Global Methylation Patterns Research Articles

Genome-wide DNA Methylation Events in TMPRSS2–ERG Fusion-Negative Prostate Cancers Implicate an EZH2-Dependent Mechanism with miR-26a Hypermethylation

Prostate cancer is the second most common cancer among men worldwide. Alterations in the DNA methylation pattern can be one of the leading causes for prostate cancer formation. This study is the first high-throughput sequencing study investigating genome-wide DNA methylation patterns in a large cohort of 51 tumor and 53 benign prostate samples using methylated DNA immunoprecipitation sequencing. Comparative analyses identified more than 147,000 cancer-associated epigenetic alterations. In addition, global methylation patterns show significant differences based on the TMPRSS2-ERG rearrangement status. We propose the hypermethylation of miR-26a as an alternative pathway of ERG rearrangement-independent EZH2 activation. The observed increase in differential methylation events in fusion-negative tumors can explain the tumorigenic process in the absence of genomic rearrangements. In contrast to TMPRSS2-ERG -rearranged tumors, the pathomechanism for gene fusion-negative tumors is completely unclear. Using a sequencing-based approach, our work uncovers significant global epigenetic alterations in TMPRSS2-ERG gene fusion-negative tumors and provides a mechanistic explanation for the tumor formation process.

Quest for Genes and Mechanisms Linking the Human Chromosome 9p21.3 Locus to Cardiovascular Disease

In the past several years, advances in molecular genetics have been adopted as tools to dissect the cause and pathophysiology of common complex diseases and their associated risk factors in a hypothesis-free approach, using the genome-wide association study design and high-density, single nucleotide polymorphism (SNP) genotyping arrays. Starting in 2007 with the discovery of the chromosome 9p21.3 (Chr9p21.3) locus (see recent review),1 major progress has been made in terms of identification of novel susceptibility loci for coronary artery disease (CAD; ie, genomic regions that are strongly and consistently associated with CAD).2,3 These CAD susceptibility loci are of major interest because they are expected to harbor genes constituting important nodes in biological pathways underlying CAD and hence may serve as shortcuts to discovery of hitherto unknown disease mechanisms. However, causal variants will only occasionally be among those that are directly typed in genome-wide association scans, and the interval within which the etiologic variant(s) are expected to lie can be sizeable, and often contains several genes. Thus, the challenge for the next several years will be to move from statistical association based on genetic markers to identification and functional characterization of individual risk-associated genes, gene variants, biological pathways, and proteins and to elucidation of their roles in the resulting vascular pathology. Article see p 1896 In the rapidly expanding group of robustly associated CAD loci, the Chr9p21.3 locus contributes the largest increase in risk, 1 and 2 copies of the risk allele (allele frequency of 0.50) being associated with a rise in CAD risk of around 20% to 40% and 40% to 70%, respectively. Interestingly, this substantial increase in risk is independent of established risk factors, which suggests that the Chr9p21.3 locus operates through an …

Hotspot Mutations in H3F3A and IDH1 Define Distinct Epigenetic and Biological Subgroups of Glioblastoma

Glioblastoma (GBM) is a brain tumor that carries a dismal prognosis and displays considerable heterogeneity. We have recently identified recurrent H3F3A mutations affecting two critical amino acids (K27 and G34) of histone H3.3 in one-third of pediatric GBM. Here, we show that each H3F3A mutation defines an epigenetic subgroup of GBM with a distinct global methylation pattern, and that they are mutually exclusive with IDH1 mutations, which characterize a third mutation-defined subgroup. Three further epigenetic subgroups were enriched for hallmark genetic events of adult GBM and/or established transcriptomic signatures. We also demonstrate that the two H3F3A mutations give rise to GBMs in separate anatomic compartments, with differential regulation of transcription factors OLIG1, OLIG2, and FOXG1, possibly reflecting different cellular origins.

