DNA Methylation Signatures In Blood Research Articles

Abstract A016: Early pancreatic cancer detection using extracellular vesicle DNA methylation signatures in blood

Abstract In 2020, there were 495,773 new cases and 466,003 deaths for pancreatic cancer worldwide. Furthermore, pancreatic cancer is emerging to be the leading cause of cancer related deaths in the U.S. by the year 2030, surpassing lung and colon cancer. The majority of pancreatic cancer patients (∼80%) are diagnosed at the most advanced (metastatic) stage, for which the 5-year survival is 3%. Dramatically, studies show that if it can be detected at an early stage when surgical removal is feasible, the 5-year survival rate significantly increased to 37%. This data highlights the crucial importance of early detection for pancreatic cancer treatment with curative intent. Unfortunately, currently there are no reliable techniques for routine screening to identify pancreatic cancer at early stages. In this Abstract, we developed and validated the performance of those DNA methylation markers to detect pancreatic cancer in a cohort of pancreatic cancer patients. Firstly, we collected a large amount of genome-wide methylation microarray data (targeting over 450,000 methylation sites) of tissue samples from several publicly available databases, including samples of solid pancreatic tumors (n = 384), noncancerous pancreatic tissues (n = 126) and healthy whole blood (n = 453). The raw microarray data were preprocessed using a pipeline implemented in R, which removes inaccurate measurements and corrects for bias caused by technical issues. A two-stage feature selection procedure was designed and implemented in Python, which makes use of various filters and a multivariate embedded method, support vector machine recursive feature elimination (SVM-RFE). The features were evaluated using two metrics, classification performance and stability, based on a tradeoff of which we selected a set of differentially methylated loci for discriminating the samples. The set of prospective markers includes 129 hypermethylated and 142 hypomethylated tumor-specific loci, 8 hypermethylated and 8 hypomethylated pancreatic tissue specific loci. To validate the performance of those pancreatic cancer-specific methylation markers, as a feasibility study, we isolated extracellular vesicles from cancer cells and plasma of cancer patients using lipid nanoprobe (LNP). Extracellular vesicles (EVs) are lipid-bilayer-enclosed vesicles of sub- micrometer size that are secreted by virtually all cell types. In a pilot study encompassing 11 pancreatic cancer patients (Stage I-IV) and age-matched 10 healthy donors (HD), we employed quantitative methylation-specific polymerase chain reaction assays (qMSP) to examine the promoter methylation status of the genes within plasma EV DNA. The receiver operating characteristic (ROC) curves were employed to assess the performance of a combined promoter methylation signature in EV DNA for the detection of pancreatic cancer. Our findings indicate that the promoter methylation of genes can be specific to pancreatic cancer, suggesting the efficient packaging of methylation information from genomic DNA into EVs. Citation Format: Hongzhang He, Siyang Zheng. Early pancreatic cancer detection using extracellular vesicle DNA methylation signatures in blood [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A016.

Consistent cord blood DNA methylation signatures of gestational age between South Asian and white European cohorts

BackgroundEpigenetic modifications, particularly DNA methylation (DNAm) in cord blood, are an important biological marker of how external exposures during gestation can influence the in-utero environment and subsequent offspring development. Despite the recognized importance of DNAm during gestation, comparative studies to determine the consistency of these epigenetic signals across different ethnic groups are largely absent. To address this gap, we first performed epigenome-wide association studies (EWAS) of gestational age (GA) using newborn cord blood DNAm comparatively in a white European (n = 342) and a South Asian (n = 490) birth cohort living in Canada. Then, we capitalized on established cord blood epigenetic GA clocks to examine the associations between maternal exposures, offspring characteristics and epigenetic GA, as well as GA acceleration, defined as the residual difference between epigenetic and chronological GA at birth.ResultsIndividual EWASs confirmed 1,211 and 1,543 differentially methylated CpGs previously reported to be associated with GA, in white European and South Asian cohorts, respectively, with a similar distribution of effects. We confirmed that Bohlin’s cord blood GA clock was robustly correlated with GA in white Europeans (r = 0.71; p = 6.0 × 10–54) and South Asians (r = 0.66; p = 6.9 × 10–64). In both cohorts, Bohlin’s clock was positively associated with newborn weight and length and negatively associated with parity, newborn female sex, and gestational diabetes. Exclusive to South Asians, the GA clock was positively associated with the newborn ponderal index, while pre-pregnancy weight and gestational weight gain were strongly predictive of increased epigenetic GA in white Europeans. Important predictors of GA acceleration included gestational diabetes mellitus, newborn sex, and parity in both cohorts.ConclusionsThese results demonstrate the consistent DNAm signatures of GA and the utility of Bohlin’s GA clock across the two populations. Although the overall pattern of DNAm is similar, its connections with the mother's environment and the baby's anthropometrics can differ between the two groups. Further research is needed to understand these unique relationships.

