Illumina HumanMethylation450 BeadChip Research Articles

Hidradenitis suppurativa associated telomere-methylome dysregulations in blood.

Hidradenitis suppurativa (HS) is a chronic debilitating disease with a significant burden of both organic and psychological comorbidities. It has been shown that certain telomere-related genes (TRGs) affect a wide range of diseases, including HS and its associated comorbidities, but their exact role in HS pathogenesis is still unknown. To determine whether TRG methylomes can be used as biomarkers in HS. Using the Illumina HumanMethylation450 BeadChip array, we examined methylation variations associated with TRGs in HS cases and age-, sex- and ethnicity-matched healthy controls. The study utilized integrated bioinformatics statistical methods, such as a false discovery rate (FDR), the area under the receiver operating characteristic curve (AUC) and principal component analysis. There were a total of 585 different differentially methylated CpG sites identified in 585 TRGs associated with HS (474 hypomethylated and 111 hypermethylated) (FDR p-value < 0.05). A number of these CpGs have been identified as being involved in increased pain sensitivity including EPAS1, AHR, CSNK1D, DNMT1, IKBKAP, NOS3, PLCB1 and PRDM16 genes; GABRB3 as a potential alcohol addiction marker; DDB1, NSMCE2 and HNRNPA2B1 associated with cancers. Pathway analysis identified 67 statistically significant pathways, including DNA repair, telomere maintenance, mismatch repair and cell cycle control (p < 0.001). The disruption of TRGs leads to the shortening of telomeres, which is associated with HS progression, ageing, cellular senescence and an increased risk of various diseases, including cancer and associated comorbidities, such as metabolic syndrome, cardiovascular disease and inflammatory disorders. Further research is necessary to better understand the underlying mechanisms and establish causal links between TRGs and HS. The present study is the first effort to comprehend potential pathomechanisms of sporadic HS cases concentrating on PBMC methylome since ours.

Multi-tissue epigenetic analysis identifies distinct associations underlying insulin resistance and Alzheimer’s disease at CPT1A locus

BackgroundInsulin resistance (IR) is a major risk factor for Alzheimer’s disease (AD) dementia. The mechanisms by which IR predisposes to AD are not well-understood. Epigenetic studies may help identify molecular signatures of IR associated with AD, thus improving our understanding of the biological and regulatory mechanisms linking IR and AD.MethodsWe conducted an epigenome-wide association study of IR, quantified using the homeostatic model assessment of IR (HOMA-IR) and adjusted for body mass index, in 3,167 participants from the Framingham Heart Study (FHS) without type 2 diabetes at the time of blood draw used for methylation measurement. We identified DNA methylation markers associated with IR at the genome-wide level accounting for multiple testing (P < 1.1 × 10−7) and evaluated their association with neurological traits in participants from the FHS (N = 3040) and the Religious Orders Study/Memory and Aging Project (ROSMAP, N = 707). DNA methylation profiles were measured in blood (FHS) or dorsolateral prefrontal cortex (ROSMAP) using the Illumina HumanMethylation450 BeadChip. Linear regressions (ROSMAP) or mixed-effects models accounting for familial relatedness (FHS) adjusted for age, sex, cohort, self-reported race, batch, and cell type proportions were used to assess associations between DNA methylation and neurological traits accounting for multiple testing.ResultsWe confirmed the strong association of blood DNA methylation with IR at three loci (cg17901584–DHCR24, cg17058475–CPT1A, cg00574958–CPT1A, and cg06500161–ABCG1). In FHS, higher levels of blood DNA methylation at cg00574958 and cg17058475 were both associated with lower IR (P = 2.4 × 10−11 and P = 9.0 × 10–8), larger total brain volumes (P = 0.03 and P = 9.7 × 10−4), and smaller log lateral ventricular volumes (P = 0.07 and P = 0.03). In ROSMAP, higher levels of brain DNA methylation at the same two CPT1A markers were associated with greater risk of cognitive impairment (P = 0.005 and P = 0.02) and higher AD-related indices (CERAD score: P = 5 × 10−4 and 0.001; Braak stage: P = 0.004 and P = 0.01).ConclusionsOur results suggest potentially distinct epigenetic regulatory mechanisms between peripheral blood and dorsolateral prefrontal cortex tissues underlying IR and AD at CPT1A locus.

