HumanMethylation450 BeadChip Array Research Articles

Early prediction of pancreatic cancer through detection of methylated circulating DNA in blood.

e16353 Background: Pancreatic cancer represents a significant oncological challenge, contributing markedly to cancer-associated mortalities. This is attributed to the asymptomatic nature of early-stage disease, its inherently aggressive phenotype, and a notably suboptimal response to existing systemic therapies. Hence, the formulation of efficacious early diagnostic modalities for pancreatic cancer amenable to surgical resection is imperative to augment patient prognoses and enhance survival outcomes. Methods: The Infinium MethylationEPIC v2.0 BeadChip platform facilitated the examination of methylation profiles within plasma samples, while the HumanMethylation450 BeadChip array was employed for analogous analyses in corresponding tissue specimens from Taiwanese pancreatic cancer cohorts, aiding in gene selection. Subsequent methylation status verification was conducted via quantitative Methylation-Specific PCR (qMSP) on both genomic DNA and circulating cell-free DNA samples. For external methylation confirmation within a Western demographic, data from The Cancer Genome Atlas (TCGA) Portal were utilized, with further validation conducted using plasma samples from U.S. pancreatic cancer patients. Total 105 plasma from pancreatic cancer patients and healthy subjects were analyzed to calculate the candidate the accuracy, sensitivity, specificity of biomarkers for predict the pancreatic cancer. Results: The test exhibited an accuracy of 90.4%, sensitivity of 92.3%, and specificity of 90.1% in the detection of pancreatic cancer. Detection levels increased from 0% in healthy individuals to 87.5% in Stage I and 100% in Stages II, III, and IV. The CA19-9 marker displayed limited specificity with a high rate of false positives. Moreover, the test dynamically evaluated tumor burden by monitoring circulating methylated ZFP30 and FBXL7 in the plasma of breast cancer patients post-treatment. Conclusions: The analysis of plasma for methylated ZFP30 and FBXL7 provides an innovative and accurate noninvasive method that outperforms the existing blood biomarker CA19-9 for the prediction and ongoing assessment of tumor burden following treatment in pancreatic cancer.

Novel insights into the whole-blood DNA methylome of asthma in ethnically diverse children and youth.

The epigenetic mechanisms of asthma remain largely understudied in African Americans and Hispanics/Latinos, two populations disproportionately affected by asthma. We aimed to identify markers, regions and processes with differential patterns of DNA methylation (DNAm) in whole blood by asthma status in ethnically diverse children and youth, and to assess their functional consequences. DNAm levels were profiled with the Infinium MethylationEPIC or HumanMethylation450 BeadChip arrays among 1226 African Americans or Hispanics/Latinos and assessed for differential methylation per asthma status at the CpG and region (differentially methylated region (DMR)) level. Novel associations were validated in blood and/or nasal epithelium from ethnically diverse children and youth. The functional and biological implications of the markers identified were investigated by combining epigenomics with transcriptomics from study participants. 128 CpGs and 196 DMRs were differentially methylated after multiple testing corrections, including 92.3% and 92.8% novel associations, respectively. 41 CpGs were replicated in other Hispanics/Latinos, prioritising cg17647904 (NCOR2) and cg16412914 (AXIN1) as asthma DNAm markers. Significant DNAm markers were enriched in previous associations for asthma, fractional exhaled nitric oxide, bacterial infections, immune regulation or eosinophilia. Functional annotation highlighted epigenetically regulated gene networks involved in corticosteroid response, host defence and immune regulation. Several implicated genes are targets for approved or experimental drugs, including TNNC1 and NDUFA12. Many differentially methylated loci previously associated with asthma were validated in our study. We report novel whole-blood DNAm markers for asthma underlying key processes of the disease pathophysiology and confirm the transferability of previous asthma DNAm associations to ethnically diverse populations.

Wildfire-related PM2.5 and DNA methylation: An Australian twin and family study

BackgroundWildfire-related fine particulate matter (PM2.5) has many adverse health impacts, but its impacts on human epigenome are unknown. We aimed to evaluate the associations between long-term exposure to wildfire-related PM2.5 and blood DNA methylation, and whether the associations differ from those with non-wildfire-related PM2.5. MethodsWe studied 479 Australian women comprising 132 twin pairs and 215 of their sisters. Blood-derived DNA methylation was measured using the HumanMethylation450 BeadChip array. Data on 3-year (year of blood collection and previous two years) average wildfire-related and non-wildfire-related PM2.5 at 0.01°×0.01° spatial resolution were created by combining information from satellite observations, chemical transport models, and ground-based observations. Exposure data were linked to each participant’s home address, assuming the address did not change during the exposure window. For DNA methylation of each cytosine-guanine dinucleotide (CpG), and for global DNA methylation represented by the average of all measured CpGs or CpGs in repetitive elements, we evaluated their associations with wildfire- or non-wildfire-related PM2.5 using a within-sibship analysis controlling for factors shared between siblings and other important covariates. Differentially methylated regions (DMRs) were defined by comb-p and DMRcate. ResultsThe 3-year average wildfire-related PM2.5 (range: 0.3 to 7.6 µg/m3, mean: 1.6 µg/m3) was negatively, but not significantly (p-values greater than 0.05) associated with all seven global DNA methylation measures. There were 26 CpGs and 33 DMRs associated with wildfire-related PM2.5 (Bonferroni adjusted p-value < 0.05) mapped to 47 genes enriched for pathways related to inflammatory regulation and platelet activation. These genes have been related to many human diseases or phenotypes e.g., cancer, mental disorders, diabetes, obesity, asthma, blood pressure. These CpGs, DMRs and enriched pathways did not overlap with the 1 CpG and 7 DMRs associated with non-wildfire-related PM2.5. ConclusionsLong-term exposure to wildfire-related PM2.5 was associated with various blood DNA methylation signatures in Australian women, and these were distinct from those associated with non-wildfire-related PM2.5.