Genetic variation and pesticide exposure influence blood DNA methylation signatures in females with early-stage Parkinson’s disease

Although sex, genetics, and exposures can individually influence risk for sporadic Parkinson’s disease (PD), the joint contributions of these factors to the epigenetic etiology of PD have not been comprehensively assessed. Here, we profiled sex-stratified genome-wide blood DNAm patterns, SNP genotype, and pesticide exposure in agricultural workers (71 early-stage PD cases, 147 controls) and explored replication in three independent samples of varying demographics (n = 218, 222, and 872). Using a region-based approach, we found more associations of blood DNAm with PD in females (69 regions) than in males (2 regions, Δβadj| ≥0.03, padj ≤ 0.05). For 48 regions in females, models including genotype or genotype and pesticide exposure substantially improved in explaining interindividual variation in DNAm (padj ≤ 0.05), and accounting for these variables decreased the estimated effect of PD on DNAm. The results suggested that genotype, and to a lesser degree, genotype-exposure interactions contributed to variation in PD-associated DNAm. Our findings should be further explored in larger study populations and in experimental systems, preferably with precise measures of exposure.

Cord blood DNA methylation signatures associated with preeclampsia are enriched for cardiovascular pathways: insights from the VDAART trial

Preeclampsia has been associated with maternal epigenetic changes, in particular DNA methylation changes in the placenta. It has been suggested that preeclampsia could also cause DNA methylation changes in the neonate. We examined DNA methylation in relation to gene expression in the cord blood of offspring born to mothers with preeclampsia. This study included 128 mother-child pairs who participated in the Vitamin D Antenatal Asthma Reduction Trial (VDAART), where assessment of preeclampsia served as secondary outcome. We performed an epigenome-wide association study of preeclampsia and cord blood DNA methylation (Illumina 450K chip). We then examined gene expression of the same subjects for validation and replicated the gene signatures in independent DNA methylation datasets. Lastly, we applied functional enrichment and network analyses to identify biological pathways that could potentially be involved in preeclampsia. In the cord blood samples (n=128), 263 CpGs were differentially methylated (FDR <0.10) in preeclampsia (n=16), of which 217 were annotated. Top pathways in the functional enrichment analysis included apelin signaling pathway and other endothelial and cardiovascular pathways. Of the 217 genes, 13 showed differential expression (p's<0.001) in preeclampsia and 11 had been previously related to preeclampsia (p's<0.0001). These genes were linked to apelin, cGMP and Notch signaling pathways, all having a role in angiogenic process and cardiovascular function. Preeclampsia is related to differential cord blood DNA methylation signatures of cardiovascular pathways, including the apelin signaling pathway. The association of these cord blood DNA methylation signatures with offspring's long-term morbidities due to preeclampsia should be further investigated. VDAART is funded by National Heart, Lung, and Blood Institute grants of R01HL091528 and UH3OD023268. HMK is supported by Jane and Aatos Erkko Foundation, Paulo Foundation, and the Pediatric Research Foundation. HM is supported by K01 award from NHLBI (1K01HL146977-01A1). PK is supported by K99HL159234 from NIH/NHLBI.

Hippocampal and peripheral blood DNA methylation signatures correlate at the gene and pathway level in a mouse model of autism.

Autism spectrum disorders (ASD) are polygenic multifactorial disorders influenced by environmental factors. ASD-related differential DNA methylation has been found in human peripheral tissues, such as placenta, paternal sperm, buccal epithelium, and blood. However, these data lack direct comparison of DNA methylation levels with brain tissue from the same individual to determine the extent that peripheral tissues are surrogates for behavior-related disorders. Here, whole genome methylation profiling at all the possible sites throughout the mouse genome (>25 million) from both brain and blood tissues revealed novel insights into the systemic contributions of DNA methylation to ASD. Sixty-six differentially methylated regions (DMRs) share the same genomic coordinates in these two tissues, many of which are linked to risk genes for neurodevelopmental disorders and intellectual disabilities (e.g. Prkch, Ptn, Hcfc1, Mid1, and Nfia). Gene ontological pathways revealed a significant number of common terms between brain and blood (N = 65 terms), and nearly half (30/65) were associated with brain/neuronal development. Furthermore, seven DMR-associated genes among these terms contain methyl-sensitive transcription factor sequence motifs within the DMRs of both tissues; four of them (Cux2, Kcnip2, Fgf13, and Mrtfa) contain the same methyl-sensitive transcription factor binding sequence motifs (HES1/2/5, TBX2 and TFAP2C), suggesting DNA methylation influences the binding of common transcription factors required for gene expression. Together, these findings suggest that peripheral blood is a good surrogate tissue for brain and support that DNA methylation contributes to altered gene regulation in the pathogenesis of ASD.

