P13.15 DNA methylation abnormalities in non-promoter regulatory regions are associated with invasive behavior in pituitary tumors

Abstract BACKGROUND Despite histologically benign, pituitary tumors (PT) may invade important adjacent neurovascular structures which can incur in significant comorbidities preventing a complete surgical resection and contributing to resistance to medical treatment. DNA methylation clearly stratified PT based on their functional status i.e. nonfunctioning PTs (NFPTs) from functioning PT (FPTs). However associations of methylation aberrations with invasive behavior is less clear. MATERIAL AND METHODS In order to evaluate whether DNA methylation alterations in regulatory regions other than promoter and coding regions are associated with invasive behavior we performed a meta-analysis of the genome-wide methylome of three public available PT cohorts plus our own (Illumina HumanMethylation platforms- 450K/EPIC). Pituitary specimens comprised of 43 invasive pituitary tumors (InvPT) and 37 noninvasive (NInvPT); 12 FPT and 68 NFPTs, in addition to 20 non-tumor pituitaries. RNA-seq data were available for one cohort (n=23, 12 InvPT,11NInvPT) and integrated with DNA methylation. Invasiveness criteria was based on Knosp grade >= 2 and/or sphenoid or dural invasion. RESULTS Wilcoxon Rank-sum test; Δβ=0.15; p-value <0.001 identified 58 differentially methylated CpG sites in InvPT that were mainly hypomethylated (95%) in relation to NInvPT. NInvPT methylation profile was similar to non-tumor specimens, despite its heterogeneity. Thirty-four percent (n=20) of the differentially methylated CpG sites were located within predicted enhancer regions distributed in intronic (40%), intergenic (40%) and promoter (20%) regions. Predicted enhancer-target genes were enriched for actin filament cell movement, response to starvation, growth factor stimulus and protein autophosporilation pathways. Among them, ZNF625 and INO80E were found mostly negative correlated among methylation and expression data (-0.50 and -0.48, respectively), besides DOC2A found to be one potentially differentially expressed gene under enhancer control (log2FC > 0.2, pvalue <0.05). CONCLUSION Our results suggest that methylation alterations in predicted regulatory regions, such as enhancers, annotated in non-promoter regions (introns and intergenic) may contribute to the invasive behavior of PT.

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Association of Assisted Reproductive Technologies with offspring cord blood DNA methylation across cohorts

10.1101/2020.06.18.20134940 ◽

2020 ◽

Author(s):

Doretta Caramaschi ◽

James Jungius ◽

Christian M. Page ◽

Boris Novakovic ◽

Richard Saffery ◽

...

Keyword(s):