Maternal adverse childhood experiences (ACEs) and DNA methylation of newborns in cord blood

BackgroundAdverse childhood experiences (ACEs) increase the risk of poor health outcomes later in life. Psychosocial stressors may also have intergenerational health effects by which parental ACEs are associated with mental and physical health of children. Epigenetic programming may be one mechanism linking parental ACEs to child health. This study aimed to investigate epigenome-wide associations of maternal preconception ACEs with DNA methylation patterns of children. In the Center for the Health Assessment of Mothers and Children of Salinas study, cord blood DNA methylation was measured using the Illumina HumanMethylation450 BeadChip. Preconception ACEs, which occurred during the mothers’ childhoods, were collected using a standard ACE questionnaire including 10 ACE indicators. Maternal ACE exposures were defined in this study as (1) the total number of ACEs; (2) the total number of ACEs categorized as 0, 1–3, and > 4; and (3) individual ACEs. Associations of ACE exposures with differential methylated positions, regions, and CpG modules determined using weighted gene co-expression network analysis were evaluated adjusting for covariates.ResultsData on maternal ACEs and cord blood DNA methylation were available for 196 mother/newborn pairs. One differential methylated position was associated with maternal experience of emotional abuse (cg05486260/FAM135B gene; q value < 0.05). Five differential methylated regions were significantly associated with the total number of ACEs, and 36 unique differential methylated regions were associated with individual ACEs (Šidák p value < 0.05). Fifteen CpG modules were significantly correlated with the total number of ACEs or individual ACEs, of which 8 remained significant in fully adjusted models (p value < 0.05). Significant modules were enriched for pathways related to neurological and immune development and function.ConclusionsMaternal ACEs prior to conception were associated with cord blood DNA methylation of offspring at birth. Although there was limited overlap between differential methylated regions and CpGs in modules associated with ACE exposures, statistically significant regions and networks were related to genes involved in neurological and immune function. Findings may provide insights to pathways linking psychosocial stressors to health. Further research is needed to understand the relationship between changes in DNA methylation and child health.

DNA methylation analysis identifies key transcription factors involved in mesenchymal stem cell osteogenic differentiation

BackgroundKnowledge about regulating transcription factors (TFs) for osteoblastogenesis from mesenchymal stem cells (MSCs) is limited. Therefore, we investigated the relationship between genomic regions subject to DNA-methylation changes during osteoblastogenesis and the TFs known to directly interact with these regulatory regions.ResultsThe genome-wide DNA-methylation signature of MSCs differentiated to osteoblasts and adipocytes was determined using the Illumina HumanMethylation450 BeadChip array. During adipogenesis no CpGs passed our test for significant methylation changes. Oppositely, during osteoblastogenesis we identified 2462 differently significantly methylated CpGs (adj. p < 0.05). These resided outside of CpGs islands and were significantly enriched in enhancer regions. We confirmed the correlation between DNA-methylation and gene expression. Accordingly, we developed a bioinformatic tool to analyse differentially methylated regions and the TFs interacting with them. By overlaying our osteoblastogenesis differentially methylated regions with ENCODE TF ChIP-seq data we obtained a set of candidate TFs associated to DNA-methylation changes. Among them, ZEB1 TF was highly related with DNA-methylation. Using RNA interference, we confirmed that ZEB1, and ZEB2, played a key role in adipogenesis and osteoblastogenesis processes. For clinical relevance, ZEB1 mRNA expression in human bone samples was evaluated. This expression positively correlated with weight, body mass index, and PPARγ expression.ConclusionsIn this work we describe an osteoblastogenesis-associated DNA-methylation profile and, using these data, validate a novel computational tool to identify key TFs associated to age-related disease processes. By means of this tool we identified and confirmed ZEB TFs as mediators involved in the MSCs differentiation to osteoblasts and adipocytes, and obesity-related bone adiposity.

Abstract P304: Association of Epigenetic Age Acceleration With Cognition in the Bogalusa Heart Study

Background: Despite known associations of epigenetic age acceleration (EAA), or increased DNA methylation (DNAm)-based age relative to chronological age, with Alzheimer’s disease, few studies have investigated the relation of EAA with midlife cognitive function. We examined cross-sectional associations of EAA with cognition among the 1,252 middle-aged participants of the Bogalusa Heart Study 2013-2016 study visit. Methods: Whole-blood DNA methylation data was generated using the Illumina HumanMethylation450 BeadChip. Four EAA measures, including extrinsic EAA (EEAA), intrinsic EAA (IEAA), PhenoAA and GrimAA, were derived from the methylation data. Cognitive function was assessed by a standard battery of eight neurocognitive tests covering five cognition domains, and a global cognition score was calculated. Associations of EAA measures with cognitive function were assessed by 3 multivariable models sequentially adding (i) demographics (age, sex, race, education) and vocabulary; (ii) lifestyle behaviors (smoking, alcohol drinking, and depression), and (iii) clinical measures (blood pressure, lipids, body mass index, glycated hemoglobin, medication, and white blood cell count. Results: After Bonferroni correction (α=1.39E-3), PhenoAA and GrimAA were significantly associated with decreased processing speed (lower Digit coding test score) and decreased working memory (higher trail making test A score). GrimAA was also associated with lower global cognition score ( Table ). EEAA was associated with decreased processing speed (lower Digit coding test). After adjusting for lifestyle and clinical measures, such associations were slightly attenuated but remained at least nominally significant. IEAA was not associated with any cognitive test results. Conclusions: EAA measured by PhenoAA, GrimAA, and EEAA were significantly associated with decreased processing speed, working memory, and/or global cognition. The associations were independent of known cognitive decline risk factors.