Abstract 4079: Proanthocyanidins reactivate silenced tumor suppressor genes p16INK4 a and Cip1/p21 by reducing DNA methylation and increasing histone acetylation in human non-small cell lung cancer cells

Abstract Non-small cell lung cancer (NSCLC) represents α80% of all types of lung cancer and includes squamous cell carcinoma, adenocarcinoma and large-cell carcinomas. The risk of NSCLC is greatly affected by epigenetic modulations in lung cells. Cancer epigenetics deal with important processes including DNA methylation and histone modification patterns that can either activate oncogenes or inactivate tumor suppressor genes, the crucial steps in tumor development. The study of epigenetic regulations has become a new target for early detection, risk assessment, prognosis and cancer therapy. Therefore, to develop more effective therapeutic agents for lung cancer, we sought to determine the effect of grape seed proanthocyanidins (GSPs), which have been shown to have anti-carcinogenic effects in some tumor models, on epigenetic modulation in NSCLC cells. For this purpose two genetically different NSCLC cell lines, A549 (p53 wild type) and H1299 (p53 mutant), were selected and treated with lower concentrations of GSPs (5, 10, 20µg/ml) for six days. The status of DNA global methylation patterns was determined by immunostaining using a monoclonal antibody specific to 5-methylcytosine (5-mC) and global DNA methylation assay. Treatment of cells with GSPs resulted in a dose-dependent decrease in 5-mC-positive cells as compared to non-GSPs-treated controls. These data were verified by dot-blot analysis. Global methylation levels of DNA as obtained from quantitative DNA methylation assay reduced to 36±5% in A549 cells and 31±5% in H1299 cells with the treatment of GSPs. The expression levels of the DNA methyltransferases, e.g., DNMT1, DNMT3a and DNMT3b, were higher in non-GSPs treated cells and reduced with GSPs treatment as analyzed by western blotting and real-time PCR. The hypermethylation of DNA in NSCLC cells may lead to silencing of tumor suppressor genes, thereby contributing to the risk of lung cancer. Therefore, we analyzed the methylation status of the various tumor suppressor genes, like p16INK4a, Cip1/p21 and RASSF1A through methylation specific PCR and the relative repression levels of their mRNA were analyzed using real-time PCR, which indicated that the DNA methylation levels of these tumor suppressor genes were reduced in GSPs treated cells, and that resulted in re-expression of the mRNA and proteins of silenced tumor suppressor genes compared to non-GSPs treated cells. GSPs decreased histone deacetylase activity and increased the levels of histone acetylation which included the acetylated lysine 9 and 14 on histone H3 (H3-Lys 9 and 14) and acetylated lysine 5 and 12 on histone H4 but decreased the levels of methylated H3-Lys 9. Together, our study provides new insight into the epigenetic mechanism of action of GSPs that may have important implications for epigenetic therapy of lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4079. doi:1538-7445.AM2012-4079

Global methylation patterns in idiopathic pulmonary fibrosis.

BackgroundIdiopathic Pulmonary Fibrosis (IPF) is characterized by profound changes in the lung phenotype including excessive extracellular matrix deposition, myofibroblast foci, alveolar epithelial cell hyperplasia and extensive remodeling. The role of epigenetic changes in determining the lung phenotype in IPF is unknown. In this study we determine whether IPF lungs exhibit an altered global methylation profile.Methodology/Principal FindingsImmunoprecipitated methylated DNA from 12 IPF lungs, 10 lung adenocarcinomas and 10 normal histology lungs was hybridized to Agilent human CpG Islands Microarrays and data analysis was performed using BRB-Array Tools and DAVID Bioinformatics Resources software packages. Array results were validated using the EpiTYPER MassARRAY platform for 3 CpG islands. 625 CpG islands were differentially methylated between IPF and control lungs with an estimated False Discovery Rate less than 5%. The genes associated with the differentially methylated CpG islands are involved in regulation of apoptosis, morphogenesis and cellular biosynthetic processes. The expression of three genes (STK17B, STK3 and HIST1H2AH) with hypomethylated promoters was increased in IPF lungs. Comparison of IPF methylation patterns to lung cancer or control samples, revealed that IPF lungs display an intermediate methylation profile, partly similar to lung cancer and partly similar to control with 402 differentially methylated CpG islands overlapping between IPF and cancer. Despite their similarity to cancer, IPF lungs did not exhibit hypomethylation of long interspersed nuclear element 1 (LINE-1) retrotransposon while lung cancer samples did, suggesting that the global hypomethylation observed in cancer was not typical of IPF.Conclusions/SignificanceOur results provide evidence that epigenetic changes in IPF are widespread and potentially important. The partial similarity to cancer may signify similar pathogenetic mechanisms while the differences constitute IPF or cancer specific changes. Elucidating the role of these specific changes will potentially allow better understanding of the pathogenesis of IPF.

