DNA Methylation Changes In Blood Research Articles

Epigenome-Wide Association Studies of COPD and Lung Function: A Systematic Review.

Chronic Obstructive Pulmonary Disease (COPD) results from gene-environment interactions over the lifetime. These interactions are captured by epigenetic changes, such as DNA methylation. This systematic review synthesizes evidence from epigenome-wide association studies (EWAS) related to COPD and lung function. Systematic literature search on PubMed, Embase and CINAHL databases, identified 1947 articles that investigated epigenetic changes associated with COPD/lung function; 17 of them met our eligibility criteria from which data was manually extracted. Differentially methylated positions (DMPs) and/or annotated genes, were considered replicated if identified by ≥2 studies with a p<1 x 10-4. Ten studies profiled DNA methylation changes in blood and 7 in respiratory samples, including surgically resected lung tissue (n=3), small airways epithelial brushings (n=2), bronchoalveolar lavage (n=1) and sputum (n=1). Main results showed: (1) high variability in study design, covariates and effect sizes, which prevented a formal meta-analysis; (2) in blood samples, 51 DMPs were replicated in relation to lung function and 12 related to COPD; (3) in respiratory samples, 42 DMPs were replicated in relation to COPD but none in relation to lung function; and, (4) in COPD vs. control studies, 123 genes (2.6% of total) were shared between ≥1 blood and ≥1 respiratory sample and associated with chronic inflammation, ion transport and coagulation. There is high heterogeneity across published COPD/lung function EWAS studies. A few genes (n=123; 2.6%) were replicated in blood and respiratory samples, suggesting that blood can recapitulate some changes in respiratory tissues. These findings have implications for future research.

DNA methylation of ELOVL2 gene as an epigenetic marker of age among Egyptian population

BackgroundCellular and molecular changes occur during aging, decreasing organ function. The aging process was measured by several biomarkers, including DNA methylation (DNAm), an epigenetic change regulating gene expression, which is highly accurate at predicting biological age. DNAm is heritable and therefore varies between different populations.AimTo assess blood DNA methylation changes as epigenetic clocks in the male and female Egyptian population. Pyrosequencing was used to measure the methylation of nine CpG sites in blood samples from 100 healthy Egyptians (18–69 years) using a cross-sectional study. Two age predicted models based on the ELOVL2 gene were compared in three age categories and correlated in all age groups despite decreasing accuracy with increasing age.ResultsThe mean absolute deviation (MAD) using the 1st and 2nd age predicted models for 18–40 years was 1.06 and 2.7, respectively; for 41–60 years, it was 4.4 and 3.8, respectively; and for > 60 years, it was 7.7 and 7.0, respectively. No significant differences in DNA methylation were found between the sexes.ConclusionDNA methylation of the ELOVL2 gene can be used as an accurate biomarker for age estimation. Additionally, this method has the potential to be more accurate than traditional methods of age estimation.

Whole blood DNA methylation changes are associated with anti-TNF drug concentration in patients with Crohn's disease.

Anti-TNF treatment failure in patients with inflammatory bowel disease (IBD) is common and frequently related to low drug concentrations. In order to identify patients who may benefit from dose optimisation at the outset of anti-TNF therapy, we sought to define epigenetic biomarkers in whole blood at baseline associated with anti-TNF drug concentrations at week 14. DNA methylation from 1,104 whole blood samples from 385 patients in the Personalised Anti-TNF Therapy in Crohn's disease (PANTS) study were assessed using the Illumina EPIC Beadchip (v1.0) at baseline, weeks 14, 30 and 54. We compared DNA methylation profiles in anti-TNF-treated patients who experienced primary non-response at week 14 and if they were assessed at subsequent time points, were not in remission at week 30 or 54 (infliximab n = 99, adalimumab n = 94), with patients who responded at week 14 and when assessed at subsequent time points, were in remission at week 30 or 54 (infliximab n = 99, adalimumab n = 93). Overall, between baseline and week 14, we observed 4,999 differentially methylated probes (DMPs) annotated to 2376 genes following anti-TNF treatment. Pathway analysis identified 108 significant gene ontology terms enriched in biological processes related to immune system processes and responses.Epigenome-wide association (EWAS) analysis identified 323 DMPs annotated to 210 genes at baseline associated with higher anti-TNF drug concentrations at week 14. Of these, 125 DMPs demonstrated shared associations with other common traits (proportion of shared CpGs compared to DMPs) including body mass index (23.2%), followed by CRP (11.5%), smoking (7.4%), alcohol consumption per day (7.1%) and IBD type (6.8%). EWAS of primary non-response to anti-TNF identified 20 DMPs that were associated with both anti-TNF drug concentration and primary non-response to anti-TNF with a strong correlation of the coefficients (Spearman's rho = -0.94, p < 0.001). Baseline DNA methylation profiles may be used as a predictor for anti-TNF drug concentration at week 14 to identify patients who may benefit from dose optimisation at the outset of anti-TNF therapy.

