Biomarker correlates with response to NY-ESO-1 TCR T cells in patients with synovial sarcoma.

Autologous T cells transduced to express a high affinity T-cell receptor specific to NY-ESO-1 (letetresgene autoleucel, lete-cel) show promise in the treatment of metastatic synovial sarcoma, with 50% overall response rate. The efficacy of lete-cel treatment in 45 synovial sarcoma patients (NCT01343043) has been previously reported, however, biomarkers predictive of response and resistance remain to be better defined. This post-hoc analysis identifies associations of response to lete-cel with lymphodepleting chemotherapy regimen (LDR), product attributes, cell expansion, cytokines, and tumor gene expression. Responders have higher IL-15 levels pre-infusion (p = 0.011) and receive a higher number of transduced effector memory (CD45RA- CCR7-) CD8 + cells per kg (p = 0.039). Post-infusion, responders have increased IFNγ, IL-6, and peak cell expansion (p < 0.01, p < 0.01, and p = 0.016, respectively). Analysis of tumor samples post-treatment illustrates lete-cel infiltration and a decrease in expression of macrophage genes, suggesting remodeling of the tumor microenvironment. Here we report potential predictive and pharmacodynamic markers of lete-cel response that may inform LDR, cell dose, and strategies to enhance anticancer efficacy.

DNA methylation in the adipose tissue and whole blood of Agent Orange-exposed Operation Ranch Hand veterans: a pilot study.

BACKGROUND: Between 1962 and 1971, the US Air Force sprayed Agent Orange across Vietnam, exposing many soldiers to this dioxin-containing herbicide. Several negative health outcomes have been linked to Agent Orange exposure, but data is lacking on the effects this chemical has on the genome. Therefore, we sought to characterize the impact of Agent Orange exposure on DNA methylation in the whole blood and adipose tissue of veterans enrolled in the Air Force Health Study (AFHS). METHODS: We received adipose tissue (n = 37) and whole blood (n = 42) from veterans in the AFHS. Study participants were grouped as having low, moderate, or high TCDD body burden based on their previously measured serum levels of dioxin. DNA methylation was assessed using the Illumina 450 K platform. RESULTS: Epigenome-wide analysis indicated that there were no FDR-significantly methylated CpGs in either tissue with TCDD burden. However, 3 CpGs in the adipose tissue (contained within SLC9A3, LYNX1, and TNRC18) were marginally significantly (q < 0.1) hypomethylated, and 1 CpG in whole blood (contained within PTPRN2) was marginally significantly (q < 0.1) hypermethylated with high TCDD burden. Analysis for differentially methylated DNA regions yielded SLC9A3, among other regions in adipose tissue, to be significantly differentially methylated with higher TCDD burden. Comparing whole blood data to a study of dioxin exposed adults from Alabama identified a CpG within the gene SMO that was hypomethylated with dioxin exposure in both studies. CONCLUSION: We found limited evidence of dioxin associated DNA methylation in adipose tissue and whole blood in this pilot study of Vietnam War veterans. Nevertheless, loci in the genes of SLC9A3 in adipose tissue, and PTPRN2 and SMO in whole blood, should be included in future exposure analyses.

Fetal Renal DNA Methylation and Developmental Programming of Stress-Induced Hypertension in Growth-Restricted Male Mice.

Fetal growth restriction (FGR) is associated with developmental programming of adult onset hypertension, which may be related to differences in nephron development. Prior studies showed that maternal nutrient restriction is associated with reduced nephrogenesis in rodents, especially in male progeny. We hypothesized that maternal genetic risk for FGR may similarly affect fetal kidney development, leading to adult onset hypertension. We employed an angiotensinogen (AGT) gene titration transgenic (TG) construct with 3 copies of the mouse AGT gene that mimics a common human genotype (AGT A[-6]G) associated with FGR. We investigated whether FGR in 2-copy (wild type, [WT]) progeny from 3-copy TG dams leads to developmental programming differences in kidney development and adult blood pressure compared with age- and sex-matched controls. Progeny were tested in the late fetal period (e17.5), neonatal period (2 weeks of age), and as young adults (12 weeks). We measured weights, tested for renal oxidative stress, compared renal DNA methylation profiles, counted the number of glomeruli, and measured adult blood pressure ± stress. Progeny from TG dams were growth restricted with evidence of renal oxidative stress, males showed fetal renal DNA hypermethylation, they had fewer glomeruli, and they developed stress-induced hypertension as adults. Their female siblings did not share this pathology and instead resembled progeny from WT dams. Surprisingly, glomerular counts in the neonatal period were not different between sexes or maternal genotypes. In turn, we suspect that differences in fetal renal DNA methylation may affect the long-term viability of glomeruli, rather than reducing nephrogenesis.