Integrated analysis reveals common DNA methylation patterns of alcohol-associated cancers: A pan-cancer analysis.

Background: The role of alcohol in carcinogenesis has received increasing attention in recent years. Evidence shows its impacts on various aspects, including epigenetics alteration. The DNA methylation patterns underlying alcohol-associated cancers are not fully understood. Methods: We investigated the aberrant DNA methylation patterns in four alcohol-associated cancers based on the Illumina HumanMethylation450 BeadChip. Pearson coefficient correlations were identified between differential methylated CpG probes and annotated genes. Transcriptional factor motifs were enriched and clustered using MEME Suite, and a regulatory network was constructed. Results: In each cancer, differential methylated probes (DMPs) were identified, and 172 hypermethylated and 21 hypomethylated pan-cancer DMPs (PDMPs) were examined further. Annotated genes significantly regulated by PDMPs were investigated and enriched in transcriptional misregulation in cancers. The CpG island chr19:58220189-58220517 was hypermethylated in all four cancers and silenced in the transcription factor ZNF154. Various biological effects were exerted by 33 hypermethylated and seven hypomethylated transcriptional factor motifs grouped into five clusters. Eleven pan-cancer DMPs were identified to be associated with clinical outcomes in the four alcohol-associated cancers, which might provide a potential point of view for clinical outcome prediction. Conclusion: This study provides an integrated insight into DNA methylation patterns in alcohol-associated cancers and reveals the corresponding features, influences, and potential mechanisms.

Sperm DNA methylome abnormalities occur both pre- and post-treatment in men with Hodgkin disease and testicular cancer

BackgroundCombination chemotherapy has contributed to increased survival from Hodgkin disease (HD) and testicular cancer (TC). However, questions concerning the quality of spermatozoa after treatment have arisen. While studies have shown evidence of DNA damage and aneuploidy in spermatozoa years following anticancer treatment, the sperm epigenome has received little attention. Our objectives here were to determine the impact of HD and TC, as well as their treatments, on sperm DNA methylation. Semen samples were collected from community controls (CC) and from men undergoing treatment for HD or TC, both before initiation of chemotherapy and at multiple times post-treatment. Sperm DNA methylation was assessed using genome-wide and locus-specific approaches.ResultsImprinted gene methylation was not affected in the sperm of HD or TC men, before or after treatment. Prior to treatment, using Illumina HumanMethylation450 BeadChip (450 K) arrays, a subset of 500 probes was able to distinguish sperm samples from TC, HD and CC subjects; differences between groups persisted post-treatment. Comparing altered sperm methylation between HD or TC patients versus CC men, twice as many sites were affected in TC versus HD men; for both groups, the most affected CpGs were hypomethylated. For TC patients, the promoter region of GDF2 contained the largest region of differential methylation. To assess alterations in DNA methylation over time/post-chemotherapy, serial samples from individual patients were compared. With restriction landmark genome scanning and 450 K array analyses, some patients who underwent chemotherapy showed increased alterations in DNA methylation, up to 2 to 3 years post-treatment, when compared to the CC cohort. Similarly, a higher-resolution human sperm-specific assay that includes assessment of environmentally sensitive regions, or “dynamic sites,” also demonstrated persistently altered sperm DNA methylation in cancer patients post-treatment and suggested preferential susceptibility of “dynamic” CpG sites.ConclusionsDistinct sperm DNA methylation signatures were present pre-treatment in men with HD and TC and may help explain increases in birth defects reported in recent clinical studies. Epigenetic defects in spermatozoa of some cancer survivors were evident even up to 2 years post-treatment. Abnormalities in the sperm epigenome both pre- and post-chemotherapy may contribute to detrimental effects on future reproductive health.

Kidney tumors associated with germline mutations of FH and SDHB show a CpG island methylator phenotype (CIMP).

