Infinium HumanMethylation450 BeadChip Research Articles

Genome-Wide DNA Methylation Signatures Predict the Early Asymptomatic Doxorubicin-Induced Cardiotoxicity in Breast Cancer.

Simple SummaryThis study examined whether the DNA methylation state of peripheral blood mononuclear cells (PBMCs) could predict cardiotoxicity caused by doxorubicin (DOX)-based chemotherapy in breast cancer patients. The results showed a significant difference in the pattern of DNA methylation of PBMCs associated with a risk of cardiotoxicity. These preliminary findings have the potential to further the goal of personalized medicine and tailor the treatment of breast cancer with DOX-based chemotherapy to reduce the toxicity to the heart.Chemotherapy with doxorubicin (DOX) may cause unpredictable cardiotoxicity. This study aimed to determine whether the methylation signature of peripheral blood mononuclear cells (PBMCs) prior to and after the first cycle of DOX-based chemotherapy could predict the risk of cardiotoxicity in breast cancer patients. Cardiotoxicity was defined as a decrease in left ventricular ejection fraction (LVEF) by >10%. DNA methylation of PBMCs from 9 patients with abnormal LVEF and 10 patients with normal LVEF were examined using Infinium HumanMethylation450 BeadChip. We have identified 14,883 differentially methylated CpGs at baseline and 18,718 CpGs after the first cycle of chemotherapy, which significantly correlated with LVEF status. Significant differentially methylated regions (DMRs) were found in the promoter and the gene body of SLFN12, IRF6 and RNF39 in patients with abnormal LVEF. The pathway analysis found enrichment for regulation of transcription, mRNA splicing, pathways in cancer and ErbB2/4 signaling. The preliminary results from this study showed that the DNA methylation profile of PBMCs may predict the risk of DOX-induced cardiotoxicity prior to chemotherapy. Further studies with larger cohorts of patients are needed to confirm these findings.

Aryl Hydrocarbon Receptor Repressor Is Hypomethylated in Psoriasis and Promotes Psoriasis-like Inflammation in HaCaT Cells.

It is known that DNA hypomethylation of aryl hydrocarbon receptor repressor (AhRR), one of the epigenetic markers of environmental pollutants, causes skin diseases. However, the function and mechanisms are still unknown. We aimed to determine whether AhRR is hypomethylated in PBMC of psoriasis patients, as well as to examine the expression of psoriasis-related inflammatory cytokines and antimicrobial peptides after 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treatment in HaCaT cells overexpressing or silencing AhRR. AhRR was determined by qPCR, Western blot, immunohistochemistry, and immunocytochemistry in skin tissue and HaCaT cells. DNA methylation of AhRR was performed by Infinium Human Methylation450 BeadChip in PBMC of psoriasis patients and methylation-specific PCR (MSP) in HaCaT cells. NF-κB pp50 translocation and activity were performed by immunocytochemistry and luciferase reporter assay, respectively. We verified AhRR gene expression in the epidermis from psoriasis patients and healthy controls. AhRR hypomethylation in PBMC of psoriasis patients and pAhRR-HaCaT cells was confirmed. The expression level of AhRR was increased in both TCDD-treated HaCaT cells and pAhRR-HaCaT cells. NF-κB pp50 translocation and activity increased with TCDD. Our results showed that AhRR was hypomethylated and overexpressed in the lesional skin of patients with psoriasis, thereby increasing AhRR gene expression and regulating pro-inflammatory cytokines through the NF-κB signaling pathway in TCDD-treated HaCaT cells.

Distinct DNA Methylation Patterns of Subependymal Giant Cell Astrocytomas in Tuberous Sclerosis Complex