BMI change during childhood, DNA methylation change at TXNIP, and glucose change during midlife.

This study investigated whether changes in DNA methylation (DNAm) at TXNIP are associated with glycemic changes and whether such an association differs with early-life adiposity changes. A total of 594 Bogalusa Heart Study participants who had blood DNAm measurements at two time points in midlife were included. Of them, 353 participants had at least four BMI measurements during childhood and adolescence. The incremental area under the curve was calculated as a measure of long-term trends of BMI during childhood and adolescence. Increase in DNAm at TXNIP was significantly associated with decrease in fasting plasma glucose (FPG) independent of covariates (p < 0.001). The study found that the strength of this relationship was significantly modified by a trend of increasing BMI during childhood and adolescence (p-interaction = 0.003). Each 1% increase in DNAm at TXNIP was associated with a 2.90- (0.77) mg/dL decrease in FPG among participants with the highest tertile of BMI incremental area under the curveand a 0.96- (0.38) mg/dL decrease among those with the middle tertile, whereas no association was observed among participants with the lowest tertile. These results indicate that changes in blood DNAm at TXNIP are significantly associated with changes in FPG in midlife, and this association was modified by BMI trendsduring childhood and adolescence.

DOP88 Understanding the molecular mechanisms of anti-TNF treatment failure: Whole blood DNA methylation changes associated with primary non-response to anti-TNF treatment in patients with Crohn’s disease

Abstract Background Anti-TNF treatment failure in patients with IBD is common and frequently related to low drug concentrations. DNA methylation sites from whole blood have been identified as effective biomarkers predicting treatment response to methotrexate and etanercept in patients with rheumatoid arthritis. We sought to define differences in DNA methylation associated with primary non-response (PNR) to anti-TNF treatment in patients with Crohn’s disease. Methods The Personalised Anti-TNF Therapy in Crohn’s disease (PANTS) study is a prospective UK-wide study investigating anti-TNF treatment failure in patients with active luminal Crohn’s disease. DNA methylation from whole blood was assessed using the Illumina EPIC Beadchip at baseline, weeks 14, 30 and 52. We compared DNA methylation profiles in a subset of adalimumab- and infliximab-treated patients who experienced PNR at week 14 and were not in remission at week 54 (infliximab = 99, adalimumab = 94) with patients who responded at week 14 and were in remission at week 54 (infliximab = 99, adalimumab = 93). Low anti-TNF drug concentration was defined as an infliximab level &amp;lt;3mg/L and adalimumab level &amp;lt;5mg/L. Epigenome-wide association (EWAS) analysis was performed using linear mixed models, where p values &amp;lt; 9 x 10-8 were considered significant. Because we observed significant changes in CD4 and CD8 T cells, B cells, NK cells, monocytes and granulocytes following anti-TNF treatment we adjusted our models for derived cell proportions (Figure 1). Results Overall, between baseline and week 14, we observed 4999 differentially methylated probes (DMPs) annotated to 2376 genes. Pathway analysis identified 108 significant gene ontology terms enriched in biological processes related to immune system processes and responses (Figure 2). EWAS analysis at baseline identified 48 DMPs annotated to 36 genes associated with PNR (Figure 3). The majority of DMPs (40/48) were hypermethylated (p &amp;lt; 0.001): of those annotated to genes, 25 were located in the gene body, 5 in the 5’ untranslated regions (UTR), 4 in transcription start sites and the remaining in 3’UTR. When EWAS analysis at baseline was stratified by anti-TNF drug concentration, 27 DMPs annotated to 17 genes were associated with PNR and low drug concentration. Of these, 11 DMPs were independently associated with low anti-TNF drug concentration at week 14 (Figure 4). We did not identify any DMPs associated with PNR with adequate drug concentration. Conclusion Our data suggests that baseline DNA methylation profiles may be used as a predictor for anti-TNF drug concentration at week 14.

The Role of Epigenetic Clocks in Explaining Educational Inequalities in Mortality: A Multicohort Study and Meta-analysis.