Germline mutations within the Krebs cycle enzyme genes fumarate hydratase (FH) or succinate dehydrogenase (SDHB, SDHC, SDHD) are associated with an increased risk of aggressive and early metastasizing variants of renal cell carcinoma (RCC). These RCCs express significantly increased levels of intracellular fumarate or succinate that inhibit 2-oxoglutarate-dependent dioxygenases, such as the TET enzymes that regulate DNA methylation. This study evaluated the genome-wide methylation profiles of 34 RCCs from patients with RCC susceptibility syndromes and 11 associated normal samples using the Illumina HumanMethylation450 BeadChip. All the HLRCC (FH mutated) and SDHB-RCC (SDHB mutated) tumors demonstrated a distinct CpG island methylator phenotype (CIMP). HLRCC tumors demonstrated an extensive and relatively uniform level of hypermethylation that showed some correlation with tumor size. SDHB-RCC demonstrated a lesser and more varied pattern of hypermethylation that overlapped in part with the HLRCC hypermethylation. Combined methylation and mRNA expression analysis of the HLRCC tumors demonstrated hypermethylation and transcription downregulation of genes associated with the HIF pathway, HIF3A and CITED4, the WNT pathway, SFRP1, and epithelial-to-mesenchymal transition and MYC expression, OVOL1. These observations were confirmed in the TCGA CIMP-RCC tumors. A selected panel of probes could identify the CIMP tumors and differentiate between HLRCC and SDHB-RCC tumors. This panel accurately detected all CIMP-RCC tumors within the TCGA RCC cohort, identifying them as HLRCC -like, and could potentially be used to create a liquid biopsy-based screening tool. The CIMP signature in these aggressive tumors could provide both a useful biomarker for diagnosis and a target for novel therapies.

Epigenetic signatures of insulin resistance associated with Alzheimer’s Disease and related traits

AbstractBackgroundInsulin resistance (IR) is a major risk factor for Alzheimer’s disease (AD) and is primarily driven by obesity, another AD risk factor. The mechanism by which IR predisposes to AD is unknown. Epigenetic studies may help identify molecular signatures of IR associated with AD, thus contributing to an improved understanding of the biological and regulatory mechanisms of IR in AD.MethodWe conducted an epigenome‐wide association study of IR, quantified using the homeostasis model assessment of IR, in 3,167 Framingham Heart Study (FHS) participants without diabetes. We selected epigenetic markers (outcome) associated with IR at the genome‐wide level (P&lt;10‐7: 0.05/450,000) and evaluated their association with AD, vascular dementia, dementia, and brain volumes (hippocampal, total brain‐TBV, intracranial‐ICV, and lateral ventricular) in up to 3,040 FHS participants. We defined significant associations using P&lt;0.002 (0.05/3/7). All analyses were performed using blood DNA methylation measured using the Illumina HumanMethylation450 BeadChip and linear mixed‐effects models adjusted for age, sex, and batch, while allowing for heterogeneous variance across batches and accounting for familial relatedness. Models including IR or brain volume as a covariate were additionally adjusted for body mass index or ICV respectively.ResultWe confirmed the association of blood DNA methylation with IR at three loci (1p32:cg17901584‐DHCR24, 11q13:cg17058475‐CPT1A, and 21q22:cg06500161‐ABCG1). Blood DNA methylation at cg17058475‐CPT1A was significantly associated with TBV (P=0.0007). The carnitine palmitoyl transferase (CPT) system, crucial for the mitochondrial beta‐oxidation of long‐chain fatty acids, is involved in metabolic syndrome, type 2 diabetes, cardiovascular and neurological diseases including AD. Blood DNA methylation levels at cg17901584‐DHCR24 and cg06500161‐ABCG1 were nominally associated with AD (P=0.03) or vascular dementia (P=0.04) respectively. Both DHCR24 and ABCG1 are involved in cholesterol synthesis or transport. Reduced expression of DHCR24 occurs in AD patient’s temporal cortex while CSF capacity to promote cell cholesterol efflux via ABCG1 is specifically impaired in AD and this impairment correlates with the main AD neurobiochemical markers.ConclusionOur methylation analysis has shed light on genes potentially implicated in both IR and AD. Future work includes association analyses using brain omics from the AD Knowledge Portal.

Shifts in the immunoepigenomic landscape of monocytes in response to a diabetes-specific social support intervention: a pilot study among Native Hawaiian adults with diabetes

