Investigating the potential of single-cell DNA methylation data to detect allele-specific methylation and imprinting.

Allele-specific methylation (ASM) is an epigenetic modification whereby one parental allele becomes methylated and the other unmethylated at a specific locus. ASM is most often driven by the presence of nearby heterozygous variants that influence methylation, but also occurs somatically in the context of genomic imprinting. In this study, we investigate ASM using publicly available single-cell reduced representation bisulfite sequencing (scRRBS) data on 608 B cells sampled from six healthy B cell samples and 1,230 cells from 11 chronic lymphocytic leukemia (CLL) samples. We developed a likelihood-based criterion to test whether a CpG exhibited ASM, based on the distributions of methylated and unmethylated reads both within and across cells. Applying our likelihood ratio test, 65,998 CpG sites exhibited ASM in healthy B cell samples according to a Bonferroni criterion (p < 8.4 × 10-9), and 32,862 CpG sites exhibited ASM in CLL samples (p < 8.5 × 10-9). We also called ASM at the sample level. To evaluate the accuracy of our method, we called heterozygous variants from the scRRBS data, which enabled variant-based calls of ASM within each cell. Comparing sample-level ASM calls to the variant-based measures of ASM, we observed a positive predictive value of 76%-100% across samples. We observed high concordance of ASM across samples and an overrepresentation of ASM in previously reported imprinted genes and genes with imprinting binding motifs. Our study demonstrates that single-cell bisulfite sequencing is a potentially powerful tool to investigate ASM, especially as studies expand to increase the number of samples and cells sequenced.

Pruning and thresholding approach for methylation risk scores in multi-ancestry populations.

Recent efforts have focused on developing methylation risk scores (MRS), a weighted sum of the individual's DNA methylation (DNAm) values of pre-selected CpG sites. Most of the current MRS approaches that utilize Epigenome-wide association studies (EWAS) summary statistics only include genome-wide significant CpG sites and do not consider co-methylation. New methods that relax the p-value threshold to include more CpG sites and account for the inter-correlation of DNAm might improve the predictive performance of MRS. We paired informed co-methylation pruning with P-value thresholding to generate pruning and thresholding (P+T) MRS and evaluated its performance among multi-ancestry populations. Through simulation studies and real data analyses, we demonstrated that pruning provides an improvement over simple thresholding methods for prediction of phenotypes. We demonstrated that European-derived summary statistics can be used to develop P+T MRS among other populations such as African populations. However, the prediction accuracy of P+T MRS may differ across multi-ancestry population due to environmental/cultural/social differences.

Longitudinal DNA methylation profiling of the rectal mucosa identifies cell-specific signatures of disease status, severity and clinical outcomes in ulcerative colitis cell-specific DNA methylation signatures of UC.

BACKGROUND: In peripheral blood, DNA methylation (DNAm) patterns in inflammatory bowel disease patients reflect inflammatory status rather than disease status. Here, we examined DNAm in diseased rectal mucosa from ulcerative colitis (UC) patients, focusing on constituent cell types with the goal of identifying therapeutic targets for UC other than the immune system. We profiled DNAm of rectal mucosal biopsies of pediatric UC at diagnosis (n = 211) and non-IBD control (n = 85) patients and performed epigenome-wide association studies (EWAS) of specific cell types to understand DNAm changes in epithelial, immune and fibroblast cells across disease states, course, and clinical outcomes. We also examined longitudinal analysis on follow-up samples (n = 73), and comparisons were made among patients with clinical outcomes including those undergoing colectomy versus those who did not. Additionally, we included RNA-seq from the same subjects to assess the impact of CpG sites on the transcription of nearby genes during the disease course. RESULTS: At diagnosis, UC rectal mucosa exhibited a lower proportion of epithelial cells and fibroblasts, and higher proportion of immune cells, in conjunction with variation in the DNAm pattern. While treatment had significant effects on the methylation signature of immune cells, its effects on fibroblasts and epithelial cells were attenuated. Individuals who required colectomy exhibited cell composition and DNAm patterns at follow-up more similar to disease onset than patients who did not require colectomy. Combining these results with gene expression profiles, we identify CpG sites whose methylation patterns are most consistent with a contribution to poor disease outcomes and could thus be potential therapeutic targets. CONCLUSIONS: Cell-specific epigenetic changes in the rectal mucosa in UC are associated with disease severity and outcome. Current therapeutics may more effectively target the immune than the epithelial and fibroblast compartments.