Peripheral Blood DNA Methylation Signatures and Response to Tofacitinib in Moderate-to-severe Ulcerative Colitis.

Predictive biomarkers for treatment efficacy of ulcerative colitis [UC] treatments are lacking. Here, we performed a longitudinal study investigating the association and potential predictive power of genome-wide peripheral blood [PB] DNA methylation signatures and response to tofacitinib treatment in UC. We recruited moderate-to-severe UC patients starting tofacitinib treatment, and measured PB DNA methylation profiles at baseline [T1], after 8 weeks [T2], and in a subset [n = 8] after a median of 20 weeks [T3] using the Illumina Infinium HumanMethylation EPIC BeadChip. After 8 weeks, we distinguished responders [R] from non-responders [NR] based on a centrally read endoscopic response [decrease in endoscopic Mayo score ≥1 or Ulcerative Colitis Endoscopic Index of Severity ≥2] combined with corticosteroid-free clinical and/or biochemical response. T1 PB samples were used for biomarker identification, and T2 and publicly available intraclass correlation [ICC] data were used for stability analyses. RNA-sequencing was performed to understand the downstream effects of the predictor CpG loci. In total, 16 R and 15 NR patients, with a median disease duration of 7 [4-12] years and overall comparable patient characteristics at baseline, were analysed. We identified a panel of 53 differentially methylated positions [DMPs] associated with response to tofacitinib [AUROC 0.74]. Most DMPs [77%] demonstrated both short- and long-term hyperstability [ICC ≥0.90], irrespective of inflammatory status. Gene expression analysis showed lower FGFR2 [pBH = 0.011] and LRPAP1 [pBH = 0.020], and higher OR2L13 [pBH = 0.016] expression at T1 in R compared with NR. Our observations demonstrate the utility of genome-wide PB DNA methylation signatures to predict response to tofacitinib.

OP03 Highly stable epigenome-wide peripheral blood DNA methylation signatures accurately predict endoscopic response to adalimumab, vedolizumab and ustekinumab in Crohn’s disease patients: The EPIC-CD study

Abstract Background Despite the proven efficacy of biological treatments in Crohn’s disease (CD), many patients fail to respond or lose response over time. Therefore, predictive biomarkers for treatment efficacy would be of extreme value. Previous epigenome-wide association studies associated differential DNA methylation with CD-specific phenotypes, suggesting a potential use in classification and prediction of response to treatment. Methods We prospectively collected and measured longitudinal peripheral blood DNA methylation profiles of 184 adult CD patients prior to (T1) and after a median of 28 weeks (T2) following biological treatment with Adalimumab (ADA), Vedolizumab (VEDO) or Ustekinumab (USTE) in a discovery (n=88) and independently collected internal validation cohort (n=96) using the Illumina EPIC BeadChip array. Response (R) was defined as the combination of endoscopic response (≥50% reduction in SES-CD score) and steroid-free clinical response (≥3 point drop in HBI or HBI ≤4 AND no systemic steroids) and/or biochemical response (≥50% reduction in C-reactive protein (CRP) and fecal calprotectin or a CRP ≤5 g/mL and fecal calprotectin ≤250 µg/g). Biomarker identification and classification analyses were performed using stability selection gradient boosting on samples taken at T1 whereas samples taken at T2 and intraclass correlation (ICC) data were used to assess long-term stability of our identified CpG loci1. Results A total of 58 ADA-patients (NR=29, NNR=29), 64 VEDO-patients (NR=36, NNR=28) and 62 USTE-patients (NR=30, NNR=32) were included. Prior to treatment, at T1, we identified distinct panels of 100 ADA-, 22 VEDO- and 68 USTE-associated CpG loci that, in combination, predict clinical- and endoscopic response with high accuracy (AUC ADA=0.73, VEDO=0.89 and USTE=0.94) upon validation. Notably, for these CpG loci, methylation levels did not significantly differ between T1 and T2, implicating stability during both induction and maintenance treatment, irrespective of inflammatory status and therapeutic intervention. In addition, the majority of these CpG loci (&amp;gt;60%) demonstrated long-term hyper stability (ICC-values ≥0.90). Furthermore, genes annotated to the CpGs of interest suggest drug specific involvement in TNF-signaling, endothelial cell-cell adhesion, integrin dependent T-cell homing, the innate immune system and Th17/Treg differentiation, corroborating to the mode of action of each drug. Conclusion Here, we report on 3 validated panels of highly stable, epigenetic biomarkers that predict clinical and endoscopic response in CD patients treated with ADA, VEDO or USTE. Additional external- and clinical validation as part of EPIC-CD and the OMICROHN clinical trial are currently ongoing.

