Methylome-wide Association Research Articles

Methylome-wide association study of first-episode schizophrenia reveals a hypermethylated CpG site in the promoter region of the TNIK susceptibility gene

Accumulating evidence suggests that epigenetics plays an important role in the etiology of schizophrenia. Here, we performed a methylome-wide association study (MWAS) of first-onset schizophrenia patients and controls from the Han Chinese population using microarray technology. The DNA methylation profiles revealed 4494 differentially methylated CpG sites. Gene ontology (GO) analysis showed that the functions of differentially methylated genes were primarily involved in enzymatic activity, cytoskeleton organization and cell adhesion, and the TNIK (encoding TRAF2- and NCK-interacting kinase) gene was enriched in most of these terms. By combining the MWAS results with those of previous genome-wide association studies (GWASs), we identified 72 candidate genes located in 49 human genome loci. Among the overlapping genes, the most significantly methylated CpG sites were in the transcriptional start site (TSS) 200 region (cg21413905, Punadjusted = 3.20 × 10−5) of TNIK. TNIK was listed in the top 50 differentially methylated loci. The results of pyrosequencing and TNIK mRNA expression were consistent with those of the microarray study. Bioinformatics analyses, dual-luciferase reporter assays and chromatin immunoprecipitation (ChIP) studies showed that TNIK interacted with genes associated with schizophrenia and NRF1 was identified as a novel transcription factor (TF) that binds to TNIK in its TSS200 region. Thus, the regulatory function of NRF1 may be influenced by the status of the methylated CpG site in this region. In summary, our study provides new insights into the epigenetic mechanisms that regulate schizophrenia. Studies of the functions of TNIK methylation should be performed in vitro and in vivo to provide a better understanding of the pathophysiology of schizophrenia.

Analysis of DNA methylation associates the cystine\u2013glutamate antiporter SLC7A11 with risk of Parkinson\u2019s disease

An improved understanding of etiological mechanisms in Parkinson’s disease (PD) is urgently needed because the number of affected individuals is projected to increase rapidly as populations age. We present results from a blood-based methylome-wide association study of PD involving meta-analysis of 229 K CpG probes in 1,132 cases and 999 controls from two independent cohorts. We identify two previously unreported epigenome-wide significant associations with PD, including cg06690548 on chromosome 4. We demonstrate that cg06690548 hypermethylation in PD is associated with down-regulation of the SLC7A11 gene and show this is consistent with an environmental exposure, as opposed to medications or genetic factors with effects on DNA methylation or gene expression. These findings are notable because SLC7A11 codes for a cysteine-glutamate anti-porter regulating levels of the antioxidant glutathione, and it is a known target of the environmental neurotoxin β-methylamino-L-alanine (BMAA). Our study identifies the SLC7A11 gene as a plausible biological target in PD.

Abstract P143: Methylome-Wide Association Of DNA Methylation And Aircraft Noise Exposure In The Women’s Health Initiative