Educational inequalities in all-cause mortality have been observed for decades. However, the underlying biological mechanisms are not well known. We aimed to assess the role of DNA methylation changes in blood captured by epigenetic clocks in explaining these inequalities. Data were from 8 prospective population-based cohort studies, representing 13 021 participants. First, educational inequalities and their portion explained by Horvath DNAmAge, Hannum DNAmAge, DNAmPhenoAge, and DNAmGrimAge epigenetic clocks were assessed in each cohort via counterfactual-based mediation models, on both absolute (hazard difference) and relative (hazard ratio) scales, and by sex. Second, estimates from each cohort were pooled through a random effect meta-analysis model. Men with low education had excess mortality from all causes of 57 deaths per 10 000 person-years (95% confidence interval [CI]: 38, 76) compared with their more advantaged counterparts. For women, the excess mortality was 4 deaths per 10 000 person-years (95% CI: -11, 19). On the relative scale, educational inequalities corresponded to hazard ratios of 1.33 (95% CI: 1.12, 1.57) for men and 1.15 (95% CI: 0.96, 1.37) for women. DNAmGrimAge accounted for the largest proportion, approximately 50%, of the educational inequalities for men, while the proportion was negligible for women. Most of this mediation was explained by differential effects of unhealthy lifestyles and morbidities of the World Health Organization (WHO) risk factors for premature mortality. These results support DNA methylation-based epigenetic aging as a signature of educational inequalities in life expectancy emphasizing the need for policies to address the unequal social distribution of these WHO risk factors.

Gender-affirming hormone therapy induces specific DNA methylation changes in blood

BackgroundDNA methylation is an epigenetic mark that is influenced by underlying genetic profile, environment, and ageing. In addition to X-linked DNA methylation, sex-specific methylation patterns are widespread across autosomal chromosomes and can be present from birth or arise over time. In individuals where gender identity and sex assigned at birth are markedly incongruent, as in the case of transgender people, feminization or masculinization may be sought through gender-affirming hormone therapy (GAHT). GAHT is a cornerstone of transgender care, yet no studies to date have investigated its effect on genome-wide methylation. We profiled genome-wide DNA methylation in blood of transgender women (n = 13) and transgender men (n = 13) before and during GAHT (6 months and 12 months into feminizing or masculinizing hormone therapy).ResultsWe identified several thousand differentially methylated CpG sites (DMPs) (Δβ ≥ 0.02, unadjusted p value < 0.05) and several differentially methylated regions (DMRs) in both people undergoing feminizing and masculinizing GAHT, the vast majority of which were progressive changes over time. X chromosome and sex-specific autosomal DNA methylation patterns established in early development are largely refractory to change in association with GAHT, with only 3% affected (Δβ ≥ 0.02, unadjusted p value < 0.05). The small number of sex-specific DMPs that were affected by GAHT were those that become sex-specific during the lifetime, known as sex-and-age DMPs, including DMRs in PRR4 and VMP1 genes. The GAHT-induced changes at these sex-associated probes consistently demonstrated a shift towards the methylation signature of the GAHT-naïve opposite sex, and we observed enrichment of previously reported adolescence-associated methylation changes.ConclusionWe provide evidence for GAHT inducing a unique blood methylation signature in transgender people. This study advances our understanding of the complex interplay between sex hormones, sex chromosomes, and DNA methylation in the context of immunity. We highlight the need to broaden the field of ‘sex-specific’ immunity beyond cisgender males and cisgender females, as transgender people on GAHT exhibit a unique molecular profile.

Blood DNA Methylation Predicts Diabetic Kidney Disease Progression in High Fat Diet-Fed Mice.

Diabetic kidney disease (DKD) progresses at different rates among patients with type 2 diabetes mellitus (T2D). Early identification of patients with a higher risk of DKD progression is essential to improve prognosis. Epigenetic modifications, particularly DNA methylation, have been independently implicated in T2D and chronic kidney disease. The current study aimed to determine changes in blood DNA methylation that reflects and predicts DKD progression. C57BL/6 mice were fed a high-fat diet (HFD) from weaning and subclassified into two groups, HFD-1 and HFD-2, according to urinary kidney injury marker KIM-1/creatinine ratios (low vs. high) and histological abnormalities (mild–moderate vs. advanced). DNA methylation profiles were determined by reduced representative bisulfide sequencing (RRBS). Our results confirmed early and established DKD at week 9 and week 32, respectively. At week 32, advanced kidney injury was associated with dysregulation of methylation and demethylation enzymes in the kidney. Blood RRBS revealed 579 and 203 differentially methylated sites (DMS) between HFD-1 and HFD-2 animals at week 32 and week 9, respectively, among which 11 were common. The DMS in blood and kidney at week 32 were both related to organ development, neurogenesis, cell junction, and Wnt signalling, while the DMS in blood at week 9 suggested a specific enrichment of kidney development processes. In conclusion, our data strongly support the implication of early blood DNA methylation modifications and DKD progression in T2D that could be used to improve the disease’s prognostication.