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.

Blood DNA methylation signatures of lifestyle exposures: tobacco and alcohol consumption

BackgroundSmoking and alcohol consumption may compromise health by way of epigenetic modifications. Epigenetic signatures of alcohol and tobacco consumption could provide insights into the reversibility of phenotypic changes incurred with differing levels of lifestyle exposures. This study describes and validates two novel epigenetic signatures of tobacco (EpiTob) and alcohol (EpiAlc) consumption and investigates their association with disease outcomes.MethodsThe epigenetic signatures, EpiTob and EpiAlc, were developed using data from the Swiss Kidney Project on Genes in Hypertension (SKIPOGH) (N = 689). Epigenetic and phenotypic data available from the 1921 (N = 550) and 1936 (N = 1091) Lothian Birth Cohort (LBC) studies, and two publicly available datasets on GEO Accession (GSE50660, N = 464; and GSE110043, N = 94) were used to validate the signatures. A multivariable logistic regression model, adjusting for age and sex, was used to assess the association between self-reported tobacco or alcohol consumption and the respective epigenetic signature, as well as to estimate the association between CVD and epigenetic signatures. A Cox proportional hazard model was used to estimate the risk of mortality in association with the EpiTob and EpiAlc signatures.ResultsThe EpiTob signature was positively associated with self-reported tobacco consumption for current or never smokers with explained variance ranging from 0.49 (LBC1921) to 0.72 (LBC1936) (pseudo-R2). In the SKIPOGH, LBC1921 and LBC1936 cohorts, the epigenetic signature for alcohol consumption explained limited variance in association with self-reported alcohol status [i.e., non-drinker, moderate drinker, and heavy drinker] (pseudo-R2 = 0.05, 0.03 and 0.03, respectively), although this improved considerably when measuring self-reported alcohol consumption with standardized units consumed per week (SKIPOGH R2 = 0.21; LBC1921 R2 = 0.31; LBC1936 R2 = 0.41). Both signatures were associated with history of CVD in SKIPOGH and LBC1936, but not in LBC1921. The EpiTob signature was associated with increased risk of all-cause and lung-cancer specific mortality in the 1936 and 1921 LBC cohorts.ConclusionsThis study found the EpiTob and EpiAlc signatures to be well-correlated with self-reported exposure status and associated with long-term health outcomes. Epigenetic signatures of lifestyle exposures may reduce measurement issues and biases and could aid in risk stratification for informing early-stage targeted interventions.

Methylome-wide Association Study of Patients with Recent-onset Psychosis.

ObjectiveDysregulation of gene expression through epigenetic mechanisms may have a vital role in the pathogenesis of schizophrenia (SZ). In this study, we investigated the association of altered methylation patterns with SZ symptoms and early trauma in patients and healthy controls.MethodsThe present study was conducted to identify methylation changes in CpG sites in peripheral blood associated with recent-onset (RO) psychosis using methylome-wide analysis. Lifestyle factors, such as smoking, alcohol, exercise, and diet, were controlled.ResultsWe identified 2,912 differentially methylated CpG sites in patients with RO psychosis compared to controls. Most of the genes associated with the top 20 differentially methylated sites had not been reported in previous methylation studies and were involved in apoptosis, autophagy, axonal growth, neuroinflammation, protein folding, etc. The top 15 significantly enriched Kyoto Encyclopedia of Genes and Genomes pathways included the oxytocin signaling pathway, long-term depression pathway, axon guidance, endometrial cancer, long-term potentiation, mitogen-activated protein kinase signaling pathway, and glutamatergic pathway, among others. In the patient group, significant associations of novel methylated genes with early trauma and psychopathology were observed.ConclusionOur results suggest an association of differential DNA methylation with the pathophysiology of psychosis and early trauma. Blood DNA methylation signatures show promise as biomarkers of future psychosis.

Blood DNA methylation signatures are associated with social determinants of health among survivors of childhood cancer