Epigenome-wide meta-analysis of BMI in nine cohorts: Examining the utility of epigenetically predicted BMI.

This study sought to examine the association between DNA methylation and body mass index (BMI) and the potential of BMI-associated cytosine-phosphate-guanine (CpG) sites to provide information about metabolic health. We pooled summary statistics from six trans-ethnic epigenome-wide association studies (EWASs) of BMI representing nine cohorts (n = 17,034), replicated these findings in the Women's Health Initiative (WHI, n = 4,822), and developed an epigenetic prediction score of BMI. In the pooled EWASs, 1,265 CpG sites were associated with BMI (p < 1E-7) and 1,238 replicated in the WHI (FDR < 0.05). We performed several stratified analyses to examine whether these associations differed between individuals of European and African descent, as defined by self-reported race/ethnicity. We found that five CpG sites had a significant interaction with BMI by race/ethnicity. To examine the utility of the significant CpG sites in predicting BMI, we used elastic net regression to predict log-normalized BMI in the WHI (80% training/20% testing). This model found that 397 sites could explain 32% of the variance in BMI in the WHI test set. Individuals whose methylome-predicted BMI overestimated their BMI (high epigenetic BMI) had significantly higher glucose and triglycerides and lower HDL cholesterol and LDL cholesterol compared to accurately predicted BMI. Individuals whose methylome-predicted BMI underestimated their BMI (low epigenetic BMI) had significantly higher HDL cholesterol and lower glucose and triglycerides. This study confirmed 553 and identified 685 CpG sites associated with BMI. Participants with high epigenetic BMI had poorer metabolic health, suggesting that the overestimation may be driven in part by cardiometabolic derangements characteristic of metabolic syndrome.

Outcomes Stratification of Head and Neck Cancer Using Pre- and Post-treatment DNA Methylation From Peripheral Blood.

PURPOSE: Established prognostic factors for head and neck squamous cell carcinoma (HNSCC) mostly consist of clinical and tumor features assessed before treatment. We report a novel application of DNA methylation in peripheral blood before and after radiation therapy to further improve outcomes stratification. METHODS AND MATERIALS: Peripheral blood samples from patients with nonmetastatic HNSCC were obtained for methylation analysis 1 week before and 1 month after radiation therapy. Patients were randomized 1:1 to a Discovery Cohort or a Validation Cohort. In the Discovery Cohort, associations between genome-wide methylation change (posttreatment minus pretreatment) and recurrence-free survival (RFS) as well as overall survival (OS) were evaluated using Cox regression. A methylation risk score (MRS) was then constructed from methylation levels at the top associated sites, filtered for residing within the regulatory regions of genes expressed in cells of hematopoietic lineage. The prognostic value of MRS was separately assessed in the Discovery and Validation Cohorts. RESULTS: Between December 2013 and September 2018, 115 patients participated in this study. Human papilloma virus negative status, oral cavity cancer, gastrostomy tube insertion, and higher neutrophil count before radiation therapy were associated with shorter RFS and OS (P < .05). Genes downstream of the methylation sites comprising MRS are HIF1A, SF1, LGALS9, and FUT5, involved in hypoxia response, blood cell maturation, and immune modulation. High MRS (in the top third) was significantly associated with worse RFS (hazard ratio [HR], 7.1; 95% confidence interval [CI], 1.4-35.5; P = .016) and OS (HR, 15.9; 95% CI, 1.6-153.6; P = .017) in the Discovery Cohort, independent of the aforementioned risk factors. These findings were replicated in the Validation Cohort, for which high MRS also independently predicted worse RFS (HR, 10.2; 95%, CI 2.4-43.4; P = .002) and OS (HR, 3.7; 95% CI, 1.3-10.4; P = .015). CONCLUSIONS: We successfully trained and validated a signature of DNA methylation in peripheral blood before and after radiation therapy that stratified outcomes among patients with HNSCC, implicating the potential for genomics-tailored surveillance and consolidation treatment.

Clonal hematopoiesis of indeterminate potential, DNA methylation, and risk for coronary artery disease.