Tuberous sclerosis complex (TSC) is a monogenic disorder caused by mutations in either the TSC1 or TSC2 gene, two key regulators of the mechanistic target of the rapamycin complex pathway. Phenotypically, this leads to growth and formation of hamartomas in several organs, including the brain. Subependymal giant cell astrocytomas (SEGAs) are low-grade brain tumors commonly associated with TSC. Recently, gene expression studies provided evidence that the immune system, the MAPK pathway and extracellular matrix organization play an important role in SEGA development. However, the precise mechanisms behind the gene expression changes in SEGA are still largely unknown, providing a potential role for DNA methylation. We investigated the methylation profile of SEGAs using the Illumina Infinium HumanMethylation450 BeadChip (SEGAs n = 42, periventricular control n = 8). The SEGA methylation profile was enriched for the adaptive immune system, T cell activation, leukocyte mediated immunity, extracellular structure organization and the ERK1 & ERK2 cascade. More interestingly, we identified two subgroups in the SEGA methylation data and show that the differentially expressed genes between the two subgroups are related to the MAPK cascade and adaptive immune response. Overall, this study shows that the immune system, the MAPK pathway and extracellular matrix organization are also affected on DNA methylation level, suggesting that therapeutic intervention on DNA level could be useful for these specific pathways in SEGA. Moreover, we identified two subgroups in SEGA that seem to be driven by changes in the adaptive immune response and MAPK pathway and could potentially hold predictive information on target treatment response.

Adipose tissue and blood leukocytes ACE2 DNA methylation in obesity and after weight loss.

Obesity was consistently associated with a poor prognosis in patients with COVID-19. Epigenetic mechanisms were proposed as the link between obesity and comorbidities risk. To evaluate the methylation levels of angiotensin-converting enzyme 2 (ACE2) gene, the main entry receptor of SARS-CoV-2, in different depots of adipose tissue (AT) and leukocytes (PBMCs) in obesity and after weight loss therapy based on a very-low-calorie ketogenic diet (VLCKD), a balanced hypocaloric diet (HCD) or bariatric surgery (BS). DNA methylation levels of ACE2 were extracted from our data sets generated by the hybridization of subcutaneous (SAT) (n=32) or visceral (VAT; n=32) adipose tissue, and PBMCs (n=34) samples in Infinium HumanMethylation450 BeadChips. Data were compared based on the degree of obesity and after 4-6months of weight loss either by following a nutritional or surgical treatment and correlated with ACE2 transcript levels. As compared with normal weight, VAT from patients with obesity showed higher ACE2methylation levels. These differences were mirrored in PBMCs but not in SAT. The observed obesity-associated methylation of ACE2 was reversed after VLCKD and HCD but not after BS. Among the studied CpG sites, cg16734967 and cg21598868, located at the promoter, were the most affected and correlated with BMI. The observed DNA methylation pattern was inversely correlated with ACE2 expression. Obesity-related VAT shows hypermethylation and downregulation of the ACE2gene that is mirrored in PBMCs and is restored after nutritional weight reduction therapy. The results warrant the necessity to further evaluate its implication for COVID-19 pathogenesis.

A panel of DNA methylation markers for the classification of consensus molecular subtypes 2 and 3 in patients with colorectal cancer

Consensus molecular subtypes (CMSs) can guide precision treatment of colorectal cancer (CRC). We aim to identify methylation markers to distinguish between CMS2 and CMS3 in patients with CRC, for which an easy test is currently lacking. To this aim, fresh‐frozen tumor tissue of 239 patients with stage I‐III CRC was analyzed. Methylation profiles were obtained using the Infinium HumanMethylation450 BeadChip. We performed adaptive group‐regularized logistic ridge regression with post hoc group‐weighted elastic net marker selection to build prediction models for classification of CMS2 and CMS3. The Cancer Genome Atlas (TCGA) data were used for validation. Group regularization of the probes was done based on their location either relative to a CpG island or relative to a gene present in the CMS classifier, resulting in two different prediction models and subsequently different marker panels. For both panels, even when using only five markers, accuracies were > 90% in our cohort and in the TCGA validation set. Our methylation marker panel accurately distinguishes between CMS2 and CMS3. This enables development of a targeted assay to provide a robust and clinically relevant classification tool for CRC patients.

Prediction of tissue origin of adenocarcinomas in the esophagogastric junction by DNA methylation.