Genome-wide characterization of cytosine-specific 5-hydroxymethylation in normal breast tissue.

Despite recent evidence that 5-hydroxymethylcytosine (5hmC) possesses roles in gene regulation distinct from 5-methylcytosine (5mC), relatively little is known regarding the functions of 5hmC in mammalian tissues. To address this issue, we utilized an approach combining both paired bisulfite (BS) and oxidative bisulfite (oxBS) DNA treatment, to resolve genome-wide patterns of 5hmC and 5mC in normal breast tissue from disease-free women. Although less abundant than 5mC, 5hmC was differentially distributed, and consistently enriched among breast-specific enhancers and transcriptionally active chromatin. In contrast, regulatory regions associated with transcriptional inactivity, such as heterochromatin and repressed Polycomb regions, were relatively depleted of 5hmC. Gene regions containing abundant 5hmC were significantly associated with lactate oxidation, immune cell function, and prolactin signaling pathways. Furthermore, genes containing abundant 5hmC were enriched among those actively transcribed in normal breast tissue. Finally, in independent data sets, normal breast tissue 5hmC was significantly enriched among CpG loci demonstrated to have altered methylation in pre-invasive breast cancer and invasive breast tumors. Primarily, our findings identify genomic loci containing abundant 5hmC in breast tissues and provide a genome-wide map of nucleotide-level 5hmC in normal breast tissue. Additionally, these data suggest 5hmC may participate in gene regulatory programs that are dysregulated during breast-related carcinogenesis.

Serum dioxin and DNA methylation in the sperm of operation ranch hand veterans exposed to Agent Orange.

BACKGROUND: Exposure to the herbicide Agent Orange during the Vietnam War was widespread and is associated with numerous adverse health outcomes. A continuing concern of veterans is the possibility that exposure to the dioxin-containing herbicide might induce adverse reproductive outcomes. We sought to assess whether exposure to Agent Orange in Vietnam was associated with changes in DNA methylation in sperm in a subset of Vietnam veterans who participated in the Air Force Health Study (AFHS). METHODS: We studied 37 members of the AFHS chosen to have no, low, medium or high exposure to Agent Orange, based upon serum dioxin levels obtained during a series of examinations. DNA from stored semen was extracted and DNA methylation assessed on the Illumina 450 K platform. RESULTS: Initial epigenome-wide analysis returned no loci that survived control for false discovery. However, the TEAD3 gene had four different CpG sites that showed loss of DNA methylation associated with dioxin exposure. Analysis assessing regional DNA methylation changes revealed 36 gene regions, including the region of the imprinted gene H19 to have altered DNA methylation associated with high exposure compared to the low exposure group. Additional comparison of our data with sperm DNA methylation data from Russian boys exposed to dioxin found an additional 5 loci that were altered in both studies and exhibited a consistent direction of association. CONCLUSIONS: Studying a small number of sperm samples from veterans enrolled in the AFHS, we did not find evidence of significant epigenome-wide alterations associated with exposure to Agent Orange. However, additional analysis showed that the H19 gene region is altered in the sperm of Agent Orange-exposed Ranch Hand veterans. Our study also replicated several findings of a prior study of dioxin-exposed Russian boys. These results provide additional candidate loci for further investigation and may have implications for the reproductive health of dioxin-exposed individuals.

Peaks, Means, and Determinants of Real-Time TVOC Exposures Associated with Cleaning and Disinfecting Tasks in Healthcare Settings.

