Role Of DNA Methylation Changes Research Articles

Global analysis of DNA methylation changes during experimented lingual carcinogenesis.

This study aims to assess the role of DNA methylation changes in tongue cancer through a comprehensive analysis of global DNA methylation alterations during experimental lingual carcinogenesis. C57BL/6J mice were subjected to 16-week oral administration of 4-nitroquinoline-1-oxide (4NQO, 50 mg/L). Lingual mucosa samples, being representative of normal tissue (week 0) and early (week 12) and advanced (week 28) tumorigenesis, were harvested for microarray and methylated DNA immunoprecipitation sequencing (MeDIP-Seq). The mRNA and promoter methylation of transforming growth factor-beta-signaling protein 1 (SMAD1) were evaluated with real-time quantitative reverse transcription polymerase chain reaction and Massarray in human lingual mucosa and tongue cancer cell lines. The cytosine guanine island (CGI) methylation level observed at 28 weeks surpassed that of both 12 weeks and 0 weeks. The promoter methylation level at 12 weeks exceeded that at 0 weeks. Notably, 208 differentially expressed genes were negatively correlated to differential methylation in promoters among 0, 12, and 28 weeks. The mRNA of SMAD1 was upregulated, concurrent with a decrease in promoter methylation levels in cell lines compared to normal mucosa. DNA methylation changed during lingual carcinogenesis. Overexpression of SMAD1 was correlated to promoter hypomethylation in tongue cancer cell lines.

Analysis of blood methylation quantitative trait loci in East Asians reveals ancestry-specific impacts on complex traits.

Methylation quantitative trait loci (mQTLs) are essential for understanding the role of DNA methylation changes in genetic predisposition, yet they have not been fully characterized in East Asians (EAs). Here we identified mQTLs in whole blood from 3,523 Chinese individuals and replicated them in additional 1,858 Chinese individuals from two cohorts. Over 9% of mQTLs displayed specificity to EAs, facilitating the fine-mapping of EA-specific genetic associations, as shown for variants associated with height. Trans-mQTL hotspots revealed biological pathways contributing to EA-specific genetic associations, including an ERG-mediated 233 trans-mCpG network, implicated in hematopoietic cell differentiation, which likely reflects binding efficiency modulation of the ERG protein complex. More than 90% of mQTLs were shared between different blood cell lineages, with a smaller fraction of lineage-specific mQTLs displaying preferential hypomethylation in the respective lineages. Our study provides new insights into the mQTL landscape across genetic ancestries and their downstream effects on cellular processes and diseases/traits.

The role of salinity on genome-wide DNA methylation dynamics in European sea bass gills.

Epigenetic modifications, like DNA methylation, generate phenotypic diversity in fish and ultimately lead to adaptive evolutionary processes. Euryhaline marine species that migrate between salinity-contrasted habitats have received little attention regarding the role of salinity on whole-genome DNA methylation. Investigation of salinity-induced DNA methylation in fish will help to better understand the potential role of this process in salinity acclimation. Using whole-genome bisulfite sequencing, we compared DNA methylation patterns in European sea bass (Dicentrarchus labrax) juveniles in seawater and after freshwater transfer. We targeted the gill as a crucial organ involved in plastic responses to environmental changes. To investigate the function of DNA methylation in gills, we performed RNAseq and assessed DNA methylome-transcriptome correlations. We showed a negative correlation between gene expression levels and DNA methylation levels in promoters, first introns and first exons. A significant effect of salinity on DNA methylation dynamics with an overall DNA hypomethylation in freshwater-transferred fish compared to seawater controls was demonstrated. This suggests a role of DNA methylation changes in salinity acclimation. Genes involved in key functions as metabolism, ion transport and transepithelial permeability (junctional complexes) were differentially methylated and expressed between salinity conditions. Expression of genes involved in mitochondrial metabolism (tricarboxylic acid cycle) was increased, whereas the expression of DNA methyltransferases 3a was repressed. This study reveals novel links between DNA methylation, mainly in promoters and first exons/introns, and gene expression patterns following salinity change.