Genome wide DNA methylation analysis identifies novel molecular subgroups and predicts survival in neuroblastoma

BackgroundNeuroblastoma is the most common malignancy in infancy, accounting for 15% of childhood cancer deaths. Outcome for the high-risk disease remains poor. DNA-methylation patterns are significantly altered in all cancer types and can be utilised for disease stratification.MethodsGenome-wide DNA methylation (n = 223), gene expression (n = 130), genetic/clinical data (n = 213), whole-exome sequencing (n = 130) was derived from the TARGET study. Methylation data were derived from HumanMethylation450 BeadChip arrays. t-SNE was used for the segregation of molecular subgroups. A separate validation cohort of 105 cases was studied.ResultsFive distinct neuroblastoma molecular subgroups were identified, based on genome-wide DNA-methylation patterns, with unique features in each, including three subgroups associated with known prognostic features and two novel subgroups. As expected, Cluster-4 (infant diagnosis) had significantly better 5-year progression-free survival (PFS) than the four other clusters. However, in addition, the molecular subgrouping identified multiple patient subsets with highly increased risk, most notably infant patients that do not map to Cluster-4 (PFS 50% vs 80% for Cluster-4 infants, P = 0.005), and allowed identification of subgroup-specific methylation differences that may reflect important biological differences within neuroblastoma.ConclusionsMethylation-based clustering of neuroblastoma reveals novel molecular subgroups, with distinct molecular/clinical characteristics and identifies a subgroup of higher-risk infant patients.

Identification of NHLRC1 as a Novel AKT Activator from a Lung Cancer Epigenome-Wide Association Study (EWAS).

Changes in DNA methylation identified by epigenome-wide association studies (EWAS) have been recently linked to increased lung cancer risk. However, the cellular effects of these differentially methylated positions (DMPs) are often unclear. Therefore, we investigated top differentially methylated positions identified from an EWAS study. This included a putative regulatory region of NHLRC1. Hypomethylation of this gene was recently linked with decreased survival rates in lung cancer patients. HumanMethylation450 BeadChip array (450K) analysis was performed on 66 lung cancer case-control pairs from the European Prospective Investigation into Cancer and Nutrition Heidelberg lung cancer EWAS (EPIC HD) cohort. DMPs identified in these pre-diagnostic blood samples were then investigated for differential DNA methylation in lung tumor versus adjacent normal lung tissue from The Cancer Genome Atlas (TCGA) and replicated in two independent lung tumor versus adjacent normal tissue replication sets with MassARRAY. The EPIC HD top hypermethylated DMP cg06646708 was found to be a hypomethylated region in multiple data sets of lung tumor versus adjacent normal tissue. Hypomethylation within this region caused increased mRNA transcription of the closest gene NHLRC1 in lung tumors. In functional assays, we demonstrate attenuated proliferation, viability, migration, and invasion upon NHLRC1 knock-down in lung cancer cells. Furthermore, diminished AKT phosphorylation at serine 473 causing expression of pro-apoptotic AKT-repressed genes was detected in these knock-down experiments. In conclusion, this study demonstrates the powerful potential for discovery of novel functional mechanisms in oncogenesis based on EWAS DNA methylation data. NHLRC1 holds promise as a new prognostic biomarker for lung cancer survival and prognosis, as well as a target for novel treatment strategies in lung cancer patients.

Hypermethylation of PRKCZ Regulated by E6 Inhibits Invasion and EMT via Cdc42 in HPV-Related Head and Neck Squamous Cell Carcinoma

Simple SummaryHPV+ and HPV- HNSCC share distinct epigenetic characteristics and clinicopathological features. The aim of our study was to assess whether DNA methylation plays a role in the progression of HPV+ HNSCC. We used a HumanMethylation450 BeadChip array (Illumina) to identify PRKCZ genes exhibiting different levels of DNA methylation between HPV+ and HPV- HNSCC. PRKCZ acts as a potent tumor promoter in HPV+ HNSCC. These findings may provide a possible explanation for the differences in the clinicopathological characteristics between HPV+ and HPV- HNSCC and promising ideas for the treatment of HPV+ HNSCC.Purpose: To study the role of target genes with aberrant DNA methylation in HPV+ HNSCC. Methods: A HumanMethylation450 BeadChip array (Illumina) was used to identify differentially methylated genes. CCK-8, flow cytometry, wound healing, and cell invasion assays were conducted to analyze the biological roles of PRKCZ. Western blot, qRT-PCR, immunohistochemistry, and animal studies were performed to explore the mechanisms underlying the functions of PRKCZ. Results: We selected PRKCZ, which is associated with HPV infection, as our target gene. PRKCZ was hypermethylated in HPV+ HNSCC patients, and PRKCZ methylation status was negatively related to the pathological grading of HNSCC patients. Silencing PRKCZ inhibited the malignant capacity of HPV+ HNSCC cells. Mechanistically, HPV might promote DNMT1 expression via E6 to increase PRKCZ methylation. Cdc42 was required for the PRKCZ-mediated mechanism of action, contributing to the occurrence of epithelial-mesenchymal transition (EMT) in HPV+ HNSCC cells. In addition, blocking PRKCZ delayed tumor growth in HPV16-E6/E7 transgenic mice. Cdc42 expression was decreased, whereas E-cadherin levels increased. Conclusion: We suggest that PRKCZ hypermethylation induces EMT via Cdc42 to act as a potent tumor promoter in HPV+ HNSCC.