Prediction of tissue origin of esophagogastric junction (EGJ) adenocarcinomas can be important for therapeutic decision, but no molecular marker is available. Here, we aimed to develop such a marker taking advantage of tissue-specific profiles of DNA methylation. DNA methylation profiles of gastric adenocarcinomas (GACs) were obtained by an Infinium HumanMethylation450 BeadChip array, and those of esophageal adenocarcinoma (EACs) were obtained from the TCGA database. DNA from formalin-fixed paraffin-embedded (FFPE) samples was analyzed by bisulfite pyrosequencing. In the screening set, 51 of 145,841 CpG sites in CpG islands were methylated at significantly higher levels in 30 GACs compared to those in 30 EACs. Among them, SLC46A3 and cg09177106 were unmethylated in all the 30 EACs. Predictive powers of these two markers were successfully confirmed in an independent validation set (18 GACs and 18 EACs) (SLC46A3, sensitivity = 77.8%, specificity = 100%; cg09177106, sensitivity = 83.3%, specificity = 94.4%), and could be applied to FFPE samples (37 GACs and 18 EACs) (SLC46A3, P = 0.0001; cg09177106, P = 0.0028). On the other hand, EAC-specific markers informative in the FFPE samples could not be isolated. Using these GAC-specific markers, nine of 46 (19.6%) TCGA EGJ adenocarcinomas were predicted to be GACs. Two GAC-specific markers, SLC46A3 and cg09177106, had a high specificity for identifying the tissue origin of EGJ adenocarcinoma.

Epigenome-wide association study detects a novel loci associated with central obesity in healthy subjects

Background and aimsCentral obesity is a condition that poses a significant risk to global health and requires the employment of novel scientific methods for exploration. The objective of this study is to use DNA methylation analysis to detect DNA methylation loci linked to obesity phenotypes, i.e. waist circumference and waist-to-hip ratio adjusted for BMI.Methods and resultsTwo-hundred and ten healthy European participants from the STANISLAS Family Study (SFS), comprising 73 nuclear families, were comprehensively assessed for methylation status using Illumina Infinium HumanMethylation450 BeadChip. An epigenome-wide association study was performed, which identified a CpG site cg16170243 located on chromosome 18q21.2 significantly associated with waist circumference, after adjusting for BMI (β = 2.32, SE = 0.41, Padj = 0.048). Cg16170243 corresponds to a 50 bp-length human methylation oligoprobe located within the AC090241.2 gene that overlaps ST8SIA5 gene. No significant association was observed with waist-to-hip ratio adjusted for BMI (Padj > 0.05).ConclusionsA novel association between DNA methylation and WC was identified, which is demonstrating that epigenetic mechanisms may have a significant impact on waist circumference ratio in healthy individuals. Further studies are warranted to address the causal effects of this association.

Epigenetic aging biomarkers and occupational exposure to benzene, trichloroethylene and formaldehyde

Epigenetic aging biomarkers are associated with increased morbidity and mortality. We evaluated if occupational exposure to three established chemical carcinogens is associated with acceleration of epigenetic aging. We studied workers in China occupationally exposed to benzene, trichloroethylene (TCE) or formaldehyde by measuring personal air exposures prior to blood collection. Unexposed controls matched by age and sex were selected from nearby factories. We measured leukocyte DNA methylation (DNAm) in peripheral white blood cells using the Infinium HumanMethylation450 BeadChip to calculate five epigenetic aging clocks and DNAmTL, a biomarker associated with leukocyte telomere length and cell replication. We tested associations between exposure intensity and epigenetic age acceleration (EAA), defined as the residuals of regressing the DNAm aging biomarker on chronological age, matching factors and potential confounders. Median differences in EAA between exposure groups were tested using a permutation test with exact p-values. Epigenetic clocks were strongly correlated with age (Spearman r > 0.8) in all three occupational studies. There was a positive exposure-response relationship between benzene and the Skin-Blood Clock EAA biomarker: median EAA was −0.91 years in controls (n = 44), 0.78 years in workers exposed to <10 ppm (n = 41; mean benzene = 1.35 ppm; p = 0.034 vs. controls), and 2.10 years in workers exposed to ≥10 ppm (n = 9; mean benzene = 27.3 ppm; p = 0.019 vs. controls; ptrend = 0.0021). In the TCE study, control workers had a median Skin-Blood Clock EAA of −0.54 years (n = 71) compared to 1.63 years among workers exposed to <10 ppm of TCE (n = 27; mean TCE = 4.22 ppm; p = 0.035). We observed no evidence of EAA associations with formaldehyde exposure (39 controls, 31 exposed). Occupational benzene and TCE exposure were associated with increased epigenetic age acceleration measured by the Skin-Blood Clock. For TCE, there was some evidence of epigenetic age acceleration for lower exposures compared to controls. Our results suggest that some chemical carcinogens may accelerate epigenetic aging.