Background: Noise pollution is common and can affect health, but mechanisms underlying this relationship remain incompletely characterized. We therefore examined the novel association between aircraft noise and DNA methylation (DNAm), an environmentally modifiable epigenetic phenomenon that affects gene expression and is associated with cardiovascular and neurological disease. Methods: We conducted a methylome-wide association study in race/ethnicity-stratified subpopulations of 4,535 post-menopausal Women’s Health Initiative participants (mean age: 64.4 years; 59.6% white; 24.7% African American; 15.7% Hispanic/Latina). We estimated DNAm in whole blood as a proportion of methylated cytosines at each Cytosine-phosphate-Guanine (CpG) site using the Illumina 450k BeadChip. In cooperation with the Federal Aviation Administration, we generated day-night-average sound level (DNL) contours around 90 major U.S. airports using the Aviation Environmental Design Tool. The DNL applies a penalty for nighttime exposure and is the primary metric for informing policy in the U.S. We estimated geocoded participant address-specific, annual, DNL aircraft noise exposures in decibels (dB) from the contours on the day of blood draw. We categorized aircraft noise exposures as non-exposed or exposed (DNL < 45 or ≥ 45dB) and used linear mixed-effects models to estimate CpG site-specific DNAm-noise associations adjusted for sociodemographic, behavioral, and technical covariates. We combined site-specific associations across race-ethnicity strata in fixed-effects, inverse variance-weighted meta-analyses and then identified the significant ( P < 1.0x10 -7 ), non-heterogeneous ( P Cochran’s Q > 0.10) associations among them. Results: Overall, DNAm was inversely associated with aircraft noise at cg21525369 (Chromosome 15; P = 6.40x10 -8 ). Cg21525369 is 56 kb upstream from ACAN , a gene encoding an extracellular matrix protein (aggrecan). Aggrecan is expressed in response to ultrasound; forms perineural nets around cochlear hair cells and auditory brainstem neurons; affects afferent extension, synaptic transmission, and neuronal vulnerability; deposits in tympanic membranes of patients with sensorineural hearing loss, and redistributes in auditory brainstem after deafferentation. Conclusions: The biologically plausible DNAm-noise association among postmenopausal U.S. women suggests aircraft noise pollution may affect methylation of DNA near a gene previously associated with hearing, although its cardiovascular, otologic, and neurological implications remain unclear pending sensitivity analysis, external replication, mediation analysis, and functional characterization.

MBD-seq - realities of a misunderstood method for high-quality methylome-wide association studies

ABSTRACTThe majority of methylome-wide association studies (MWAS) have been performed using commercially available array-based technologies such as the Infinium Human Methylation 450K and the Infinium MethylationEPIC arrays (Illumina). While these arrays offer a convenient and relatively robust assessment of the probed sites they only allow interrogation of 2-4% of all CpG sites in the human genome. Methyl-binding domain sequencing (MBD-seq) is an alternative approach for MWAS that provides near-complete coverage of the methylome at similar costs as the array-based technologies. However, despite publication of multiple positive evaluations, the use of MBD-seq for MWAS is often fiercely criticized. Here we discuss key features of the method and debunk misconceptions using empirical data. We conclude that MBD-seq represents an excellent approach for large-scale MWAS and that increased utilization is likely to result in more discoveries, advance biological knowledge, and expedite the clinical translation of methylome-wide research findings.

Cell Type–Specific Methylome-wide Association Studies Implicate Neurotrophin and Innate Immune Signaling in Major Depressive Disorder

BackgroundWe sought to characterize methylation changes in brain and blood associated with major depressive disorder (MDD). As analyses of bulk tissue may obscure association signals and hamper the biological interpretation of findings, these changes were studied on a cell type–specific level. MethodsIn 3 collections of human postmortem brain (n = 206) and 1 collection of blood samples (N = 1132) of MDD cases and controls, we used epigenomic deconvolution to perform cell type–specific methylome-wide association studies within subpopulations of neurons/glia for the brain data and granulocytes/T cells/B cells/monocytes for the blood data. Sorted neurons/glia from a fourth postmortem brain collection (n = 58) were used for validation purposes. ResultsCell type–specific methylome-wide association studies identified multiple findings in neurons/glia that were detected across brain collections and were reproducible in physically sorted nuclei. Cell type–specific analyses in blood samples identified methylome-wide significant associations in T cells, monocytes, and whole blood that replicated findings from a past methylation study of MDD. Pathway analyses implicated p75 neurotrophin receptor/nerve growth factor signaling and innate immune toll-like receptor signaling in MDD. Top results in neurons, glia, bulk brain, T cells, monocytes, and whole blood were enriched for genes supported by genome-wide association studies for MDD and other psychiatric disorders. ConclusionsWe both replicated and identified novel MDD–methylation associations in human brain and blood samples at a cell type–specific level. Our results provide mechanistic insights into how the immune system may interact with the brain to affect MDD susceptibility. Importantly, our findings involved associations with MDD in human samples that implicated many closely related biological pathways. These disease-linked sites and pathways represent promising new therapeutic targets for MDD.