Age-related changes to the genome-wide DNA methylation (DNAm) pattern observed in blood are well-documented. Clonal hematopoiesis of indeterminate potential (CHIP), characterized by the age-related acquisition and expansion of leukemogenic mutations in hematopoietic stem cells (HSCs), is associated with blood cancer and coronary artery disease (CAD). Epigenetic regulators DNMT3A and TET2 are the two most frequently mutated CHIP genes. Here, we present results from an epigenome-wide association study for CHIP in 582 Cardiovascular Health Study (CHS) participants, with replication in 2655 Atherosclerosis Risk in Communities (ARIC) Study participants. We show that DNMT3A and TET2 CHIP have distinct and directionally opposing genome-wide DNAm association patterns consistent with their regulatory roles, albeit both promoting self-renewal of HSCs. Mendelian randomization analyses indicate that a subset of DNAm alterations associated with these two leading CHIP genes may promote the risk for CAD.

Gaseous air pollutants and DNA methylation in a methylome-wide association study of an ethnically and environmentally diverse population of U.S. adults.

Epigenetic mechanisms may underlie air pollution-health outcome associations. We estimated gaseous air pollutant-DNA methylation (DNAm) associations using twelve subpopulations within Women's Health Initiative (WHI) and Atherosclerosis Risk in Communities (ARIC) cohorts (n = 8397; mean age 61.3 years; 83% female; 46% African-American, 46% European-American, 8% Hispanic/Latino). We used geocoded participant address-specific mean ambient carbon monoxide (CO), nitrogen oxides (NO2; NOx), ozone (O3), and sulfur dioxide (SO2) concentrations estimated over the 2-, 7-, 28-, and 365-day periods before collection of blood samples used to generate Illumina 450 k array leukocyte DNAm measurements. We estimated methylome-wide, subpopulation- and race/ethnicity-stratified pollutant-DNAm associations in multi-level, linear mixed-effects models adjusted for sociodemographic, behavioral, meteorological, and technical covariates. We combined stratum-specific estimates in inverse variance-weighted meta-analyses and characterized significant associations (false discovery rate; FDR<0.05) at Cytosine-phosphate-Guanine (CpG) sites without among-strata heterogeneity (PCochran's Q > 0.05). We attempted replication in the Cooperative Health Research in Region of Augsburg (KORA) study and Normative Aging Study (NAS). We observed a -0.3 (95% CI: -0.4, -0.2) unit decrease in percent DNAm per interquartile range (IQR, 7.3 ppb) increase in 28-day mean NO2 concentration at cg01885635 (chromosome 3; regulatory region 290 bp upstream from ZNF621; FDR = 0.03). At intragenic sites cg21849932 (chromosome 20; LIME1; intron 3) and cg05353869 (chromosome 11; KLHL35; exon 2), we observed a -0.3 (95% CI: -0.4, -0.2) unit decrease (FDR = 0.04) and a 1.2 (95% CI: 0.7, 1.7) unit increase (FDR = 0.04), respectively, in percent DNAm per IQR (17.6 ppb) increase in 7-day mean ozone concentration. Results were not fully replicated in KORA and NAS. We identified three CpG sites potentially susceptible to gaseous air pollution-induced DNAm changes near genes relevant for cardiovascular and lung disease. Further harmonized investigations with a range of gaseous pollutants and averaging durations are needed to determine the effect of gaseous air pollutants on DNA methylation and ultimately gene expression.

Methylation quantitative trait loci are largely consistent across disease states in Crohn's disease.

Recently, we identified 1,189 CpG sites whose DNA methylation level in blood associated with Crohn's disease. Here, we examined associations between DNA methylation and genetic variants to identify methylation quantitative trait loci across disease states in (1) 402 blood samples from 164 newly diagnosed pediatric Crohn's disease patients taken at 2 time points (diagnosis and follow-up), and 74 non-inflammatory bowel disease controls, (2) 780 blood samples from a non-Crohn's disease adult population, and (3) 40 ileal biopsies (17 Crohn's disease cases and 23 non-inflammatory bowel disease controls) from group (1). Genome-wide DNAm profiling and genotyping were performed using the Illumina MethylationEPIC and Illumina Multi-Ethnic arrays. SNP-CpG associations were identified via linear models adjusted for age, sex, disease status, disease subtype, estimated cell proportions, and genotype-based principal components. In total, we observed 535,448 SNP-CpG associations between 287,881 SNPs and 12,843 CpG sites (P < 8.21 × 10-14). Associations were highly consistent across different ages, races, disease states, and tissue types, suggesting that the majority of these methylation quantitative trait loci participate in common gene regulation. However, genes near CpGs associated with inflammatory bowel disease SNPs were enriched for 18 KEGG pathways relevant to inflammatory bowel disease-linked immune function and inflammatory responses. We observed suggestive evidence for a small number of tissue-specific associations and disease-specific associations in ileum, though larger studies will be needed to confirm these results. Our study concludes that the vast majority of blood-derived methylation quantitative trait loci are common across individuals, though a subset may be involved in processes related to Crohn's disease. Independent cohort studies will be required to validate these findings.