Dna Methylation ◽

Cord Blood ◽

Study Design ◽

Assisted Reproductive Technologies ◽

Biological Effects ◽

Meta Analysis ◽

Reproductive Technologies ◽

P Value ◽

Cpg Sites

AbstractStudy questionIs DNA methylation at birth associated with having been conceived by assisted reproductive technologies (ART)?Summary answerThis study shows does not provide strong evidence of an association of conception by ART with variation in infant blood cell DNA methylation.What is known alreadyAssisted reproductive technologies (ART) are procedures used to help infertile/subfertile couples conceive. Due to its importance in gene regulation during early development programming, DNA methylation and its perturbations associated with ART could reveal new insights into the biological effects of ART and potential adverse offspring outcomes.Study designWe investigated the association of DNA methylation and ART using a case-control study design (N=205 ART cases and N=2439 non-ART controls in discovery cohorts; N=149 ART cases and N=58 non-ART controls in replication cohort).Participants/materials, settings, methodWe assessed the association between ART and DNA methylation at birth in cord blood (205 ART conceptions and 2439 naturally conceived controls) at >450000 CpG sites across the genome in two sub-samples of the UK Avon Longitudinal Study of Parents and Children (ALSPAC) and two sub-samples of the Norwegian Mother, Father and Child Cohort Study (MoBa) by meta-analysis. We explored replication of findings in the Australian Clinical review of the Health of adults conceived following Assisted Reproductive Technologies (CHART) study (N=149 ART conceptions and N=58 controls).Main results and the role of chanceThe ALSPAC and MoBa meta-analysis revealed evidence of association between conception by ART and DNA methylation (false-discovery-rate-corrected p-value < 0.05) at 5 CpG sites which are annotated to 2 genes. Methylation at 3 of these sites has been previously linked to cancer, aging, HIV infection and neurological diseases. None of these associations replicated in the CHART cohort. There was evidence of a functional role of ART-induced hypermethylation at CpG sites located within regulatory regions as shown by putative transcription factor binding and chromatin remodelling.Limitations, reasons for cautionsWhile insufficient power is likely, heterogeneity in types of ART and between populations may also contribute. Larger studies might identify replicable variation in DNA methylation at birth due to ART.Wider implications of the findingsART-conceived newborns present with divergent DNA methylation in cord blood white cells. If these associations are true and causal, they might have long-term consequences for offspring health.

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EPIGENETIC ALTERATIONS ASSOCIATED WITH PREMATURE OVARIAN FAILURE

Clinical & Investigative Medicine ◽

10.25011/cim.v31i4.4804 ◽

2008 ◽

Vol 31 (4) ◽

pp. 11

Author(s):

Manda Ghahremani ◽

Courtney W Hannah ◽

Maria Peneherrera ◽

Karla L Bretherick ◽

Margo R Fluker ◽

...

Keyword(s):

Dna Methylation ◽

Premature Ovarian Failure ◽

Promoter Region ◽

Gene Promoter ◽

Ovarian Failure ◽

P Value ◽

Epigenetic Alterations ◽

Methylation Array ◽

Cpg Sites ◽

Deficient Mice

Background/Purpose: Premature ovarian failure (POF) affects 1% of women with a largely idiopathic and poorly understood etiology. The objective of this study was to identify specific epigenetic alterations by measuring DNA methylation of gene regulatory regions in women with POF vs. controls. Methods: Blood samples were collected from idiopathic POFpatients (Amenorrhea for at least 3 months and 2 serum FSH levels of > 40mIU/ml obtained > 1 month apart prior to age 40) and control women (CW) (healthy pregnancy after age 37 with out a pregnancy loss). Genomic DNA was extracted from EDTA anticoagulated blood and bisulfite converted for analysis using the Illumina Golden Gate Methylation Panel which measures DNA methylation at 1506 CpG sites in the promoter regions of 807 genes in 10 POF and 12 CW. Candidate genes with altered epigenetic marks between POF and CW at a nominal P-value < 0.05 were identified using a t-testcomparison within the Illumina bead studio software. Genes of interest were further analyzed for quantitative methylation at specific CpG sites using pyrosequencing in 30 POF and 30 CW. Results: Comparison of DNA methylation profiles of our initial POF and CW groups identified several genes with statistically significanthyper- or hypo- methylation in the POF group (P < 0.05), including the Androgen Receptor (AR)promoter region, which was significantly hypermethylated. To further validate these results, DNA methylation of the AR gene promoter was quantified bypryosequencing in a larger group of POF and CW. Pyrosequencing further confirmed a significantly higher DNA methylation of the AR promoter region inPOF vs. CW (P=0.007). Conclusions: This is a novel study identifying epigenetic alterations in POF. The hypermethylation of the AR gene in POF patients may cause decreased level of the AR in these women. This is especially interesting given a recent report of induced POF in AR deficient mice^1. Specific epigenetic markers, as identified by DNA methylation array profiling in blood, may serve as useful biomarkers for POF and other fertility disorders. However, it will need to be determined if these methylation changes are present prior to diagnosis, or are a consequence of menopause itself. Reference: 1.Hiroko S. et al. Premature ovarian failure in androgenreceptor deficient mice. PNAS;103:224-9

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DNA methylation is associated with lung function in never smokers

Respiratory Research ◽

10.1186/s12931-019-1222-8 ◽

2019 ◽

Vol 20 (1) ◽

Cited By ~ 3

Author(s):

Maaike de Vries ◽

◽

Ivana Nedeljkovic ◽

Diana A. van der Plaat ◽

Alexandra Zhernakova ◽

...