Epigenome-wide association study of human frontal cortex identifies differential methylation in Lewy body pathology

Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) are closely related progressive disorders with no available disease-modifying therapy, neuropathologically characterized by intraneuronal aggregates of misfolded α-synuclein. To explore the role of DNA methylation changes in PD and DLB pathogenesis, we performed an epigenome-wide association study (EWAS) of 322 postmortem frontal cortex samples and replicated results in an independent set of 200 donors. We report novel differentially methylated replicating loci associated with Braak Lewy body stage near TMCC2, SFMBT2, AKAP6 and PHYHIP. Differentially methylated probes were independent of known PD genetic risk alleles. Meta-analysis provided suggestive evidence for a differentially methylated locus within the chromosomal region affected by the PD-associated 22q11.2 deletion. Our findings elucidate novel disease pathways in PD and DLB and generate hypotheses for future molecular studies of Lewy body pathology.

The Role of Epigenetic Clocks in Explaining Educational Inequalities in Mortality: A Multicohort Study and Meta-analysis.

Educational inequalities in all-cause mortality have been observed for decades. However, the underlying biological mechanisms are not well known. We aimed to assess the role of DNA methylation changes in blood captured by epigenetic clocks in explaining these inequalities. Data were from 8 prospective population-based cohort studies, representing 13 021 participants. First, educational inequalities and their portion explained by Horvath DNAmAge, Hannum DNAmAge, DNAmPhenoAge, and DNAmGrimAge epigenetic clocks were assessed in each cohort via counterfactual-based mediation models, on both absolute (hazard difference) and relative (hazard ratio) scales, and by sex. Second, estimates from each cohort were pooled through a random effect meta-analysis model. Men with low education had excess mortality from all causes of 57 deaths per 10 000 person-years (95% confidence interval [CI]: 38, 76) compared with their more advantaged counterparts. For women, the excess mortality was 4 deaths per 10 000 person-years (95% CI: -11, 19). On the relative scale, educational inequalities corresponded to hazard ratios of 1.33 (95% CI: 1.12, 1.57) for men and 1.15 (95% CI: 0.96, 1.37) for women. DNAmGrimAge accounted for the largest proportion, approximately 50%, of the educational inequalities for men, while the proportion was negligible for women. Most of this mediation was explained by differential effects of unhealthy lifestyles and morbidities of the World Health Organization (WHO) risk factors for premature mortality. These results support DNA methylation-based epigenetic aging as a signature of educational inequalities in life expectancy emphasizing the need for policies to address the unequal social distribution of these WHO risk factors.

The role of blood DNA methylation in smoking-related cancers

BACKGROUND AND AIM: Smoking is a well-known carcinogen consistently associated with differential DNA methylation in diverse populations. Dysregulations of DNA methylation have been associated with lung cancer. Smoking and lung cancer are common among many indigenous communities of the United States, leading to health disparities. This study investigated the potential mediating role of DNA methylation changes in the association of smoking with incident smoking-related cancers. METHODS: DNA methylation was measured in blood collected at baseline (1989-1991) using the EPIC 850K platform in 2351 American Indian participants of the Strong Heart Study. Cancer incidence was available through 2017 (97 cases for lung cancer, 222 cases for a combined smoking-related cancer endpoint including lung, esophageal, stomach, colorectal, liver, pancreatic and kidney cancers). Iterative Sure Independence Screening paired with adaptive elastic-net was used to select differentially methylated CpGs associated with cancer. Mediation analysis using Aalen additive hazard models was then applied to the selected CpGs. RESULTS:At total of 71 CpGs were associated with lung cancer and smoking-related cancer. For lung cancer, we found significant mediated effects for seven CpGs for current versus former or never smoking, and for eight CpGs for cumulative smoking. For smoking-related cancers, we found significant mediated associations for eight CpGs for current versus former or never smoking, and for one CpG for cumulative smoking. CpGs annotated to the AHRR and the PRSS23 genes (well-known smoking-related genes in DNA methylation studies), showed up to 70 % and up to 48 % of relative mediated effects, respectively, without accounting for multiple mediation. CONCLUSIONS:Our study provides strong evidence that a large extent of the association of smoking with smoking-related cancers, especially in lung cancer, can be explained by differential DNA methylation changes in well-known smoking related genes. Replication is ongoing in the Framingham Heart Study and the Rotterdam Study. KEYWORDS: Smoking, DNA methylation, cancer, American Indians

Epigenetic Changes Induced by Maternal Factors during Fetal Life: Implication for Type 1 Diabetes.