BackgroundNative Hawaiians are disproportionately affected by type 2 diabetes mellitus (DM), a chronic metabolic, non-communicable disease characterized by hyperglycemia and systemic inflammation. Unrelenting systemic inflammation frequently leads to a cascade of multiple comorbidities associated with DM, including cardiovascular disease, microvascular complications, and renal dysfunction. Yet few studies have examined the link between chronic inflammation at a cellular level and its relationship to standard DM therapies such as diabetes-specific lifestyle and social support education, well recognized as the cornerstone of clinical standards of diabetes care. This pilot study was initiated to explore the association of monocyte inflammation using epigenetic, immunologic, and clinical measures following a 3-month diabetes-specific social support program among high-risk Native Hawaiian adults with DM.ResultsFrom a sample of 16 Native Hawaiian adults with DM, monocytes enriched from peripheral blood mononuclear cells (PBMCs) of 8 individuals were randomly selected for epigenomic analysis. Using the Illumina HumanMethylation450 BeadChip microarray, 1,061 differentially methylated loci (DML) were identified in monocytes of participants at baseline and 3 months following a DM-specific social support program (DM-SSP). Gene ontology analysis showed that these DML were enriched within genes involved in immune, metabolic, and cardiometabolic pathways, a subset of which were also significantly differentially expressed. Ex vivo analysis of immune function showed improvement post-DM-SSP compared with baseline, characterized by attenuated interleukin 1β and IL-6 secretion from monocytes. Altered cytokine secretion in response to the DM-SSP was significantly associated with changes in the methylation and gene expression states of immune-related genes in monocytes between intervention time points.ConclusionsOur pilot study provides preliminary evidence of changes to inflammatory monocyte activity, potentially driven by epigenetic modifications, 3 months following a DM-specific SSP intervention. These novel alterations in the trajectory of monocyte inflammatory states were identified at loci that regulate transcription of immune and metabolic genes in high-risk Native Hawaiians with DM, suggesting a relationship between improvements in psychosocial behaviors and shifts in the immunoepigenetic patterns following a diabetes-specific SSP. Further research is warranted to investigate how social support influences systemic inflammation via immunoepigenetic modifications in chronic inflammatory diseases such as DM.

DNA methylation changes in response to neoadjuvant chemotherapy are associated with breast cancer survival

BackgroundLocally advanced breast cancer is a heterogeneous disease with respect to response to neoadjuvant chemotherapy (NACT) and survival. It is currently not possible to accurately predict who will benefit from the specific types of NACT. DNA methylation is an epigenetic mechanism known to play an important role in regulating gene expression and may serve as a biomarker for treatment response and survival. We investigated the potential role of DNA methylation as a prognostic marker for long-term survival (> 5 years) after NACT in breast cancer.MethodsDNA methylation profiles of pre-treatment (n = 55) and post-treatment (n = 75) biopsies from 83 women with locally advanced breast cancer were investigated using the Illumina HumanMethylation450 BeadChip. The patients received neoadjuvant treatment with epirubicin and/or paclitaxel. Linear mixed models were used to associate DNA methylation to treatment response and survival based on clinical response to NACT (partial response or stable disease) and 5-year survival, respectively. LASSO regression was performed to identify a risk score based on the statistically significant methylation sites and Kaplan–Meier curve analysis was used to estimate survival probabilities using ten years of survival follow-up data. The risk score developed in our discovery cohort was validated in an independent validation cohort consisting of paired pre-treatment and post-treatment biopsies from 85 women with locally advanced breast cancer. Patients included in the validation cohort were treated with either doxorubicin or 5-FU and mitomycin NACT.ResultsDNA methylation patterns changed from before to after NACT in 5-year survivors, while no significant changes were observed in non-survivors or related to treatment response. DNA methylation changes included an overall loss of methylation at CpG islands and gain of methylation in non-CpG islands, and these changes affected genes linked to transcription factor activity, cell adhesion and immune functions. A risk score was developed based on four methylation sites which successfully predicted long-term survival in our cohort (p = 0.0034) and in an independent validation cohort (p = 0.049).ConclusionOur results demonstrate that DNA methylation patterns in breast tumors change in response to NACT. These changes in DNA methylation show potential as prognostic biomarkers for breast cancer survival.

Predicting risk of lung function impairment and all-cause mortality using a DNA methylation-based classifier of tobacco smoke exposure

BackgroundThe Epigenetic Smoking Status Estimator (EpiSmokEr) predicts smoking phenotypes based on DNA methylation at 121 CpG sites. ObjectiveEvaluate associations of EpiSmokEr-predicted versus self-reported smoking phenotypes with lung function and all-cause mortality in a cohort of older adults. MethodsThe prospective Normative Aging Study collected DNA methylation measurements from 1999 to 2012 with follow-up through 2016. The R package EpiSmokEr derived predicted smoking phenotypes based on DNA methylation levels assayed by the Illumina HumanMethylation450 Beadchip. Spirometry was collected every 3–5 years. Airflow limitation was defined as forced expiratory volume in 1 s/forced vital capacity <0.7. Vital status was monitored through periodic mailings. ResultsAmong 784 participants contributing 5414 person-years of follow-up, the EpiSmokEr-predicted smoking phenotypes matched the self-reported phenotypes for 228 (97%) never smokers and 22 (71%) current smokers. In contrast, EpiSmokEr classified 407 (79%) self-reported former smokers as never smokers. Nonetheless, the EpiSmokEr-predicted former smoking phenotype was more strongly associated with incident airflow limitation (hazard ratio [HR] = 3.15, 95% confidence interval [CI] = 1.50–6.59) and mortality (HR = 2.11, 95% CI = 1.56–2.85) compared to the self-reported former smoking phenotype (airflow limitation: HR = 2.21, 95% CI = 1.13–4.33; mortality: HR = 1.08, 95% CI = 0.86–1.36). Risk of airflow limitation and death did not differ among self-reported never smokers and former smokers who were classified as never smokers. The discriminative accuracy of EpiSmokEr-predicted phenotypes for incident airflow limitation and mortality was improved compared to self-reported phenotypes. ConclusionsThe DNA methylation-based EpiSmokEr classifier may be a useful surrogate of smoking-induced lung damage and may identify former smokers most at risk of adverse smoking-related health effects.