Association between dietary patterns reflecting one-carbon metabolism nutrients intake before pregnancy and placental DNA methylation

ABSTRACT The preconception period represents an important window for foetal and epigenetic programming. Some micronutrients (B vitamins, choline, betaine, methionine) implicated in one-carbon metabolism (OCM) are essential for major epigenetic processes that take place in early pregnancy. However, few studies have evaluated the implication of the micronutrients in placental DNA methylation. We investigated whether intake of OCM nutrients in the year before pregnancy was associated with placental DNA methylation in the EDEN mother–child cohort. Maternal dietary intake was assessed with a food-frequency questionnaire. Three dietary patterns, ‘varied and balanced diet,’ ‘vegetarian tendency,’ and ‘bread and starchy food,’ were used to characterize maternal OCM dietary intake. The Illumina Infinium HumanMethylation450 BeadChip was used to measure placental DNA methylation of 573 women included in the analyses. We evaluated the association of dietary patterns with global DNA methylation. Then, we conducted an agnostic epigenome-wide association study (EWAS) and investigated differentially methylated regions (DMRs) associated with each dietary pattern. We found no significant association between the three dietary patterns and global DNA methylation or individual CpG sites. DMR analyses highlighted associations between the ‘varied and balanced’ or ‘vegetarian tendency’ pattern and DMRs located at genes previously implicated in functions essential for embryonic development, such as neurodevelopment. The ‘bread and starchy food’ pattern was associated with regions related to genes whose functions involve various metabolic and cell synthesis-related processes. In mainly well-nourished French women without major deficiencies, OCM intake before pregnancy was not associated with major variation in DNA methylation.

Methylation markers associated with air pollution exposure and cardiometabolic health in the Framingham Offspring Study

BACKGROUND AND AIM: We conducted a systematic literature review to identify methylation (CpG) sites associated with air pollution exposure. We assessed if these CpG sites were associated with air pollution and cardiometabolic health outcomes in the Framingham Offspring Study. We hypothesized that associated CpG sites could provide mechanistic insight into cardiometabolic health effects of air pollution. METHODS: We used data from 480 Framingham Offspring Study participants with epigenome-wide methylation profiles (Illumina Infinium HumanMethylation450 BeadChip on blood-derived DNA) assessed at the eighth examination cycle (2005-2008; 70% men; mean age=71 years). We examined cross-sectional associations between 2358 CpG sites identified through our systematic literature review and (1) short-term air pollutant exposures (1- and 28-day average particle number concentration, nitrogen dioxide, black carbon (BC), and particulate matter (PM2.5); linear regression); (2) annual average BC and PM2.5 exposure (from 2000; linear regression); and (3) cardiovascular disease or cardiometabolic multi-morbidity (CVD/CMD; logistic regression). We assessed longitudinal associations between methylation markers and cardiometabolic outcomes (CVD/CMD at the ninth examination cycle or CVD-related mortality; logistic regression). All models adjusted for age, sex, body mass index, and current smoking. We a priori chose p0.001 as the threshold for statistical significance (recognizing findings could still be due to chance). RESULTS:We identified 16 CpGs associated with ≥1 air pollutant. One CpG corresponding to a gene encoding serine/threonine protein kinases was associated with both 28-day BC and PM2.5. Thirteen CpGs associated cross-sectionally with CVD/CMD, and 42 CpGs associated longitudinally with CVD/CMD/CVD-related mortality (11 overlapping CpGs; 0 overlapping with air pollutant-associated CpGs). Four CpGs associated with air pollutants and seven CpGs associated cross-sectionally with CVD/CMD correspond to genes involving serine/threonine kinases. CpGs associated with air pollutants and health outcomes also corresponded to genes involved in inflammation/immunity (5 CpGs) and transcription (12 CpGs). CONCLUSIONS:Serine/threonine protein kinases may affect biologic pathways relating air pollution exposure to cardiometabolic health. KEYWORDS: epigenomics, molecular epidemiology, long-term exposure, short-term exposure, cardiovascular diseases