DNA methylation regulates lineage-specifying genes in primary lymphatic and blood endothelial cells

During embryonic development, the lymphatic system emerges by transdifferentiation from the cardinal vein. Although lymphatic and blood vasculature share a close molecular and developmental relationship, they display distinct features and functions. However, even after terminal differentiation, transitions between blood endothelial cells (BEC) and lymphatic endothelial cells (LEC) have been reported. Since phenotypic plasticity and cellular differentiation processes frequently involve epigenetic mechanisms, we hypothesized that DNA methylation might play a role in regulating cell type-specific expression in endothelial cells. By analyzing global gene expression and methylation patterns of primary human dermal LEC and BEC, we identified a highly significant set of genes, which were differentially methylated and expressed. Pathway analyses of the differentially methylated and upregulated genes in LEC revealed involvement in developmental and transdifferentiation processes. We further identified a set of novel genes, which might be implicated in regulating BEC-LEC plasticity and could serve as therapeutic targets and/or biomarkers in vascular diseases associated with alterations in the endothelial phenotype.

Epigenetic and antioxidant effects of dietary isothiocyanates and selenium: potential implications for cancer chemoprevention

There is evidence from epidemiological studies suggesting that increased consumption of cruciferous vegetables may protect against specific cancers more effectively than total fruit and vegetable intake. These beneficial effects are attributed to the glucosinolate breakdown products, isothiocyanates (ITC). Similarly, selenium (Se) consumption has also been inversely associated with cancer risk and as an integral part of many selenoproteins may influence multiple pathways in the development of cancer. This paper will briefly review the current state of knowledge concerning the effect of Se and ITC in cancer development with a particular emphasis on its antioxidant properties, and will also address whether alterations in DNA methylation may be a potential mechanism whereby these dietary constituents protect against the carcinogenic process. Furthermore, we will discuss the advantages of combining ITC and Se to benefit from their complementary mechanisms of action to potentially protect against the alterations leading to neoplasia. Based on this review it may be concluded that an understanding of the impact of ITC and Se on aberrant DNA methylation in relation to factors modulating gene-specific and global methylation patterns, in addition to the effect of these food constituents as modulators of key selenoenzymes, such as gastrointestinal glutathione peroxidase-2 (GPx2) and thioredoxin reductase-1 (TrxR1), may provide insights into the potential synergy among various components of a plant-based diet that may counteract the genetic and epigenetic alterations that initiate and sustain neoplasia.

Unique DNA methylome profiles in CpG island methylator phenotype colon cancers

A subset of colorectal cancers was postulated to have the CpG island methylator phenotype (CIMP), a higher propensity for CpG island DNA methylation. The validity of CIMP, its molecular basis, and its prognostic value remain highly controversial. Using MBD-isolated genome sequencing, we mapped and compared genome-wide DNA methylation profiles of normal, non-CIMP, and CIMP colon specimens. Multidimensional scaling analysis revealed that each specimen could be clearly classified as normal, non-CIMP, and CIMP, thus signifying that these three groups have distinctly different global methylation patterns. We discovered 3780 sites in various genomic contexts that were hypermethylated in both non-CIMP and CIMP colon cancers when compared with normal colon. An additional 2026 sites were found to be hypermethylated in CIMP tumors only; and importantly, 80% of these sites were located in CpG islands. These data demonstrate on a genome-wide level that the additional hypermethylation seen in CIMP tumors occurs almost exclusively at CpG islands and support definitively that these tumors were appropriately named. When these sites were examined more closely, we found that 25% were adjacent to sites that were also hypermethylated in non-CIMP tumors. Thus, CIMP is also characterized by more extensive methylation of sites that are already prone to be hypermethylated in colon cancer. These observations indicate that CIMP tumors have specific defects in controlling both DNA methylation seeding and spreading and serve as an important first step in delineating molecular mechanisms that control these processes.

