Differentially Methylated Loci Research Articles

Developmental cannabidiol exposure increases anxiety and modifies genome-wide brain DNA methylation in adult female mice

BackgroundUse of cannabidiol (CBD), the primary non-psychoactive compound found in cannabis, has recently risen dramatically, while relatively little is known about the underlying molecular mechanisms of its effects. Previous work indicates that direct CBD exposure strongly impacts the brain, with anxiolytic, antidepressant, antipsychotic, and other effects being observed in animal and human studies. The epigenome, particularly DNA methylation, is responsive to environmental input and can direct persistent patterns of gene regulation impacting phenotype. Epigenetic perturbation is particularly impactful during embryogenesis, when exogenous exposures can disrupt critical resetting of epigenetic marks and impart phenotypic effects lasting into adulthood. The impact of prenatal CBD exposure has not been evaluated; however, studies using the psychomimetic cannabinoid Δ9-tetrahydrocannabinol (THC) have identified detrimental effects on psychological outcomes in developmentally exposed adult offspring. We hypothesized that developmental CBD exposure would have similar negative effects on behavior mediated in part by the epigenome. Nulliparous female wild-type Agouti viable yellow (Avy) mice were exposed to 20 mg/kg CBD or vehicle daily from two weeks prior to mating through gestation and lactation. Coat color shifts, a readout of DNA methylation at the Agouti locus in this strain, were measured in F1 Avy/a offspring. Young adult F1 a/a offspring were then subjected to tests of working spatial memory and anxiety/compulsive behavior. Reduced-representation bisulfite sequencing was performed on both F0 and F1 cerebral cortex and F1 hippocampus to identify genome-wide changes in DNA methylation for direct and developmental exposure, respectively.ResultsF1 offspring exposed to CBD during development exhibited increased anxiety and improved memory behavior in a sex-specific manner. Further, while no significant coat color shift was observed in Avy/a offspring, thousands of differentially methylated loci (DMLs) were identified in both brain regions with functional enrichment for neurogenesis, substance use phenotypes, and other psychologically relevant terms.ConclusionsThese findings demonstrate for the first time that despite positive effects of direct exposure, developmental CBD is associated with mixed behavioral outcomes and perturbation of the brain epigenome.

Genome-Wide Assessment of DNA Methylation in Chicken Cardiac Tissue Exposed to Different Incubation Temperatures and CO2 Levels.

Temperature and CO2 concentration during incubation have profound effects on broiler chick development, and numerous studies have identified significant effects on hatch heart weight (HW) as a result of differences in these parameters. Early life environment has also been shown to affect broiler performance later in life; it has thus been suggested that epigenetic mechanisms may mediate long-term physiological changes induced by environmental stimuli. DNA methylation is an epigenetic modification that can confer heritable changes in gene expression. Using reduced-representation bisulfite sequencing (RRBS), we assessed DNA methylation patterns in cardiac tissue of 84 broiler hatchlings incubated at two egg shell temperatures (EST; 37.8°C and 38.9°C) and three CO2 concentrations (0.1%, 0.4%, and 0.8%) from day 8 of incubation onward. We assessed differential methylation between EST treatments and identified 2,175 differentially methylated (DM) CpGs (1,121 hypermethylated, 1,054 hypomethylated at 38.9° vs. 37.8°) in 269 gene promoters and 949 intragenic regions. DM genes (DMGs) were associated with heart developmental processes, including cardiomyocyte proliferation and differentiation. We identified enriched binding motifs among DM loci, including those for transcription factors associated with cell proliferation and heart development among hypomethylated CpGs that suggest increased binding ability at higher EST. We identified 9,823 DM CpGs between at least two CO2 treatments, with the greatest difference observed between 0.8 and 0.1% CO2 that disproportionately impacted genes involved in cardiac muscle development and response to low oxygen levels. Using HW measurements from the same chicks, we performed an epigenome-wide association study (EWAS) for HW, and identified 23 significantly associated CpGs, nine of which were also DM between ESTs. We found corresponding differences in transcript abundance between ESTs in three DMGs (ABLIM2, PITX2, and THRSP). Hypomethylation of an exonic CpG in PITX2 at 38.9°C was associated with increased expression, and suggests increased cell proliferation in broiler hatchlings incubated at higher temperatures. Overall, these results identified numerous epigenetic associations between chick incubation factors and heart development that may manifest in long-term differences in animal performance.

Subacute cannabidiol alters genome-wide DNA methylation in adult mouse hippocampus.