Elevated methylation of the vault RNA2-1 promoter in maternal blood is associated with preterm birth

BackgroundPreterm birth, defined as parturition before 37 completed weeks of gestation, is associated with an increased risk of neonatal complications and death, as well as poor health and disease later in life. Epigenetics could contribute to the mechanism underlying preterm birth.ResultsGenome-wide DNA methylation analysis of whole blood cells from 10 women (5 term and 5 preterm deliveries) was performed using an Illumina Infinium HumanMethylation450 BeadChips array. We identified 1,581 differentially methylated CpG sites in promoter regions between term and preterm birth. Although the differences were not significant after correcting for multiple tests, seven CpGs on the genomically imprinted vault RNA2-1 (VTRNA2-1; also known as non-coding RNA, nc886 or miR-886) showed the largest differences (range: 26–39 %). Pyrosequencing verification was performed with blood samples from pregnant women recruited additionally (39 term and 43 preterm deliveries). In total, 28 (34.1 %) samples showed hypomethylation of the VTRNA2-1 promoter (< 13 % methylation), while 54 (65.9 %) samples showed elevated methylation levels between 30 and 60 %. Elevated methylation of VTRNA2-1 promoter was associated with an increased risk of preterm birth after adjusting for maternal age, season of delivery, parity and white blood cell count. The mRNA expression of VTRNA2-1 was 0.51-fold lower in women with preterm deliveries (n = 20) compared with women with term deliveries (n = 20).ConclusionsVTRNA2-1 is a noncoding transcript to environmentally responsive epialleles. Our results suggest that elevated methylation of the VTRNA2-1 promoter may result in increased risk of PTB caused by the pro-inflammatory cytokines. Further studies are needed to confirm the association of VTRNA2-1 methylation with preterm birth in a large population, and to elucidate the underlying mechanism.

Aging biological markers in a cohort of antipsychotic-naïve first-episode psychosis patients

Schizophrenia is a severe and multifactorial disorder with an unknown causative pathophysiology. Abnormalities in neurodevelopmental and aging processes have been reported. Relative telomere length (RTL) and DNA methylation age (DMA), well-known biomarkers for estimating biological age, are both commonly altered in patients with schizophrenia compared to healthy controls. However, few studies investigated these aging biomarkers in first-episode psychosis (FEP) and in antipsychotic-naïve patients. To cover the existing gap regarding DMA and RTL in FEP and antipsychotic treatment, we aimed to verify whether those aging markers could be associated with psychosis and treatment response. Thus, we evaluated these measures in the blood of FEP antipsychotic-naïve patients and healthy controls (HC), as well as the response to antipsychotics after 10 weeks of treatment with risperidone. RTL was measured in 392 subjects, being 80 FEP and 312 HC using qPCR, while DMA was analyzed in a subset of 60 HC, 60 FEP patients (antipsychotic-naïve) and 59 FEP-10W (after treatment) using the “Multi-tissue Predictor”and the Infinium HumanMethylation450 BeadChip Kit. We observed diminished DMA and longer RTL in FEP patients before treatment compared to healthy controls, indicating a decelerated aging process in those patients. We found no statistical difference between responder and non-responder patients at baseline for both markers. An increased DMA was observed in patients after 10 weeks of treatment, however, after adjusting for blood cell composition, no significant association remained. Our findings indicate a decelerated aging process in the early phases of the disease.

Abstract 2094: Correaltions between genome-wide DNA methylation profiles and genomic driver aberrations during multistage lung adenocaricinogenesis