Comprehensive Genome Methylation Analysis in Bladder Cancer: Identification and Validation of Novel Methylated Genes and Application of These as Urinary Tumor Markers

Epigenetic alterations are common and can now be addressed in a parallel fashion. We investigated the methylation in bladder cancer with respect to location in genome, consistency, variation in metachronous tumors, impact on transcripts, chromosomal location, and usefulness as urinary markers. A microarray assay was utilized to analyze methylation in 56 samples. Independent validation was conducted in 63 samples by a PCR-based method and bisulfite sequencing. The methylation levels in 174 urine specimens were quantified. Transcript levels were analyzed using expression microarrays and pathways were analyzed using dedicated software. Global methylation patterns were established within and outside CpG islands. We validated methylation of the eight tumor markers genes ZNF154 (P < 0.0001), HOXA9 (P < 0.0001), POU4F2 (P < 0.0001), EOMES (P = 0.0005), ACOT11 (P = 0.0001), PCDHGA12 (P = 0.0001), CA3 (P = 0.0002), and PTGDR (P = 0.0110), the candidate marker of disease progression TBX4 (P < 0.04), and other genes with stage-specific methylation. The methylation of metachronous tumors was stable and targeted to certain pathways. The correlation to expression was not stringent. Chromosome 21 showed most differential methylation (P < 0.0001) and specifically hypomethylation of keratins, which together with keratin-like proteins were epigenetically regulated. In DNA from voided urine, we detected differential methylation of ZNF154 (P < 0.0001), POU4F2 (P < 0.0001), HOXA9 (P < 0.0001), and EOMES (P < 0.0001), achieving 84% sensitivity and 96% specificity. We initiated a detailed mapping of the methylome in metachronous bladder cancer. Novel genes with tumor, chromosome, as well as pathway-specific differential methylation in bladder cancer were identified. The methylated genes were promising cancer markers for early detection of bladder cancer.

Aberrant Epigenetic and Genetic Marks Are Seen in Myelodysplastic Leukocytes and Reveal Dock4 as a Candidate Pathogenic Gene on Chromosome 7q

Myelodysplastic syndromes (MDS) are characterized by abnormal and dysplastic maturation of all blood lineages. Even though epigenetic alterations have been seen in MDS marrow progenitors, very little is known about the molecular alterations in dysplastic peripheral blood cells. We analyzed the methylome of MDS leukocytes by the HELP assay and determined that it was globally distinct from age-matched controls and was characterized by numerous novel, aberrant hypermethylated marks that were located mainly outside of CpG islands and preferentially affected GTPase regulators and other cancer-related pathways. Additionally, array comparative genomic hybridization revealed that novel as well as previously characterized deletions and amplifications could also be visualized in peripheral blood leukocytes, thus potentially reducing the need for bone marrow samples for future studies. Using integrative analysis, potentially pathogenic genes silenced by genetic deletions and aberrant hypermethylation in different patients were identified. DOCK4, a GTPase regulator located in the commonly deleted 7q31 region, was identified by this unbiased approach. Significant hypermethylation and reduced expression of DOCK4 in MDS bone marrow stem cells was observed in two large independent datasets, providing further validation of our findings. Finally, DOCK4 knockdown in primary marrow CD34(+) stem cells led to decreased erythroid colony formation and increased apoptosis, thus recapitulating the bone marrow failure seen in MDS. These findings reveal widespread novel epigenetic alterations in myelodysplastic leukocytes and implicate DOCK4 as a pathogenic gene located on the 7q chromosomal region.