Brain DNA Methylation Patterns in CLDN5 Associated With Cognitive Decline.

BACKGROUND: Cognitive trajectory varies widely and can distinguish people who develop dementia from people who remain cognitively normal. Variation in cognitive trajectory is only partially explained by traditional neuropathologies. We sought to identify novel genes associated with cognitive trajectory using DNA methylation profiles from human postmortem brain. METHODS: We performed a brain epigenome-wide association study of cognitive trajectory in 636 participants from the ROS (Religious Orders Study) and MAP (Rush Memory and Aging Project) using DNA methylation profiles of the dorsolateral prefrontal cortex. To maximize our power to detect epigenetic associations, we used the recently developed Gene Association with Multiple Traits test to analyze the 5 measured cognitive domains simultaneously. RESULTS: We found an epigenome-wide association for differential methylation of sites in the CLDN5 locus and cognitive trajectory (p = 9.96 × 10-7) that was robust to adjustment for cell type proportions (p = 8.52 × 10-7). This association was primarily driven by association with declines in episodic (p = 4.65 × 10-6) and working (p = 2.54 × 10-7) memory. This association between methylation in CLDN5 and cognitive decline was significant even in participants with no or little signs of amyloid-ß and neurofibrillary tangle pathology. CONCLUSIONS: Differential methylation of CLDN5, a gene that encodes an important protein of the blood-brain barrier, is associated with cognitive trajectory beyond traditional Alzheimer's disease pathologies. The association between CLDN5 methylation and cognitive trajectory in people with low pathology suggests an early role for CLDN5 and blood-brain barrier dysfunction in cognitive decline and Alzheimer's disease.

Differential DNA methylation and changing cell-type proportions as fibrotic stage progresses in NAFLD.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is characterized by changes in cell composition that occur throughout disease pathogenesis, which includes the development of fibrosis in a subset of patients. DNA methylation (DNAm) is a plausible mechanism underlying these shifts, considering that DNAm profiles differ across tissues and cell types, and DNAm may play a role in cell-type differentiation. Previous work investigating the relationship between DNAm and fibrosis in NAFLD has been limited by sample size and the number of CpG sites interrogated. RESULTS: Here, we performed an epigenome-wide analysis using Infinium MethylationEPIC array data from 325 individuals with NAFLD, including 119 with severe fibrosis and 206 with no histological evidence of fibrosis. After adjustment for latent confounders, we identified 7 CpG sites whose DNAm associated with fibrosis (p < 5.96 × 10-8). Analysis of RNA-seq data collected from a subset of individuals (N = 56) revealed that gene expression at 288 genes associated with DNAm at one or more of the 7 fibrosis-related CpGs. DNAm-based estimates of cell-type proportions showed that estimated proportions of natural killer cells increased, while epithelial cell proportions decreased with disease stage. Finally, we used an elastic net regression model to assess DNAm as a biomarker of fibrotic stage and found that our model predicted fibrosis with a sensitivity of 0.93 and provided information beyond a model based solely on cell-type proportions. CONCLUSION: These findings are consistent with DNAm as a mechanism underpinning or marking fibrosis-related shifts in cell composition and demonstrate the potential of DNAm as a possible biomarker of NAFLD fibrosis.

Examining the association between adiposity and DNA methylation: A systematic review and meta-analysis.