Abstract The aim of this study was to clarify correlations between epigenomic and genomic alterations during multistage lung adenocarcinogenesis. Single-CpG resolution genome-wide DNA methylation analysis with the Infinium HumanMethylation27 BeadChip was performed using 162 paired samples of non-cancerous lung tissue (N) and corresponding tumorous tissue (T) from patients with lung adenocarcinomas. Correlations between DNA methylation data on the one hand and clinicopathological parameters and genomic driver mutations, i.e. mutations of EGFR, KRAS, BRAF, and HER2 and fusion involving ALK, RET, and ROS1, were examined. In N samples, which were considered to be at precancerous stages, DNA methylation levels in 12,629 probes were significantly correlated with recurrence-free survival. DNA methylation profiles at the precancerous N stages may determine the prognostic outcome. Principal component analysis revealed that distinct DNA methylation profiles at the precancerous N stage tended not to induce specific genomic driver mutations. Most of the genes showing significant DNA methylation alterations during transition from N to T were shared by two or more driver aberration groups, indicating that, in general, each driver mutation does not induce a specific DNA methylation profile. Commonly shared DNA methylation alterations which could potentially result in expression abnormalities were enriched among transcriptional factors. In contrast, only 67 genes showed EGFR mutation-specific or “pan-negative”-specific DNA methylation alterations. Among the 67 genes, most of the DNA methylation alterations were correlated with clinicopathological parameters reflecting tumor aggressiveness. We further focused on the ZNF132 gene, for which DNA hypermethylation had been observed only in the “pan-negative”-type lung adenocarcinomas and was significantly correlated with their vascular invasion. 5-aza-2'-deoxycytidine treatment restored the expression levels of ZNF132 mRNA in lung adenocarcinoma cell lines, indicating that ZNF132 had been silenced due to DNA hypermethylation. Knockdown of ZNF132 using siRNA transfection led to increased cell migration ability, rather than increased cell growth or reduced apoptosis. We concluded that DNA hypermethylation of the ZNF132 gene participates in the clinicopathological aggressiveness of “pan-negative”-type lung adenocarcinomas. In addition, DNA methylation alterations at the precancerous stage may determine tumor aggressiveness, and such alterations that accumulate after driver mutation may additionally modify clinicopathological features through alterations of gene expression. Citation Format: Kenichi Hamada, Ying Tian, Mao Fujimoto, Takahashi Yoriko, Takashi Kohno, Koji Tsuta, Shun-ichi Watanabe, Teruhiko Yoshida, Hisao Asamura, Yae Kanai, Eri Arai. Correaltions between genome-wide DNA methylation profiles and genomic driver aberrations during multistage lung adenocaricinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2094.

The early-life exposome and epigenetic age acceleration in children

The early-life exposome influences future health and accelerated biological aging has been proposed as one of the underlying biological mechanisms. We investigated the association between more than 100 exposures assessed during pregnancy and in childhood (including indoor and outdoor air pollutants, built environment, green environments, tobacco smoking, lifestyle exposures, and biomarkers of chemical pollutants), and epigenetic age acceleration in 1,173 children aged 7 years old from the Human Early-Life Exposome project. Age acceleration was calculated based on Horvath’s Skin and Blood clock using child blood DNA methylation measured by Infinium HumanMethylation450 BeadChips. We performed an exposure-wide association study between prenatal and childhood exposome and age acceleration. Maternal tobacco smoking during pregnancy was nominally associated with increased age acceleration. For childhood exposures, indoor particulate matter absorbance (PMabs) and parental smoking were nominally associated with an increase in age acceleration. Exposure to the organic pesticide dimethyl dithiophosphate and the persistent pollutant polychlorinated biphenyl-138 (inversely associated with child body mass index) were protective for age acceleration. None of the associations remained significant after multiple-testing correction. Pregnancy and childhood exposure to tobacco smoke and childhood exposure to indoor PMabs may accelerate epigenetic aging from an early age.

Abstract MP42: Adherence To Healthy Dietary Patterns In Pregnancy And Placental Dna Methylation- An Epigenome Wide Association Study