Residential surrounding greenness and DNA methylation: an epigenome-wide association study

BACKGROUND AND AIM: DNA methylation is a potential biological mechanism through which residential greenness affects health, but little is known about its association with greenness and whether the association could be modified by genetic background. We aimed to evaluate the association between surrounding greenness and genome-wide DNA methylation and potential gene-greenness interaction effects on DNA methylation. METHODS: We measured blood-derived DNA methylation using the HumanMethylation450 BeadChip array (Illumina) for 479 Australian women, including 66 monozygotic, 66 dizygotic twin pairs, and 215 sisters of these twins. Surrounding greenness was represented by Normalized Difference Vegetation Index (NDVI) and Enhanced Vegetation Index (EVI) within 300, 500, 1000 or 2000 metres surrounding participants’ home addresses. For each cytosine-guanine dinucleotide (CpG), the association between its methylation level and NDVI or EVI were evaluated by generalized estimating equations, adjusting for age, education, marital status, area-level socioeconomic status, smoking behavior, cell-type proportions and familial clustering. We used comb-p and DMRcate to identify significant differentially methylated regions (DMRs). For each significant CpG, we evaluated the interaction effects of greenness and single-nucleotide polymorphisms (SNPs) within ±1Mb window on its methylation level. RESULTS:We found associations between surrounding greenness and blood DNA methylation for one CpG (cg04720477, mapped to the promoter region of CNP gene) with false discovery rate [FDR]0.05, and for another 9 CpGs with 0.05≤FDR0.10. For two of these CpGs, we found 33 SNPs significantly (FDR0.05) modified the greenness-methylation association. There were 35 significant DMRs related to surrounding greenness that were identified by both comb-p (Sidak p-value0.01) and DMRcate (FDR0.01). Those CpGs and DMRs were mapped to genes related to many human diseases, such as mental health disorders and neoplasms as well as nutritional and metabolic diseases. CONCLUSIONS:Surrounding greenness was associated with blood DNA methylation of many loci across human genome, and this association could be modified by genetic variations. KEYWORDS: Green space, Epigenomics, Molecular epidemiology, Environmental epidemiology

Ambient temperature and genome-wide DNA methylation: A twin and family study in Australia

Little is known about the association between ambient temperature and DNA methylation, which is a potential biological process through which ambient temperature affects health. This study aimed to evaluate the association between ambient temperature and DNA methylation across human genome. We included 479 Australian women, including 132 twin pairs and 215 sisters of these twins. Blood-derived DNA methylation was measured using the HumanMethylation450 BeadChip array. Data on average ambient temperature during eight different exposure windows [lag0d (the blood draw day), lag0-7d (the current day and previous seven days prior to blood draw), lag0-14d, lag0-21d, lag0-28d, lag0-90d, lag0-180d, and lag0-365d)] was linked to each participant's home address. For each cytosine-guanine dinucleotide (CpG), we evaluated the association between its methylation level and temperature using generalized estimating equations (GEE), adjusting for important covariates. We used comb-p and DMRcate to identify differentially methylated regions (DMRs). We identified 31 CpGs at which blood DNA methylation were significantly associated with ambient temperature with false discovery rate [FDR] < 0.05. There were 82 significant DMRs identified by both comb-p (Sidak p-value < 0.01) and DMRcate (FDR < 0.01). Most of these CpGs and DMRs only showed association with temperature during one specific exposure window. These CpGs and DMRs were mapped to 85 genes. These related genes have been related to many human chronic diseases or phenotypes (e.g., diabetes, arthritis, breast cancer, depression, asthma, body height) in previous studies. The signals of short-term windows (lag0d and lag0-21d) showed enrichment in biological processes related to cell adhesion. In conclusion, short-, medium-, and long-term exposures to ambient temperature were all associated with blood DNA methylation, but the target genomic loci varied by exposure window. These differential methylation signals may serve as potential biomarkers to understand the health impacts of temperature.