Cleaning and disinfecting tasks and product use are associated with elevated prevalence of asthma and respiratory symptoms among healthcare workers; however, the levels of exposure that pose a health risk remain unclear. The objective of this study was to estimate the peak, average, and determinants of real-time total volatile organic compound (TVOC) exposure associated with cleaning tasks and product-use. TVOC exposures were measured using monitors equipped with a photoionization detector (PID). A simple correction factor was applied to the real-time measurements, calculated as a ratio of the full-shift average TVOC concentrations from a time-integrated canister and the PID sample, for each sample pair. During sampling, auxiliary information, e.g. tasks, products used, engineering controls, was recorded on standardized data collection forms at 5-min intervals. Five-minute averaged air measurements (n = 10 276) from 129 time-series comprising 92 workers and four hospitals were used to model the determinants of exposures. The statistical model simultaneously accounted for censored data and non-stationary autocorrelation and was fit using Markov-Chain Monte Carlo within a Bayesian context. Log-transformed corrected concentrations (cTVOC) were modeled, with the fixed-effects of tasks and covariates, that were systematically gathered during sampling, and random effect of person-day. The model-predicted geometric mean (GM) cTVOC concentrations ranged from 387 parts per billion (ppb) for the task of using a product containing formaldehyde in laboratories to 2091 ppb for the task of using skin wipes containing quaternary ammonium compounds, with a GM of 925 ppb when no products were used. Peak exposures quantified as the 95th percentile of 15-min averages for these tasks ranged from 3172 to 17 360 ppb. Peak and GM task exposures varied by occupation and hospital unit. In the multiple regression model, use of sprays was associated with increasing exposures, while presence of local exhaust ventilation, large room volume, and automatic sterilizer use were associated with decreasing exposures. A detailed understanding of factors affecting TVOC exposure can inform targeted interventions to reduce exposures and can be used in epidemiologic studies as metrics of short-duration peak exposures.

A Bayesian Approach for Summarizing and Modeling Time-Series Exposure Data with Left Censoring.

OBJECTIVE: Direct reading instruments are valuable tools for measuring exposure as they provide real-time measurements for rapid decision making. However, their use is limited to general survey applications in part due to issues related to their performance. Moreover, statistical analysis of real-time data is complicated by autocorrelation among successive measurements, non-stationary time series, and the presence of left-censoring due to limit-of-detection (LOD). A Bayesian framework is proposed that accounts for non-stationary autocorrelation and LOD issues in exposure time-series data in order to model workplace factors that affect exposure and estimate summary statistics for tasks or other covariates of interest. METHOD: A spline-based approach is used to model non-stationary autocorrelation with relatively few assumptions about autocorrelation structure. Left-censoring is addressed by integrating over the left tail of the distribution. The model is fit using Markov-Chain Monte Carlo within a Bayesian paradigm. The method can flexibly account for hierarchical relationships, random effects and fixed effects of covariates. The method is implemented using the rjags package in R, and is illustrated by applying it to real-time exposure data. Estimates for task means and covariates from the Bayesian model are compared to those from conventional frequentist models including linear regression, mixed-effects, and time-series models with different autocorrelation structures. Simulations studies are also conducted to evaluate method performance. RESULTS: Simulation studies with percent of measurements below the LOD ranging from 0 to 50% showed lowest root mean squared errors for task means and the least biased standard deviations from the Bayesian model compared to the frequentist models across all levels of LOD. In the application, task means from the Bayesian model were similar to means from the frequentist models, while the standard deviations were different. Parameter estimates for covariates were significant in some frequentist models, but in the Bayesian model their credible intervals contained zero; such discrepancies were observed in multiple datasets. Variance components from the Bayesian model reflected substantial autocorrelation, consistent with the frequentist models, except for the auto-regressive moving average model. Plots of means from the Bayesian model showed good fit to the observed data. CONCLUSION: The proposed Bayesian model provides an approach for modeling non-stationary autocorrelation in a hierarchical modeling framework to estimate task means, standard deviations, quantiles, and parameter estimates for covariates that are less biased and have better performance characteristics than some of the contemporary methods.

Normal breast tissue DNA methylation differences at regulatory elements are associated with the cancer risk factor age.