Background: Accumulating evidence support the intergenerational impacts of diet in pregnancy. The underlying mechanisms, however, remain unclear. Placental epigenetic mechanisms may be involved although data from human epidemiological studies are sparse. We aimed to investigate associations of dietary quality in pregnancy with epigenome-wide placental DNA methylation in a multiracial pregnancy cohort. Methods: DNA methylation was measured using the Illumina Infinium Human Methylation450 Beadchip on placentas obtained at delivery from 301 pregnant women who participated in the Eunice Kennedy Shriver National Institute of Child Health and Human Development Fetal Growth Studies-Singleton cohort. Dietary information during periconception and early first trimester was collected using food frequency questionnaires, and diet in the second and third trimester was collected using a 24-hour dietary recall during four study visits. Scores for adherence to three healthy dietary patterns, alternate Healthy Eating Index (aHEI), alternate Mediterranean Diet (aMED), and Dietary Approaches to Stop Hypertension (DASH), were calculated. For associations of each dietary pattern score with methylation, we conducted analyses using robust linear regression models after the adjustment for age, pre-pregnancy body mass index, race/ethnicity, physical activity, total energy intakes, and population stratification. Genes annotating the top significant CpG sites (false discovery rate (FDR) adjusted P&lt;0.05) were queried for enrichment of functional pathways using the Ingenuity Pathway Analysis tool. Results: Adherence to aHEI was significantly associated with methylation of 8 CpG sites, with the most significant association manifested in cg16724319- MDH1B (P=1.9x10 -10 ). Adherence to aMED was related to methylation of 14 CpG sites, with the most significant association manifested in cg07835181- CLCN7 (P=1.7x10 -11 ). DASH was significantly related to 33 CpG sites, with the most significant association manifested in cg26292547- REV3L (P=4.4x10 -10 ). Further, genes annotating the significant CpG sites were enriched in pathways related to cardiovascular and nervous system development and function, cancer, organismal injury and abnormalities, and reproductive system diseases. Conclusion: Findings from the epigenome wide study suggest that overall dietary quality in pregnancy is associated with placental DNA methylation changes at different loci potentially related to cardiovascular, neurological, reproductive, and cancer phenotypes.

A panel of DNA methylation markers for the classification of consensus molecular subtypes 2 and 3 in patients with colorectal cancer.

3545 Background: Consensus molecular subtypes (CMSs) can guide precision treatment of colorectal cancer (CRC). Currently available assays can identify CMS1 and CMS4 cases well, while a dedicated test to distinguish CMS2 and 3 is lacking. This study aimed to identify a panel of methylation markers to distinguish between CMS2 and 3 in patients with CRC. Methods: Fresh-frozen tumor tissue of 239 patients with stage I-III CRC was included. CMS classification was performed on RNA-seq data using the single-sample-prediction parameter from the “CMSclassifier” package. Methylation profiles were obtained using the Infinium HumanMethylation450 BeadChip. We performed adaptive group-regularised logistic ridge-regression with post-hoc group-weighted elastic net marker selection to build prediction models for classification of CMS2 and CMS3 based on 15, 10 or 5 markers. Data from TCGAwas used for validation. Results: Overall methylation profiles differed between CMS2 and CMS3. Group-regularisation of the probes was done based on their location either relative to a CpG island or relative to a gene present in the CMS classifier resulting in two different prediction models and subsequently different marker panels. For both panels, even when using only 5 markers, sensitivity, specificity, and accuracy were &gt; 90%. Validation showed comparable performances. Conclusions: Our highly sensitive and specific methylation marker panel can be used to distinguish CMS2 and 3. This enables development of a qPCR DNA methylation assay in patients with CRC to provide a specific and non-invasive classification tool.

Genome-wide associations between alcohol consumption and blood DNA methylation: evidence from twin study.

Aim: Alcohol intake alters DNA methylation profiles and methylation might mediate the association between alcohol and disease, but limited number of positive CpG sites repeatedly replicated. Materials & methods: In total, 57 monozygotic (MZ) twin pairs discordant for alcohol drinking from the Chinese National Twin Registry and 158 MZ and dizygotictwin pairs in the Swedish Adoption/Twin Study of Aging were evaluated. DNA methylation was detected using the Infinium HumanMethylation450 BeadChip. Results: Among candidate CpG sites, cg07326074 was significantly correlated with drinking after adjusting for covariates in MZ twins in both datasets but not in the entire sample or dizygotic twins. Conclusion: The hypermethylation of cg07326074, located in the tumor-promoting gene C16orf59, was associated with alcohol consumption.