Obesity is associated with widespread differential DNA methylation (DNAm) patterns, though there have been limited overlap in the obesity-associated cytosine-guanine nucleotide pair (CpG) sites that have been identified in the literature. We systematically searched four databases for studies published until January 2020. Eligible studies included cross-sectional, longitudinal, or intervention studies examining adiposity and genome-wide DNAm in non-pregnant adults aged 18-75 in all tissue types. Study design and results were extracted in the descriptive review. Blood-based DNAm results in body mass index (BMI) and waist circumference (WC) were meta-analyzed using weighted sum of Z-score meta-analysis. Of the 10,548 studies identified, 46 studies were included in the systematic review with 18 and nine studies included in the meta-analysis of BMI and WC, respectively. In the blood, 77 and four CpG sites were significant in three or more studies of BMI and WC, respectively. Using a genome-wide threshold for significance, 52 blood-based CpG sites were significantly associated with BMI. These sites have previously been associated with many obesity-related diseases including type 2 diabetes, cardiovascular disease, Crohn's disease, and depression. Our study shows that DNAm at 52 CpG sites represent potential mediators of obesity-associated chronic diseases and may be novel intervention or therapeutic targets to protect against obesity-associated chronic diseases.

Epigenetic age acceleration, fatigue, and inflammation in patients undergoing radiation therapy for head and neck cancer: A longitudinal study.

BACKGROUND: The authors measured epigenetic age acceleration (EAA) during and after cancer treatment and its association with inflammation and fatigue, which is a debilitating symptom in patients with cancer. METHODS: Patients who had head and neck cancer without distant metastases were assessed before, immediately after, and at 6 months and 12 months postradiotherapy. Blood DNA methylation was assessed using a proprietary bead chip (the Illumina MethylationEPIC BeadChip). EAA was calculated using the Levine epigenetic clock (DNAmPhenoAge), adjusted for chronological age. Fatigue was assessed using the Multidimensional Fatigue Inventory-20. Inflammatory markers were measured using standard techniques. RESULTS: Most patients (N = 133) were men, White, had advanced disease, and received concurrent chemoradiation. EAA changes over time were significant, with the largest increase (4.9 years) observed immediately after radiotherapy (P < .001). Increased EAA was associated with elevated fatigue (P = .003) over time, and patients who had severe fatigue experienced 3.1 years higher EAA than those who had low fatigue (P < .001), which was more prominent (5.6 years; P = .018) for patients who had human papillomavirus-unrelated disease at 12 months posttreatment. EAA was also positively associated with inflammatory markers, including C-reactive protein (CRP) and interleukin-6 (IL-6), over time (P < .001), and patients who had high CRP and IL-6 levels exhibited increases of 4.6 and 5.9 years, respectively, in EAA compared with those who had low CRP and IL-6 levels (P < .001). CRP and IL-6 mediated the association between EAA and fatigue (CRP: 95% CI, 0.060-0.279; IL-6: 95% CI, 0.024-0.220). CONCLUSIONS: Patients with head and neck cancer experienced increased EAA, especially immediately after treatment completion. EAA was associated with greater fatigue and inflammation, including 1 year after treatment. Inflammation may be a target to reduce the impact of age acceleration on poor functional outcomes.

Ten-eleven translocation protein 1 modulates medulloblastoma progression.

BACKGROUND: Medulloblastoma (MB) is the most common malignant pediatric brain tumor that originates in the cerebellum and brainstem. Frequent somatic mutations and deregulated expression of epigenetic regulators in MB highlight the substantial role of epigenetic alterations. 5-hydroxymethylcytosine (5hmC) is a highly abundant cytosine modification in the developing cerebellum and is regulated by ten-eleven translocation (TET) enzymes. RESULTS: We investigate the alterations of 5hmC and TET enzymes in MB and their significance to cerebellar cancer formation. We show total abundance of 5hmC is reduced in MB, but identify significant enrichment of MB-specific 5hmC marks at regulatory regions of genes implicated in stem-like properties and Nanog-binding motifs. While TET1 and TET2 levels are high in MBs, only knockout of Tet1 in the smoothened (SmoA1) mouse model attenuates uncontrolled proliferation, leading to a favorable prognosis. The pharmacological Tet1 inhibition reduces cell viability and platelet-derived growth factor signaling pathway-associated genes. CONCLUSIONS: These results together suggest a potential key role of 5hmC and indicate an oncogenic nature for TET1 in MB tumorigenesis, suggesting it as a potential therapeutic target for MBs.

Association of Epigenetic Age Acceleration With Risk Factors, Survival, and Quality of Life in Patients With Head and Neck Cancer.