Integrated genetic and methylomic analyses identify shared biology between autism and autistic traits

Previous studies have identified differences in DNA methylation in autistic individuals compared to neurotypical individuals. Yet, it is unclear if this extends to autistic traits—subclinical manifestation of autism features in the general population. Here, we investigate the association between DNA methylation at birth (cord blood), and scores on the Social and Communication Disorders Checklist (SCDC), a measure of autistic traits, in 701 8-year-olds, by conducting a methylome-wide association study (MWAS). We did not identify significant CpGs associated with SCDC. The most significant CpG site was cg14379490, on chromosome 9 (MWAS beta = − 1.78 ± 0.35, p value = 5.34 × 10−7). Using methylation data for autism in peripheral tissues, we did not identify a significant concordance in effect direction of CpGs with p value < 10−4 in the SCDC MWAS (binomial sign test, p value > 0.5). In contrast, using methylation data for autism from post-mortem brain tissues, we identify a significant concordance in effect direction of CpGs with a p value < 10−4 in the SCDC MWAS (binomial sign test, p value = 0.004). Supporting this, we observe an enrichment for genes that are dysregulated in the post-mortem autism brain (one-sided Wilcoxon rank-sum test, p value = 6.22 × 10−5). Finally, integrating genome-wide association study (GWAS) data for autism (n = 46,350) with mQTL maps from cord-blood (n = 771), we demonstrate that mQTLs of CpGs associated with SCDC scores at p value thresholds of 0.01 and 0.005 are significantly shifted toward lower p values in the GWAS for autism (p < 5 × 10−3). We provide additional support for this using a GWAS of SCDC, and demonstrate a lack of enrichment in a GWAS of Alzheimer’s disease. Our results highlight the shared cross-tissue methylation architecture of autism and autistic traits, and demonstrate that mQTLs associated with differences in DNA methylation associated with childhood autistic traits are enriched for common genetic variants associated with autism and autistic traits.

Differential DNA methylation in blood as a mediator of the association between cigarette smoking and bladder cancer risk among postmenopausal women

ABSTRACTSmoking accounts for approximately 52% of bladder cancer incidence among postmenopausal women, but the underlying mechanism is poorly understood. Our study investigates whether changes in DNA methylation, as measured in blood, mediate the impact of smoking on bladder cancer risk among postmenopausal women. We conducted analyses among 206 cases and 251 controls that were current or never smokers at baseline from a previous case-control study of bladder cancer and genome-wide DNA methylation nested within the Women’s Health Initiative. Separate mediation analyses were conducted for three CpG sites demonstrating robust associations with smoking in prior methylome-wide association studies: cg05575921 (AhRR), cg03636183 (F2RL3), and cg19859270 (GPR15). We estimated causal effects using the regression-based, four-way decomposition approach, which addresses the interaction between smoking and each CpG site. The overall proportion of the excess relative risk mediated by cg05575921 was 92% (p-value = 0.004) and by cg19859270 was 79% (p-value = 0.02). The largest component of the excess relative risk of bladder cancer due to 30 pack-years of smoking history in current smokers was the mediated interaction for both cg05575921 (72%, p = 0.02) and cg19859270 (72%, p-value = 0.04), where the mediated interaction is the effect of smoking on bladder cancer that both acts through differential methylation and depends on smoking history. There was little evidence that smoking was mediated through cg03636183. Our results suggest that differential methylation of cg05575921 and cg19859270 mediate the effects of smoking on bladder cancer, potentially revealing downstream effects of smoking relevant for carcinogenesis.