Methylation biomarkers of polybrominated diphenyl ethers (PBDEs) and association with breast cancer risk at the time of menopause.

Exposure to polybrominated diphenyl ethers (PBDEs) may influence risk of developing post-menopausal breast cancer. Although mechanisms are poorly understood, epigenetic regulation of gene expression may play a role. To identify DNA methylation (DNAm) changes associated with PBDE serum levels and test the association of these biomarkers with breast cancer risk. We studied 397 healthy women (controls) and 133 women diagnosed with breast cancer (cases) between ages 40 and 58years who participated in the California Teachers Study. PBDE levels were measured in blood. Infinium Human Methylation EPIC Bead Chips were used to measure DNAm. Using multivariable linear regression models, differentially methylated CpG sites (DMSs) and regions (DMRs) associated with serum PBDE levels were identified using controls. For top-ranked DMSs and DMRs, targeted next-generation bisulfite sequencing was used to measure DNAm for 133 invasive breast cancer cases and 301 age-matched controls. Conditional logistic regression was used to evaluate associations between DMSs and DMRs and breast cancer risk. We identified 15 DMSs and 10 DMRs statistically significantly associated with PBDE levels (FDR<0.05). Methylation changes in a DMS at BMP8B and DMRs at TP53 and A2M-AS1 were statistically significantly (FDR<0.05) associated with breast cancer risk. We show for the first time that serum PBDE levels are associated with differential methylation and that PBDE-associated DNAm changes in blood are associated with breast cancer risk.

Longitudinal change in blood DNA epigenetic signature after smoking cessation

ABSTRACT Cigarette smoking is associated with epigenetic changes that may be reversible following smoking cessation. Whole blood DNA methylation was evaluated in Framingham Heart Study Offspring (n = 169) and Third Generation (n = 30) cohort participants at two study visits 6 years apart and in Atherosclerosis Risk in Communities (ARIC) study (n = 222) participants at two study visits 20 years apart. Changes in DNA methylation (delta β values) at 483,565 cytosine-phosphate-guanine (CpG) sites and differentially methylated regions (DMRs) were compared between participants who were current, former, or never smokers at both visits (current-current, former-former, never-never, respectively), versus those who quit in the interim (current-former). Interim quitters had more hypermethylation at four CpGs annotated to AHRR, one CpG annotated to F2RL3, and one intergenic CpG (cg21566642) compared with current-current smokers (FDR < 0.02 for all), and two significant DMRs were identified. While there were no significant differentially methylated CpGs in the comparison of interim quitters and former-former smokers, 106 DMRs overlapping with small nucleolar RNA were identified. As compared with all non-smokers, current-current smokers additionally had more hypermethylation at two CpG sites annotated to HIVEP3 and TMEM126A, respectively, and another intergenic CpG (cg14339116). Gene transcripts associated with smoking cessation were implicated in immune responses, cell homoeostasis, and apoptosis. Smoking cessation is associated with early reversion of blood DNA methylation changes at CpG sites annotated to AHRR and F2RL3 towards those of never smokers. Associated gene expression suggests a role of longitudinal smoking-related DNA methylation changes in immune response processes.

Impact of vitamin C supplementation on placental DNA methylation changes related to maternal smoking: association with gene expression and respiratory outcomes