Keyword(s):

Dna Methylation ◽

Lung Function ◽

Ribosome Biogenesis ◽

Meta Analysis ◽

Smoking History ◽

Rotterdam Study ◽

Main Risk Factor ◽

Cpg Sites ◽

Never Smokers ◽

Integrative Omics

Abstract Background Active smoking is the main risk factor for COPD. Here, epigenetic mechanisms may play a role, since cigarette smoking is associated with differential DNA methylation in whole blood. So far, it is unclear whether epigenetics also play a role in subjects with COPD who never smoked. Therefore, we aimed to identify differential DNA methylation associated with lung function in never smokers. Methods We determined epigenome-wide DNA methylation levels of 396,243 CpG-sites (Illumina 450 K) in blood of never smokers in four independent cohorts, LifeLines COPD&C (N = 903), LifeLines DEEP (N = 166), Rotterdam Study (RS)-III (N = 150) and RS-BIOS (N = 206). We meta-analyzed the cohort-specific methylation results to identify differentially methylated CpG-sites with FEV1/FVC. Expression Quantitative Trait Methylation (eQTM) analysis was performed in the Biobank-based Integrative Omics Studies (BIOS). Results A total of 36 CpG-sites were associated with FEV1/FVC in never smokers at p-value< 0.0001, but the meta-analysis did not reveal any epigenome-wide significant CpG-sites. Of interest, 35 of these 36 CpG-sites have not been associated with lung function before in studies including subjects irrespective of smoking history. Among the top hits were cg10012512, cg02885771, annotated to the gene LTV1 Ribosome Biogenesis factor (LTV1), and cg25105536, annotated to Kelch Like Family Member 32 (KLHL32). Moreover, a total of 11 eQTMS were identified. Conclusions With the identification of 35 CpG-sites that are unique for never smokers, our study shows that DNA methylation is also associated with FEV1/FVC in subjects that never smoked and therefore not merely related to smoking.

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DNA methylation mediates the effect of cocaine use on HIV severity

10.1101/2020.05.11.20027458 ◽

2020 ◽

Author(s):

Chang Shu ◽

Amy C. Justice ◽

Xinyu Zhang ◽

Zuoheng Wang ◽

Dana B. Hancock ◽

...

Keyword(s):

Dna Methylation ◽

Cohort Study ◽

Mediation Effect ◽

Hiv Positive ◽

P Value ◽

Mediation Effects ◽

Cocaine Use ◽

Cpg Sites ◽

Hiv Progression ◽

Immunodeficiency Virus

Background: Cocaine use accelerates human immunodeficiency virus (HIV) progression and worsens HIV outcomes. We assessed whether DNA methylation in blood mediates the association between cocaine use and HIV severity in a veteran population. Methods: We analyzed 1,435 HIV-positive participants from the Veterans Aging Cohort Study Biomarker Cohort (VACS-BC). HIV severity was measured by the Veteran Aging Cohort Study (VACS) index. We assessed the effect of cocaine use on VACS index and mortality among the HIV-positive participants. We selected candidate mediators that were associated with both persistent cocaine use and VACS index by epigenome-wide association (EWA) scans at a liberal p-value cutoff of 0.001. Mediation analysis of the candidate CpG sites between cocaine effect and the VACS index was conducted, and the joint mediation effect of multiple CpGs was estimated. A two-step epigenetic Mendelian randomization (MR) analysis was conducted as validation. Results: More frequent cocaine use was significantly associated with a higher VACS index (β=1.00, p=2.7E-04), and cocaine use increased the risk of 10-year mortality (hazard ratio=1.10, p=0.011) with adjustment for confounding factors. Fifteen candidate mediator CpGs were selected from the EWA scan. Twelve of these CpGs showed significant mediation effects, with each explaining 11.3%-29.5% of the variation. The mediation effects for 3 of the 12 CpGs were validated by the two-step epigenetic MR analysis. The joint mediation effect of the 12 CpGs accounted for 47.2% of cocaine effect on HIV severity. Genes harboring these 12 CpGs are involved in the antiviral response (IFIT3, IFITM1, NLRC5, PLSCR1, PARP9) and HIV progression (CX3CR1, MX1). Conclusions: We identified 12 DNA methylation CpG sites that appear to play a mediation role in the association between cocaine use and HIV severity.