Residential surrounding greenness and DNA methylation: An epigenome-wide association study

BackgroundDNA methylation is a potential biological mechanism through which residential greenness affects health, but little is known about its association with greenness and whether the association could be modified by genetic background. We aimed to evaluate the association between surrounding greenness and genome-wide DNA methylation and potential gene-greenness interaction effects on DNA methylation. MethodsWe measured blood-derived DNA methylation using the HumanMethylation450 BeadChip array (Illumina) for 479 Australian women, including 66 monozygotic, 66 dizygotic twin pairs, and 215 sisters of these twins. Surrounding greenness was represented by Normalized Difference Vegetation Index (NDVI) and Enhanced Vegetation Index (EVI) within 300, 500, 1000 or 2000 m surrounding participants’ home addresses. For each cytosine-guanine dinucleotide (CpG), the associations between its methylation level and NDVI or EVI were evaluated by generalized estimating equations, after adjusting for age, education, marital status, area-level socioeconomic status, smoking behavior, cell-type proportions, and familial clustering. We used comb-p and DMRcate to identify significant differentially methylated regions (DMRs). For each significant CpG, we evaluated the interaction effects of greenness and single-nucleotide polymorphisms (SNPs) within ±1 Mb window on its methylation level. ResultsWe found associations between surrounding greenness and blood DNA methylation for one CpG (cg04720477, mapped to the promoter region of CNP gene) with false discovery rate [FDR] < 0.05, and for another 9 CpGs with 0.05 ≤ FDR < 0.10. For two of these CpGs, we found 33 SNPs significantly (FDR < 0.05) modified the greenness-methylation association. There were 35 significant DMRs related to surrounding greenness that were identified by both comb-p (Sidak p-value < 0.01) and DMRcate (FDR < 0.01). Those CpGs and DMRs were mapped to genes related to many human diseases, such as mental health disorders and neoplasms as well as nutritional and metabolic diseases. ConclusionsSurrounding greenness was associated with blood DNA methylation of many loci across human genome, and this association could be modified by genetic variations.

Increased DNA methylation variability in rheumatoid arthritis-discordant monozygotic twins

BackgroundRheumatoid arthritis is a common autoimmune disorder influenced by both genetic and environmental factors. Epigenome-wide association studies can identify environmentally mediated epigenetic changes such as altered DNA methylation, which may also be influenced by genetic factors. To investigate possible contributions of DNA methylation to the aetiology of rheumatoid arthritis with minimum confounding genetic heterogeneity, we investigated genome-wide DNA methylation in disease-discordant monozygotic twin pairs.MethodsGenome-wide DNA methylation was assessed in 79 monozygotic twin pairs discordant for rheumatoid arthritis using the HumanMethylation450 BeadChip array (Illumina). Discordant twins were tested for both differential DNA methylation and methylation variability between rheumatoid arthritis and healthy twins. The methylation variability signature was then compared with methylation variants from studies of other autoimmune diseases and with an independent healthy population.ResultsWe have identified a differentially variable DNA methylation signature that suggests multiple stress response pathways may be involved in the aetiology of the disease. This methylation variability signature also highlighted potential epigenetic disruption of multiple RUNX3 transcription factor binding sites as being associated with disease development. Comparison with previously performed epigenome-wide association studies of rheumatoid arthritis and type 1 diabetes identified shared pathways for autoimmune disorders, suggesting that epigenetics plays a role in autoimmunity and offering the possibility of identifying new targets for intervention.ConclusionsThrough genome-wide analysis of DNA methylation in disease-discordant monozygotic twins, we have identified a differentially variable DNA methylation signature, in the absence of differential methylation in rheumatoid arthritis. This finding supports the importance of epigenetic variability as an emerging component in autoimmune disorders.

Aberrant DNA methylation of ADAMTS16 in colorectal and other epithelial cancers

BackgroundADAMs (a disintegrin and metalloproteinase) have long been associated with tumor progression. Recent findings indicate that members of the closely related ADAMTS (ADAMs with thrombospondin motifs) family are also critically involved in carcinogenesis. Gene silencing through DNA methylation at CpG loci around e.g. transcription start or enhancer sites is a major mechanism in cancer development. Here, we aimed at identifying genes of the ADAM and ADAMTS family showing altered DNA methylation in the development or colorectal cancer (CRC) and other epithelial tumors.MethodsWe investigated potential changes of DNA methylation affecting ADAM and ADAMTS genes in 117 CRC, 40 lung cancer (LC) and 15 oral squamous-cell carcinoma (SCC) samples. Tumor tissue was analyzed in comparison to adjacent non-malignant tissue of the same patients. The methylation status of 1145 CpGs in 51 ADAM and ADAMTS genes was measured with the HumanMethylation450 BeadChip Array. ADAMTS16 protein expression was analyzed in CRC samples by immunohistochemistry.ResultsIn CRC, we identified 72 CpGs in 18 genes which were significantly affected by hyper- or hypomethylation in the tumor tissue compared to the adjacent non-malignant tissue. While notable/frequent alterations in methylation patterns within ADAM genes were not observed, conspicuous changes were found in ADAMTS16 and ADAMTS2. To figure out whether these differences would be CRC specific, additional LC and SCC tissue samples were analyzed. Overall, 78 differentially methylated CpGs were found in LC and 29 in SCC. Strikingly, 8 CpGs located in the ADAMTS16 gene were commonly differentially methylated in all three cancer entities. Six CpGs in the promoter region were hypermethylated, whereas 2 CpGs in the gene body were hypomethylated indicative of gene silencing. In line with these findings, ADAMTS16 protein was strongly expressed in globlet cells and colonocytes in control tissue but not in CRC samples. Functional in vitro studies using the colorectal carcinoma cell line HT29 revealed that ADAMTS16 expression restrained tumor cell proliferation.ConclusionsWe identified ADAMTS16 as novel gene with cancer-specific promoter hypermethylation in CRC, LC and SCC patients implicating ADAMTS16 as potential biomarker for these tumors. Moreover, our results provide evidence that ADAMTS16 may have tumor suppressor properties.