ABSTRACT Social epigenomics is an emerging field in which social scientist collaborate with computational biologists, especially epigeneticists, to address the underlying pathway for biological embedding of life experiences. This social epigenomics study included long-term childhood cancer survivors enrolled in the St. Jude Lifetime Cohort. DNA methylation (DNAm) data were generated using the Illumina EPIC BeadChip, and three social determinants of health (SDOH) factors were assessed: self-reported educational attainment, personal income, and an area deprivation index based on census track data. An epigenome-wide association study (EWAS) was performed to evaluate the relation between DNAm at each 5’-cytosine-phosphate-guanine-3’ (CpG) site and each SDOH factor based on multivariable linear regression models stratified by ancestry (European ancestry, n = 1,618; African ancestry, n = 258). EWAS among survivors of European ancestry identified 130 epigenome-wide significant SDOH–CpG associations (P < 9 × 10−8), 25 of which were validated in survivors of African ancestry (P < 0.05). Thirteen CpGs were associated with all three SDOH factors and resided at pleiotropic loci in cigarette smoking–related genes (e.g., CLDND1 and CPOX). After accounting for smoking and body mass index, these associations remained significant with attenuated effect sizes. Seven of 13 CpGs were associated with gene expression level based on 57 subsamples with blood RNA sequencing data available. In conclusion, DNAm signatures, many resembling the effect of tobacco use, were associated with SDOH factors among survivors of childhood cancer, thereby suggesting that biologically distal SDOH factors influence health behaviours or related factors, the epigenome, and subsequently survivors’ health.

Prenatal Particulate Matter Exposure Is Associated with Saliva DNA Methylation at Age 15: Applying Cumulative DNA Methylation Scores as an Exposure Biomarker.

Exposure in utero to particulate matter (PM2.5 and PM10) is associated with maladaptive health outcomes. Although exposure to prenatal PM2.5 and PM10 has cord blood DNA methylation signatures at birth, signature persistence into childhood and saliva cross-tissue applicability has not been tested. In the Fragile Families and Child Wellbeing Study, a United States 20-city birth cohort, average residential PM2.5 and PM10 during the three months prior to birth was estimated using air quality monitors with inverse distance weighting. Saliva DNA methylation at ages 9 (n = 749) and 15 (n = 793) was measured using the Illumina HumanMethylation 450 k BeadArray. Cumulative DNA methylation scores for particulate matter were estimated by weighting participant DNA methylation at each site by independent meta-analysis effect estimates and standardizing the sums. Using a mixed-effects regression analysis, we tested the associations between cumulative DNA methylation scores at ages 9 and 15 and PM exposure during pregnancy, adjusted for child sex, age, race/ethnicity, maternal income-to-needs ratio, nonmartial birth status, and saliva cell-type proportions. Our study sample was 50.5% male, 56.3% non-Hispanic Black, and 19.8% Hispanic, with a median income-to-needs ratio of 1.4. Mean exposure levels for PM2.5 were 27.9 μg/m3/day (standard deviation: 7.0; 23.7% of observations exceeded safety standards) and for PM10 were 15.0 μg/m3/day (standard deviation: 3.1). An interquartile range increase in PM2.5 exposure (10.73 μg/m3/day) was associated with a −0.0287 standard deviation lower cumulative DNA methylation score for PM2.5 (95% CI: −0.0732, 0.0158, p = 0.20) across all participants. An interquartile range increase in PM10 exposure (3.20 μg/m3/day) was associated with a −0.1472 standard deviation lower cumulative DNA methylation score for PM10 (95% CI: −0.3038, 0.0095, p = 0.06) across all participants. The PM10 findings were driven by the age 15 subset where an interquartile range increase in PM10 exposure was associated with a −0.024 standard deviation lower cumulative DNA methylation score for PM10 (95% CI: −0.043, −0.005, p = 0.012). Findings were robust to adjustment for PM exposure at ages 1 and 3. In utero PM10-associated DNA methylation differences were identified at age 15 in saliva. Benchmarking the timing and cell-type generalizability is critical for epigenetic exposure biomarker assessment.

Particulate matter exposure in utero is associated with childhood saliva DNA methylation at ages 9 and 15

Background: Exposure in utero to particulate matter (PM2.5 and PM10) is associated with health outcomes. Prenatal PM2.5 and PM10 exposures have cord blood DNA methylation signatures at birth, however signature persistence into childhood and saliva cross-tissue applicability have not been tested. Methods: In the Fragile Families and Childhood Wellbeing Study, a United States 20-city (200,000+) birth cohort, trimester-specific PM2.5 and PM10 was measured using air quality monitor inverse distance weighting. Saliva DNA methylation at ages 9 (n=835) and 15 (n=861) was measured using the Illumina HumanMethylation 450k BeadArray. Cumulative DNA methylation scores for particulate matter were estimated by mean centering participant DNA methylation, weighting by meta-analysis effect estimates, and standardizing the sums. Using multivariable linear regression, we tested cumulative DNA methylation scores at ages 9 and 15 for associations with pregnancy PM levels, adjusted for sex, income to needs ratio, city, nonmartial birth status, race/ethnicity, and cell type proportions. Results: Our study sample was 50.5% male, 20% non-Hispanic white, 55% non-Hispanic black, 21% Hispanic, and median income to needs ratio of 1.4. In the third trimester, mean PM2.5 exposure levels were 26.97 ug/m3/day (standard deviation: 6.9) and PM10 were 14.47 ug/m3/day (standard deviation: 3.2). At age 9, a one-standard deviation increase in cumulative PM2.5 DNA methylation score was associated with 0.44 ug/m3/day (95% confidence interval: -0.07-0.96) higher third trimester PM2.5 exposure. At age 9, a one-standard deviation increase in cumulative PM10 DNA methylation score was associated with 0.76 ug/m3/day (95% confidence interval: 0.56-0.98) higher third trimester PM10 exposure. Findings were consistent at age 15 and robust to adjustment for exposure at ages 1 and 3. Conclusion: In utero PM associated DNA methylation differences persist until ages 9 and 15 and can be detected in saliva. Benchmarking the persistence and cell type generalizability is critical for epigenetic exposure biomarker assessment.