BackgroundMaternal smoking during pregnancy (MSDP) affects development of multiple organ systems including the placenta, lung, brain, and vasculature. In particular, children exposed to MSDP show lifelong deficits in pulmonary function and increased risk of asthma and wheeze. Our laboratory has previously shown that vitamin C supplementation during pregnancy prevents some of the adverse effects of MSDP on offspring respiratory outcomes. Epigenetic modifications, including DNA methylation (DNAm), are a likely link between in utero exposures and adverse health outcomes, and MSDP has previously been associated with DNAm changes in blood, placenta, and buccal epithelium. Analysis of placental DNAm may reveal critical targets of MSDP and vitamin C relevant to respiratory health outcomes.ResultsDNAm was measured in placentas obtained from 72 smokers enrolled in the VCSIP RCT: NCT03203603 (37 supplemented with vitamin C, 35 with placebo) and 24 never-smokers for reference. Methylation at one CpG, cg20790161, reached Bonferroni significance and was hypomethylated in vitamin C supplemented smokers versus placebo. Analysis of spatially related CpGs identified 93 candidate differentially methylated regions (DMRs) between treatment groups, including loci known to be associated with lung function, oxidative stress, fetal development and growth, and angiogenesis. Overlap of nominally significant differentially methylated CpGs (DMCs) in never-smokers versus placebo with nominally significant DMCs in vitamin C versus placebo identified 9059 candidate “restored CpGs” for association with placental transcript expression and respiratory outcomes. Methylation at 274 restored candidate CpG sites was associated with expression of 259 genes (FDR < 0.05). We further identified candidate CpGs associated with infant lung function (34 CpGs) and composite wheeze (1 CpG) at 12 months of age (FDR < 0.05). Increased methylation in the DIP2C, APOH/PRKCA, and additional candidate gene regions was associated with improved lung function and decreased wheeze in offspring of vitamin C-treated smokers.ConclusionsVitamin C supplementation to pregnant smokers ameliorates changes associated with maternal smoking in placental DNA methylation and gene expression in pathways potentially linked to improved placental function and offspring respiratory health. Further work is necessary to validate candidate loci and elucidate the causal pathway between placental methylation changes and outcomes of offspring exposed to MSDP.Clinical trial registration ClinicalTrials.gov, NCT01723696. Registered November 6, 2012. https://clinicaltrials.gov/ct2/show/record/NCT01723696.

High polygenic burden is associated with blood DNA methylation changes in individuals with suicidal behavior

Suicidal behavior is result of the interaction of several contributors, including genetic and environmental factors. The integration of approaches considering the polygenic component of suicidal behavior, such as polygenic risk scores (PRS) and DNA methylation is promising for improving our understanding of the complex interplay between genetic and environmental factors in this behavior. The aim of this study was the evaluation of DNA methylation differences between individuals with high and low genetic burden for suicidality. The present study was divided into two phases. In the first phase, genotyping with the Psycharray chip was performed in a discovery sample of 568 Mexican individuals, of which 149 had suicidal behavior (64 individuals with suicidal ideation, 50 with suicide attempt and 35 with completed suicide). Then, a PRS analysis based on summary statistics from the Psychiatric Genomic Consortium was performed in the discovery sample. In a second phase, we evaluated DNA methylation differences between individuals with high and low genetic burden for suicidality in a sub-sample of the discovery sample (target sample) of 94 subjects. We identified 153 differentially methylated sites between individuals with low and high-PRS. Among genes mapped to differentially methylated sites, we found genes involved in neurodevelopment (CHD7, RFX4, KCNA1, PLCB1, PITX1, NUMBL) and ATP binding (KIF7, NUBP2, KIF6, ATP8B1, ATP11A, CLCN7, MYLK, MAP2K5). Our results suggest that genetic variants might increase the predisposition to epigenetic variations in genes involved in neurodevelopment. This study highlights the possible implication of polygenic burden in the alteration of epigenetic changes in suicidal behavior.

Prenatal smoke effect on mouse offspring Igf1 promoter methylation from fetal stage to adulthood is organ and sex specific.

Prenatal smoke exposure (PSE) is associated with reduced birth weight, impaired fetal development, and increased risk for diseases later in life. Changes in DNA methylation may be involved, as multiple large-scale epigenome-wide association studies showed that PSE is robustly associated with DNA methylation changes in blood among offspring in early life. Insulin-like growth factor-1 (IGF1) is important in growth, differentiation, and repair processes after injury. However, no studies investigated the organ-specific persistence of PSE-induced methylation change of Igf1 into adulthood. Based on our previous studies on the PSE effect on Igf1 promoter methylation in fetal and neonatal mouse offspring, we now have extended our studies to adulthood. Our data show that basal Igf1 promoter methylation generally increased in the lung but decreased in the liver (except for 2 persistent CpG sites in both organs) across three different developmental stages. PSE changed Igf1 promoter methylation in all three developmental stages, which was organ and sex specific. The PSE effect was less pronounced in adult offspring compared with the fetal and neonatal stages. In addition, the PSE effect in the adult stage was more pronounced in the lung compared with the liver. For most CpG sites, an inverse correlation was found for promoter methylation and mRNA expression when the data of all three stages were combined. This was more prominent in the liver. Our findings provide additional evidence for sex- and organ-dependent prenatal programming, which supports the developmental origins of health and disease (DOHaD) hypothesis.