Lowly methylated region analysis identifies EBF1 as a potential epigenetic modifier in breast cancer

ABSTRACTBreast cancer (BC) encompasses heterogeneous pathologies with different subtypes exhibiting distinct molecular changes, including those related to DNA methylation. However, the role of these changes in mediating BC heterogeneity is poorly understood. Lowly methylated regions (LMRs), non-CpG island loci that usually contain transcription factor (TF) binding sites, have been suggested to act as regulatory elements that define cellular identity. In this study, we aimed to identify the key subtype-specific TFs that may lead to LMR generation and shape the BC methylome and transcription program. We initially used whole-genome bisulfite sequencing (WGBS) data available at The Cancer Genome Atlas (TCGA) portal to identify subtype-specific LMRs. Differentially methylated regions (DMRs) within the BC PAM50 subtype-specific LMRs were selected by comparing tumors and normal tissues in a larger TCGA cohort assessed by HumanMethylation450 BeadChip (450K) arrays and TF enrichment analyses were performed. To assess the impact of LMRs on gene expression, TCGA RNA sequencing data were downloaded and Pearson correlations between methylation levels of loci presenting subtype-specific TF motifs and expression of the nearest genes were calculated. WGBS methylome data revealed a large number of LMRs for each of the BC subtypes. Analysis of these LMRs in the 450K datasets available for a larger sample set identified 7,765, 5,657, and 19 differentially methylated positions (DMPs) between normal adjacent tissues and tumor tissues from basal, luminal, and HER2-enriched subtypes, respectively. Unsupervised clustering showed that the discriminatory power of the top DMPs was remarkably strong for basal BC. Interestingly, in this particular subtype, we found 4,409 differentially hypomethylated positions grouped into 1,185 DMRs with a strong enrichment for the early B-cell factor 1 (EBF1) motifs. The methylation levels of the DMRs containing EBF1 motifs showed a strong negative correlation with the expression of 719 nearby genes, including BTS2 and CD74, two oncogenes known to be specific for basal BC subtype and for poor outcome. This study identifies LMRs specific to the three main BC subtypes and reveals EBF1 as a potentially important regulator of BC subtype-specific methylation and gene expression program.

Hypermethylation of BEND5 contributes to cell proliferation and is a prognostic marker of colorectal cancer.

Aberrant hypermethylation of CpG islands in tumor suppressor genes (TSGs) contributes to colorectal tumorigenesis. To identify new colorectal cancer (CRC) screening marker, we investigated DNA methylation alterations in novel TSGs. Using HumanMethylation450 BeadChip arrays, CpG regions in BEND5 were the most highly methylated among all genomic regions in 26 colorectal tumors compared to paired non-neoplastic tissues from a Taiwan cohort. Therefore, BEND5 was selected for further analysis. Quantitative methylation-specific real-time PCR revealed that 86.7% (117/135) of CRC patients exhibited hypermethylated BEND5. Real-time reverse transcription PCR identified that BEND5 mRNA expression was downregulated in 68% (32/47) of the analyzed samples. BEND5 hypermethylation was associated with poor overall survival (OS) in Taiwan patients with early-stage CRC (P = 0.037). In a CRC tissue set from South Korea, OS was higher in patients with high BEND5 protein expression than in those with low BEND5 protein expression (P = 0.037) by using immunohistochemistry assays. Consistently, BEND5 hypermethylation was associated with poor OS in patients with early-stage CRC in The Cancer Genome Atlas (TCGA) data set (P = 0.003). Multivariate Cox proportional hazards regression analysis further supported that hypermethylation of BEND5 genes was significantly associated with OS in Taiwan and TCGA CRC patients (P = 0.023 and 0.033, respectively). Finally, the cell model assay with transient transfection of BEND5 or si-BEND5 knockdown indicated that BEND5 inhibited cancer cell proliferation. In conclusion, epigenetic alteration in the candidate TSG BEND5 contributes to colorectal cancer development and is a prognostic marker of CRC.

Nightshift work, chronotype, and genome-wide DNA methylation in blood

ABSTRACTMolecular mechanisms underlying the negative health effects of shift work are poorly understood, which remains a barrier to developing intervention strategies to protect the long-term health of shift workers. We evaluated genome-wide differences in DNA methylation (measured in blood) between 111 actively employed female nightshift and 86 actively employed female dayshift workers from the Seattle metropolitan area. We also explored the effect of chronotype (i.e., measure of preference for activity earlier or later in the day) on DNA methylation among 110 of the female nightshift workers and an additional group of 131 male nightshift workers. Methylation data were generated using the Illumina Infinium HumanMethylation450 BeadChip (450K) Array. After applying the latest methylation data processing methods, we compared methylation levels at 361,210 CpG loci between the groups using linear regression models adjusted for potential confounders and applied the false-discovery rate (FDR) ≤ 0.05 to account for multiple comparisons. No statistically significant associations at the genome-wide level were observed with shift work or chronotype, though based on raw P values and absolute effect sizes, there were suggestive associations in genes that have been previously linked with cancer (e.g., BACH2, JRK, RPS6KA2) and type-2 diabetes (e.g., KCNQ1). Given that our study was underpowered to detect moderate effects, examining these suggestive results in well-powered independent studies or in pooled data sets may improve our understanding of the pathways underlying the negative health effects of shift work and the influence of personal factors such as chronotype. Such an approach may help identify potential interventions that can be used to protect the long-term health of shift workers.