BACKGROUND: The underlying biological mechanisms through which epidemiologically defined breast cancer risk factors contribute to disease risk remain poorly understood. Identification of the molecular changes associated with cancer risk factors in normal tissues may aid in determining the earliest events of carcinogenesis and informing cancer prevention strategies. METHODS: Here we investigated the impact cancer risk factors have on the normal breast epigenome by analyzing DNA methylation genome-wide (Infinium 450 K array) in cancer-free women from the Susan G. Komen Tissue Bank (n = 100). We tested the relation of established breast cancer risk factors, age, body mass index, parity, and family history of disease, with DNA methylation adjusting for potential variation in cell-type proportions. RESULTS: We identified 787 cytosine-guanine dinucleotide (CpG) sites that demonstrated significant associations (Q value <0.01) with subject age. Notably, DNA methylation was not strongly associated with the other evaluated breast cancer risk factors. Age-related DNA methylation changes are primarily increases in methylation enriched at breast epithelial cell enhancer regions (P = 7.1E-20), and binding sites of chromatin remodelers (MYC and CTCF). We validated the age-related associations in two independent populations, using normal breast tissue samples (n = 18) and samples of normal tissue adjacent to tumor tissue (n = 97). The genomic regions classified as age-related were more likely to be regions altered in both pre-invasive (n = 40, P = 3.0E-03) and invasive breast tumors (n = 731, P = 1.1E-13). CONCLUSIONS: DNA methylation changes with age occur at regulatory regions, and are further exacerbated in cancer, suggesting that age influences breast cancer risk in part through its contribution to epigenetic dysregulation in normal breast tissue.

A path analysis of multiple neurotoxic chemicals and cognitive functioning in older US adults (NHANES 1999-2002).

BACKGROUND: Polychlorinated biphenyls (PCBs) and metals (lead and cadmium) are neurotoxic and affect neurobehavioral performance. Yet little is known about the association between exposure to multiple neurotoxic compounds and cognitive functioning in older adults. METHODS: Using data from two consecutive cycles of the National Health and Nutrition and Examination Survey (1999-2002), path analysis was used to simultaneously evaluate the association between whole blood concentrations of 14 neurotoxic compounds and cognitive functioning measured by the Digit Symbol Coding Test of the Weschler Adult Intelligence Scale, 3rd Edition in participants 60-84 years of age (N = 498). Effect modification was assessed for age (above/below the mean) and sex. RESULTS: The final path model fit 5 compounds (i.e. PCB 74, PCB 118, PCB 146, PCB 153, and lead). After controlling for co-exposures and confounders, PCB 146 (ß = -0.16, 95% CI: -0.29, -0.02, p = 0.02) and lead (ß = -0.10, 95% CI: -0.20, -0.006, p = 0.04) were negatively associated with DSC scores in 60-84 year olds. Whereas, PCB 153 was positively associated with DSC scores (ß =0.20, 95% CI: 0.05, 0.35; p = 0.01). CONCLUSIONS: This cross-sectional analysis which controlled for collinear exposure to several neurotoxic compounds demonstrated an association between non-dioxin like polychlorinated biphenyl exposure, specifically PCB 146, and lower cognitive functioning, in older adults. Lead exposure was also weakly associated with lower cognitive functioning. Additional studies are needed to determine the causality of the observed associations.

5-Hydroxymethylcytosine localizes to enhancer elements and is associated with survival in glioblastoma patients.

Glioblastomas exhibit widespread molecular alterations including a highly distorted epigenome. Here, we resolve genome-wide 5-methylcytosine and 5-hydroxymethylcytosine in glioblastoma through parallel processing of DNA with bisulfite and oxidative bisulfite treatments. We apply a statistical algorithm to estimate 5-methylcytosine, 5-hydroxymethylcytosine and unmethylated proportions from methylation array data. We show that 5-hydroxymethylcytosine is depleted in glioblastoma compared with prefrontal cortex tissue. In addition, the genomic localization of 5-hydroxymethylcytosine in glioblastoma is associated with features of dynamic cell-identity regulation such as tissue-specific transcription and super-enhancers. Annotation of 5-hydroxymethylcytosine genomic distribution reveal significant associations with RNA regulatory processes, immune function, stem cell maintenance and binding sites of transcription factors that drive cellular proliferation. In addition, model-based clustering results indicate that patients with low-5-hydroxymethylcytosine patterns have significantly poorer overall survival. Our results demonstrate that 5-hydroxymethylcytosine patterns are strongly related with transcription, localizes to disease-critical genes and are associated with patient prognosis.