Organ-specific autoimmune diseases, such as type 1 diabetes, are believed to result from T-cell-mediated damage of the target tissue. The immune-mediated tissue injury, in turn, is known to depend on complex interactions between genetic and environmental factors. Nevertheless, the mechanisms whereby environmental factors contribute to the pathogenesis of autoimmune diseases remain elusive and represent a major untapped target to develop novel strategies for disease prevention. Given the impact of the early environment on the developing immune system, epigenetic changes induced by maternal factors during fetal life have been linked to a likelihood of developing an autoimmune disease later in life. In humans, DNA methylation is the epigenetic mechanism most extensively investigated. This review provides an overview of the critical role of DNA methylation changes induced by prenatal maternal conditions contributing to the increased risk of immune-mediated diseases on the offspring, with a particular focus on T1D. A deeper understanding of epigenetic alterations induced by environmental stressors during fetal life may be pivotal for developing targeted prevention strategies of type 1 diabetes by modifying the maternal environment.

Invasive and Noninvasive Nonfunctioning Gonadotroph Pituitary Tumors Differ in DNA Methylation Level of LINE-1 Repetitive Elements.

Purpose: Epigenetic dysregulation plays a role in pituitary tumor pathogenesis. Some differences in DNA methylation were observed between invasive and noninvasive nonfunctioning gonadotroph tumors. This study sought to determine the role of DNA methylation changes in repetitive LINE-1 elements in nonfunctioning gonadotroph pituitary tumors. Methods: We investigated LINE-1 methylation levels in 80 tumors and normal pituitary glands with bisulfite-pyrosequencing. Expression of two LINE-1 open reading frames (L1-ORF1 and L1-ORF2) was analyzed with qRT-PCR in tumor samples and mouse gonadotroph pituitary cells treated with DNA methyltransferase inhibitor. Immunohistochemical staining against L1-ORF1p was also performed in normal pituitary glands and tumors. Results: Hypomethylation of LINE-1 was observed in pituitary tumors. Tumors characterized by invasive growth revealed lower LINE-1 methylation level than noninvasive ones. LINE-1 methylation correlated with overall DNA methylation assessed with HM450K arrays and negatively correlated with L1-ORF1 and L1-ORF2 expression. Treatment of αT3-1 gonadotroph cells with 5-Azacytidine clearly increased the level of L1-ORF1 and L1-ORF2 mRNA; however, its effect on LβT2 cells was less pronounced. Immunoreactivity against L1-ORF1p was higher in tumors than normal tissue. No difference in L1-ORF1p expression was observed in invasive and noninvasive tumors. Conclusion: Hypomethylation of LINE-1 is related to invasive growth and influences transcriptional activity of transposable elements.

Integrated Analysis of DNA Methylation, Hydroxymethylation, and Gene Expression Data Using ME-Class2.

There is increasing interest in understanding the pathological role of DNA methylation changes in disease by profiling genome-wide methylation changes. This includes both 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). The typical profiling study is designed to measure 5mC and/or 5hmC levels alongside gene expression in a set of samples and controls to determine a list of candidate genes whose 5mC and/or 5hmC changes are associated with expression changes. We recently showed that ME-Class2 substantially outperforms other bioinformatic approaches at accurately identify genes with highly associated methylation and expression changes. ME-Class2 further illuminated how synergistic changes in 5mC and 5hmC potentially contribute to gene silencing and activation. Here we present a detailed protocol for using ME-Class2 to analyze genome-wide methylation (5mC and/or 5hmC) and expression data. Further, we provide advice about extending ME-Class2 to study the relationships between other epigenetic marks.