Methylome-wide association study provides evidence of particulate matter air pollution-associated DNA methylation

BackgroundDNA methylation (DNAm) may contribute to processes that underlie associations between air pollution and poor health. Therefore, our objective was to evaluate associations between DNAm and ambient concentrations of particulate matter (PM) ≤2.5, ≤10, and 2.5–10 μm in diameter (PM2.5; PM10; PM2.5–10). MethodsWe conducted a methylome-wide association study among twelve cohort- and race/ethnicity-stratified subpopulations from the Women's Health Initiative and the Atherosclerosis Risk in Communities study (n = 8397; mean age: 61.5 years; 83% female; 45% African American; 9% Hispanic/Latino American). We averaged geocoded address-specific estimates of daily and monthly mean PM concentrations over 2, 7, 28, and 365 days and 1 and 12 months before exams at which we measured leukocyte DNAm in whole blood. We estimated subpopulation-specific, DNAm-PM associations at approximately 485,000 Cytosine-phosphate-Guanine (CpG) sites in multi-level, linear, mixed-effects models. We combined subpopulation- and site-specific estimates in fixed-effects, inverse variance-weighted meta-analyses, then for associations that exceeded methylome-wide significance and were not heterogeneous across subpopulations (P < 1.0 × 10−7; PCochran's Q > 0.10), we characterized associations using publicly accessible genomic databases and attempted replication in the Cooperative Health Research in the Region of Augsburg (KORA) study. ResultsAnalyses identified significant DNAm-PM associations at three CpG sites. Twenty-eight-day mean PM10 was positively associated with DNAm at cg19004594 (chromosome 20; MATN4; P = 3.33 × 10−8). One-month mean PM10 and PM2.5–10 were positively associated with DNAm at cg24102420 (chromosome 10; ARPP21; P = 5.84 × 10−8) and inversely associated with DNAm at cg12124767 (chromosome 7; CFTR; P = 9.86 × 10−8). The PM-sensitive CpG sites mapped to neurological, pulmonary, endocrine, and cardiovascular disease-related genes, but DNAm at those sites was not associated with gene expression in blood cells and did not replicate in KORA. ConclusionsAmbient PM concentrations were associated with DNAm at genomic regions potentially related to poor health among racially, ethnically and environmentally diverse populations of U.S. women and men. Further investigation is warranted to uncover mechanisms through which PM-induced epigenomic changes may cause disease.

Methylome-Wide Association Study in Peripheral White Blood Cells Focusing on Central Obesity and Inflammation.

Epigenetic signatures such as DNA methylation may be associated with specific obesity traits in different tissues. The onset and development of some obesity-related complications are often linked to visceral fat accumulation. The aim of this study was to explore DNA methylation levels in peripheral white blood cells to identify epigenetic methylation marks associated with waist circumference (WC). DNA methylation levels were assessed using Infinium Human Methylation 450K and MethylationEPIC beadchip (Illumina) to search for putative associations with WC values of 473 participants from the Methyl Epigenome Network Association (MENA) project. Statistical analysis and Ingenuity Pathway Analysis (IPA) were employed for assessing the relationship between methylation and WC. A total of 669 CpGs were statistically associated with WC (FDR < 0.05, slope ≥ |0.1|). From these CpGs, 375 CpGs evidenced a differential methylation pattern between females with WC ≤ 88 and > 88 cm, and 95 CpGs between males with WC ≤ 102 and > 102 cm. These differentially methylated CpGs are located in genes related to inflammation and obesity according to IPA. Receiver operating characteristic (ROC) curves of the top four significant differentially methylated CpGs separated by sex discriminated individuals with presence or absence of abdominal fat. ROC curves of all the CpGs from females and one CpG from males were validated in an independent sample (n = 161). These methylation results add further insights about the relationships between obesity, adiposity-associated comorbidities, and DNA methylation where inflammation processes may be involved.

Independent Methylome-Wide Association Studies of Schizophrenia Detect Consistent Case-Control Differences.