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Neonatal Lead (Pb) Exposure and DNA Methylation Profiles in Dried Bloodspots

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph17186775 ◽

2020 ◽

Vol 17 (18) ◽

pp. 6775

Author(s):

Luke Montrose ◽

Jaclyn M. Goodrich ◽

Masako Morishita ◽

Joseph Kochmanski ◽

Zachary Klaver ◽

...

Keyword(s):

Dna Methylation ◽

Inductively Coupled Plasma ◽

Housing Stock ◽

The United States ◽

P Value ◽

Inductively Coupled ◽

Cpg Sites ◽

Differentially Methylated Genes ◽

The World ◽

Pb Exposure

Lead (Pb) exposure remains a major concern in the United States (US) and around the world, even following the removal of Pb from gasoline and other products. Environmental Pb exposures from aging infrastructure and housing stock are of particular concern to pregnant women, children, and other vulnerable populations. Exposures during sensitive periods of development are known to influence epigenetic modifications which are thought to be one mechanism of the Developmental Origins of Health and Disease (DOHaD) paradigm. To gain insights into early life Pb exposure-induced health risks, we leveraged neonatal dried bloodspots in a cohort of children from Michigan, US to examine associations between blood Pb levels and concomitant DNA methylation profiles (n = 96). DNA methylation analysis was conducted via the Infinium MethylationEPIC array and Pb levels were assessed via high resolution inductively coupled plasma mass spectrometry (HR-ICP-MS). While at-birth Pb exposure levels were relatively low (average 0.78 µg/dL, maximum of 5.27 ug/dL), we identified associations between DNA methylation and Pb at 33 CpG sites, with the majority (82%) exhibiting reduced methylation with increasing Pb exposure (q < 0.2). Biological pathways related to development and neurological function were enriched amongst top differentially methylated genes by p-value. In addition to increases/decreases in methylation, we also demonstrate that Pb exposure is related to increased variability in DNA methylation at 16 CpG sites. More work is needed to assess the accuracy and precision of metals assessment using bloodspots, but this study highlights the utility of this unique resource to enhance environmental epigenetics research around the world.

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DNA Methylation Influences miRNA Expression in Gonadotroph Pituitary Tumors

Life ◽

10.3390/life10050059 ◽

2020 ◽

Vol 10 (5) ◽

pp. 59

Author(s):

Joanna Boresowicz ◽

Paulina Kober ◽

Natalia Rusetska ◽

Maria Maksymowicz ◽

Agnieszka Paziewska ◽

...