The expression of hematopoietic progenitor cell antigen CD34 is regulated by DNA methylation in a site-dependent manner in gastrointestinal stromal tumours.

The anatomic site-dependent expression of hematopoietic progenitor cell antigen CD34 is a feature of gastrointestinal stromal tumours (GISTs). The basis for the differential CD34 expression is only incompletely understood. This study aimed at understanding the regulation of CD34 in GISTs and clarification of its site-dependent expression. Two sample sets of primary GISTs were interrogated including 52 fresh-frozen and 134 paraffin-embedded and formalin-fixed specimens. DNA methylation analysis was performed by HumanMethylation450 BeadChip array in three cell lines derived from gastric and intestinal GISTs, and differentially methylated CpG sites were established upstream of CD34. The methylation degree was further quantified by pyrosequencing, and inverse correlation with CD34 mRNA and protein abundance was revealed. The gene's expression could be activated upon induction of DNA hypomethylation with 5-aza-2'-deoxycytidine in GIST-T1 cells. In patient samples, a strong inverse correlation of DNA methylation degree with immunohistochemically evaluated CD34 expression was documented. Both CD34 expression and DNA methylation levels were specific to the tumours' anatomic location and mutation status. A constant decrease in methylation levels was observed ranging from almost 100% hypermethylation in intestinal GISTs from duodenum to hypomethylation in rectum. CD34 was heavily methylated in gastric PDGFRA-mutant GISTs in comparison to hypomethylated KIT-mutant counterparts. Next to CD34 hypermethylation, miR-665 was predicted and experimentally confirmed to target CD34 mRNA in GIST-T1 cells. Our results suggest that CD34 expression in GISTs may undergo a complex control by DNA methylation and miR-665. Differential methylation and expression of CD34 in GISTs along the gastrointestinal tract axis and in tumours that harbour different gain-of-function mutations suggest the origin from different cell populations in the gastrointestinal tract.

Array probe density and pathobiological relevant CpG calling bias in human disease and physiological DNA methylation profiling.

The HumanMethylation450 BeadChip array (450K; Infinium) is a widely used tool in epigenomics. A recognized concern in the 450K platform is the potential effect of the number of probes/gene (PG) on ranking differentially methylated (DM) CpGs (DM-CpGs) before testing for enrichment of gene ontology categories. We previously showed in a fatty acid (FA)-induced DNA methylation profiling study that when DM-CpGs are ranked by the number of called DM-CpGs-to-PG ratio, the 150 top-ranking gene list is enriched in pathways that overlap with the corresponding Affymetrix array-based expression data. In this study, a comparative analysis of thirteen 450K-based studies representing FA-stimulated cellular models, aging, diseased and normal tissues, revealed that the 150 top-ranking DM-CpGs are in high PG genes. This points to a significant false-negative rate in the low PG gene set when delta-beta-based ranking is performed. We show that PG is not related to the density of methylation-prone sites, as it does not follow gene length or GC content. Conversely, ranking genes by the number of DM-CpGs-to-PG ratio and analysing the 150 top-ranking entries yields significantly enriched gene disease- or tissue-specific function categories that are increased both in number and in the degree of overlap with expression data compared with delta-beta-only ranking or to the previously published gometh-based pipeline. The 15 top-ranking loci list is also significantly enriched in non-coding RNAs, a greatly underrepresented transcript type in 450K. In summary, the proposed simple normalization method yields pathobiologically relevant DM-CpGs. This method is relevant for the newly developed MethylationEPIC (Infinium) microarray.