Validation of a DNA methylation reference panel for the estimation of nucleated cells types in cord blood.

Cord blood is widely used as surrogate tissue in epigenome-wide association studies of prenatal conditions. Cell type composition variation across samples can be an important confounder of epigenome-wide association studies in blood that constitute a mixture of cells. We evaluated a newly developed cord blood reference panel to impute cell type composition from DNA methylation levels, including nucleated red blood cells (nRBCs). We estimated cell type composition from 154 unique cord blood samples with available DNA methylation data as well as direct measurements of nucleated cell types. We observed high correlations between the estimated and measured composition for nRBCs (r = 0.92, R2 = 0.85), lymphocytes (r = 0.77, R2 = 0.58), and granulocytes (r = 0.72, R2 = 0.52), and a moderate correlation for monocytes (r = 0.51, R2 = 0.25) as well as relatively low root mean square errors from the residuals ranging from 1.4 to 5.4%. These results validate the use of the cord blood reference panel and highlight its utility and limitations for epidemiological studies.

Reference-free deconvolution of DNA methylation data and mediation by cell composition effects.

BACKGROUND: Recent interest in reference-free deconvolution of DNA methylation data has led to several supervised methods, but these methods do not easily permit the interpretation of underlying cell types. RESULTS: We propose a simple method for reference-free deconvolution that provides both proportions of putative cell types defined by their underlying methylomes, the number of these constituent cell types, as well as a method for evaluating the extent to which the underlying methylomes reflect specific types of cells. We demonstrate these methods in an analysis of 23 Infinium data sets from 13 distinct data collection efforts; these empirical evaluations show that our algorithm can reasonably estimate the number of constituent types, return cell proportion estimates that demonstrate anticipated associations with underlying phenotypic data; and methylomes that reflect the underlying biology of constituent cell types. CONCLUSIONS: Our methodology permits an explicit quantitation of the mediation of phenotypic associations with DNA methylation by cell composition effects. Although more work is needed to investigate functional information related to estimated methylomes, our proposed method provides a novel and useful foundation for conducting DNA methylation studies on heterogeneous tissues lacking reference data.

Regions of variable DNA methylation in human placenta associated with newborn neurobehavior.

The placenta regulates the in utero environment and functionally impacts fetal development. Candidate gene studies identified variation in placental DNA methylation is associated with newborn neurologic and behavioral outcomes including movement quality, lethargic behavior, attention, and arousal. We sought to identify novel regions of variable DNA methylation associated with newborn attention, lethargy, quality of movement, and arousal by performing an epigenome-wide association study in 335 infants from a US birth cohort. Methylation status was quantified using the Illumina HumanMethylation450 BeadChip array and associations to newborn outcomes assessed by the NICU Network Neurobehavioral Scales (NNNS) were identified while incorporating established bioinformatics algorithms to control for confounding by cell type composition. Methylation of CpGs within FHIT (cg15970800) and ANKRD11 (cg16710656) demonstrated genome-wide significance (P < 1.8 × 10(-7)) in specific associations with infant attention. CpGs whose differential methylation was associated with all 4 neurobehavioral outcomes were common to 50 genes involved in biological processes relating to cellular adhesion and nervous system development. Comprehensive methylation profiling identified relationships between methylation of FHIT and ANKRD11, which have been previously linked to neurodevelopment and behavioral outcomes in genetic association studies. Subtle changes in DNA methylation of these genes within the placenta may impact normal variation of a newborn's ability to alter and track visual and auditory stimuli. Gene ontology analysis suggested that those genes with variable methylation related to these outcomes are over-represented in biological pathways involved in brain development and placental physiology, supportive of our hypothesis for a key role of the placenta in neurobehavioral outcomes.

Cross-Sectional Study of Polybrominated Flame Retardants and Self-Reported Attention Deficit Hyperactivity Disorder in US Youth Aged 12-15 (NHANES 2003-2004).