DNA methylation signatures of Prostate Cancer in peripheral T-cells

BackgroundProstate Cancer (PCa) is the second most common cancer in men where advancements have been made for early detection using imaging techniques, however these are limited by lesion size. Immune surveillance has emerged as an effective approach for early detection and to monitor disease progression. In recent studies, we have shown that host peripheral blood immune cells undergo changes in DNA methylation in liver and breast cancer.MethodsIn the current study, we examined the DNA methylation status of peripheral blood T cells of men with positive biopsy for PCa versus men with negative biopsy having benign prostate tissue, defined as controls. T cells DNA was isolated and subjected to Illumina Infinium methylation EPIC array and validated using Illumina amplicon sequencing and pyrosequencing platforms.ResultsDifferential methylation of 449 CG sites between control and PCa T cell DNA showed a correlation with Gleason score (p < 0.05). Two hundred twenty-three differentially methylated CGs between control and PCa (∆ß +/− 10%, p < 0.05), were enriched in pathways involved in immune surveillance system. Three CGs which were found differentially methylated following DMP (Differentially methylated probes) analysis of ChAMP remained significant after BH (Benjamini-Hochberg) correction, of which, 2 CGs were validated. Predictive ability of combination of these 3 CGs (polygenic methylation score, PMS) to detect PCa had high sensitivity, specificity and overall accuracy. PMS also showed strong positive correlation with Gleason score and tumor volume of PCa patients.ConclusionsResults from the current study provide for the first-time a potential role of DNA methylation changes in peripheral T cells in PCa. This non-invasive methodology may allow for early intervention and stratification of patients into different prognostic groups to reduce PCa associated morbidity from repeat invasive prostate biopsies and design therapeutic strategy to reduce PCa associated mortality.

Epigenetic modifications: An important mechanism in diabetic disturbances.

In the search for explanations of diabetes pathomechanisms, especially the development of its vascular complications (micro- and macrovascular ), although current, good metabolic control of diabetes, attention was drawn to the role of epigenetic inheritance associated with epigenetic modifications of histone proteins and DNA in hyperglycemia conditions. This study showed the significant role of DNA methylation and histone epigenetic modifications (a different nature and a different degree) in the transmission of information that is not connected with gene inheritance but concerns the persistent changes induced by hyperglycemia..Attention was paid to the role of DNA methylation of pancreatic cells in the pathogenesis of type 1 diabetes, but also type 2. The important role of DNA methylation changes in a so-called intrauterine growth restriction (IUGR) as reason of subsequent development of diabetes was particularly emphasized. In the pathogenesis of type 2 diabetes and its complications, especially microvascular complications, the greatest share and importance of epigenetic modifications on mitochondrial DNA metylation are the most important. The multidirectionality Complicaand complexity of epigenetic modifications of histone proteins indicate their importance in the development of diabetic disturbances. An especially important role is attributed to methylation and acetylation of histone proteins, in particular on arginine and lysine, whose changes occur most frequently. Moreover, epigenetic modifications of the enzymes, especially methylases, responsible for these processes are the underlying. It has been indicated that the identification of epigenetic differences within the DNA or histone proteins may be a useful prognostic biomarker of susceptibility to the disease development in the future. Moreover, they may become a potential target for future therapeutic interventions for clinical disorders in diabetes.

Epigenetics and Bone Remodeling.

Bone remodeling is a diverse field of study with many direct clinical applications; past studies have implicated epigenetic alterations as key factors of both normal bone tissue development and function and diseases of pathologic bone remodeling. The purpose of this article is to review the most important recent advances that link epigenetic changes to the bone remodeling field. Epigenetics describes three major phenomena: DNA modification via methylation, histone side chain modifications, and short non-coding RNA sequences which work in concert to regulate gene transcription in a heritable fashion. Recent findings include the role of DNA methylation changes of Wnt, RANK/RANKL, and other key signaling pathways, epigenetic regulation of osteoblast and osteoclast differentiation, and others. Although much work has been done, much is still unknown. Future epigenome-wide studies should focus on extending the tissue coverage, integrating multiple epigenetic analyses with transcriptome data, and working to uncover epigenetic changes linked with early events in aberrant bone remodeling.

Promoter methylation patterns of ABCB1, ABCC1 and ABCG2 in human cancer cell lines, multidrug-resistant cell models and tumor, tumor-adjacent and tumor-distant tissues from breast cancer patients.