Methylome-wide association studies (MWASs) are promising complements to sequence variation studies. We used existing sequencing-based methylation data, which assayed the majority of all 28 million CpGs in the human genome, to perform an MWAS for schizophrenia in blood, while controlling for cell-type heterogeneity with a recently generated platform-specific reference panel. Next, we compared the MWAS results with findings from 3 existing large-scale array-based schizophrenia methylation studies in blood that assayed up to ~450 000 CpGs. Our MWAS identified 22 highly significant loci (P < 5 × 10-8) and 852 suggestively significant loci (P < 1 × 10-5). The top finding (P = 5.62 × 10-11, q = 0.001) was located in MFN2, which encodes mitofusin-2 that regulates Ca2+ transfer from the endoplasmic reticulum to mitochondria in cooperation with DISC1. The second-most significant site (P = 1.38 × 10-9, q = 0.013) was located in ALDH1A2, which encodes an enzyme for astrocyte-derived retinoic acid-a key neuronal morphogen with relevance for schizophrenia. Although the most significant MWAS findings were not assayed on the arrays, we observed significant enrichment of overlapping findings with 2 of the 3 array datasets (P = 0.0315, 0.0045, 0.1946). Overrepresentation analysis of Gene Ontology terms for the genes in the significant overlaps suggested high similarity in the biological functions detected by the different datasets. Top terms were related to immune and/or stress responses, cell adhesion and motility, and a broad range of processes essential for neurodevelopment.

47 - METHYLOME-WIDE ASSOCIATION STUDIES FOR MAJOR DEPRESSIVE DISORDER IN BLOOD OVERLAP WITH METHYLATION RESULTS FROM BRAIN AND LARGE-SCALE GWAS

Background Epigenetic modifications such as DNA methylation provide stability and diversity to the cellular phenotype and aberrant methylation has been implicated in processes underlying psychiatric disorders. Therefore, studies combining DNA methylation and genotype information provide a promising approach to study disorders where genotype information alone has failed to reveal the full etiology. Methods We applied an optimized MBD-seq protocol to assay the complete CpG methylome in cases with Major Depressive Disorder (MDD) and controls using blood samples (N=1,132) from Netherlands Study of Depression and Anxiety and brain samples (N=64) from the Victorian Brain Bank Network. Data were analyzed with RaMWAS, a novel Bioconductor package specifically designed for Methylome-Wide Association Studies (MWAS). To study the overlap between top MWAS findings in blood and brain, we used a permutation based enrichment test (shiftR) that accounted for the dependency between adjacent CpG sites. Furthermore, we utilized the methylation data in combination with existing genotype information from the same individuals in a MWAS of CpGs created or destroyed by SNPs. Next, we tested whether top results from this CpG-SNP MWAS overlapped with recent large-scale GWAS to identify robust associations with genomic loci of importance for MDD etiology. Results The MWAS in blood identified five methylome-wide significant sites (P Discussion This work involves the largest MWAS for MDD performed to date. Our integrated analysis with brain tissue, genotype information and GWAS results highlighted biological functions of potential value for MDD etiology. Part of the associated methylation marks in blood overlapped with MWAS finding in brain. As blood can easily be collected in a clinical setting, these loci may be of direct value as potential diagnostic biomarkers for MDD.

A Whole Methylome Study of Ethanol Exposure in Brain and Blood: An Exploration of the Utility of Peripheral Blood as Proxy Tissue for Brain in Alcohol Methylation Studies.

Recent reviews have highlighted the potential use of blood-based methylation biomarkers as diagnostic and prognostic tools of current and future alcohol use and addiction. Due to the substantial overlap that often exists between methylation patterns across different tissues, including blood and brain, blood-based methylation may track methylation changes in brain; however, little work has explored the overlap in alcohol-related methylation in these tissues. To study the effects of alcohol on the brain methylome and identify possible biomarkers of these changes in blood, we performed a methylome-wide association study in brain and blood from 40 male DBA/2J mice that received either an acute ethanol (EtOH) or saline intraperitoneal injection. To investigate all 22 million CpGs in the mouse genome, we enriched for the methylated genomic fraction using methyl-CpG binding domain (MBD) protein capture followed by next-generation sequencing (MBD-seq). We performed association tests in blood and brain separately followed by enrichment testing to determine whether there was overlapping alcohol-related methylation in the 2 tissues. The top result for brain was a CpG located in an intron of Ttc39b (p=5.65×10-08 ), and for blood, the top result was located in Espnl (p=5.11×10-08 ). Analyses implicated pathways involved in inflammation and neuronal differentiation, such as CXCR4, IL-7, and Wnt signaling. Enrichment tests indicated significant overlap among the top results in brain and blood. Pathway analyses of the overlapping genes converge on MAPKinase signaling (p=5.6×10-05 ) which plays a central role in acute and chronic responses to alcohol and glutamate receptor pathways, which can regulate neuroplastic changes underlying addictive behavior. Overall, we have shown some methylation changes in brain and blood after acute EtOH administration and that the changes in blood partly mirror the changes in brain suggesting the potential for DNA methylation in blood to be biomarkers of alcohol use.