DNA Methylation in Peripheral Blood Cells of Pigs Cloned by Somatic Cell Nuclear Transfer

To date, the genome-wide DNA methylation status of cloned pigs has not been investigated. Due to the relatively low success rate of pig cloning by somatic cell nuclear transfer, a better understanding of the epigenetic reprogramming and the global methylation patterns associated with development in cloned pigs is required. In this study we applied methylation-specific digital karyotyping tag sequencing by Solexa technology and investigated the genome-wide DNA methylation profiles of peripheral blood cells in cloned pigs with normal phenotypes in comparison with their naturally bred controls. In the result, we found that globally there was no significant difference of DNA methylation patterns between the two groups. Locus-specifically, some genes involved in embryonic development presented a generally increased level of methylation. Our findings suggest that in cloned pigs with normal phenotypes, the DNA methylation pattern is quite normal, and that DNA methylation changes in some genomic regions are compatible with normal development.

DNMT3A mutations in myeloproliferative neoplasms

Since the discovery of the gain-of-function mutation JAK2 V617F in 2005, an increasing number of mutations have been described in myeloproliferative neoplasms (MPN) and related myeloid malignancies.1, 2, 3, 4 For instance, use of array-based techniques such as comparative genomic hybridization and single-nucleotide polymorphism (SNP) analysis led to the identification of genes involved in epigenetic regulation such as tet-oncogene family member 2 (TET2), additional sex combs like 1 (ASXL1) and enhancer of zeste 2 (EZH2).5, 6, 7 Inactivating mutations in TET2, ASXL1 and EZH2 are considered to promote myeloid tumorigenesis because of epigenetic modulation of target genes. More recently, a whole-genome sequencing study in acute myeloid leukemia (AML) uncovered recurrent mutations in 22% of AML patients in another epigenetic regulator, the DNA methyltransferase 3A gene DNMT3A.8 In this study, DNMT3A mutations were associated with poor outcome and, thus, are of clinical relevance. However, exact mechanisms of action of DNMT3A mutations are still unclear because global methylation patterns and 5-methylcytosine content in AML genomes were not significantly altered. Nevertheless, mutations of TET2, ASXL1, EZH2 and DNMT3A occur in a significant number of patients with myeloid malignancies underlining the pathogenic relevance of epigenetic changes.

Abstract 4801: Comprehensive genome methylation analysis in bladder cancer; identification and validation of novel methylated genes and application of these as urinary tumor markers

Abstract Cancer of the urinary bladder is one of the major cancers being the fifth most common neoplasm in the industrialised countries. Urothelial carcinomas account for more than 90% of all bladder cancers. The majority of bladder cancers are non-muscle invasive cancers characterized by frequent recurrences after resection and up to 25% will develop an aggressive phenotype. We investigated the methylation in bladder cancer with respect to location in genome, consistency, variation in metachronous tumors, impact on transcripts, chromosomal location, and usefulness as urinary markers of bladder cancer. In this study, a microarray assay with more than 27.000 CpG sites was utilized to analyze methylation in 56 samples. Independent validations were performed in 63 samples by methylation-sensitive high-resolution melting (MS-HRM) and bisulfite sequencing. Transcript levels were analyzed using expression microarrays, and pathways using dedicated software. DNA from 40 urinary specimens was analyzed by MS-HRM. Our results show that global methylation patterns were established within and outside CpG-islands. Differential methylation (p&amp;lt;0.0001 to p&amp;lt;0.05) was identified in 1588 genes between normals and cancers, in 1019 genes with stage specific methylation, and in 149 genes as progression markers. We validated methylation of eight genes, the tumor markers ZNF154 (p&amp;lt;0.0001), HOXA9 (p&amp;lt;0.0001), and POU4F2 (p&amp;lt;0.0001) and the progression marker TBX4 (p&amp;lt;0.002). The methylation of metachronous tumors was relatively stable and targeted to certain pathways. The correlation to expression was not stringent. Chromosome 21 showed most differential methylation (p&amp;lt;0.0001) and specifically hypomethylation of keratins, which together with keratin like proteins were epigenetically regulated. In DNA from voided urine we detected differential methylation of ZNF154 (p&amp;lt;0.0001), POU4F2 (p&amp;lt;0.0001), and HOXA9 (p&amp;lt;0.0044), achieving 94% sensitivity and 100% specificity. Our results suggest that both hypo- and hyper methylation is stage dependent, over-represented at chromosome 21, and has a restricted influence on transcript levels in general, but clearly on keratins. The methylated genes are highly promising urinary cancer markers. 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 4801. doi:10.1158/1538-7445.AM2011-4801