Triglyceride-Associated Epigenetic Markers and Diet and Lifestyle Habits in the Framingham Heart Study

ObjectivesThe current evidence has shown that environmental and lifestyle factors (e.g., diet, physical activity, tobacco smoking, alcohol) are associated with DNA methylation patterns. However, the mechanisms underlying the relation between diet and other exposures and epigenetic profiles are not well understood. To reduce such knowledge gap, we investigated the links between lifestyle, including diet, and methylation marks with plasma triglyceride concentrations (TG).MethodsWe first conducted an epigenome-wide association study (EWAS) (Illumina HumanMethylation450 BeadChip) for TG in the Framingham Heart Study Offspring population (n = 2,178). We then examined the relationships between dietary and lifestyle-related variables, collected over 13 years, and differential DNA methylation marks associated with the last TG measures (exam 8). Second, we conducted a mediation analysis to examine the potential causal relationship between diet-related variables and TG.ResultsThe EWAS analyses revealed 28 TG-associated DNA methylation sites at 19 regions (e.g., ABCG1, CPT1A, DHCR24, PHGDH, SLC7A11, SLC43A1, SREBF1, TXNIP). Within those methylation sites, we identified 427 significant associations between these DNA methylation sites and one or more dietary and lifestyle-related variables after accounting for multiple testing. The two most significant and consistent associations between TG-associated DNA methylation markers and diet were alcohol (g/day) and carbohydrate intake (% total energy), with P-values ranging from 10^(−4) to 10^(−56). Mediation analyses demonstrated that alcohol and carbohydrate intake independently affect TG via DNA methylation markers as mediators. For seven of the 19 identified differential DNA-methylation regions, higher alcohol intake was associated with decreased methylation and higher TG. In contrast, increased carbohydrate intake was associated with higher DNA methylation at two epigenetic loci (CPT1A and SLC7A11) and lower TG.ConclusionsOur findings indicate that dietary factors (i.e., alcohol and carbohydrate) are associated with specific DNA methylation markers that could mediate some of the observed associations between diet and cardiometabolic risk factors.Funding SourcesThis work was funded by the US Department of Agriculture, under agreement no. 8050-51,000-107-000D.

Effects of stressful life-events on DNA methylation in panic disorder and major depressive disorder

BackgroundPanic disorder (PD) is characterized by recurrent panic attacks and higher affection of women as compared to men. The lifetime prevalence of PD is about 2–3% in the general population leading to tremendous distress and disability. Etiologically, genetic and environmental factors, such as stress, contribute to the onset and relapse of PD. In the present study, we investigated epigenome-wide DNA methylation (DNAm) in respond to a cumulative, stress-weighted life events score (wLE) in patients with PD and its boundary to major depressive disorder (MDD), frequently co-occurring with symptoms of PD.MethodsDNAm was assessed by the Illumina HumanMethylation450 BeadChip. In a meta-analytic approach, epigenome-wide DNAm changes in association with wLE were first analyzed in two PD cohorts (with a total sample size of 183 PD patients and 85 healthy controls) and lastly in 102 patients with MDD to identify possible overlapping and opposing effects of wLE on DNAm. Additionally, analysis of differentially methylated regions (DMRs) was conducted to identify regional clusters of association.ResultsTwo CpG-sites presented with p-values below 1 × 10−05 in PD: cg09738429 (p = 6.40 × 10−06, located in an intergenic shore region in next proximity of PYROXD1) and cg03341655 (p = 8.14 × 10−06, located in the exonic region of GFOD2). The association of DNAm at cg03341655 and wLE could be replicated in the independent MDD case sample indicating a diagnosis independent effect. Genes mapping to the top hits were significantly upregulated in brain and top hits have been implicated in the metabolic system. Additionally, two significant DMRs were identified for PD only on chromosome 10 and 18, including CpG-sites which have been reported to be associated with anxiety and other psychiatric phenotypes.ConclusionThis first DNAm analysis in PD reveals first evidence of small but significant DNAm changes in PD in association with cumulative stress-weighted life events. Most of the top associated CpG-sites are located in genes implicated in metabolic processes supporting the hypothesis that environmental stress contributes to health damaging changes by affecting a broad spectrum of systems in the body.