DNA methylation biomarkers of myocardial infarction and cardiovascular disease

BackgroundThe epigenetic landscape underlying cardiovascular disease (CVD) is not completely understood and the clinical value of the identified biomarkers is still limited. We aimed to identify differentially methylated loci associated with acute myocardial infarction (AMI) and assess their validity as predictive and causal biomarkers.ResultsWe designed a case–control, two-stage, epigenome-wide association study on AMI (ndiscovery = 391, nvalidation = 204). DNA methylation was assessed using the Infinium MethylationEPIC BeadChip. We performed a fixed-effects meta-analysis of the two samples. 34 CpGs were associated with AMI. Only 12 of them were available in two independent cohort studies (n ~ 1800 and n ~ 2500) with incident coronary and cardiovascular disease (CHD and CVD, respectively). The Infinium HumanMethylation450 BeadChip was used in those two studies. Four of the 12 CpGs were validated in association with incident CHD: AHRR-mapping cg05575921, PTCD2-mapping cg25769469, intergenic cg21566642 and MPO-mapping cg04988978. We then assessed whether methylation risk scores based on those CpGs improved the predictive capacity of the Framingham risk function, but they did not. Finally, we aimed to study the causality of those associations using a Mendelian randomization approach but only one of the CpGs had a genetic influence and therefore the results were not conclusive.ConclusionsWe have identified 34 CpGs related to AMI. These loci highlight the relevance of smoking, lipid metabolism, and inflammation in the biological mechanisms related to AMI. Four were additionally associated with incident CHD and CVD but did not provide additional predictive information.

DNA methylation and gene expression integration in cardiovascular disease

BackgroundThe integration of different layers of omics information is an opportunity to tackle the complexity of cardiovascular diseases (CVD) and to identify new predictive biomarkers and potential therapeutic targets. Our aim was to integrate DNA methylation and gene expression data in an effort to identify biomarkers related to cardiovascular disease risk in a community-based population. We accessed data from the Framingham Offspring Study, a cohort study with data on DNA methylation (Infinium HumanMethylation450 BeadChip; Illumina) and gene expression (Human Exon 1.0 ST Array; Affymetrix). Using the MOFA2 R package, we integrated these data to identify biomarkers related to the risk of presenting a cardiovascular event.ResultsFour independent latent factors (9, 19, 21—only in women—and 27), driven by DNA methylation, were associated with cardiovascular disease independently of classical risk factors and cell-type counts. In a sensitivity analysis, we also identified factor 21 as associated with CVD in women. Factors 9, 21 and 27 were also associated with coronary heart disease risk. Moreover, in a replication effort in an independent study three of the genes included in factor 27 were also present in a factor identified to be associated with myocardial infarction (CDC42BPB, MAN2A2 and RPTOR). Factor 9 was related to age and cell-type proportions; factor 19 was related to age and B cells count; factor 21 pointed to human immunodeficiency virus infection-related pathways and inflammation; and factor 27 was related to lifestyle factors such as alcohol consumption, smoking and body mass index. Inclusion of factor 21 (only in women) improved the discriminative and reclassification capacity of the Framingham classical risk function and factor 27 improved its discrimination.ConclusionsUnsupervised multi-omics data integration methods have the potential to provide insights into the pathogenesis of cardiovascular diseases. We identified four independent factors (one only in women) pointing to inflammation, endothelium homeostasis, visceral fat, cardiac remodeling and lifestyles as key players in the determination of cardiovascular risk. Moreover, two of these factors improved the predictive capacity of a classical risk function.

Telomere length and epigenetic age acceleration in adolescents with anxiety disorders

Evidence on the relationship between genetics and mental health are flourishing. However, few studies are evaluating early biomarkers that might link genes, environment, and psychopathology. We aimed to study telomere length (TL) and epigenetic age acceleration (AA) in a cohort of adolescents with and without anxiety disorders (N = 234). We evaluated a representative subsample of participants at baseline and after 5 years (n = 76) and categorized them according to their anxiety disorder diagnosis at both time points: (1) control group (no anxiety disorder, n = 18), (2) variable group (anxiety disorder in one evaluation, n = 38), and (3) persistent group (anxiety disorder at both time points, n = 20). We assessed relative mean TL by real-time quantitative PCR and DNA methylation by Infinium HumanMethylation450 BeadChip. We calculated AA using the Horvath age estimation algorithm and analyzed differences among groups using generalized linear mixed models. The persistent group of anxiety disorder did not change TL over time (p = 0.495). The variable group had higher baseline TL (p = 0.003) but no accelerated TL erosion in comparison to the non-anxiety control group (p = 0.053). Furthermore, there were no differences in AA among groups over time. Our findings suggest that adolescents with chronic anxiety did not change telomere length over time, which could be related to a delay in neuronal development in this period of life.