Use of cannabidiol (CBD), the most abundant non-psychoactive compound found in cannabis (Cannabis sativa), has recently increased as a result of widespread availability of CBD-containing products. CBD is FDA-approved for the treatment of epilepsy and exhibits anxiolytic, antipsychotic, prosocial, and other behavioral effects in animal studies and clinical trials, however, the underlying mechanisms governing these phenotypes are still being elucidated. The epigenome, particularly DNA methylation, is responsive to environmental input and can govern persistent patterns of gene regulation affecting phenotype across the life course. In order to understand the epigenomic activity of cannabidiol exposure in the adult brain, 12-week-old male wild-type a/a Agouti viable yellow (Avy ) mice were exposed to either 20 mg/kg CBD or vehicle daily by oral administration for 14 days. Hippocampal tissue was collected and reduced-representation bisulfite sequencing (RRBS) was performed. Analyses revealed 3,323 differentially methylated loci (DMLs) in CBD-exposed animals with a small skew toward global hypomethylation. Genes for cell adhesion and migration, dendritic spine development, and excitatory postsynaptic potential were found to be enriched in a gene ontology term analysis of DML-containing genes, and disease ontology enrichment revealed an overrepresentation of DMLs in gene sets associated with autism spectrum disorder, schizophrenia, and other phenotypes. These results suggest that the epigenome may be a key substrate for CBD's behavioral effects and provides a wealth of gene regulatory information for further study.

Genome-wide epigenetic analyses in Japanese immigrant plantation workers with Parkinson\u2019s disease and exposure to organochlorines reveal possible involvement of glial genes and pathways involved in neurotoxicity

BackgroundParkinson’s disease (PD) is a disease of the central nervous system that progressively affects the motor system. Epidemiological studies have provided evidence that exposure to agriculture-related occupations or agrichemicals elevate a person’s risk for PD. Here, we sought to examine the possible epigenetic changes associated with working on a plantation on Oahu, HI and/or exposure to organochlorines (OGC) in PD cases.ResultsWe measured genome-wide DNA methylation using the Illumina Infinium HumanMethylation450K BeadChip array in matched peripheral blood and postmortem brain biospecimens in PD cases (n = 20) assessed for years of plantation work and presence of organochlorines in brain tissue. The comparison of 10+ to 0 years of plantation work exposure detected 7 and 123 differentially methylated loci (DML) in brain and blood DNA, respectively (p < 0.0001). The comparison of cases with 4+ to 0–2 detectable levels of OGCs, identified 8 and 18 DML in brain and blood DNA, respectively (p < 0.0001). Pathway analyses revealed links to key neurotoxic and neuropathologic pathways related to impaired immune and proinflammatory responses as well as impaired clearance of damaged proteins, as found in the predominantly glial cell population in these environmental exposure-related PD cases.ConclusionsThese results suggest that distinct DNA methylation biomarker profiles related to environmental exposures in PD cases with previous exposure can be found in both brain and blood.

Association of DNA-Methylation Profiles With Immune Responses Elicited in Breast Cancer Patients Immunized With a Carbohydrate-Mimicking Peptide: A Pilot Study.

Immune response to a given antigen, particularly in cancer patients, is complex and is controlled by various genetic and environmental factors. Identifying biomarkers that can predict robust response to immunization is an urgent need in clinical cancer vaccine development. Given the involvement of DNA methylation in the development of lymphocytes, tumorigenicity and tumor progression, we aimed to analyze pre-vaccination DNA methylation profiles of peripheral blood mononuclear cells (PBMCs) from breast cancer subjects vaccinated with a novel peptide-based vaccine referred to as P10s-PADRE. This pilot study was performed to evaluate whether signatures of differentially methylated (DM) loci can be developed as potential predictive biomarkers for prescreening subjects with cancer who will most likely generate an immune response to the vaccine. Genomic DNA was isolated from PBMCs of eight vaccinated subjects, and their DNA methylation profiles were determined using Infinium® MethylationEPIC BeadChip array from Illumina. A linear regression model was applied to identify loci that were differentially methylated with respect to anti-peptide antibody titers and with IFN-γ production. The data were summarized using unsupervised-learning methods: hierarchical clustering and principal-component analysis. Pathways and networks involved were predicted by Ingenuity Pathway Analysis. We observed that the profile of DM loci separated subjects in regards to the levels of immune responses. Canonical pathways and networks related to metabolic and immunological functions were found to be involved. The data suggest that it is feasible to correlate methylation signatures in pre-treatment PBMCs with immune responses post-treatment in cancer patients going through standard-of-care chemotherapy. Larger and prospective studies that focus on DM loci in PBMCs is warranted to develop pre-screening biomarkers before BC vaccination.Clinical Trial Registration: www.ClinicalTrials.gov, Identifier: NCT02229084.

Whole Genome DNA Methylation Analysis of Active Pulmonary Tuberculosis Disease Identifies Novel Epigenotypes: PARP9/miR-505/RASGRP4/GNG12 Gene Methylation and Clinical Phenotypes.