Whole Blood DNA Methylation Signatures of Diet Are Associated With Cardiovascular Disease Risk Factors and All-Cause Mortality.

DNA methylation patterns associated with habitual diet have not been well studied. Diet quality was characterized using a Mediterranean-style diet score and the Alternative Healthy Eating Index score. We conducted ethnicity-specific and trans-ethnic epigenome-wide association analyses for diet quality and leukocyte-derived DNA methylation at over 400 000 CpGs (cytosine-guanine dinucleotides) in 5 population-based cohorts including 6662 European ancestry, 2702 African ancestry, and 360 Hispanic ancestry participants. For diet-associated CpGs identified in epigenome-wide analyses, we conducted Mendelian randomization (MR) analysis to examine their relations to cardiovascular disease risk factors and examined their longitudinal associations with all-cause mortality. We identified 30 CpGs associated with either Mediterranean-style diet score or Alternative Healthy Eating Index, or both, in European ancestry participants. Among these CpGs, 12 CpGs were significantly associated with all-cause mortality (Bonferroni corrected P<1.6×10-3). Hypermethylation of cg18181703 (SOCS3) was associated with higher scores of both Mediterranean-style diet score and Alternative Healthy Eating Index and lower risk for all-cause mortality (P=5.7×10-15). Ten additional diet-associated CpGs were nominally associated with all-cause mortality (P<0.05). MR analysis revealed 8 putatively causal associations for 6 CpGs with 4 cardiovascular disease risk factors (body mass index, triglycerides, high-density lipoprotein cholesterol concentrations, and type 2 diabetes mellitus; Bonferroni corrected MR P<4.5×10-4). For example, hypermethylation of cg11250194 (FADS2) was associated with lower triglyceride concentrations (MR, P=1.5×10-14).and hypermethylation of cg02079413 (SNORA54; NAP1L4) was associated with body mass index (corrected MR, P=1×10-6). Habitual diet quality was associated with differential peripheral leukocyte DNA methylation levels of 30 CpGs, most of which were also associated with multiple health outcomes, in European ancestry individuals. These findings demonstrate that integrative genomic analysis of dietary information may reveal molecular targets for disease prevention and treatment.

DNA methylation signature on phosphatidylethanol, not on\xa0self-reported alcohol consumption, predicts hazardous alcohol consumption in two distinct populations

The process of diagnosing hazardous alcohol drinking (HAD) is based on self-reported data and is thereby vulnerable to bias. There has been an interest in developing epigenetic biomarkers for HAD that might complement clinical assessment. Because alcohol consumption has been previously linked to DNA methylation (DNAm), we aimed to select DNAm signatures in blood to predict HAD from two demographically and clinically distinct populations (Ntotal = 1,549). We first separately conducted an epigenome-wide association study (EWAS) for phosphatidylethanol (PEth), an objective measure of alcohol consumption, and for self-reported alcohol consumption in Cohort 1. We identified 83 PEth-associated CpGs, including 23 CpGs previously associated with alcohol consumption or alcohol use disorder. In contrast, no CpG reached epigenome-wide significance on self-reported alcohol consumption. Using a machine learning approach, two CpG subsets from EWAS on PEth and on self-reported alcohol consumption from Cohort 1 were separately tested for the prediction of HAD in Cohort 2. We found that a subset of 143 CpGs selected from the EWAS on PEth showed an excellent prediction of HAD with the area under the receiver operating characteristic curve (AUC) of 89.4% in training set and 73.9% in validation set of Cohort 2. However, CpGs preselected from the EWAS on self-reported alcohol consumption showed a poor prediction of HAD with AUC 75.2% in training set and 57.6% in validation set. Our results demonstrate that an objective measure for alcohol consumption is a more informative phenotype than self-reported data for revealing epigenetic mechanisms. The PEth-associated DNAm signature in blood could serve as a robust biomarker for alcohol consumption.

Blood DNA methylation signatures to detect dementia prior to overt clinical symptoms.