Overexpression of ABCB1, ABCC1 and ABCG2 in tumor tissues is considered a major cause of limited efficacy of anticancer drugs. Gene expression of ABC transporters is regulated by multiple mechanisms, including changes in the DNA methylation status. Most of the studies published so far only report promoter methylation levels for either ABCB1 or ABCG2, and data on the methylation status for ABCC1 are scarce. Thus, we determined the promoter methylation patterns of ABCB1, ABCC1 and ABCG2 in 19 human cancer cell lines. In order to contribute to the elucidation of the role of DNA methylation changes in acquisition of a multidrug resistant (MDR) phenotype, we also analyzed the promoter methylation patterns in drug-resistant sublines of the cancer cell lines GLC-4, SW1573, KB-3-1 and HL-60. In addition, we investigated if aberrant promoter methylation levels of ABCB1, ABCC1 and ABCG2 occur in tumor and tumor-surrounding tissues from breast cancer patients.Our data indicates that hypomethylation of the ABCC1 promoter is not cancer type-specific but occurs in cancer cell lines of different origins. Promoter methylation was found to be an important mechanism in gene regulation of ABCB1 in parental cancer cell lines and their drug-resistant sublines. Overexpression of ABCC1 in MDR cell models turned out to be mediated by gene amplification, not by changes in the promoter methylation status of ABCC1. In contrast to the promoters of ABCC1 and ABCG2, the promoter of ABCB1 was significantly higher methylated in tumor tissues than in tumor-adjacent and tumor-distant tissues from breast cancer patients.

DNA methylation, bacteria and airway inflammation: latest insights.

DNA methylation is an epigenetic mechanism that has been implicated in the pathogenesis of chronic inflammatory diseases by regulating differentiation, proliferation, apoptosis, and activation of immune cells. Changes in the methylation status of relevant genes have been linked to the origin, perpetuation, and severity of airway diseases. The DNA methylation profile can be also modified by the action of viral and bacterial colonization. Bacteria and specially Staphylococcus aureus toxins are recognized inflammatory amplifying factors in both lower and upper airway chronic diseases. This review summarizes the existent knowledge about the role of DNA methylation changes in chronic airway diseases and the contribution of bacterial infection on this event. It has been demonstrated that changes in DNA methylation, either intrinsic or induced by allergen or infection, may be linked to the pathogenesis of asthma and allergy. These changes in methylation may suppress the production of anti-inflammatory mediators and increase the survival and activation of pro-inflammatory cells, as well as modify the immune response in response to bacterial infection, increasing their survival and pathogenicity within the infected organism. Understanding the intrinsic epigenetic mechanisms, as well as the effect of environment -for example, bacterial infection in the pathogenesis of airways diseases - will greatly improve the management and the diagnosis of these diseases.

DNA methylation map of human atherosclerosis.

Epigenetic alterations may contribute to the development of atherosclerosis. In particular, DNA methylation, a reversible and highly regulated DNA modification, could influence disease onset and progression because it functions as an effector for environmental influences, including diet and lifestyle, both of which are risk factors for cardiovascular diseases. To address the role of DNA methylation changes in atherosclerosis, we compared a donor-matched healthy and atherosclerotic human aorta sample using whole-genome shotgun bisulfite sequencing. We observed that the atherosclerotic portion of the aorta was hypermethylated across many genomic loci in comparison with the matched healthy counterpart. Furthermore, we defined specific loci of differential DNA methylation using a set of donor-matched aortic samples and a high-density (>450 000 CpG sites) DNA methylation microarray. The functional importance in the disease was corroborated by crossing the DNA methylation signature with the corresponding expression data of the same samples. Among the differentially methylated CpGs associated with atherosclerosis onset, we identified genes participating in endothelial and smooth muscle functions. These findings provide new clues toward a better understanding of the molecular mechanisms of atherosclerosis. Our data identify an atherosclerosis-specific DNA methylation profile that highlights the contribution of different genes and pathways to the disorder. Interestingly, the observed gain of DNA methylation in the atherosclerotic lesions justifies efforts to develop DNA demethylating agents for therapeutic benefit.

THU0454 Changes in DNA Methylation Related to Smoking Are Associated with Ra. an Epigenome Wide Association Study in Monozygotic RA Discordant Twin Pairs

BackgroundThere is increasing evidence for a pathogenetic role of DNA methylation changes in RA. Monozygotic (MZ) twins provide a unique opportunity to study the epigenetic effects in complex diseases like...