Alcohol consumption is associated with widespread changes in blood DNA methylation: Analysis of cross-sectional and longitudinal data.

DNA methylation may be one of the mechanisms by which alcohol consumption is associated with the risk of disease. We conducted a large-scale, cross-sectional, genome-wide DNA methylation association study of alcohol consumption and a longitudinal analysis of repeated measurements taken several years apart. Using the Illumina HumanMethylation450 BeadChip, DNA methylation was measured in blood samples from 5606 Melbourne Collaborative Cohort Study (MCCS) participants. For 1088 of them, these measures were repeated using blood samples collected a median of 11 years later. Associations between alcohol intake and blood DNA methylation were assessed using linear mixed-effects regression models. Independent data from the London Life Sciences Prospective Population (LOLIPOP) (N = 4042) and Cooperative Health Research in the Augsburg Region (KORA) (N = 1662) cohorts were used to replicate associations discovered in the MCCS. Cross-sectional analyses identified 1414 CpGs associated with alcohol intake at P < 10-7 , 1243 of which had not been reported previously. Of these novel associations, 1078 were replicated (P < .05) using LOLIPOP and KORA data. Using the MCCS data, we also replicated 403 of 518 previously reported associations. Interaction analyses suggested that associations were stronger for women, non-smokers, and participants genetically predisposed to consume less alcohol. Of the 1414 CpGs, 530 were differentially methylated (P < .05) in former compared with current drinkers. Longitudinal associations between the change in alcohol intake and the change in methylation were observed for 513 of the 1414 cross-sectional associations. Our study indicates that alcohol intake is associated with widespread changes in DNA methylation across the genome. Longitudinal analyses showed that the methylation status of alcohol-associated CpGs may change with alcohol consumption changes in adulthood.

Peripheral DNA methylation, cognitive decline and brain aging: pilot findings from the Whitehall II imaging study.

Aim:The present study investigated the link between peripheral DNA methylation (DNAm), cognitive impairment and brain aging.Methods:We tested the association between blood genome-wide DNAm profiles using the Illumina 450K arrays, cognitive dysfunction and brain MRI measures in selected participants of the Whitehall II imaging sub-study.Results:Eight differentially methylated regions were associated with cognitive impairment. Accelerated aging based on the Hannum epigenetic clock was associated with mean diffusivity and global fractional anisotropy. We also identified modules of co-methylated loci associated with white matter hyperintensities. These co-methylation modules were enriched among pathways relevant to β-amyloid processing and glutamatergic signaling.Conclusion:Our data support the notion that blood DNAm changes may have utility as a biomarker for cognitive dysfunction and brain aging.

An epigenome-wide study of cord blood DNA methylations in relation to prenatal perfluoroalkyl substance exposure: The Hokkaido study

BackgroundPrenatal exposure to perfluoroalkyl substances (PFASs) influences fetal development and later in life. ObjectiveTo investigate cord blood DNA methylation changes associated with prenatal exposure to PFASs. MethodsWe assessed DNA methylation in cord blood samples from 190 mother-child pairs from the Sapporo cohort of the Hokkaido Study (discovery cohort) and from 37 mother-child pairs from the Taiwan Maternal and Infant Cohort Study (replication cohort) using the Illumina HumanMethylation 450 BeadChip. We examined the associations between methylation and PFAS levels in maternal serum using robust linear regression models and identified differentially methylated positions (DMPs) and regions (DMRs). ResultsWe found four DMPs with a false discovery rate below 0.05 in the discovery cohort. Among the top 20 DMPs ranked by the lowest P-values for perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) exposure, four DMPs showed the same direction of effect and P-value < 0.05 in the replication assay: cg16242615 mapped to ZBTB7A, cg21876869 located in the intergenic region (IGR) of USP2-AS1, cg00173435 mapped to TCP11L2, and cg18901140 located in IGR of NTN1. For DMRs, we found a region associated with PFOA exposure with family-wise error rate < 0.1 located in ZFP57, showing the same direction of effect in the replication cohort. Among the top five DMRs ranked by the lowest P-values that were associated with exposure to PFOS and PFOA, in addition to ZFP57, DMRs in the CYP2E1, SMAD3, SLC17A9, GFPT2, DUSP22, and TCERG1L genes showed the same direction of effect in the replication cohort. ConclusionWe suggest that prenatal exposure to PFASs may affect DNA methylation status at birth. Longitudinal studies are needed to examine whether methylation changes observed are associated with differential health outcomes.