We hypothesized that DNA methylation patterns may contribute to the development of active pulmonary tuberculosis (TB). Illumina’s DNA methylation 450 K assay was used to identify differentially methylated loci (DML) in a discovery cohort of 12 active pulmonary TB patients and 6 healthy subjects (HS). DNA methylation levels were validated in an independent cohort of 64 TB patients and 24 HS. Microarray analysis identified 1028 DMLs in TB patients versus HS, and 3747 DMLs in TB patients after versus before anti-TB treatment, while autophagy was the most enriched signaling pathway. In the validation cohort, PARP9 and miR505 genes were hypomethylated in the TB patients versus HS, while RASGRP4 and GNG12 genes were hypermethylated, with the former two further hypomethylated in those with delayed sputum conversion, systemic symptoms, or far advanced lesions. MRPS18B and RPTOR genes were hypomethylated in TB patients with pleural involvement. RASGRP4 gene hypermethylation and RPTOR gene down-regulation were associated with high mycobacterial burden. TB patients with WIPI2/GNG12 hypermethylation or MRPS18B/FOXO3 hypomethylation had lower one-year survival. In vitro ESAT6 and CFP10 stimuli of THP-1 cells resulted in DNA de-methylation changes of the PARP9, RASGRP4, WIPI2, and FOXO3 genes. In conclusions, aberrant DNA methylation over the PARP9/miR505/RASGRP4/GNG12 genes may contribute to the development of active pulmonary TB disease and its clinical phenotypes, while aberrant DNA methylation over the WIPI2/GNG12/MARPS18B/FOXO3 genes may constitute a determinant of long-term outcomes.

Acute enterovirus infections significantly alter host cellular DNA methylation status

Acute infections with enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) usually cause Hand, foot and mouth disease (HFMD) among infants and young children with several large outbreaks worldwide. Unfortunately, the molecular mechanisms underlying enterovirus infections remain largely unknown. In this study, we analyzed the genome-wide DNA methylation patterns of host cells in response to EV71 and CVA16 infections using the Illumina Infinium HumanMethylation450 BeadChip. Of over 480,000 loci studied, significant differential methylation was observed between EV71 infected-cells and control cells at 3957 CpG sites, out of which 2478 were hypermethylated and 1479 were hypomethylated, whereas CVA16 infection resulted in methylation level changes of 5194 CpG sites with 4288 hypermethylated and 906 hypomethylated. These differential methylated loci displayed a wide range of genomic distributions in chromosomes, inside and surrounding areas (shores and shelves) of CpG islands, as well as functional gene regions including promoter, gene body and 3’UTR. Based on methylation alterations, 1189 genes were identified to be potentially co-associated with the replication processes of two enteroviruses. GO function annotation and enrichment analysis of 1189 common differentially methylated genes reflected a broad spectrum of biological regulatory events during viral infection. KEGG pathway analysis indicated the involvement of diverse signaling pathways including viral myocarditis, Notch signaling and antigen processing and presentation. Our present study provides a novel insight into enterovirus-host interaction network at epigenetic profile, thus contributing to improved understanding of HFMD pathogenesis.

Genome-wide analysis of DNA methylation profile identifies differentially methylated loci associated with human intervertebral disc degeneration.

BackgroundEnvironmental and endogenous factors under genetic predisposition are considered to initiate the human intervertebral disc (IVD) degeneration. DNA methylation is an essential mechanism to ensure cell-specific gene expression for normal development and tissue stability. Aberrant epigenetic alterations play a pivotal role in several diseases, including osteoarthritis. However, epigenetic alternations, including DNA methylation, in IVD degeneration have not been evaluated. The purpose of this study was to comprehensively compare the genome-wide DNA methylation profiles of human IVD tissues, specifically nucleus pulpous (NP) tissues, with early and advanced stages of disc degeneration.MethodsHuman NP tissues were used in this study. The samples were divided into two groups: early stage degeneration (n = 8, Pfirrmann’s MRI grade: I-III) and advanced stage degeneration (n = 8, grade: IV). Genomic DNA was processed for genome-wide DNA methylation profiling using the Infinium MethylationEPIC BeadChip array. Extraction of raw methylation data, clustering and scatter plot of each group values of each sample were performed using a methylation module in GenomeStudio software. The identification of differentially methylated loci (DMLs) and the Gene Ontology (GO) analysis were performed using R software with the ChAMP package.ResultsUnsupervised hierarchical clustering revealed that early and advanced stage degenerated IVD samples segregated into two main clusters by their DNA methylome. A total of 220 DMLs were identified between early and advanced disc degeneration stages. Among these, four loci were hypomethylated and 216 loci were hypermethylated in the advanced disc degeneration stage. The GO enrichment analysis of genes containing DMLs identified two significant GO terms for biological processes, hemophilic cell adhesion and cell-cell adhesion.ConclusionsWe conducted a genome-wide DNA methylation profile comparative study and observed significant differences in DNA methylation profiles between early and advanced stages of human IVD degeneration. These results implicate DNA methylation in the process of human IVD degeneration.

Analysis of DNA Methylation Patterns Associated with In Vitro Propagated Globe Artichoke Plants Using an EpiRADseq-Based Approach.