DNA methylome alterations in chemical carcinogenesis

Carcinogenesis, a complex multifactorial process of the transformation of normal cells into malignant cells, is characterized by many biologically significant and interdependent alterations triggered by the mutational and/or non-mutational (i.e., epigenetic) events. One of these events, specific to all types of cancer, is alterations in DNA methylation. This review summarizes the current knowledge of the role of DNA methylation changes induced by various genotoxic chemicals (carcinogenic agents that interact with DNA) and non-genotoxic carcinogens (chemicals causing tumor by mechanisms other than directly damaging DNA) in the lung, colorectal, liver, and hematologic carcinogenesis. It also emphasizes the potential role for epigenetic changes to serve as markers for carcinogen exposure and carcinogen risk assessment.

Abstract 3142: Hypermethylation of the tissue inhibitor of metalloproteinases-2 gene in multiple myeloma

Abstract Multiple myeloma (MM) is a B-cell neoplasm that is characterized by the accumulation of malignant plasma cells in the bone marrow (BM) as well as abundant BM angiogenesis. Aberrant methylation of CpG islands near gene promoter regions is the most widely studied epigenetic abnormality in human malignancies and is associated with loss of gene function. There is increasing evidence for a role of the tissue inhibitor of metalloproteinases 2 (TIMP-2) gene as a tumor suppressor. Overexpression of TIMP-2 may result in decreased invasive potential, suppression of tumor growth and vascularization as well as inhibition of angiogenesis. We could previously show that TIMP-2 may become hypermethylated in association with transcriptional silencing in indolent and aggressive non-Hodgkin's lymphomas. Recently, downregulation of TIMP-2 was also reported in MM, and this is thought to contribute to increased BM angiogenesis. In this study, we determined the methylation status of the promoter-associated CpG island of TIMP-2 in primary MM samples and investigated correlations between TIMP-2 methylation and clinical parameters. Methylation of the promoter-associated CpG island of TIMP-2 was analyzed by methylation-specific polymerase chain reaction (MSP) in samples from 84 MM patients (median age 65 years [range 40-94]; 48 males, 36 females; 79 BM samples, 5 peripheral blood samples). Overall survival curves were plotted according to the method of Kaplan and Meier and compared using the log-rank test. Correlations between variables were analyzed using the Fisher's exact two-sided test and independent t-test, respectively. MSP analysis revealed aberrant methylation of the TIMP-2 promoter region in 4/84 MM patient samples. We found an association of TIMP-2 hypermethylation with plasma cell leukemia (p&amp;lt;0.0001) and decreased platelet counts (111 G/l vs. 258 G/l; p=0.04). There was a trend towards an inferior overall survival in MM patients with a hypermethylated TIMP-2 gene, but this difference was not statistically significant (p=0.09). We conclude that promoter hypermethylation of TIMP-2 is a novel epigenetic event in MM that may contribute to disease progression and could serve as a prognostic biomarker. Further studies are warranted to elucidate the functional consequences of epigenetic dysregulation of TIMP-2 in the pathogenesis of MM. Additionally, the increasing evidence for the role of DNA methylation changes in MM may serve as a basis for the use of epigenetically targeted therapeutic approaches in malignant plasma cell disorders. 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 3142. doi:1538-7445.AM2012-3142

Epigenetic Dysregulation of the Tissue Inhibitor of Metalloproteinases-2 Gene in Multiple Myeloma