Keyword(s):

Dna Methylation ◽

Mirna Expression ◽

Target Genes ◽

Expression Profiles ◽

Pituitary Tumors ◽

Target Prediction ◽

Aberrant Methylation ◽

Expression Levels ◽

Putative Target ◽

Encoding Genes

microRNAs are involved in pathogenesis of cancer. DNA methylation plays a role in transcription of miRNA-encoding genes and may contribute to changed miRNA expression in tumors. This issue was not investigated in pituitary neuroendocrine tumors (PitNETs) previously. DNA methylation patterns, assessed with HumanMethylation450K arrays in 34 PitNETs and five normal pituitaries, were used to determine differentially methylated CpGs located at miRNA genes. It showed aberrant methylation in regions encoding for 131 miRNAs. DNA methylation data and matched miRNA expression profiles, determined with next-generation sequencing (NGS) of small RNAs, were correlated in 15 PitNETs. This showed relationship between methylation and expression levels for 12 miRNAs. DNA methylation and expression levels of three of them (MIR145, MIR21, and MIR184) were determined in the independent group of 80 tumors with pyrosequencing and qRT-PCR and results confirmed both aberrant methylation in PitNETs and correlation between methylation and expression. Additionally, in silico target prediction was combined with analysis of established miRNA profiles and matched mRNA expression pattern, assessed with amplicon-based NGS to indicate putative target genes of epigenetically deregulated miRNAs. This study reveals aberrant DNA methylation in miRNA-encoding genes in gonadotroph PitNETs. Methylation changes affect expression level of miRNAs that regulate putative target genes with tumorigenesis-relevant functions.

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DNA methylation profiling identifies epigenetic differences between early versus late stages of diabetic chronic kidney disease

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfaa226 ◽

2020 ◽

Author(s):

Ashani Lecamwasam ◽

Boris Novakovic ◽

Braydon Meyer ◽

Elif I Ekinci ◽

Karen M Dwyer ◽

...

Keyword(s):

Chronic Kidney Disease ◽

Dna Methylation ◽

Kidney Disease ◽

Secretory Protein ◽

Differentially Methylated Region ◽

P Value ◽

Operating Characteristics ◽

Cross Sectional ◽

Cpg Sites ◽

Methylation Patterns

Abstract Background We investigated a cross-sectional epigenome-wide association study of patients with early and late diabetes-associated chronic kidney disease (CKD) to identify possible epigenetic differences between the two groups as well as changes in methylation across all stages of diabetic CKD. We also evaluated the potential of using a panel of identified 5′-C-phosphate-G-3′ (CpG) sites from this cohort to predict the progression of diabetic CKD. Methods This cross-sectional study recruited 119 adults. DNA was extracted from blood using the Qiagen QIAampDNA Mini Spin Kit. Genome-wide methylation analysis was performed using Illumina Infinium MethylationEPIC BeadChips (HM850K). Intensity data files were processed and analysed using the minfi and MissMethyl packages for R. We examined the degree of methylation of CpG sites in early versus late diabetic CKD patients for CpG sites with an unadjusted P-value <0.01 and an absolute change in methylation of 5% (n = 239 CpG sites). Results Hierarchical clustering of the 239 CpG sites largely separated the two groups. A heat map for all 239 CpG sites demonstrated distinct methylation patterns in the early versus late groups, with CpG sites showing evidence of progressive change. Based on our differentially methylated region (DMR) analysis of the 239 CpG sites, we highlighted two DMRs, namely the cysteine-rich secretory protein 2 (CRISP2) and piwi-like RNA-mediated gene silencing 1 (PIWIL1) genes. The best predictability for the two groups involved a receiver operating characteristics curve of eight CpG sites alone and achieved an area under the curve of 0.976. Conclusions We have identified distinct DNA methylation patterns between early and late diabetic CKD patients as well as demonstrated novel findings of potential progressive methylation changes across all stages (1–5) of diabetic CKD at specific CpG sites. We have also identified associated genes CRISP2 and PIWIL1, which may have the potential to act as stage-specific diabetes-associated CKD markers, and showed that the use of a panel of eight identified CpG sites alone helps to increase the predictability for the two groups.

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Use of DNA methylation to distinguish gallbladder carcinoma.

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.15_suppl.e15605 ◽

2019 ◽

Vol 37 (15_suppl) ◽

pp. e15605-e15605

Author(s):

Xiaoqing Jiang ◽

Bin Li ◽

Zhiquan Qiu ◽

Yong Yu ◽

Zhishuai Li ◽

...