Globe artichoke represents one of the main horticultural species of the Mediterranean basin, and ‘Spinoso sardo’ is the most widespread and economically relevant varietal type in Sardinia, Italy. In the last decades, in vitro culture of meristematic apices has increased the frequency of aberrant plants in open-field production. These off-type phenotypes showed highly pinnate-parted leaves and late inflorescence budding, and emerged from some branches of the true-to-type ‘Spinoso sardo’ plants. This phenomenon cannot be foreseen and is reversible through generations, suggesting the occurrence of epigenetic alterations. Here, we report an exploratory study on DNA methylation patterns in off-type/true-to-type globe artichoke plants, using a modified EpiRADseq technology, which allowed the identification of 2897 differentially methylated loci (DML): 1998 in CG, 458 in CHH, and 441 in CHG methylation contexts of which 720, 88, and 152, respectively, were in coding regions. Most of them appeared involved in primary metabolic processes, mostly linked to photosynthesis, regulation of flower development, and regulation of reproductive processes, coherently with the observed phenotype. Differences in the methylation status of some candidate genes were integrated with transcriptional analysis to test whether these two regulation levels might interplay in the emergence and spread of the ‘Spinoso sardo’ non-conventional phenotype.

Abstract 472: Tumor DNA methylation and microRNA expression in breast cancer among American women of African or European ancestry

Abstract American women of African ancestry (AA) are more likely than those of European ancestry (EA) to be diagnosed with aggressive, estrogen receptor (ER) negative breast cancer, and to die of this disease. The reasons remain largely unknown. Epigenetic mechanisms, particularly differential DNA methylation and altered microRNA expression, may contribute to this racial disparity. We previously conducted genome-wide DNA methylation profiling of fresh-frozen breast tumor samples from 58 AA and 80 EA women, with subsequent analyses restricted to CpGs within coding genes. In this study, we focused on CpGs within loci encoding microRNAs, and sought to identify differentially methylated loci (DML) by ER status and by race. We identified a total of 297 DML (Δβ &amp;gt;0.1; FDR&amp;lt;0.05) between ER negative and ER positive breast tumors. Of these, 113 were differentially methylated regardless of race; 65 were specific to EAs; and 119 were specific to AAs. The top DML common to both races were located within miR-155HG, -365-1, -135b, -196b, and -190b, while the top race-specific DML were located within miR-1256, -1224, -1249, -9-3, -657/-338, and miR-25 (EA), or within miR-2053, -526a1, -518B, -489, and -125b1 (AA). We also identified 25 DML (Δβ &amp;gt;0.1; P&amp;lt;0.05) between AA and EA tumors. Among these 25 DML, all of which were hypomethylated in AAs versus EAs, three were specific to ER+ tumors (miR-548G/FILIP1L,-572, -661/PLEC1), 21 were specific to ER- tumors (e.g., miR-2053, -1323, -125b1), and one was common to both tumor subtypes (miR-662). In order to assess how DNA methylation may impact gene expression, we performed miRSeq on tumors from a subset of included women (29 AA and 29 EA). We observed that altered methylation at certain DML was inversely associated with microRNA expression; for example, three DML in the promoter region of miR-190b (Spearman's rho=-0.65, -0.68 and 0.55, respectively; all P&amp;lt;1.2x10-6), and two in miR-135b (rho=-0.56 and -0.59, respectively; both P&amp;lt;1.2x10-07). Interestingly, multiple loci within the promoter region of miR-190b were hypermethylated in ER- compared to ER+ tumors, and we further observed that miR-190b expression was significantly downregulated in ER- versus ER+ tumors (log2FC=-2.45 and -4.92 in EAs and AAs, respectively; FDR&amp;lt;0.05). Lower expression of miR-190 has been linked previously to reduced breast cancer survival, potentially reflecting effects on IGF-related pathways. In summary, based on genome-wide profiling of tumor DNA methylation and microRNA expression, our results suggest that DNA methylation patterns differ by tumor subtype and by race, and that this altered methylation may affect gene expression. These findings will provide the basis for further functional analyses, which are likely to improve our understanding of race-related tumor biological differences and lead to specific targeted preventative and therapeutic strategies. Citation Format: Zhihong Gong, Dan Wang, Jie Wang, Matthew F. Buas, Song Liu, Steven A. Belinsky, Michael J. Higgins, Christine B. Ambrosone. Tumor DNA methylation and microRNA expression in breast cancer among American women of African or European ancestry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 472.