Abstract 5089 Background:Multiple myeloma (MM) is a B-cell neoplasm characterized by the accumulation of malignant plasma cells in the bone marrow (BM) as well as abundant BM angiogenesis. Aberrant methylation of CpG islands near gene promoter regions is the most widely studied epigenetic abnormality in human malignancies and is associated with loss of gene expression. There is increasing evidence for a role of the tissue inhibitor of metalloproteinases 2 (TIMP2) gene as a tumor suppressor. Overexpression of TIMP2 may result in decreased invasive potential, suppression of tumor growth and vascularization as well as inhibition of angiogenesis. We could previously show that TIMP2 may become hypermethylated in association with transcriptional silencing in indolent and aggressive non-Hodgkin's lymphomas. Recently, downregulation of TIMP2 was also reported in MM, and this is thought to contribute to increased BM angiogenesis. In this study, we determined the methylation status of the promoter-associated CpG island of TIMP2 in primary MM samples and investigated correlations between TIMP2 methylation and clinical parameters. Methods:Methylation of the promoter-associated CpG island of TIMP2 was analyzed by methylation-specific polymerase chain reaction (MSP) in samples from 76 MM patients (median age 65 years [range 40–94]; 44 males, 32 females; 71 BM samples, 5 peripheral blood samples). Overall survival curves were plotted according to the method of Kaplan and Meier and compared using the log-rank test. Correlations between variables were analyzed using the Fisher’s exact two-sided test and independent t-test, respectively. Results:MSP analysis revealed that there was aberrant methylation of the TIMP2 promoter region in 4/76 MM patient samples. We found an association of TIMP2 hypermethylation with plasma cell leukemia (p<0.0001) and decreased platelet counts (257.1 G/l vs. 111.5 G/l; p=0.04). There was a trend towards an inferior overall survival in MM cases with a hypermethylated TIMP2 gene, but this difference was not statistically significant (p=0.11). Conclusions:Our study shows that promoter hypermethylation of TIMP2 is a novel epigenetic event in MM that may contribute to disease progression and could serve as a prognostic biomarker. Further studies are warranted to elucidate the functional consequences of epigenetic dysregulation of TIMP2 in the pathogenesis of MM. Additionally, the increasing evidence for the role of DNA methylation changes in MM may serve as a basis for the use of epigenetically targeted therapeutic approaches in malignant plasma cell disorders. Disclosures:Herman:MDxHealth: Consultancy.

Epigenetic Inactivation of the Secreted Frizzled-Related Protein Gene Family in Multiple Myeloma.

Multiple myeloma (MM) is a B-cell neoplasm that is characterized by the accumulation of malignant plasma cells in the bone marrow. Previous molecular studies have largely focused on acquired genetic aberrations in MM. There is increasing evidence that in addition to genetic aberrations epigenetic processes play a major role in carcinogenesis. Aberrant methylation of CpG islands near gene promoter regions is the most widely studied epigenetic abnormality in human malignancies and is associated with loss of gene function. This epigenetic event acts as an alternative to mutations and deletions to disrupt tumor suppressor gene function. The Wnt pathway has been recognized to be essential for normal organ development, and a role for Wnt signal transduction at several stages of lymphocyte differentiation and in the self-renewal of hematopoietic stem cells could be demonstrated. Alterations in the Wnt pathway have been shown to contribute to the pathogenesis of various human malignancies. Epigenetic silencing of the family of secreted frizzled-related proteins (SFRPs), which act as Wnt antagonists, was recently reported in several solid tumors and in acute lymphoblastic leukemia. In order to investigate the potential role of abnormal Wnt signaling in MM, we determined the methylation status of the promoter-associated CpG islands of SFRP-1, 2, 4 and 5 in the MM cell lines U266, LP-1, RPMI-8226 and OPM-2. Methylation-specific polymerase chain reaction (MSP) analysis revealed that promoter hypermethylation of the SFRP genes was a frequent event in MM cell lines (SFRP-1: 2/4, SFRP-2: 2/4, SFRP-4: 1/4 and SFRP-5: 3/4). Aberrant methylation of SFRP-1 and SFRP-2 in MM cell lines was associated with transcriptional silencing, as determined by real-time reverse transcriptase polymerase chain reaction. Treatment of cell lines that carry a hypermethylated SFRP-1 and SFRP-2 gene, respectively, with the demethylating agent 5-aza-2′-deoxycytidine resulted in gene reexpression. We then analyzed by MSP the methylation status of SFRP-1, 2, 4 and 5 in 76 specimens obtained from MM patients. The frequency of aberrant methylation among the primary patient samples was 38.2% (29/76) for SFRP-1, 59.2% (45/76) for SFRP-2, 2.6% (2/76) for SFRP-4 and 7.6% (6/76) for SFRP-5. There was a high incidence of concomitant hypermethylation of SFRP-1 and SRFP-2. We conclude that promoter hypermethylation of the SFRP genes is a novel epigenetic event in MM that may contribute to aberrant activation of the Wnt pathway. Further studies are warranted to elucidate the functional consequences of aberrant Wnt signaling by downregulation of the SFRP genes in the pathogenesis of MM. Additionally, the increasing evidence for the important role of DNA methylation changes in malignant plasma cell disorders may serve as a basis for the use of epigenetically targeted therapeutic approaches in MM in the future.