Methylome-wide association findings for major depressive disorder overlap in blood and brain and replicate in independent brain samples.

We present the first large-scale methylome-wide association studies (MWAS) for major depressive disorder (MDD) to identify sites of potential importance for MDD etiology. Using a sequencing-based approach that provides near-complete coverage of all 28 million common CpGs in the human genome, we assay methylation in MDD cases and controls from both blood (N=1,132) and postmortem brain tissues (N=61 samples from Brodmann Area 10, BA10). The MWAS for blood identified several loci with P 1.91×10−8-4.39×10−8 and a resampling approach showed that the cumulative association was significant (P=4.03×10−10) with the signal coming from the top 25,000 MWAS markers. Furthermore, a permutation based analysis showed significant overlap (P=5.4×10−3) between the MWAS findings in blood and brain (BA10). This overlap was significantly enriched for a number of features including being in eQTLs in blood and frontal cortex, CpG islands and shores, and exons. The overlapping sites were also enriched for active chromatin states in brain including genic enhancers and active transcription start sites. Furthermore, three loci located in GABBR2, RUFY3 and in an intergenic region on chromosome 2 replicated with the same direction of effect in the second brain tissue (BA25, N=60) from the same individuals and in two independent brain collections (BA10, N=81 and 64). GABBR2 inhibits neuronal activity through G protein-coupled second-messenger systems and RUFY3 is implicated in the establishment of neuronal polarity and axon elongation. In conclusion, we identified and replicated methylated loci associated with MDD that are involved in biological functions of likely importance to MDD etiology.

Convergence of evidence from a methylome-wide CpG-SNP association study and GWAS of major depressive disorder

DNA methylation is an epigenetic modification that provides stability and diversity to the cellular phenotype. It is influenced by both genetic sequence variation and environmental factors, and can therefore potentially account for variation of heritable phenotypes and disorders. Therefore, methylome-wide association studies (MWAS) are promising complements to genome-wide association studies (GWAS) of genetic variants. Of particular interest are methylation sites (CpGs) that are created or destroyed by the alleles of single-nucleotide polymorphisms (SNPs), as these so-called CpG-SNPs may show variation in methylation levels on top of what can be explained by the sequence variation. Using sequencing-based data from 1132 major depressive disorder (MDD) cases and controls, we performed a MWAS of 970,414 common CpG-SNPs. The analysis identified 27 suggestively significant (P < 1.00 × 10−5) CpG-SNPs associations. Furthermore, the MWAS results were over-represented (odds ratios ranging 1.36–5.00; P ranging 4.9 × 10−3–8.1 × 10−2) among findings from three recent GWAS for MDD-related phenotypes. Overlapping loci included, e.g., ROBO2, ASIC2, and DCC. As the CpG-SNP analysis accounts for the number of alleles that creates CpGs, the methylation differences could not be explained by differences in allele frequencies. Thus, the results show that the MWAS and GWASs provide independent lines of evidence for the involvement of these loci in MDD. In conclusion, our methylation study of MDD contributes novel information about loci of relevance that complements previous findings and generates new hypothesis about MDD etiology, such as that the functional effects of genetic association may be partly mediated and/or enhanced by the methylation status in these loci.

Interaction between plasma homocysteine and the MTHFR c.677C > T polymorphism is associated with site-specific changes in DNA methylation in humans.