IntroductionThis study determined whether blood DNA methylation (DNAm) patterns differentiate individuals with presymptomatic dementia compared to controls.MethodsDNAm was measured in 73 individuals prior to dementia diagnosis and 87 cognitively healthy controls matched for age, sex, smoking, education, and baseline cognition. DNAm was also measured at 3 years follow‐up in 25 dementia cases, and 24 controls.ResultsCases and controls differed in DNAm (unadjusted P < .01) at the time of diagnosis (n = 28,787 probes), and pre‐diagnosis (n = 15,111 probes), with cg01404610 (General transcription factor IIA subunit 1 gene) significant after correction for multiple testing. Overall, 1150 probes overlapped between analyses (methylation differences from –10.6% to +11.0%), and effect sizes increased from pre‐diagnosis to diagnosis.DiscussionDiscernible blood DNAm signatures are in dementia cases before the appearance of overt clinical symptoms. Blood‐based methylation may serve as a potential biomarker of dementia, but further investigation is needed to determine their true clinical utility.

Diet quality associated peripheral blood DNA methylation signatures

AbstractLeukocyte DNA methylation patterns associated with habitual diet may reveal molecular mechanisms involved in the pathogenesis of diet-related chronic diseases and highlight targets for prevention and treatment. We aimed to examine peripheral blood derived leukocyte DNA methylation signatures associated with diet quality. We meta-analyzed epigenome-wide associations between diet quality and DNA methylation levels at over 400,000 cytosine-guanine dinucleotides (CpGs). We conducted analysis primarily in 6,662 European ancestry (EA) participants and secondarily in a group additionally including 3,062 participants of non-European ancestry from five population-based cohort studies. DNA methylation profiles were measured in whole blood, CD4 + T-cells, or CD14 + monocytes. We used food frequency questionnaires to assess habitual intake and constructed two diet quality scores: the Mediterranean-style diet score (MDS) and Alternative Healthy Eating Index (AHEI). Our primary analysis identified 32 diet-associated CpGs, 12 CpGs for MDS and 24 CpGs for AHEI (at FDR &lt; 0.05, corresponding p-values = 1.2×10-6 and 3.1×10-6, respectively) in EA participants. Four of these CpGs were associated with both MDS and AHEI. In addition, Mendelian randomization analysis indicated that seven diet-associated CpGs were causally linked to at least one of the CVD risk factors. For example, hypermethylation of cg11250194 (FADS2), which was associated with higher diet quality scores, was also associated with lower fasting triglycerides concentrations (p-value = 1.5×10-14) and higher high-density lipoprotein cholesterol concentrations (p-value = 1.7×10-8). Transethnic meta-analysis identified nine additional CpGs associated with diet quality (either MDS or AHEI) at FDR &lt; 0.05. Overall quality of habitual diet was associated with differential peripheral leukocyte DNA methylation levels of 32 CpGs in EA participants. The diet-associated CpGs may serve as biomarkers and targets for preventive measures in CVD health. Future studies are warranted to examine diet-associated DNA methylation patterns in larger, ethnically diverse study samples.

Functional DNA methylation signatures for autism spectrum disorder genomic risk loci: 16p11.2 deletions and CHD8 variants

BackgroundAutism spectrum disorder (ASD) is a common and etiologically heterogeneous neurodevelopmental disorder. Although many genetic causes have been identified (> 200 ASD-risk genes), no single gene variant accounts for > 1% of all ASD cases. A role for epigenetic mechanisms in ASD etiology is supported by the fact that many ASD-risk genes function as epigenetic regulators and evidence that epigenetic dysregulation can interrupt normal brain development. Gene-specific DNAm profiles have been shown to assist in the interpretation of variants of unknown significance. Therefore, we investigated the epigenome in patients with ASD or two of the most common genomic variants conferring increased risk for ASD. Genome-wide DNA methylation (DNAm) was assessed using the Illumina Infinium HumanMethylation450 and MethylationEPIC arrays in blood from individuals with ASD of heterogeneous, undefined etiology (n = 52), and individuals with 16p11.2 deletions (16p11.2del, n = 9) or pathogenic variants in the chromatin modifier CHD8 (CHD8+/−, n = 7).ResultsDNAm patterns did not clearly distinguish heterogeneous ASD cases from controls. However, the homogeneous genetically-defined 16p11.2del and CHD8+/− subgroups each exhibited unique DNAm signatures that distinguished 16p11.2del or CHD8+/− individuals from each other and from heterogeneous ASD and control groups with high sensitivity and specificity. These signatures also classified additional 16p11.2del (n = 9) and CHD8 (n = 13) variants as pathogenic or benign. Our findings that DNAm alterations in each signature target unique genes in relevant biological pathways including neural development support their functional relevance. Furthermore, genes identified in our CHD8+/− DNAm signature in blood overlapped differentially expressed genes in CHD8+/− human-induced pluripotent cell-derived neurons and cerebral organoids from independent studies.ConclusionsDNAm signatures can provide clinical utility complementary to next-generation sequencing in the interpretation of variants of unknown significance. Our study constitutes a novel approach for ASD risk-associated molecular classification that elucidates the vital cross-talk between genetics and epigenetics in the etiology of ASD.