Abstract 5328: Intergenic DNA methylation differences in ER- breast tumors from African American versus European American women

Abstract Incidence of estrogen receptor (ER)-negative breast cancer, a subtype with worse prognosis, is higher among African American (AA) versus European American (EA) women. The reasons for this gap remain poorly defined, but likely include differences in the prevalence and biological effects of known risk factors for ER- disease (eg. parity without breastfeeding and early menarche). These exposures may influence risk of ER- cancer via multiple mechanisms, one of which is epigenetic regulation of gene expression. Altered expression of genes controlling the proliferation and/or differentiation of luminal progenitor cells in the breast is likely to affect the propensity of such cells to give rise to ER- versus ER+ cancer. We previously conducted genome-wide DNA methylation profiling of 224 ER- tumor samples from 141 AA and 83 EA women in the Women's Circle of Health Study, and identified 374 differentially methylated loci (DML) by race. Our initial analysis focused on DMLs mapping to annotated genes, and revealed that a key pro-luminal transcription factor gene, FOXA1, was hypermethylated and repressed in AA compared to EA tumors. Moreover, parity without breastfeeding, more common among AAs, was associated with increased FOXA1 methylation. In this study, motivated by the biological importance of the non-coding genome, we focused on ~100 intergenic DML, and used paired 450K and RNA-seq data to assess the relationship between CpG methylation and RNA expression of genes located up to 50 kb away. Methylation at ten DML was significantly correlated with paired gene expression (FDR q&amp;lt;0.05). For seven of these loci, lower methylation tracked with higher expression of the paired gene. One such CpG, hypomethylated in AA versus EA tumors, mapped to a putative enhancer element located 19 kb upstream of, and predicted to interact with, the promoter of LEMD1, a pro-tumorigenic cancer-testis antigen. Analysis of an independent set of 207 ER- breast cancers from TCGA confirmed that LEMD1 RNA expression was higher in AA compared to EA tumors (P=0.01, log2FC=0.95). Our findings suggest that epigenetic differences between AA and EA ER- tumors are linked to altered gene expression and may hold functional significance in breast cancer pathogenesis. Further studies are needed to extend these results to normal breast tissues from cancer-free women, and explore a potential role for FOXA1/LEMD1 epigenetic regulation in luminal progenitor cell homeostasis. Citation Format: Matthew F. Buas, Jianhong Chen, Qiang Hu, Song Liu, Zhihong Gong, Michael J. Higgins, Christine B. Ambrosone. Intergenic DNA methylation differences in ER- breast tumors from African American versus European American women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5328.

Abstract P2-04-02: Comparison of DNA methylation patterns in normal breast tissue from women with and without breast cancer

Abstract BACKGROUND: Increasing evidence suggests that epigenetic mechanisms play critical roles in the development of breast cancer. However, precise DNA methylation signatures associated with breast cancer susceptibility remain unknown. We sought to compare DNA methylation changes in the normal breast tissue of women with and without breast cancer to identify patterns of aberrant DNA methylation in women with breast cancer. METHODS:Samples of normal breast tissue were collected from four cohorts of women: age &amp;lt; 50 years with and without breast cancer, and age ≥50 years with and without breast cancer. Normal breast tissue from healthy women was obtained from the Komen Tissue Bank at IU Simon Cancer Center and from women presenting for reduction mammoplasty at Yale New Haven Hospital. Normal breast tissue from women with breast cancer was obtained from patients undergoing adjuvant total mastectomy at Yale Breast Center. DNA was extracted using Qiagen AllPrep Universal kit. Raw data files in idat format were imported to Partek Genomics Suite 6.6 for normalization and differential methylation analysis. Raw intensities were normalized using With Array Normalization (SWAN) method. Principal component analysis (PCA) were performed as quality control. Differentially methylated loci (DML) between control and breast cancer groups were detected when False discovery rate (FDR) &amp;lt; 0.05 and fold change &amp;gt; 1.5. Functional enrichment analysis of genes with DML in the gene body were conducted using METACORE™. Pathways with FDR &amp;lt; 0.05 were selected. RESULTS: Ninety-three normal breast tissue samples from 89 subjects were analyzed (breast cancer=40, unaffected=53). Comparison of DNA methylation patterns between women with and without breast cancer revealed 200 DMLs. The majority of DMLs (186) were hyper-methylated in breast cancer patients, and 48 DMLs locate in enhancers of genes. 170 DMLs locate in 134 genes, enriched in two pathways: (1) Cell adhesion_Endothelial cell contacts by junctional mechanisms, and (2) Neurophysiological process_Constitutive and regulated NMDA receptor trafficking. Genes associated with cell adhesion and cell contacts included: ACTN2, GJA4, GJA7 and MAGI1. Two hyper-methylated loci were found in enhancers of ACTN2. In addition, one hyper-methylated locus in GJA4, one hyper-methylated and one hypo-methylated loci in GJA7, and two hyper-methylated loci in MAGI1 were detected in breast cancer patients. Genes associated with NMDA receptor trafficking include: TPK1, ADCY4 and LIN7C. One and two loci were found in TPK1 and ADCY4, respectively, that were hyper-methylated in normal breast tissue from cancer patients in the gene body, while a hypo-methylated locus in breast cancer patients was identified in LIN7C. CONCLUSIONS: Comparison of DNA methylation patterns of normal breast tissue from women with and without breast cancer reveal specific mechanistic pathways and genes that are differentially methylated in women with breast cancer. DNA methylation of normal breast tissue deserves further study as a potential biomarker for breast cancer risk stratification and may lend new insight into mechanisms of breast cancer development. Citation Format: Hofstatter EW, Zhu Y, Horvath S, Chagpar AB, Wali VB, Bossuyt V, Storniolo AM, Hatzis C, Patwardhan G, Von Wahlde M-K, Butler M, Epstein L, Stavris K, Sturrock T, Au A, Kwei S, Pusztai L. Comparison of DNA methylation patterns in normal breast tissue from women with and without breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-04-02.