Abstract 74: Analysis of Genome-Wide Blood DNA Methylation Profile in Moyamoya Disease

Background: Moyamoya disease (MMD) is a rare cerebrovascular disorder of unknown cause. Epigenetic regulations such as methylation play key role in gene expression and cerebrovascular diseases, however, methylation changes in MMD has not been previously reported. To understand the methylation profile of MMD, we performed a genome-wide DNA methylation analysis of whole blood from MMD patients. Method: Twenty-eight female adult patients and age-/sex- matched 10 healthy control volunteers (34.2 ± 1.3y) were included. All patients developed TIA or headache and were diagnosed as typical MMD (n=19 and 9), predominantly with Suzuki’s angiographical stage 3. DNA was extracted from frozen whole blood and processed with bisulfide conversion. Genome-wide DNA methylation profile was screened using Illumina 850K EPIC microarray. Blood DNA methylome and clinical characteristics were analyzed. Results: Self-declared race was predominantly Asian or Caucasian (n=14 and 13 in MMD, n=6 and 3 in control). Eleven patients showed asymptomatic cerebral infarct. MMD susceptible variant (rs112735431) was confirmed only in 4 Asian MMD. We observed distinct blood DNA methylation profile between Asian and Caucasian. There was no significant correlation between the methylation profile and symptom, infarct lesion, nor MMD susceptible variant. Our data revealed 51% of differentially methylated CpG probes (DMPs) in Caucasian MMD (P&lt;0.01), located at CpG island or shore (51% of all the 5782 DMPs), and 3742 genes were regulated by these island/shore locating DMPs. IPA disease and bio-function profiler indicated that these genes involved in the adhesion of T lymphocytes and monocytes, as well as transmigration of peripheral blood lymphocytes. Conclusion: Our results indicate that significant methylation changes occur in the blood of MMD patients. In Caucasian MMD, circulating T lymphocytes and monocytes may have a role in disease development, since these cells are known to participate in thickened fibrocellular intima in MMD. Ongoing studies are identifying the correlation of DNA methylation in blood and gene expression in the vascular tissue. Blood DNA methylation changes may be related with the risk of Caucasian MMD.

P2.01-036 Identification of a Novel Oncogenic Ubiquitin Ligase from a Lung Cancer Epigenome-Wide Association Study (EWAS)

Lung cancer (LC) is the leading cancer-related cause of death worldwide with a 5-year survival rate of only 8%. Smoking is the main risk factor for lung malignancies, but not every smoker develops LC. DNA methylation alterations in tumor tissue lead to genome instability and thus can contribute to malignant cell transformation. Smoking-associated blood DNA methylation changes have been shown to indicate LC risk. We aimed to investigate differential blood DNA methylation in healthy smokers for the identification of novel oncogenes. We performed Illumina 450K epigenome-wide DNA methylation arrays for 66 smoking prediagnostic lung cancer case-control pairs. The blood DNA samples for this nested case-control design came from the European Prospective Investigation into Cancer and Nutrition (EPIC) Heidelberg cohort. Differentially methylated candidate CpGs were then tested in lung tumor versus adjacent normal tissue for differential methylation in multiple patient sample sets. Additionally, we tested whether differential methylation leads to differential gene expression in lung tumor versus adjacent normal tissue. The top candidate CpG proximal gene was evaluated in functional LC cell based assays to investigate the consequences of its elevated expression in LC. The top differentially methylated candidate CpGs from EPIC were also found differentially methylated in lung tumor versus adjacent normal tissue. The two top hypermethylated CpGs were located within differentially methylated regions (DMRs) and the proximal or associated genes were differentially expressed in lung tumors. The top DMR revealed the regulation of gene expression by DNA methylation of a downstream ubiquitin E3 ligase. Deregulated expression of that gene was associated with LC cell proliferation, migration and glucose uptake in vitro. The gene was found to be involved in the activation of AKT by mTORC2. When the gene was knocked down, apoptotic genes which are suppressed by activated AKT in LC cells were re-expressed. Expression of a cell cycle promoting regulator stimulated by activated AKT was found repressed in LC knock down cells. We identified differential DNA methylation in blood from healthy smokers who later developed LC. The top differentially methylated CpGs were also differentially methylated in lung tumor versus adjacent normal tissue samples. The top DMR lead to expression of a proximal ubiquitin E3 ligase. We showed, that this gene is crucial for the mTORC2-mediated activation of AKT. This ubiquitin E3 ligase has not previously been associated with cancer but our findings identify it as a novel epigenetically deregulated LC oncogene.