P108 LONGITUDINAL MQTL STUDY IN BLOOD FROM PEDIATRIC CROHN’S DISEASE PATIENTS

Crohn’s disease (CD) is a relapsing-remitting inflammatory disorder with flares of inflammation. We have recently shown that CD associated DNA methylation signatures in blood are a transient consequence of inflammation. Here, we investigated how genetic associations of DNA methylation change during the course of the disease, as a result of varying clinical features in pediatric CD patients. We used a subset of 164 pediatric CD patients from the RISK inception cohort. DNA was extracted from whole blood samples at two time points – at diagnosis and 1-3 years after diagnosis. Genome-wide genotyping and DNA methylation profiling was performed using the Illumina’s Multi-Ethnic Genotyping array and HumanMethylationEPIC ‘850K’ array, respectively. The effect size for each variant-CpG association (within ±500 kb; cis-methylation quantitative trait loci (mQTLs)) was estimated using linear mixed effects model implemented in GEMMA with age, gender, cell-type proportions and genetic relationship matrix as covariates. A total of ~613,000 genetic variants were tested against ~625,000 CpG sites at two time points, separately. At baseline, we identified a total of 62,970 mQTLs comprising 35,942 unique SNPs and 13,053 CpGs (Bonferroni P < 3.25 x 10-10). Among these mQTLs were SNPs previously found to be associated with CD. Of the 142 CD variants from large, meta-analyses of genome-wide association studies, 21 SNPs showed an association with a total of 39 CpGs. Notably, rs1819333 and rs1250546 depicted the strongest association with CpG sites annotated to known CD susceptibility genes, RNASET2 and RSP6KA2, and ZMIZ1, respectively. On the other hand, when we examined the plasma CRP levels, patients had elevated levels of CRP at diagnosis which were significantly lower during the time of the follow-up (P = 8.4 x 10-9). However, when we compared the mQTL effects from follow-up to baseline, we noted an extremely strong positive correlation suggesting the non-inflammatory nature of the identified CD-mQTLs (R2=0.99; P < 2.2e-16) [Figure 1]. Longitudinal stability of the relationship between genetic variation and DNA methylation despite effective treatment of the inflammation symptoms strongly suggests that these mQTL are not being driven by inflammation. Whether or not the associations are causally related to IBD remains an open question. Our findings also support the utility of using DNA methylation to refine the functional consequences of genetic variation in CD. Figure 1: Effects of all baseline significant mQTLs compared with follow-up showing perfect correlation suggessting non-inflammatory nature.

Abstract 50: DNA Methylation Signatures in Blood Distinguish Patients at Risk of Post-operative Atrial Fibrillation Following Cardiopulmonary Bypass

Post-operative atrial fibrillation (AF) is the most common complication for cardiac surgery, with an incidence of 27-40% and is associated with increased 30-day mortality. Several models exist to predict post-op AF using clinical data, however none have been widely implemented, and all have room for improvement (average area under ROC curve is ~0.70). We tested whether DNA methylation, assayed from pre-operative blood draws, could add predictive value. We enrolled a cohort of 55 adult patients undergoing cardiopulmonary bypass, with a 36% incidence of post-op AF. Reduced representative bisulfite sequencing provided single-base resolution for an average of 3 million CpGs with 10x sequencing coverage per sample. We first tested whether these CpGs were correlated with AF. Of the 809,569 CpGs that passed thresholds in all samples, 16,774 were significantly correlated with AF (corrected p-value &lt;=0.01 using methylKit in R). We used the R package Glmnet to perform logistic regression on the 16,774 CpGs in 40 of the 55 patients using 5-fold cross validation to build a model and selecting the 381 CpGs which were included in 95% of the 5,000 models. Our assumption being that these 381 CpGs were predictive no matter the subset of patients selected in the randomly generated groups employed during cross validation. Using these 381 CpGs as inputs we built a final model that ultimately incorporated 51 CpGs. None of the 51 CpGs were significantly correlated with age (corrected p-value &lt;=0.01), ruling out age as a confounding factor. We tested our model on the remaining 15 patients. Our model correctly predicted 5 of the 5 AF cases, and 9 of the 10 AF-free cases. Validation of our model in a larger cohort is ongoing, and future directions will incorporate our epigenomic signature with readily available clinical risk factors to further optimize the model.