Methylation at CpG sites related to growth differentiation factor-15 was not prospectively associated with cardiovascular death in discordant monozygotic twins

Myocardial infarction patients had decreased methylation at four growth differentiating factor-15 (GDF-15) related CpG sites (cg13033858, cg16936953, cg17150809, and cg18608055). These sites had not been studied for their association with cardiovascular disease (CVD) deaths. Thus, we aimed to assess the associations independent of genes, shared environment, and traditional CVD risk factors. Nineteen white, male, monozygotic twin pairs discordant for CVD deaths were included from the National Heart, Lung and Blood Institute Twin Study (NHLBI) initiated in 1969. Data on vital status was collected through December 31, 2014. Methylation of buffy coat DNA at exam 3 (1986–87) was measured using the Illumina HumanMethylation450 BeadChip. Principal component analysis was used to generate a score representing blood leukocyte composition and baseline CVD risk factors and predominated with natural killer cells, CD4+ T cells, and Framingham risk score. Conditional logistic regression demonstrated that methylation at the four CpG sites was not associated with CVD deaths before (all p > 0.05, bootstrapped p > 0.05) and after adjustment for the score (all p > 0.05). Joint influences of cg16936953 and the score were statistically significant (p < 0.05). In conclusion, joint influences of methylation at the site cg16936953 and the score are prospectively associated with CVD deaths independent of germline and common environment.ClinicalTrials.gov Identifier for NHLBI Twin Study: NCT00005124.

KDmarkers: A biomarker database for investigating epigenetic methylation and gene expression levels in Kawasaki disease

Kawasaki disease (KD) is a form of acute systemic vasculitis that primarily affects children and has become the most common cause of acquired heart disease. While the etiopathogenesis of KD remains unknown, the diagnostic criteria of KD have been well established. Nevertheless, the diagnosis of KD is currently based on subjective clinical symptoms, and no molecular biomarker is yet available. We have previously performed and combined methylation array (Illumina HumanMethylation450 BeadChip) and transcriptome array (Affymetrix GeneChip Human Transcriptome Array 2.0) to identify genes that are differentially methylated/expressed in KD patients compared with control subjects. We have found that decreased methylation levels combined with elevated gene expression can indicate genes (e.g., toll-like receptors and CD177) involved in the disease mechanisms of KD. In this study, we constructed a database called KDmarkers to allow researchers to access these valuable potential KD biomarkers identified via methylation array and transcriptome array. KDmarkers provides three search modes. First, users can search genes differentially methylated and/or differentially expressed in KD patients compared with control subjects. Second, users can check the KD patient groups in which a given gene is differentially methylated and/or differentially expressed. Third, users can explore the DNA methylation levels and gene expression levels in all samples (KD patients and controls) for a particular gene of interest. We further demonstrated that the results in KDmarkers are strongly associated with KD immune responses. All analysis results can be downloaded for downstream experimental designs. KDmarkers is available online at https://cosbi.ee.ncku.edu.tw/KDmarkers/.

Comprehensive DNA Methylation Analysis of Human Neuroblastoma Cells Treated With Haloperidol and Risperidone.

Accumulating evidence suggests that the epigenetic alterations induced by antipsychotics contribute to the therapeutic efficacy. However, global and site-specific epigenetic changes by antipsychotics and those shared by different classes of antipsychotics remain poorly understood. We conducted a comprehensive DNA methylation analysis of human neuroblastoma cells cultured with antipsychotics. The cells were cultured with low and high concentrations of haloperidol or risperidone for 8 days. DNA methylation assay was performed with the Illumina HumanMethylation450 BeadChip. We found that both haloperidol and risperidone tended to cause hypermethylation changes and showed similar DNA methylation changes closely related to neuronal functions. A total of 294 differentially methylated probes (DMPs), including 197 hypermethylated and 97 hypomethylated DMPs, were identified with both haloperidol and risperidone treatment. Gene ontology analysis of the hypermethylated probe-associated genes showed enrichment of genes related to the regulation of neurotransmitter receptor activity and lipoprotein lipase activity. Pathway analysis identified that among the DMP-associated genes, SHANK1 and SHANK2 were the major genes in the neuropsychiatric disorder-related pathways. Our data would be valuable for understanding the mechanisms of action of antipsychotics from an epigenetic viewpoint.