Epigenome-wide analyses of prenatal perfluoroalkyl acids exposure on cord blood DNA methylation: the Hokkaido study

Prenatal exposure to perfluoroalkyl acids (PFAAs) can modify fetal metabolic pathways leading to obesity in later years. One possible mechanism is through epigenetic modification in utero. However, whether prenatal PFAA exposure can influence infant DNA methylation has not been rigorously evaluated in epidemiological studies. Here, we assessed epigenome–wide cord blood DNA methylation changes in relation to prenatal PFAA exposure. Mother-child pairs (N=164) from the Hokkaido Study on Environment and Children’s Health were included in the study. Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) levels in maternal serum during pregnancy were measured by liquid chromatography-tandem mass spectrometry. Cord blood methylation was determined by Illumina HumanMethylation 450 BeadChip. Robust linear regression was applied for the associations between DNA methylation and PFAAs. We selected CpGs with either i) false–discovery rate &lt; 0.05, or ii) partial regression coefficient ≥ 0.075 (PFOS) or ≥ 0.05 (PFOA), and then identified genes that showed coherent hypo– or hypermethylation at more than 3 CpGs on a locus. Among 368 and 616 CpGs associated, 26 CpGs on 7 genes and 43 CpGs on 11 genes were identified in relation to PFOS and PFOA exposure, respectively. We found several genes associated with energy metabolism: i.e. In association with PFOS, RPH3AL cg17193961 (β=0.09, p=0.01) and 2 other CpGs on the locus were hypermethylated, which encodes a protein playing a key role in insulin secretion; With PFOA, GALNT2 cg24250902 (β=-0.06, p= 1.3x10-6) and 3 CpGs were hypomethylated, which encodes glycosyltransferase and may influence triglyceride levels. We also found hypomethylation of a histone deacetylase gene, HDAC4 cg20784693 (β=-0.10, p= 2.9x10-3) and 3 CpGs, which involves glucose homeostasis. Our results suggest prenatal PFAA exposure can epigenetically modify genes involved in energy metabolism. Further study is required to replicate our findings.

Integrative DNA methylome analysis of pan-cancer biomarkers in cancer discordant monozygotic twin-pairs.

BackgroundA key focus in cancer research is the discovery of biomarkers that accurately diagnose early lesions in non-invasive tissues. Several studies have identified malignancy-associated DNA methylation changes in blood, yet no general cancer biomarker has been identified to date. Here, we explore the potential of blood DNA methylation as a biomarker of pan-cancer (cancer of multiple different origins) in 41 female cancer discordant monozygotic (MZ) twin-pairs sampled before or after diagnosis using the Illumina HumanMethylation450 BeadChip.ResultsWe analysed epigenome-wide DNA methylation profiles in 41 cancer discordant MZ twin-pairs with affected individuals diagnosed with tumours at different single primary sites: the breast, cervix, colon, endometrium, thyroid gland, skin (melanoma), ovary, and pancreas. No significant global differences in whole blood DNA methylation profiles were observed. Epigenome-wide analyses identified one novel pan-cancer differentially methylated position at false discovery rate (FDR) threshold of 10 % (cg02444695, P = 1.8 × 10−7) in an intergenic region 70 kb upstream of the SASH1 tumour suppressor gene, and three suggestive signals in COL11A2, AXL, and LINC00340. Replication of the four top-ranked signals in an independent sample of nine cancer-discordant MZ twin-pairs showed a similar direction of association at COL11A2, AXL, and LINC00340, and significantly greater methylation discordance at AXL compared to 480 healthy concordant MZ twin-pairs. The effects at cg02444695 (near SASH1), COL11A2, and LINC00340 were the most promising in biomarker potential because the DNA methylation differences were found to pre-exist in samples obtained prior to diagnosis and were limited to a 5-year period before diagnosis. Gene expression follow-up at the top-ranked signals in 283 healthy individuals showed correlation between blood methylation and gene expression in lymphoblastoid cell lines at PRL, and in the skin tissue at AXL. A significant enrichment of differential DNA methylation was observed in enhancer regions (P = 0.03).ConclusionsWe identified DNA methylation signatures in blood associated with pan-cancer, at or near SASH1, COL11A2, AXL, and LINC00340. Three of these signals were present up to 5 years prior to cancer diagnosis, highlighting the potential clinical utility of whole blood DNA methylation analysis in cancer surveillance.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-016-0172-y) contains supplementary material, which is available to authorized users.