One-carbon metabolism provides a direct link among dietary folate/vitamin B12 exposure, the activity of the enzyme methylenetetrahydrofolate reductase (MTHFR), and epigenetic regulation of the genome via DNA methylation. Previously, it has been shown that the common c.677C > T polymorphism in MTHFR influences global DNA methylation status through a direct interaction with folate status and (indirectly) with total homocysteine (tHcy) levels. To build on that and other more recent observations that have further highlighted associations among MTHFR c.677C > T, tHcy, and aberrations in DNA methylation, we investigated whether the interaction between mildly elevated plasma tHcy and the c.677C > T polymorphism is associated with site-specific changes in DNA methylation in humans. We used data on plasma tHcy levels, c.677C > T polymorphism, and site-specific DNA methylation levels for a total of 915 white women and 335 men from the TwinsUK registry ( n = 610) and the Rotterdam study ( n = 670). We performed methylome-wide association analyses in each cohort to model the interaction between levels of tHcy and c.677C > T genotypes on DNA methylation β values. Our meta-analysis identified 13 probes significantly associated with rs1801133 × tHcy levels [false-discovery rate (FDR) < 0.05]. The most significant associations were with a cluster of probes at the AGTRAP-MTHFR-NPPA/B gene locus on chromosome 1 (FDR = 1.3E-04), with additional probes on chromosomes 2, 3, 4, 7, 12, 16, and 19. Our top 2 hits on chromosome 1 were functionally associated with variability in expression of the TNF receptor superfamily member 8 ( TNFRSF8) gene/locus on that chromosome. This is the first study, to our knowledge, to provide a direct link between perturbations in 1-carbon metabolism, through an interaction of tHcy and the activity of MTHFR enzyme on epigenetic regulation of the genome via DNA methylation.-Nash, A. J., Mandaviya, P. R., Dib, M.-J., Uitterlinden, A. G., van Meurs, J., Heil, S. G., Andrew, T., Ahmadi, K. R. Interaction between plasma homocysteine and the MTHFR c.677C>T polymorphism is associated with site-specific changes in DNA methylation in humans.

Abstract 3320: Discovery and development of DNA methylation biomarkers in human papillomavirus related oropharyngeal squamous cell carcinoma

Abstract Purpose: Human papillomavirus (HPV) related oropharyngeal squamous cell carcinoma (OPSCC) has an improved prognosis compared to cancers associated with tobacco and alcohol exposure, and also exhibits a different composition of epigenetic alterations. In this methylome-wide association study, we identified DNA methylation-based biomarkers with potential utility in screening for HPV positive OPSCC. Experimental Design: In the discovery stage, genome wide DNA methylation was measured using methyl-CpG binding domain protein-enriched genome sequencing (MBD-seq) in 50 HPV positive OPSCC tissues and 25 normal tissues. We defined 51 differentially methylated regions (DMRs) with maximal methylation specificity to cancer samples. We used TCGA methylation array data to evaluate the performance of the proposed candidates. Top 20 candidates were validated using quantitative methylation specific PCR (QMSP) in independent 24 HPV related OPSCCs and 22 controls. Results: Supervised hierarchical clustering of 51 DMRs based on TCGA methylation data found that HPV positive OPSCC had significantly higher DNA methylation levels compared to normal samples and head and neck squamous cell carcinoma (HNSCC) without HPV or with different tumor sites. The methylation levels of all top 20 candidates in HPV positive OPSCC were significantly higher than those in normal samples in TCGA data. 16 of these 20 candidates were found by QMSP to have significant higher methylation levels compared with controls. 2 candidates (CCDC181 and OR6S1) had a sensitivity of 100%, while 10 candidates (KCNA3, ITGA4, BEND4, ELMO1, SFMBT2, HOXB4, ZNF439, ZNF93, VSTM2B and ZNF773) had a specificity of 100%. Our 20 candidates were found by the receiver operating characteristic analysis to have a prediction accuracy ranging from 56.3% to 99.8%. Conclusions: This study discovered 20 epigenetic markers for detection of HPV related OPSCC, which can potentially be applied to develop a population based screening test and improve disease management. Citation Format: Shuling Ren, Daria A. Gaykalova, Theresa W. Guo, Alexander V. Favorov, Elana J. Fertig, Zubair Khan, Peter M. DeJong, Mizuo Ando, Chao Liu, Akihiro Sakai, Takahito Fukusumi, Sunny Haft, Sayed Sadat, J Silvio Gutkind, Wayne M. Koch, Joseph A. Califano. Discovery and development of DNA methylation biomarkers in human papillomavirus related oropharyngeal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3320.