Abstract 3937: Global methylation patterns, genome copy number alterations and expression analysis distinguish two distinct subclasses of hepatocellular carcinoma

Abstract Background: Hepatocellular carcinoma (HCC) is a devastating disease ranking as the third most frequent cause of cancer deaths worldwide. In the US, the incidence of HCC tripled between 1975 and 2005 and at present day survival rates are as low as 20% due to limited treatment options. Future therapeutic advances rely on a greater understanding of the full range of genetic and epigenetic defects displayed by this cancer. Recent advances in whole genome methylation profiling have allowed us to investigate these alterations in a cohort of HCC and normal liver samples. Methods: Using the Illumina Infinium platform, we analyzed the methylation patterns in over 50 human HCC and normal liver samples obtained from surgical resection and transplant specimens. We also investigated the expression and gene copy number (CGH) status on the same cohort using the Agilent platform. Findings: We have been able to identify two very distinct groups of HCC based on their methylation status. In each class of tumors, there are both hypo- and hypermethylated genes relative to normal liver. Several of these tumor-specific alterations have been validated by pyrosequencing. In one class of tumors, many genes that are normally repressed by polycomb group proteins in stem cells are hypermethylated, consistent with the tumor cell of origin being a progenitor stem cell. The other class of tumors does not show this association and are likely to originate from a different cell of origin. Intriguingly, these two classes also correspond to distinct groups based on both DNA copy number analysis and expression analysis. Conclusion: Combining these three independent methods of global analysis; expression profiling, epigenome analysis and CGH data, we have identified a robust new classification scheme for HCC. This approach should provide insight for future therapeutic advances. 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 3937. doi:10.1158/1538-7445.AM2011-3937

Effects of Altered Maternal Folic Acid, Vitamin B12 and Docosahexaenoic Acid on Placental Global DNA Methylation Patterns in Wistar Rats

Potential adverse effects of excess maternal folic acid supplementation on a vegetarian population deficient in vitamin B12 are poorly understood. We have previously shown in a rat model that maternal folic acid supplementation at marginal protein levels reduces brain omega-3 fatty acid levels in the adult offspring. We have also reported that reduced docosahexaenoic acid (DHA) levels may result in diversion of methyl groups towards DNA in the one carbon metabolic pathway ultimately resulting in DNA methylation. This study was designed to examine the effect of normal and excess folic acid in the absence and presence of vitamin B12 deficiency on global methylation patterns in the placenta. Further, the effect of maternal omega 3 fatty acid supplementation on the above vitamin B12 deficient diets was also examined. Our results suggest maternal folic acid supplementation in the absence of vitamin B12 lowers plasma and placental DHA levels (p<0.05) and reduces global DNA methylation levels (p<0.05). When this group was supplemented with omega 3 fatty acids there was an increase in placental DHA levels and subsequently DNA methylation levels revert back to the levels of the control group. Our results suggest for the first time that DHA plays an important role in one carbon metabolism thereby influencing global DNA methylation in the placenta.

Intensity matters: brain, behaviour and the epigenome of prenatally stressed rats

There is a general consensus that prenatal stress alters offspring brain development, however, the details are often inconsistent. Hypothesising that variation to the level of stress would produce different maternal experiences; this study was designed to examine offspring outcomes following a single prenatal stress paradigm at two different intensities. Pregnant Long Evans rats received mild, high, or no-stress from gestational days 12–16. Offspring underwent early behavioural testing and global methylation patterns were analysed from brain tissue of the frontal cortex and hippocampus. The two different prenatal stress intensities produced significantly different and often, opposite effects in the developing brain. Mild prenatal stress decreased brain weight in both males and females, whereas extreme stress increased female brain weight. Mild prenatal stress slowed development of sensorimotor abilities and decreased locomotion, whereas high prenatal stress also slowed development of sensorimotor learning but increased locomotion. Finally, mild prenatal stress increased global DNA methylation levels in the frontal cortex and hippocampus whereas high prenatal stress was associated with a dramatic decrease. The data from this study provide evidence to support a dose-dependent effect of prenatal stress on multiple aspects of brain development, potentially contributing to long-term outcomes.