Keyword(s):

Dna Methylation ◽

Malignant Disease ◽

Gallbladder Disease ◽

White Blood Cells ◽

Stage Iv ◽

P Value ◽

Targeted Bisulfite Sequencing ◽

Gallbladder Diseases ◽

Cpg Sites ◽

A Genome

e15605 Background: Gallbladder cancer (GBC), an uncommon malignancy with a high mortality rate, is often diagnosed late due to lack of early symptoms and the relative hidden nature of the gallbladder. Despite the advancements in imaging technologies, there is no reliable screening test for GBC. The role of aberrant DNA methylation in the process of tumorigenesis both at individual genes and a genome-wide scale has been well elucidated. It occurs very early in cancer development, thus capable of serving as a screening marker. Methods: Panel Design: Methylation data of tumor samples (12 types, n = 4,772), adjacent normal (8 types, n = 411), and normal white blood cells (n = 656) from TCGA and GSE were compared. Differentially methylated sites were derived using a Bayesian hierarchical model-DSS with an adjusted p-value < 0.05. Our panel covers 80,672 CpG sites, spanning 1.05Mb of human genome. This panel contains 12,196 GBC relevant CpG sites. We performed targeted bisulfite sequencing on 23 GBC patients (6 stage II-III, 17 stage IV) and 13 patients with non-malignant gallbladder diseases (cholecystitis and gallstones). Of the 23 GBC patients, we obtained adjacent normal tissue from 7 of them. Basic clinical features such as age, gender, of patients with GBC and patients with non-malignant gallbladder diseases were comparable. Results: Among the 12,196 GBC relevant CpG sites, 10,216 sites were statistically significantly hypermethylated and 275 sites were statistically significantly hypomethylated comparing to patients with non-malignant gallbladder disease as well as adjacent normal gallbladder tissues. Subsequently, we used the derived differentially methylated CpG sites to construct a linear regression model, achieving an area under curve of 99%. Collectively, the methylation levels were comparable between tissues with non-malignant disease and adjacent normal. Interestingly, when considering the 275 hypomethylated markers alone, we observed that the methylation level of adjacent normal tissues is significantly higher than tissues with non-malignant disease. Conclusions: Collectively, our panel can effectively distinguish GBC samples from non-cancerous samples, demonstrating the potential of DNA methylation in GBC screening. Furthermore, hypomethylation markers can be used to distinguish non-malignant disease from the healthy.

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Integration of public DNA methylation and expression networks via eQTMs improves prediction of functional gene-gene associations

10.1101/2021.12.17.473125 ◽

2021 ◽

Author(s):

Shuang Li ◽

Cancan Qi ◽

Patrick Deelen ◽

Floranne Boulogne ◽

Niek de Klein ◽

...

Keyword(s):

