeneity of DNA methylation patterning. This latter feature is suggestive of potential epigenetic clonal complexity and failure to properly control the boundaries of methylated regions of the genome in these tumors. Clusters A and B revealed enrichment for GC features and cluster B had increased expression of MUM1 (Fisher exact test, p=0.007 and p=0.002). Furthermore, we noted that higher expression of DNMT3B and DNMT3L were associated with hypermethylation in DLBCL samples, while higher expression level of AICDA was associated with aberrant hypomethylation in DLBCLs as compared to normal GC B-cells. Higher expression levels of epigenetic modifiers EZH2 and MBD4 was associated with greater heterogeneity of DNA methylation patterning compared to the normal methylation pattern in germinal center B cells. Collectively, the data indicate that DLBCLs are composed of entities defined by specific DNA methylation profiles that only partially overlap with the ABC and GCB classification. The DLBCL subtypes display perturbation of genes likely to play significant biological roles, and aberrant methylation patterning can be traced in part to aberrant expression of epigenetic regulators including DNA methyltransferases, AICDA and EZH2. Disclosures:No relevant conflicts of interest to declare.