Abstract 3483: Genome-wide analysis of the antisense transcriptome in cancer

Abstract Natural antisense transcripts (NATs) are the most abundant class of long noncoding RNAs (lncRNAs). Importantly, NATs are one of the most unknown ncRNAs. However, due to their highly gene locus-specific effects, NATs may provide a unique entry point for therapeutic intervention in targeted gene regulation. For this reason we are interested in studying the regulation of sense/antisense transcription in cancer related locus. In our previous work we found that, at the important cancer-related locus vimentin (VIM) there is a previously uncharacterized antisense transcript (VIM-AS1), which is transcribed head-to-head with VIM gene. This antisense transcript enhances R loop formation; it allows the binding of transcription factors as NF-kB, low nucleosome occupancy and the transcription of VIM gene. Our results are the first example of R loop mediated enhancement of gene expression involving head-to-head antisense transcription at cancer related locus. We also observed that in colon and breast cancer there was a positive expression correlation between VIM and VIM-AS1 and we have observed that hypermethylation silences both transcripts in colon primary tumors. In the present work we have seen that this positive correlation between sense and antisense transcripts is present in most of the tumor types from patients that we have obtained from Genomic Data Commons Data Portal (GDC Portal). Importantly this positive correlation correlates better in the cases in which the antisense transcript is downregulated or maintains the expression than in tumors where VIM and VIM-AS1 are up-regulated. Furthermore, we wanted to see if the cellular function is also coordinated. VIM is a component of the intermediate filament family of proteins that has an important role in epithelial mesenchymal transition. We found that specific depletion of VIM-AS1 results in a decrease of VIM expression, and now we have found that this depletion produces low migration rate and more invasion capability, as expected. On the other hand, the mechanisms used by antisense transcripts to regulate their corresponding sense mRNAs are not fully understood and in view of these results we are investigating from a genome-wide perspective the regulation of sense/antisense transcription, the involvement of R loop structures and DNA methylation in cancer. For this, we have applied a number of techniques: RNAseq strand specific, Illumina’s HumanMethylation450 Bead Chip array, RNAseq strand specific with overexpression of RNaseH (which specifically degrades the RNA in RNA/DNA hybrids), and DRIPseq (which immunoprecipitates the hybrids DNA-RNAs). The analysis of these data sheds light on the study of the role of sense/antisense transcripts in cancer-related locus. Citation Format: Raquel Boque-Sastre, Manuel Castro de Moura, Antonio Gomez, Sònia Guil, Manel Esteller. Genome-wide analysis of the antisense transcriptome in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3483. doi:10.1158/1538-7445.AM2017-3483

Unique Clinical Epidemiologic Risk Factors Are Associated with Distinct Methylation Subgroups in Newly-Diagnosed Acute Myeloid Leukemia (AML)

Abstract Background: Large case-control studies have identified common epidemiologic exposures associated with risk of AML development, including obesity [BMI ≥30 kg/m2] associated with 2-fold increase risk. However it is not known whether individual risk factors are associated with unique AML genetic lesions. We have recently identified a significant association of some common exposures including obesity with unique disease cytogenetic subgroups in a cohort of n=295 consecutive AML patients diagnosed and treated at Mayo Clinic [Cancer Epidemiol 39:1084, 2015]. Aberrant DNA methylation is a hallmark of cancer and has been reported in AML, and we therefore performed an analysis of global DNA methylation in patients in this cohort to determine whether specific epidemiologic exposures are associated with a unique pattern of DNA methylation. Methods: Patients in the initial cohort were previously reported. An available baseline AML bone marrow cytogenetic pellet from diagnosis was identified for n=148 patients (50.2%). Following IRB approval and using previously reported techniques [Leukemia 24:1283, 2010], leukemia DNA was isolated from the remnant diagnostic cytogenetic samples and processed on the HumanMethylation450 BeadChip array (Illumina). Individual CpG dinucleotides were assigned a Beta value (i.e. methylation value) ranging from 0 (unmethylated) to 1 (methylated). We excluded those with minor methylation changes (&lt;0.2 between any two samples), resulting in 383,524 CpG's. For the purposes of this first analysis we have focused only on CpG's with an inter-quartile range (IQR, i.e. difference between 75th and 25th percentiles) of greater than 0.25, comprising a total of 39,657 CpG's. An unsupervised cluster analysis of differential methylation was generated ("Heatmap", Figure). Univariate and multivariable analyses were performed adjusting for age as a continuous variable to determine the association of individual exposures with unique methylation clusters (primary endpoint), and we also evaluated the association of clinical characteristics and clinical outcomes after therapy (secondary endpoint). The median age of patients was 69 yrs (range 19-89), male 65%, and Caucasian 89%. The median BMI was 27.2 (range 16.9-59.9), and n=47 patients (32%) had BMI ≥30. Standard cytogenetic risk groups were applied [favorable (n=8), normal (n=61), intermediate abnormal (n=32) and poor (n=47)]. Results: We identified 4 distinct methylation clusters (Figure: Cluster #1, Black horizontal bar; Cluster #2, Green; Cluster #3, Blue; and Cluster #4, Red). Multivariate analysis of select variables is shown in Table. Patients in cluster #1 were significantly more likely to have a BMI ≥30 [Odds Ratio (OR) 2.49, p=0.017] and more likely to have favorable risk cytogenetics although this was not statistically significant [OR 6.71, p=0.088], while those in cluster #2 were significantly more likely to be have a history of regular NSAID use [OR 3.26, p=0.03] and less likely to have normal [OR 0.40, p=0.044] and possibly intermediate abnormal cytogenetics [OR 0.34, p=0.051]. Those in cluster #3 were significantly younger [median age 48 yrs, p&lt;0.001] and were also more likely to have prior history MDS although this was not statistically significant [OR 3.7, p=0.082]. Cluster #4 was not correlated with any distinct exposure apart from possible decreased alcohol use [OR 0.3, p=0.079]. History of past or current tobacco use, regular acetaminophen use, documented myelotoxin exposure, family history, and secondary AML did not correlate with a unique methylation cluster. There was no independent association of methylation cluster with overall survival (p=0.46), although among patients receiving intensive therapy cluster #2 was associated with lower complete remission rate [OR 0.28 (95% CI 0.09-0.86), p=0.026]. Conclusion: This first analysis of AML molecular epidemiology and clinical outcome suggests that specific disease risk factors (esp. obesity and age) are associated with unique leukemia DNA methylation clusters. In addition, aberrant methylation is associated with some clinical features and with outcome after therapy, suggesting that epidemiologic risk factors may have a prognostic impact. This provides novel insight into leukemogenesis, and will guide prospective studies of AML epidemiology and outcome. Figure. Figure. Disclosures Foran: medscape: Honoraria; pfizer: Honoraria; Millennium Pharmaceuticals, Inc.: Research Funding; novartis: Honoraria; karyopharm: Honoraria; boehringer ingelheim: Research Funding; agios: Research Funding; Cellerant: Research Funding.