Epigenome-wide association study of chronic obstructive pulmonary disease and lung function in Koreans.

Aim:To identify differentially methylated probes (DMPs) and regions (DMRs) in relation to chronic obstructive pulmonary disease (COPD) and lung function traits.Methods:We performed an epigenome-wide association study of COPD and spirometric parameters, including forced expiratory volume in 1 s (FEV1), forced vital capacity (FVC) and FEV1/FVC, in blood DNA using the Infinium HumanMethylation450 (n = 100, a Korean COPD cohort).Results:We found one significant DMP (cg03559389, DIP2C) and 104 significant DMRs after multiple-testing correction. Of these, 34 DMRs mapped to genes differential expressed with respect to the same trait. Five of the genes were associated with more than two traits: CTU2, USP36, ZNF516, KLK10 and CPT1B.Conclusion:We identified novel differential methylation loci related to COPD and lung function in blood DNA in Koreans and confirmed previous findings in non-Asians. Epigenetic modification could contribute to the etiology of these phenotypes.

Aberrant methylation patterns affect the molecular pathogenesis of rheumatoid arthritis

This study aims to investigate DNA methylation signatures in fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA), and to explore the relationship with transcription factors (TFs) that help to distinguish RA from osteoarthritis (OA). Microarray dataset of GSE46346, including six FLS samples from patients with RA and five FLS samples from patients with OA, was downloaded from the Gene Expression Omnibus database. RA and OA samples were screened for differentially methylated loci (DMLs). The corresponding differentially methylated genes (DMGs) were identified, followed by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) enrichment analysis. A transcriptional regulatory network was built with TFs and their corresponding DMGs. Overall, 280 hypomethylated loci and 561 hypermethylated loci were screened. Genes containing hypermethylated loci were enriched in pathways in cancer, ECM-receptor interaction, focal adhesion and neurotrophin signaling pathways. Genes containing hypomethylated loci were enriched in the neurotrophin signaling pathway. Moreover, we found that CCCTC-binding factor (CTCF), Yin Yang 1 (YY1), v-myc avian myelocytomatosis viral oncogene homolog (c-MYC), and early growth response 1 (EGR1) were important TFs in the transcriptional regulatory network. Therefore, DMGs might participate in the neurotrophin signaling pathway, pathways in cancer, ECM-receptor interaction and focal adhesion pathways in RA. Furthermore, CTCF, c-MYC, YY1, and EGR1 may play important roles in RA through regulating DMGs.

Genome-wide analysis of DNA methylation and their associations with long noncoding RNA/mRNA expression in non-small-cell lung cancer.

The goal of this study is to identify differentially methylated (DM) loci associated with long noncoding RNA (lncRNA)/mRNA expression in non-small-cell lung cancer (NSCLC). Microarrays were used to interrogate genome-wide methylation and expression of lncRNA/mRNA in NSCLC. We identified 113,644 DM loci between tumors and adjacent tissues. Among them, 26,310 DM loci were associated with 1685 differentially expressed genes, and 839 genes had significant correlations between methylation and expression, of which 26 hypermethylated loci in transcription start site 200 were correlated with low gene expression. We validated the correlations between methylation and expression in five genes (CDO1, C2orf40, SCARF1, ZFP106 and IFFO1) using pyrosequencing and quantitative polymerase chain reaction. We also found significant correlations between lncRNAs and mRNAs, and validated four of the correlations with quantitative polymerase chain reaction. Integrated analysis of genome-wide DNA methylation and lncRNA/mRNA expression allows us to identify new DM loci-correlated with gene expression in NSCLC.

DMAK: A curated pan-cancer DNA methylation annotation knowledgebase

ABSTRACTPan-cancer analysis can identify cell- and tissue-specific genomic loci and regions with underlying biological functions. Here we present an online curated DNA Methylation Annotation Knowledgebase, DMAK, which includes the pan-cancer analysis results for differentially-methylated loci and regions by the Reduced Representation Bisulfite Sequencing profiling technology. DMAK contains 3 modules of curated information and analysis results on 688,445 CpG sites across 19 cancer and embryonic stem cell lines from ENCODE, and further analysis of survival associations with clinical sources retrieved from TCGA. The knowledgebase covers all identified differentially-methylated CpG sites and regions of interest, further annotated genomic information, together with tumor suppressor genes information and calculated methylation level. DMAK provides meaningful clues for deriving functional association network and related clinical association results based on protein-coding genes, including tumor suppressor genes, identified from differentially methylated regions of interest. Thus DMAK constitutes a comprehensive reference source for the current epigenetic research and clinical study.