Longitudinal DNA methylation dynamics as a practical indicator in clinical epigenetics

BackgroundOne of the fundamental assumptions of DNA methylation in clinical epigenetics is that DNA methylation status can change over time with or without interplay with environmental and clinical conditions. However, little is known about how DNA methylation status changes over time under ordinary environmental and clinical conditions. In this study, we revisited the high frequency longitudinal DNA methylation data of two Japanese males (24 time-points within three months) and characterized the longitudinal dynamics.ResultsThe results showed that the majority of CpGs on Illumina HumanMethylation450 BeadChip probe set were longitudinally stable over the time period of three months. Focusing on dynamic and stable CpGs extracted from datasets, dynamic CpGs were more likely to be reported as epigenome-wide association study (EWAS) markers of various traits, especially those of immune- and inflammatory-related traits; meanwhile, the stable CpGs were enriched in metabolism-related genes and were less likely to be EWAS markers, indicating that the stable CpGs are stable both in the short-term within individuals and under various environmental and clinical conditions.ConclusionsThis study indicates that CpGs with different stabilities are involved in different functions and traits, and thus, they are potential indicators that can be applied for clinical epigenetic studies to outline underlying mechanisms.

An integrative epi-transcriptomic approach identifies the human cartilage chitinase 3-like protein 2 (CHI3L2) as a potential mediator of B12 deficiency in adipocytes

ABSTRACT Vitamin B12 has multiple biochemical functions including in the one-carbon cycle generating a methyl group for DNA methylation, and metabolism of fatty acids and amino acids to generate energy via the citric acid cycle. The aim of our study was to use a combined epigenomic and transcriptomic approach to identify novel genes mediating the effect of B12 on adipogenesis. Human pre-adipocytes (CHUB-S7) were treated with a range of B12 (0–500 nM) concentrations from the day of cell seeding until harvesting in discovery and validation experiments prior to genome-wide methylation analysis using the Illumina HumanMethylation 450Beadchip. For transcriptomic analysis, RNA-seq libraries were run on the Illumina HiSeq 2500. To further investigate the expression of any genes on human adipogenesis, a second human preadipocyte strain was studied (SGBS) by real-time quantitative PCR (qRT-PCR). A combined epigenetic and transcriptomic approach in differentiated human pre-adipocyte cell line, CHUB-S7, identified that the Human cartilage chitinase 3-like protein 2 (CHI3L2) gene was hypo-methylated and had increased expression in low B12 conditions. Furthermore, there was an approximately 1000-fold increase in CHI3L2 expression in the early days of adipocyte differentiation, which paralleled an increase of lipid droplets in differentiated SGBS cells and an increased expression level of markers of mature adipocytes. In summary, we have identified a potential role of the human cartilage chitinase 3-like protein 2 (CHI3L2) in adipocyte function in the presence of low B12 levels.

Prenatal metal exposure, cord blood DNA methylation and persistence in childhood: an epigenome-wide association study of 12 metals

BackgroundPrenatal exposure to essential and non-essential metals impacts birth and child health, including fetal growth and neurodevelopment. DNA methylation (DNAm) may be involved in pathways linking prenatal metal exposure and health. In the Project Viva cohort, we analyzed the extent to which metals (As, Ba, Cd, Cr, Cs, Cu, Hg, Mg, Mn, Pb, Se, and Zn) measured in maternal erythrocytes were associated with differentially methylated positions (DMPs) and regions (DMRs) in cord blood and tested if associations persisted in blood collected in mid-childhood. We measured metal concentrations in first-trimester maternal erythrocytes, and DNAm in cord blood (N = 361) and mid-childhood blood (N = 333, 6–10 years) with the Illumina HumanMethylation450 BeadChip. For each metal individually, we tested for DMPs using linear models (considered significant at FDR < 0.05), and for DMRs using comb-p (Sidak p < 0.05). Covariates included biologically relevant variables and estimated cell-type composition. We also performed sex-stratified analyses.ResultsPb was associated with decreased methylation of cg20608990 (CASP8) (FDR = 0.04), and Mn was associated with increased methylation of cg02042823 (A2BP1) in cord blood (FDR = 9.73 × 10–6). Both associations remained significant but attenuated in blood DNAm collected at mid-childhood (p < 0.01). Two and nine Mn-associated DMPs were identified in male and female infants, respectively (FDR < 0.05), with two and six persisting in mid-childhood (p < 0.05). All metals except Ba and Pb were associated with ≥ 1 DMR among all infants (Sidak p < 0.05). Overlapping DMRs annotated to genes in the human leukocyte antigen (HLA) region were identified for Cr, Cs, Cu, Hg, Mg, and Mn.ConclusionsPrenatal metal exposure is associated with DNAm, including DMRs annotated to genes involved in neurodevelopment. Future research is needed to determine if DNAm partially explains the relationship between prenatal metal exposures and health outcomes.