Aberrant global methylation patterns affect the molecular pathogenesis and prognosis of multiple myeloma

AbstractWe used genome-wide methylation microarrays to analyze differences in CpG methylation patterns in cells relevant to the pathogenesis of myeloma plasma cells (B cells, normal plasma cells, monoclonal gammopathy of undetermined significance [MGUS], presentation myeloma, and plasma cell leukemia). We show that methylation patterns in these cell types are capable of distinguishing nonmalignant from malignant cells and the main reason for this difference is hypomethylation of the genome at the transition from MGUS to presentation myeloma. In addition, gene-specific hypermethylation was evident at the myeloma stage. Differential methylation was also evident at the transition from myeloma to plasma cell leukemia with remethylation of the genome, particularly of genes involved in cell–cell signaling and cell adhesion, which may contribute to independence from the bone marrow microenvironment. There was a high degree of methylation variability within presentation myeloma samples, which was associated with cytogenetic differences between samples. More specifically, we found methylation subgroups were defined by translocations and hyperdiploidy, with t(4;14) myeloma having the greatest impact on DNA methylation. Two groups of hyperdiploid samples were identified, on the basis of unsupervised clustering, which had an impact on overall survival. Overall, DNA methylation changes significantly during disease progression and between cytogenetic subgroups.

Frontiers in Toxicogenomics – The Grand Challenge: To Understand How the Genome and Epigenome Interact with the Toxic Environment