Abstract 2778: Interaction of Microsatellite Instability (MSI) and tumor for Differential DNA Methylation in Colorectal carcinoma

Abstract Introduction: Colorectal Cancer (CRC) is one of the most common malignancies worldwide. The role of MSI and KRAS somatic mutation in tumor tissue is well known in CRC. In genome-wide scale, we explored whether differential methylation is associated with MSI status. Methods: We carried out a genome-wide methylation assay (Illumina 450K) for a total of 250 paired samples from 125 CRC patients (m = 72, f = 53) at different stages (stage1:25, stage II: 33 and stageIII: 67). Of them 101 had left-sided (descending colon to rectum) CRC and 30 had MSI, and 34 had somatic mutation in KRAS (rs112445441). Results: MSI was more frequent in the right-sided tumor (54% vs. 17%, p = 0.003). Frequency of KRAS mutation was not different between right and left-sided CRC (29% vs. 27%). Among the patients with microsatellite stable (MSS) CRC (n = 95), paired comparison of methylation data between tumor and corresponding normal tissue revealed a total of 1641 tumor-specific differentially methylated loci (DML) covering 686 genes that were significant at FDR 0.001 and the magnitude of difference (delta beta) was at least 20%; Similar analysis in patients with MSI (n = 30) revealed 6209 tumor-specific DML covering 2316 genes. This suggested that MSI is associated with methylation change in much larger number of genes. We could not find any methylation signature from normal colon tissue that could predict MSI or KRAS mutation in the corresponding tumor tissue. However, we identified 413 genes to be differentially methylated in tumor tissue compared to corresponding normal tissue only in presence of MSI, irrespective of KRAS mutation status, tumor staging, and location of tumor. These are “MSI-associated tumor-specific genes”. Among these, 19 DML covering17 genes showed delta-beta &amp;gt;30%. The list was enriched in genes associated with biologically relevant GO-terms like, “negative regulation of cell proliferation”, “regulation of MAP kinase activity”, “regulation of biological process” etc. We also identified 240 genes differentially methylated in tumor tissue compared to corresponding normal tissue irrespective of MSI status, KRAS mutation, tumor staging, and location of tumor. These are “general tumor-specific genes”. Among these loci, 87 DML (49 genes) showed delta-beta &amp;gt;30%. Conclusions: Our study shows evidence of association between MSI and DNA methylation in the pathogenesis of CRC. Citation Format: Muhammad G. Kibriya, Mohammed Kamal, Mustafizur Rahman, Shantanu Roy, Maruf Raza, Rupash Paul, Habibul Ahsan, Zahidul Haq, Farzana Jasmine. Interaction of Microsatellite Instability (MSI) and tumor for Differential DNA Methylation in Colorectal carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2778.

Abstract 2777: One-carbon metabolism genetic variant and genome-wide DNA methylation in breast tissues from healthy women

Abstract Altered DNA methylation is an early event in carcinogenesis. Little is known about the mechanism of altered methylation in breast tissue; possible factors include diet such as alcohol and folate intake, and genetic variation for enzymes in one carbon metabolism. Examination of the association of these factors with methylation in breast tissues from healthy women provides insight into these changes. Blood and glandular breast tissues from 81 women with no history of cancer and who underwent reduction mammoplasty were assayed. The 96-plex Illumina BeadXpress® or TaqMan® SNP Genotyping assays assessed SNPs, genome-wide DNA methylation profiling was performed using the Illumina Infinium HumanMethylation450 BeadChip.The Affymetrix GeneChip Human Trascriptome Array 2.0 was used to compare gene expression level with methylation change in fresh frozen breast tissues. Biological networks of differentially-methylated (DM) genes were assigned using the Ingenuity Pathway Analysis (IPA). Fifty-seven CpG sites were DM in comparisons of genotype for eight SNPs in FTHFD, MTHFD1, MTHFR, MTR, MTRR, and TYMS (P&amp;lt;5.0 × 10-5 for each). SNPs in FTHFD were associated with 56% of the DM CpGs. SNPs in FTHFD and MTR were associated with DM CpG sites in their own genes. Six methylation and gene expression pairs were modestly to weakly correlated (P&amp;lt;0.05), five positively correlated (HCN4, FRMD4A, FTHFD, SLC39A7, and LOC63930) and one negatively correlated (ADAMTS14). Four DM CpGs identified by SNPs in MTRR, MTHFR, and FTHFD were significantly associated with alcohol consumption and/or breast folate. Forty-five DM genes were available in the IPA database. IPA revealed enrichment for genes (91%) involved in cancers. The top-scoring network was “Energy Production, Molecular Transportation, Nucleic Acid Metabolism” (score = 32). The top molecular and cellular functions were Amino Acid Metabolism (ALDH1L1, MTR, and PTPRN2), Cell-to-Cell Signaling (DLG3, GRN, HLA-DQB1, PTPRN2, and SLC6A3), Cellular Function and Maintenance (ADAMTS14, ADMTS2, CRIPT, DCL1, DLG3, GRN, KAG2, PTPRN2, and RXRB). High concordance of methylation levels for all DM loci analyzed was found between HM450 and pyrosequencing on 75 technically validated samples (Spearman correlation r = 0.98, P&amp;lt;1.0×10-47). This is the first comprehensive study of the association between variation in one-carbon metabolism genes and genome-wide DNA methylation in histologically normal breast tissues. These SNPs, particularly FTHFD, as well as alcohol intake and folate exposure appear to affect DNA methylation in the breast of healthy women. The finding that SNPs in FTHFD and MTR are associated with their own methylation is also novel and highlights a role for these SNPs as methylation quantitative trait loci. Understanding of the role of one carbon metabolism in altered DNA methylation could provide insight into prevention of breast tumors. Citation Format: Min-Ae Song, Theodore M. Brasky, Catalin Marian, Daniel Y. Weng, Cenny Taslim, Adana A. Llanos, Ramona G. Dumitrescu, Zhenhua Liu, Joel B. Mason, Bhaskar V. Kallakury, Jo L. Freudenheim, Peter G. Shields. One-carbon metabolism genetic variant and genome-wide DNA methylation in breast tissues from healthy women. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2777.