PURPOSE: Epigenetic age acceleration (EAA) is robustly linked with mortality and morbidity. This study examined risk factors of EAA and its association with overall survival (OS), progression-free survival (PFS), and quality of life (QOL) in patients with head and neck cancer (HNC) receiving radiation therapy. METHODS AND MATERIALS: Patients without distant metastasis were enrolled and followed before and at the end of radiation therapy and at 6 and 12 months after radiation therapy. EAA was calculated with DNAmPhenoAge at all 4 time points. Risk factors included demographic characteristics, lifestyle, clinical characteristics, treatment-related symptoms, and blood biomarkers. Survival data were collected until August 2020, and QOL was measured using Functional Assessment of Cancer Therapy-HNC. RESULTS: Increased comorbidity, symptoms unrelated to human papilloma virus, and more severe treatment-related symptoms were associated with higher EAA (P = .03 to P < .001). A nonlinear association (quadratic) between body mass index (BMI) and EAA was observed: decreased BMI (<35 kg/m2; P = .04) and increased BMI (≥35 kg/m2; P = .01) were linked to higher EAA. Increased EAA (per year) was associated with worse OS (hazard ratio [HR], 1.11 [95% confidence interval {CI}, 1.03-1.18; P = .004]; HR, 1.10 [95% CI, 1.01-1.19; P = .02] for EAA at 6 and 12 months after treatment, respectively) and PFS (HR, 1.10 [95% CI, 1.02-1.19; P = .02]; HR, 1.14 [95% CI, 1.06-1.23; P < .001]; and HR, 1.08 [95% CI, 1.02-1.14; P = .01]) for EAA before, immediately after, and 6 months after radiation therapy, respectively) and QOL over time (ß = -0.61; P = .001). An average of 3.25 to 3.33 years of age acceleration across time, which was responsible for 33% to 44% higher HRs of OS and PFS, was observed in those who died or developed recurrence compared with those who did not (all P < .001). CONCLUSIONS: Compared with demographic and lifestyle factors, clinical characteristics were more likely to contribute to faster biological aging in patients with HNC. Acceleration in epigenetic age resulted in more aggressive adverse events, including OS and PFS. EAA could be considered as a marker for cancer outcomes, and decelerating aging could improve survival and QOL.

Epigenetically mediated electrocardiographic manifestations of sub-chronic exposures to ambient particulate matter air pollution in the Women's Health Initiative and Atherosclerosis Risk in Communities Study.

BACKGROUND: Short-duration exposure to ambient particulate matter (PM) air pollution is associated with cardiac autonomic dysfunction and prolonged ventricular repolarization. However, associations with sub-chronic exposures to coarser particulates are relatively poorly characterized as are molecular mechanisms underlying their potential relationships with cardiovascular disease. MATERIALS AND METHODS: We estimated associations between monthly mean concentrations of PM < 10 µm and 2.5-10 µm in diameter (PM10; PM2.5-10) with time-domain measures of heart rate variability (HRV) and QT interval duration (QT) among U.S. women and men in the Women's Health Initiative and Atherosclerosis Risk in Communities Study (nHRV = 82,107; nQT = 76,711). Then we examined mediation of the PM-HRV and PM-QT associations by DNA methylation (DNAm) at three Cytosine-phosphate-Guanine (CpG) sites (cg19004594, cg24102420, cg12124767) with known sensitivity to monthly mean PM concentrations in a subset of the participants (nHRV = 7,169; nQT = 6,895). After multiply imputing missing PM, electrocardiographic and covariable data, we estimated associations using attrition-weighted, linear, mixed, longitudinal models adjusting for sociodemographic, behavioral, meteorological, and clinical characteristics. We assessed mediation by estimating the proportions of PM-HRV and PM-QT associations mediated by DNAm. RESULTS: We found little evidence of PM-HRV association, PM-QT association, or mediation by DNAm. CONCLUSIONS: The findings suggest that among racially/ethnically and environmentally diverse U.S. populations, sub-chronic exposures to coarser particulates may not exert appreciable, epigenetically mediated effects on cardiac autonomic function or ventricular repolarization. Further investigation in better-powered studies is warranted, with additional focus on shorter duration exposures to finer particulates and non-electrocardiographic outcomes among relatively susceptible populations.