A recent advance that has revitalized toxicology studies in the past couple of decades is Frontiers in Genetics “toxicogenomics,” the study of the effects of environmental toxins on global changes within a cell or tissue as related to genomic variables. “Transcriptional toxicogenomics” is the study of global gene expression patterns after administering a toxin. Similarly, “toxicoproteomics” is the study of global changes in protein levels or post-translational modifications; while “toxicometabolomics” is the study of global changes in metabolite levels after administering a toxin. A new field called “toxicoepigenomics” or more simply, the “epigenetics of toxicology,” is the study of global epigenetic changes in a cell that are caused by exposure of a cell, tissue, or organism to a toxin or drug. Frontiers in Toxicogenomics provides a format for presenting cutting-edge reviews and studies in the frontiers of all branches of toxicogenomics. One type of study within the area of transcriptional toxicogenomics called “genetical genomics” or “genetics of gene expression,” is the combination of quantitative trait-locus (QTL) and whole-genome expression analyses (Brem et al., 2002; Broman, 2005). In genetical genomics, the investigator treats the expression of each of the ∼20,000 genes in an organism as a quantitative trait and identifies cis-expression QTL (eQTL) in cis-regulatory sequences and trans-eQTL in trans-acting factors that regulate expression of co-regulated genes in specific pathways. Genetical toxicogenomics adds a new dimension to genetical genomics by studying the effects of an environmental toxin on global gene expression patterns (Ruden et al., 2009). Such studies are theoretically very powerful because they not only tell which genes are induced in a particular tissue (such as a brain region) by the toxin, but they also allow the identification of trans-acting factors and cis-acting sequences that are regulated by the toxin. Toxicogenetical genomic studies in mice have the advantage that thousands of recombinant inbred lines have recently been generated in a collaborative cross amongst eight different wild type strains of mice (Churchill et al., 2004). Rats are sometimes preferred models for toxicological screens because they have much larger organs than mice, allowing for easier gene and protein expression analyses because much more material can be obtained compared with mice. Recombinant inbred lines have also been developed in rats, and some laboratories have conducted genetical genomics and genetical toxicogenomics analyses in rats, especially in the rat brain. The transcriptional toxicogenomic studies have mostly been done with recombinant inbred lines, such as in mice, C. elegans, and Drosophila, and utilized gene expression microarrays. However, next-generation sequencing technologies allow new types of transcriptional toxicogenomic studies to be conducted. For example, “speed QTL” analyses can be done by selecting a large population of model organisms for a particular trait, such as lifespan after feeding a toxin, for several generations and then sequencing the genome of the long-living descendents (Lai et al., 2007). The single nucleotide polymorphisms (SNPs) that are enriched in the long-living descendents are located in candidate toxin-survival genes which can be studied in follow up genetic and biochemical studies. Third generation sequencing technologies will soon make the “$1000 genome” a reality and will not only allow “personalized medicine,” which is using a person's genome sequence to optimize a drug regimen to treat a disease (Ruden, 2007), but also “personalized toxicogenomics” which uses this genome information to determine a person's susceptibility to an environmental toxin. Toxicoproteomics and toxicometabolimics are new areas that utilize state-of-the-art mass spectrometry and other sensitive assay systems that can quantify protein, peptide, and metabolic levels in a population of model organisms. Instead of gene expression being the quantitative trait, as in transcriptional toxicogenomics, protein, and metabolite levels can be used as quantitative traits. In this manner, protein QTL (pQTL) and metabolite-QTL (mQTL) can be identified in both cis- and trans-genes that regulate the levels of molecules. Toxicoepigenomics is the study of global epigenomic changes, such as DNA methylation changes, histone modifications, or microRNA changes, that are caused by exposure to toxins. In this manner, epigenetic-QTL (epiQTL) can be identified that identify cis- and trans-regulatory elements and factors that alter the epigenome in response to a toxin. It is known for instance that prenatal lead exposure causes hypomethylation of repeats in humans. It would be interesting to study global methylation patterns after exposure to lead and other toxins in large populations. In conclusion, toxicogenomics is an exciting new field that has revitalized toxicology research. No longer need toxicologists study only LD50 values (lethal dose that kills 50% of the population) and use death alone as a toxicology endpoint. Now toxicologists can perform studies with much more subtle endpoints, such as gene expression, protein or metabolite levels, and epigenomic changes. Frontiers in Toxicogenomics provides a format for present research and reviews in this exciting new field.

Lead exposure in pheochromocytoma cells induces persistent changes in amyloid precursor protein gene methylation patterns

It has been suggested that lead (Pb) exposure in early life may increase amyloid precursor protein (APP) expression and promote the pathogenesis of Alzheimer's disease in old age. The current study examined whether the DNA methylation patterns of APP gene in rat pheochromocytoma (PC12) cells changed after Pb acetate exposure. Undifferentiated PC12 cells were exposed to three doses of Pb acetate (50, 250, and 500 nM) and one control for 2 days or 1 week. The methylation patterns of APP promoter and global DNA methylation were analyzed. The DNA methyltransferase 1 (DNMT1) expression and the level of amyloid β peptide (Aβ) were also investigated. The results showed that the exposure of the three concentrations of Pb acetate could make the APP promoter hypomethylated. The global DNA methylation level and the expression of DNMT1 were changed in the 500 nM group after 2 days exposure and in the 250 and 500 nM group after 7 days exposure. Thus, Pb may exert neurotoxic effects through mechanisms that alter the global and promoter methylation patterns of APP gene. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.

Gobal DNA methylation in fetal human germ cells and germ cell tumors: correlation with differentiation and cisplatin resistance

Differences in the global methylation pattern, i.e., hyper- as well as hypo-methylation, are observed in cancer, including germ cell tumors (GCTs). Related to their precursor cells, the GCT methylation status differs according to histology. We investigated the methylation pattern of normal fetal, infantile, and adult germ cells (n = 103) and GCTs (n = 251) by immunohistochemical staining for 5-mCytidine. We found that the global methylation pattern of male germ cells changes from hypomethylation to hypermethylation, while female germ cells remain unmethylated at all stages.