Differential DNA methylation marks and gene comethylation of COPD in African-Americans with COPD exacerbations.

BackgroundChronic obstructive pulmonary disease (COPD) is the third-leading cause of death worldwide. Identifying COPD-associated DNA methylation marks in African-Americans may contribute to our understanding of racial disparities in COPD susceptibility. We determined differentially methylated genes and co-methylation network modules associated with COPD in African-Americans recruited during exacerbations of COPD and smoking controls from the Pennsylvania Study of Chronic Obstructive Pulmonary Exacerbations (PA-SCOPE) cohort.MethodsWe assessed DNA methylation from whole blood samples in 362 African-American smokers in the PA-SCOPE cohort using the Illumina Infinium HumanMethylation27 BeadChip Array. Final analysis included 19302 CpG probes annotated to the nearest gene transcript after quality control. We tested methylation associations with COPD case-control status using mixed linear models. Weighted gene comethylation networks were constructed using weighted gene coexpression network analysis (WGCNA) and network modules were analyzed for association with COPD.ResultsThere were five differentially methylated CpG probes significantly associated with COPD among African-Americans at an FDR less than 5 %, and seven additional probes that approached significance at an FDR less than 10 %. The top ranked gene association was MAML1, which has been shown to affect NOTCH-dependent angiogenesis in murine lung. Network modeling yielded the “yellow” and “blue” comethylation modules which were significantly associated with COPD (p-value 4 × 10-10 and 4 × 10-9, respectively). The yellow module was enriched for gene sets related to inflammatory pathways known to be relevant to COPD. The blue module contained the top ranked genes in the concurrent differential methylation analysis (FXYD1/LGI4, gene significance p-value 1.2 × 10-26; MAML1, p-value 2.0 × 10-26; CD72, p-value 2.1 × 10-25; and LPO, p-value 7.2 × 10-25), and was significantly associated with lung development processes in Gene Ontology gene-set enrichment analysis.ConclusionWe identified 12 differentially methylated CpG sites associated with COPD that mapped to biologically plausible genes. Network module comethylation patterns have identified candidate genes that may be contributing to racial differences in COPD susceptibility and severity. COPD-associated comethylation modules contained genes previously associated with lung disease and inflammation and recapitulated known COPD-associated genes. The genes implicated by differential methylation and WGCNA analysis may provide mechanistic targets contributing to COPD susceptibility, exacerbations, and outcomes among African-Americans.Trial registrationTrial Registration: NCT00774176, Registry: ClinicalTrials.gov, URL: www.clinicaltrials.gov, Date of Enrollment of First Participant: June 2004, Date Registered: 04 January 2008 (retrospectively registered).Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-016-0459-8) contains supplementary material, which is available to authorized users.

An epigenome-wide study of body mass index and DNA methylation in blood using participants from the Sister Study cohort.

Background/ObjectivesThe relationship between obesity and chronic disease risk is well-established; the underlying biological mechanisms driving this risk increase may include obesity-related epigenetic modifications. To explore this hypothesis, we conducted a genome-wide analysis of DNA methylation and body mass index (BMI) using data from a subset of women in the Sister Study.Subjects/MethodsThe Sister Study is a cohort of 50,884 U.S. women who had a sister with breast cancer but were free of breast cancer themselves at enrollment. Study participants completed examinations which included measurements of height and weight, and provided blood samples. Blood DNA methylation data generated with the Illumina Infinium HumanMethylation27 BeadChip array covering 27,589 CpG sites was available for 871 women from a prior study of breast cancer and DNA methylation. To identify differentially methylated CpG sites associated with body mass index, we analyzed this methylation data using robust linear regression with adjustment for age and case status. For those CpGs passing the false discovery rate significance level, we examined the association in a replication set comprised of a non-overlapping group of 187 women from the Sister Study who had DNA methylation data generated using the Infinium HumanMethylation450 BeadChip array. Analysis of this expanded 450K array identified additional BMI-associated sites which were investigated with targeted pyrosequencing.ResultsFour CpG sites reached genome-wide significance (FDR q<0.05) in the discovery set and associations for all four were significant at strict Bonferroni correction in the replication set. An additional 23 sites passed FDR in the replication set and 5 were replicated by pyrosequencing in the discovery set. Several of the genes identified including ANGPT4, RORC, SOCS3, FSD2, XYLT1, ABCG1, STK39, ASB2, and CRHR2 have been linked to obesity and obesity-related chronic diseases.ConclusionsOur findings support the hypothesis that obesity-related epigenetic differences are detectable in blood and may be related to risk of chronic disease.