Background. Animal toxicity tests and epidemiological studies suggest that exposure to PBDEs can alter attention behavior, yet few studies have examined their association with diagnosis of attention deficit hyperactivity disorder (ADHD) in adolescents. Methods. Logistic regression was used to examine the cross-sectional association between ADHD and lipid and non-lipid adjusted blood serum concentrations of 2',4-tribromodiphenyl ether (BDE-28), 2,2',4,4'-tetrabromodiphenylether (BDE-47), 2,2',4,4',5-pentabromodiphenyl ether (BDE-99), 2,2',4,4',5,5'-pentabromodiphenyl ether (BDE-100), 2,2',4,4',5,5'-hexabromodiphenyl ether (BDE-153), serum PBDEs, above/below the 75th percentile of serum PBDEs, and tertiles of serum PBDE in 12-15-year-olds (N = 292) using the National Health and Nutrition Examination Survey (NHANES) 2003-2004. Results. The ADHD weighted prevalence was 13.57%. The weighted adjusted odds ratios (AOR) and 95% confidence interval (CI) between ADHD diagnosis and lipid adjusted BDE-28, BDE-47, BDE-99, BDE-100, BDE-153, serum total PBDE, serum PBDE concentrations above the 75th percentile, and serum PBDE concentrations in the second or third tertile were 1.16 (95% CI: 0.51, 2.67), 1.36 (95% CI: 0.72, 2.56), 1.51 (95% CI: 0.70, 3.25), 1.53 (95% CI: 0.73, 3.23), 1.43 (95% CI: 0.57, 3.56), 1.41 (0.71, 2.83), 0.59 (0.10, 3.56), 6.16 (1.19, 31.90), and 0.99 (0.23, 4.29). Conclusions. We observed no association between serum PBDE concentrations and ADHD in US youths.

OxyBS: estimation of 5-methylcytosine and 5-hydroxymethylcytosine from tandem-treated oxidative bisulfite and bisulfite DNA.

UNLABELLED: The use of sodium bisulfite (BS) treatment followed by hybridization to an Illumina Infinium BeadChip (HumanMethylation450 and MethylationEPIC) is a common method for interrogating 5-methylcytosine (5mC) at single nucleotide resolution. However, standard treatment of DNA with BS does not allow disambiguation of 5mC from an additional cytosine modification, 5-hydroxymethylcytosine (5hmC). Recently, it has been demonstrated that paired BS and oxidative bisulfite (oxBS) treatment on the same sample followed by hybridization to an Infinium microarray permits the differentiation of 5hmC from 5mC. Nevertheless, estimation of 5hmC and 5mC from tandem-treated arrays has been shown to produce irregular estimates of cytosine modifications. RESULTS: We present a novel method using maximum likelihood estimation to accurately estimate the parameters of unmethylated cytosine (5C), 5mC and 5hmC from Infinium microarray data given the signal intensities from the oxBS and BS replicates. AVAILABILITY AND IMPLEMENTATION: OxyBS is an R package available on CRAN. CONTACT: Andres.Houseman@oregonstate.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

methyLiftover: cross-platform DNA methylation data integration.

UNLABELLED: : The public availability of high throughput molecular data provides new opportunities for researchers to advance discovery, replication and validation efforts. One common challenge in leveraging such data is the diversity of measurement approaches and platforms and a lack of utilities enabling cross-platform comparisons among data sources for analysis. We present a method to map DNA methylation data from bisulfite sequencing approaches to CpG sites measured with the widely used Illumina methylation bead-array platforms. Correlations and median absolute deviations support the validity of using bisulfite sequencing data in combination with Illumina bead-array methylation data. AVAILABILITY AND IMPLEMENTATION: https://github.com/Christensen-Lab-Dartmouth/methyLiftover includes source, documentation and data references. CONTACT: brock.c.christensen@dartmouth.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Hydroxymethylation is uniquely distributed within term placenta, and is associated with gene expression.