Comparison of genome-scale DNA methylation profiles in hepatocellular carcinoma by viral status

ABSTRACTHepatocellular carcinoma (HCC) incidence has steadily increased in the US over the past 30 years. Our understanding of epigenetic regulation in HCC is still limited, especially the impact of hepatitis B virus (HBV) or hepatitis C virus (HCV) infection on aberrant DNA methylation. We performed genome-wide DNA methylation profiling in 33 fresh frozen tumor samples, including 10 HBV-HCC, 13 HCV-HCC, and 10 non-infected (NIV-HCC) using the Illumina HumanMethylation450 BeadChip. Gene expression profiling was also performed using the Illumina whole-genome DASL HT Assay. Biological influences and gene networks of the differentially-methylated (DM) CpG loci were predicted using the Ingenuity Pathway Analysis. Genome-wide methylation analysis identified 7, 26, and 98 DM loci between HBV-HCC vs. HCV-HCC, HBV-HCC vs. NIV-HCC, and HCV-HCC vs. NIV-HCC, respectively, at P < 5 × 10−5 for each. Overall, the DM loci were highly enriched for enhancers (48%), promoters (37%), or CpG islands and surrounding regions (37%). Most DM loci were hypermethylated in HCV-HCC compared to HBV-HCC or NIV-HCC. The DM loci were associated with a variety of biological functions including Cell Morphology (HBV-HCC vs. NIV-HCC), Cell Death/ Survival (HBV-HCC vs. NIV-HCC), or Cellular Growth and Proliferation (HCV-HCC vs. NIV-HCC). A subset of the DM loci were correlated (either positively or negatively) with their gene expression or associated with alcohol consumption, BMI, cirrhosis, diabetes, and cigarette smoking. Our findings of differential methylation by viral infection lend insights into the potential effects of viral infection on the epigenetic regulation and further the development and progression of HCC.

Whole Genome DNA Methylation Analysis of Obstructive Sleep Apnea: IL1R2, NPR2, AR, SP140 Methylation and Clinical Phenotype.

We hypothesized that DNA methylation patterns may contribute to disease severity or the development of hypertension and excessive daytime sleepiness (EDS) in patients with obstructive sleep apnea (OSA). Illumina's (San Diego, CA, USA) DNA methylation 27-K assay was used to identify differentially methylated loci (DML). DNA methylation levels were validated by pyrosequencing. A discovery cohort of 15 patients with OSA and 6 healthy subjects, and a validation cohort of 72 patients with sleep disordered breathing (SDB). Microarray analysis identified 636 DMLs in patients with OSA versus healthy subjects, and 327 DMLs in patients with OSA and hypertension versus those without hypertension. In the validation cohort, no significant difference in DNA methylation levels of six selected genes was found between the primary snoring subjects and OSA patients (primary outcome). However, a secondary outcome analysis showed that interleukin-1 receptor 2 (IL1R2) promoter methylation (-114 cytosine followed by guanine dinucleotide sequence [CpG] site) was decreased and IL1R2 protein levels were increased in the patients with SDB with an oxygen desaturation index > 30. Androgen receptor (AR) promoter methylation (-531 CpG site) and AR protein levels were both increased in the patients with SDB with an oxygen desaturation index > 30. Natriuretic peptide receptor 2 (NPR2) promoter methylation (-608/-618 CpG sites) were decreased, whereas levels of both NPR2 and serum C type natriuretic peptide protein were increased in the SDB patients with EDS. Speckled protein 140 (SP140) promoter methylation (-194 CpG site) was increased, and SP140 protein levels were decreased in the patients with SDB and EDS. IL1R2 hypomethylation and AR hypermethylation may constitute an important determinant of disease severity, whereas NPR2 hypomethylation and SP140 hypermethylation may provide a biomarker for vulnerability to EDS in OSA. A commentary on this article appears in this issue on page 723.