Dna Methylation ◽

Gene Networks ◽

Prediction Models ◽

Meta Analysis ◽

Rna Seq ◽

Integrated Network ◽

Functional Relationships ◽

Cpg Sites ◽

Molecular Phenotypes ◽

Public Datasets

Gene co-expression networks can be used to infer functional relationships between genes, but they do not work well for all genes. We investigated whether DNA methylation can provide complementary information for such genes. We first carried out an eQTM meta-analysis of 3,574 gene expression and methylation samples from blood, brain and nasal epithelial brushed cells to identify links between methylated CpG sites and genes. This revealed 6,067 significant eQTM genes, and we observed that histone modification information is predictive of both eQTM direction and presence, enabling us to link many CpG sites to genes. We then generated a co-methylation network - MethylationNetwork - using 27,720 publicly available methylation profiles and integrated it with a public RNA-seq co-expression dataset of 31,499 samples. Here, we observed that MethylationNetwork can identify experimentally validated interacting pairs of genes that could not be identified in the RNA-seq datasets. We then developed a novel integration pipeline based on CCA and used the integrated methylation and gene networks to predict gene pairs reported in the STRING database. The integrated network showed significantly improved prediction performance compared to using a DNA co-methylation or a gene co-expression network alone. This is the first study to integrate data from two -omics layers from unmatched public samples across different tissues and diseases, and our results highlight the issues and potential of integrating public datasets from multiple molecular phenotypes. The eQTMs we identified can be used as an annotation resource for epigenome-wide association, and we believe that our integration pipeline can be used as a framework for future -omics integration analyses of public datasets. We provide supporting materials and results, including the harmonized DNA methylation data from multiple tissues and diseases in https://data.harmjanwestra.nl/comethylation/, the discovered and predicted eQTMs, the corresponding CCA components and the trained prediction models in a Zenodo repository (https://zenodo.org/record/4666994). We provide notebooks to facilitate use of the proposed pipeline in a GitHub repository (https://github.com/molgenis/methylationnetwork).

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Abstract P259: Genome-Wide DNA Methylation and Subclinical Cardiovascular Disease in the Coronary Artery Risk Development in Young Adults (CARDIA) Study

Circulation ◽

10.1161/circ.129.suppl_1.p259 ◽

2014 ◽

Vol 129 (suppl_1) ◽

Author(s):

Lifang Hou ◽

Xin Liu ◽

Yinan Zheng ◽

Wei Zhang ◽

Xiao Zhang ◽

...

Keyword(s):

Dna Methylation ◽

Young Adults ◽

Coronary Artery ◽

Vascular Function ◽

Subclinical Atherosclerosis ◽

Statistical Significance ◽

White Blood Cells ◽

Small Sample ◽

P Value ◽

Cpg Sites

Background: Coronary artery calcification (CAC) and carotid intima-media thickness (CIMT), established measures of subclinical atherosclerosis, that have been demonstrated to improve prediction of coronary artery disease (CAD) risk beyond classical risk factors (CRFs). This study examined the epigenetic mechanisms underlying the appearance of CAC and increased CIMT, which have not been previously explored. Methods: We conducted an epigenome-wide association study (EWAS) in 46 non-smoking and non-diabetic white subjects randomly selected from the Coronary Artery Risk Development in Young Adults (CARDIA) study. CAC and CIMT were measured by computed tomography (CT) and catotid artery ultrasound at examination year (Y) 20. The Illumina HumanMethylation450 BeadChip was used to measure DNA methylation in white blood cells collected at Y15. We dropped one sample with >1% of the CpG sites having a detection p-value >0.05 and then exclude ~160k CpG probes due to their ambiguously mapping to the genome or with the presence of common SNPs, etc. Both background adjustment and normalization were performed separately for Infinium I and II probes, and new values were calculated and then transformed into M-values. After correction for potential chip effect, we examined the associations between these pre-processed methylation levels at each CpG site with CAC and CIMT using multiple logistic and linear regression models, respectively. Pathway analysis was performed to explore the gene sets that were significantly associated with CAC and CIMT. Results: Several CpG sites in multiple genes were significantly associated with CAC or CIMT. Some of these genes play roles in the regulation of vascular function, such as ion binding and transport ( HRH1 , LRP1B , KCNJ9 , TRIM40 , ADAMTS3 for CAC and BRSK2 , ZNF428 , TROVE2 , C1orf86 for CIMT) and metabolic processes ( LEPR for CAC and NUP50 for CIMT). Pathway analyses revealed several common canonical pathways for CAC and CIMT including, calcium signaling, axon guidance, and focal adhesion etc., which are relevant to the occurrence of these two subclinical CADs. The statistical significance of identified CpG sties for either CAC or CIMT did not remain after correction for multiple testing, possibly due to the small sample size. Conclusion: Our preliminary findings suggest that methylomic mechanisms may play a role in the development of CAC and CIMT, and subsequently the etiology of CAD. Future replication studies in larger longitudinally studies are needed.

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