The conversion of cytosine to 5-methylcystosine (5mC) is an important regulator of gene expression. 5mC may be enzymatically converted to 5-hydroxymethylcytosine (5hmC), with a potentially distinct regulatory function. We sought to investigate these cytosine modifications and their effect on gene expression by parallel processing of genomic DNA using bisulfite and oxidative bisulfite conversion in conjunction with RNA sequencing. Although values of 5hmC across the placental genome were generally low, we identified ∼21,000 loci with consistently elevated levels of 5-hydroxymethycytosine. Absence of 5hmC was observed in CpG islands and, to a greater extent, in non-CpG island-associated regions. 5hmC was enriched within poised enhancers, and depleted within active enhancers, as defined by H3K27ac and H3K4me1 measurements. 5hmC and 5mC were significantly elevated in transcriptionally silent genes when compared with actively transcribed genes. 5hmC was positively associated with transcription in actively transcribed genes only. Our data suggest that dynamic cytosine regulation, associated with transcription, provides the most complete epigenomic landscape of the human placenta, and will be useful for future studies of the placental epigenome.-Green, B. B., Houseman, E. A., Johnson, K. C., Guerin, D. J., Armstrong, D. A., Christensen, B. C., Marsit, C. J. Hydroxymethylation is uniquely distributed within term placenta, and is associated with gene expression.

The DNA methylation profile of activated human natural killer cells.

Natural killer (NK) cells are now recognized to exhibit characteristics akin to cells of the adaptive immune system. The generation of adaptive memory is linked to epigenetic reprogramming including alterations in DNA methylation. The study herein found reproducible genome wide DNA methylation changes associated with human NK cell activation. Activation led predominately to CpG hypomethylation (81% of significant loci). Bioinformatics analysis confirmed that non-coding and gene-associated differentially methylated sites (DMS) are enriched for immune related functions (i.e., immune cell activation). Known DNA methylation-regulated immune loci were also identified in activated NK cells (e.g., TNFA, LTA, IL13, CSF2). Twenty-one loci were designated high priority and further investigated as potential markers of NK activation. BHLHE40 was identified as a viable candidate for which a droplet digital PCR assay for demethylation was developed. The assay revealed high demethylation in activated NK cells and low demethylation in naïve NK, T- and B-cells. We conclude the NK cell methylome is plastic with potential for remodeling. The differentially methylated region signature of activated NKs revealed similarities with T cell activation, but also provided unique biomarker candidates of NK activation, which could be useful in epigenome-wide association studies to interrogate the role of NK subtypes in global methylation changes associated with exposures and/or disease states.

Epigenome-Wide Assessment of DNA Methylation in the Placenta and Arsenic Exposure in the New Hampshire Birth Cohort Study (USA).

BACKGROUND: Arsenic is one of the most commonly encountered environmental toxicants, and research from model systems has suggested that one mode of its toxic activity may be through alterations in DNA methylation. In utero exposure to arsenic can affect fetal, newborn, and infant health, resulting in a range of phenotypic outcomes. OBJECTIVES: This study examined variation in placental DNA methylation and its relationship to arsenic exposure in 343 individuals enrolled in the New Hampshire Birth Cohort Study. METHODS: Linear regression models using a reference-free correction to account for cellular composition were employed to determine CpG loci affected by arsenic levels. RESULTS: Total arsenic measured in maternal urine during the second trimester was not associated with methylation in the placenta, whereas arsenic levels quantified through maternal toenail collected at birth were associated with methylation at a single CpG locus (p = 4.1 × 10-8). Placenta arsenic levels were associated with 163 differentially methylated loci (false discovery rate < 0.05), with 11 probes within the LYRM2 gene reaching genome-wide significance (p < 10-8). Measurement of LYRM2 mRNA levels indicated that methylation was weakly to moderately correlated with expression (r = 0.15, p < 0.06). In addition, we identified pathways suggesting changes in placental cell subpopulation proportions associated with arsenic exposure. CONCLUSIONS: These data demonstrate the potential for arsenic, even at levels commonly experienced in a U.S. population, to have effects on the DNA methylation status of specific genes in the placenta and thus supports a potentially novel mechanism for arsenic to affect long-term children's health. CITATION: Green BB, Karagas MR, Punshon T, Jackson BP, Robbins DJ, Houseman EA, Marsit CJ. 2016. Epigenome-wide assessment of DNA methylation in the placenta and arsenic exposure in the New Hampshire Birth Cohort Study (USA). Environ Health Perspect 124:1253-1260; http://dx.doi.org/10.1289/ehp.1510437.