Chromatin Accessibility Identifies Regulatory Elements Predictive of Gene Expression and Disease Outcome in Multiple Myeloma.

PURPOSE: Multiple myeloma is a malignancy of plasma cells. Extensive genetic and transcriptional characterization of myeloma has identified subtypes with prognostic and therapeutic implications. In contrast, relatively little is known about the myeloma epigenome. EXPERIMENTAL DESIGN: CD138+CD38+ myeloma cells were isolated from fresh bone marrow aspirate or the same aspirate after freezing for 1-6 months. Gene expression and chromatin accessibility were compared between fresh and frozen samples by RNA sequencing (RNA-seq) and assay for transpose accessible chromatin sequencing (ATAC-seq). Chromatin accessible regions were used to identify regulatory RNA expression in more than 700 samples from newly diagnosed patients in the Multiple Myeloma Research Foundation CoMMpass trial (NCT01454297). RESULTS: Gene expression and chromatin accessibility of cryopreserved myeloma recapitulated that of freshly isolated samples. ATAC-seq performed on a series of biobanked specimens identified thousands of chromatin accessible regions with hundreds being highly coordinated with gene expression. More than 4,700 of these chromatin accessible regions were transcribed in newly diagnosed myelomas from the CoMMpass trial. Regulatory element activity alone recapitulated myeloma gene expression subtypes, and in particular myeloma subtypes with immunoglobulin heavy chain translocations were defined by transcription of distal regulatory elements. Moreover, enhancer activity predicted oncogene expression implicating gene regulatory mechanisms in aggressive myeloma. CONCLUSIONS: These data demonstrate the feasibility of using biobanked specimens for retrospective studies of the myeloma epigenome and illustrate the unique enhancer landscapes of myeloma subtypes that are coupled to gene expression and disease progression.

Epigenome-wide association study of diet quality in the Women's Health Initiative and TwinsUK cohort.

BACKGROUND: Diet quality is a risk factor for chronic disease and mortality. Differential DNA methylation across the epigenome has been associated with chronic disease risk. Whether diet quality is associated with differential methylation is unknown. This study assessed whether diet quality was associated with differential DNA methylation measured across 445 548 loci in the Women's Health Initiative (WHI) and the TwinsUK cohort. DESIGN: The discovery cohort consisted of 4355 women from the WHI. The replication cohort consisted of 571 mono- and dizygotic twins from the TwinsUK cohort. DNA methylation was measured in whole blood using the Illumina Infinium HumanMethylation450 Beadchip. Diet quality was assessed using the Alternative Healthy Eating Index 2010 (AHEI-2010). A meta-analysis, stratified by study cohort, was performed using generalized linear models that regressed methylation on AHEI-2010, adjusting for cell composition, chip number and location, study characteristics, principal components of genetic relatedness, age, smoking status, race/ethnicity and body mass index (BMI). Statistical significance was defined as a false discovery rate < 0.05. Significant sites were tested for replication in the TwinsUK cohort, with significant replication defined by P < 0.05 and a consistent direction. RESULTS: Diet quality was significantly associated with differential DNA methylation at 428 cytosine-phosphate-guanine (CpG) sites in the discovery cohort. A total of 24 CpG sites were consistent with replication in the TwinsUK cohort, more than would be expected by chance (P = 2.7x10-4), with one site replicated in both the blood and adipose tissue (cg16379999 located in the body of SEL1L). CONCLUSIONS: Diet quality was associated with methylation at 24 CpG sites, several of which have been associated with adiposity, inflammation and dysglycaemia. These findings may provide insight into pathways through which diet influences chronic disease.

Genome-wide DNA methylation differences and polychlorinated biphenyl (PCB) exposure in a US population.

Exposure to polychlorinated biphenyls (PCBs), an endocrine-disrupting compound, is ubiquitous despite decades-old bans on the manufacture and use of PCBs. Increased exposure to PCBs is associated with adverse health consequences throughout life, including type 2 diabetes and cancer. PCB exposure is also associated with alterations in epigenetic marks and gene transcription, which could lead to adverse health outcomes, but many of these are population-specific. To further investigate the association between PCB and epigenetic marks, DNA methylation was measured at 787,684 CpG sites in 641 peripheral blood samples from the Michigan Polybrominated Biphenyl (PBB) Registry. 1345 CpGs were associated with increased total PCB level after controlling for age, sex, and 24 surrogate variables (FDR < 0.05). These CpGs were enriched in active promoter and transcription associated regions (p < 0.05), and in regions around the binding sites for transcription factors involved in xenobiotic metabolism and immune function (FDR < 0.05). PCB exposure also associated with proportions of CD4T, NK, and granulocyte cell types, and with the neutrophil to lymphocyte ratio (NLR) (p < 0.05), and the estimated effect sizes of PCB on the epigenome were correlated with the effect sizes previously reported in an epigenome-wide study of C-reactive protein (r = 0.29; p = 2.22e-5), supporting previous studies on the association between PCB and immune dysfunction. These results indicate that PCB exposure is associated with differences in epigenetic marks in active regions of the genome, and future work should investigate whether these may mediate the association between PCB and health consequences.

The role of the gut microbiome in cancer-related fatigue: pilot study on epigenetic mechanisms.

PURPOSE: Recent evidence supports a key role of gut microbiome in brain health. We conducted a pilot study to assess associations of gut microbiome with cancer-related fatigue and explore the associations with DNA methylation changes. METHODS: Self-reported Multidimensional Fatigue Inventory and stool samples were collected at pre-radiotherapy and one-month post-radiotherapy in patients with head and neck cancer. Gut microbiome data were obtained by sequencing the 16S ribosomal ribonucleic acid gene. DNA methylation changes in the blood were assessed using Illumina Methylation EPIC BeadChip. RESULTS: We observed significantly different gut microbiota patterns among patients with high vs. low fatigue across time. This pattern was characterized by low relative abundance in short-chain fatty acid-producing taxa (family Ruminococcaceae, genera Subdoligranulum and Faecalibacterium; all p < 0.05), with high abundance in taxa associated with inflammation (genera Family XIII AD3011 and Erysipelatoclostridium; all p < 0.05) for high-fatigue group. We identified nine KEGG Orthology pathways significantly different between high- vs. low-fatigue groups over time (all p < 0.001), including pathways related to fatty acid synthesis and oxidation, inflammation, and brain function. Gene set enrichment analysis (GSEA) was performed on the top differentially methylated CpG sites that were associated with the taxa and fatigue. All biological processes from the GSEA were related to immune responses and inflammation (FDR < 0.05). CONCLUSIONS: Our results suggest different patterns of the gut microbiota in cancer patients with high vs. low fatigue. Results from functional pathways and DNA methylation analyses indicate that inflammation is likely to be the major driver in the gut-brain axis for cancer-related fatigue.

Association between DNA methylation levels in brain tissue and late-life depression in community-based participants.

OBJECTIVE: Major depressive disorder (MDD) arises from a combination of genetic and environmental risk factors and DNA methylation is one of the molecular mechanisms through which these factors can manifest. However, little is known about the epigenetic signature of MDD in brain tissue. This study aimed to investigate associations between brain tissue-based DNA methylation and late-life MDD. METHODS: We performed a brain epigenome-wide association study (EWAS) of late-life MDD in 608 participants from the Religious Order Study and the Rush Memory and Aging Project (ROS/MAP) using DNA methylation profiles of the dorsal lateral prefrontal cortex generated using the Illumina HumanMethylation450 Beadchip array. We also conducted an EWAS of MDD in each sex separately. RESULTS: We found epigenome-wide significant associations between brain tissue-based DNA methylation and late-life MDD. The most significant and robust association was found with altered methylation levels in the YOD1 locus (cg25594636, p value = 2.55 × 10-11; cg03899372, p value = 3.12 × 10-09; cg12796440, p value = 1.51 × 10-08, cg23982678, p value = 7.94 × 10-08). Analysis of differentially methylated regions (p value = 5.06 × 10-10) further confirmed this locus. Other significant loci include UGT8 (cg18921206, p value = 1.75 × 10-08), FNDC3B (cg20367479, p value = 4.97 × 10-08) and SLIT2 (cg10946669, p value = 8.01 × 10-08). Notably, brain tissue-based methylation levels were strongly associated with late-life MDD in men more than in women. CONCLUSIONS: We identified altered methylation in the YOD1, UGT8, FNDC3B, and SLIT2 loci as new epigenetic factors associated with late-life MDD. Furthermore, our study highlights the sex-specific molecular heterogeneity of MDD.