Abstract MP61: A Multi-pollutant, Multi-ethnic, and Methylome-wide Association Study Highlights Epigenetic Effects of Exposure to Ambient Air Pollution Mixtures

Background: Ambient air pollution mixtures including fine particulates and gases have been associated with cardiovascular disease (CVD). Although mechanisms underlying the associations remain largely uncharacterized to date, examining epigenetic responses to air pollution mixtures may identify biological systems affected by exposure and provide insight into the pathophysiology and environmental epidemiology of CVD. Therefore, we undertook a multi-pollutant, methylome-wide study of associations between DNA methylation (DNAm) at 485,000 Cytosine-phosphate-Guanine (CpG) sites and pollutants regulated by the U.S. Environmental Protection Agency under the Clean Air Act. Methods: Data from the Atherosclerosis Risk in Communities Study and three sub-studies within the Women’s Health Initiative (WHI) included information on 8,567 participants, 83% of whom were women, 46% African American, and 9% Hispanic/Latino (mean age, 61.3 years). Daily mean, geocoded participant address-specific concentrations of ambient particulate matter with diameter ≤ 10μm (PM 10 ), ≤2.5μm (PM 2.5 ), and 2.5-10μm (PM 2.5-10 ); ozone (O 3 ); nitrogen dioxide (NO 2 ); nitrogen oxides (NO x ); carbon monoxide (CO); and sulfur dioxide (SO 2 ) were averaged over 2, 7, 28, and 365 days before measurement of leukocyte DNAm in whole blood. Methylome-wide, pollutant-DNAm associations were estimated separately at each averaging period in each stratum defined by race/ethnicity and study using multi-level, linear mixed models adjusting for socio-demographic, behavioral, and meteorological characteristics; estimated leukocyte proportions; and technical covariates. Estimates were combined across strata in pollutant- and period-specific, fixed-effects, inverse-variance weighted meta-analyses. Then multi-pollutant, period-specific effects were examined using the adaptive sum of powered scores (U) test (aSPU). CpG sites associated with multi-pollutant significance (aSPU P &lt; 1.0 x 10 -7 ) were characterized in silico to assess their putative function and biological plausibility. Results: Overall analyses identified a significant, 28-day mean, multi-pollutant association at cg15008743 near ZNF83 on chromosome 19 (p = 1x10 -7 ) and a highly-suggestive 28-day mean, multipollutant association at cg00489219 near ZNF621 on chromosome 3 (p = 5x10 -7 ). ZNF83 and ZNF621 encode zinc finger proteins with a potentially broad reach across biological systems given their transcription factor, metal, and DNA binding activities. ZNF621 also harbors rare genetic variants with large effects on blood pressure. Discussion: Methylome-wide associations of DNAm with EPA-regulated ambient air pollutants suggest biologically plausible pathways underpinning the increasingly well-documented ties between multi-pollutant mixtures and cardiovascular disease.

Abstract P105: Gaseous Air Pollutants and DNA Methlyation in a Methylome-wide Association Study of an Ethnically and Environmentally Diverse Population of U.S. Adults

Background: Epigenetic mechanisms underlying associations between coronary artery disease (CAD) and ambient exposures to carbon monoxide (CO), nitrogen oxides (NO 2 /NOx), ozone (O 3 ), and sulfur dioxide (SO 2 ) are incompletely understood. We therefore examined associations between these gaseous air pollutants and DNA methylation (DNAm)—a process that controls gene expression. Methods: We conducted a race/ethnicity- and study-stratified, methylome-wide association study of 12 Women’s Health Initiative and Atherosclerosis Risk in Communities subpopulations (N=8567, mean age 61.3 years, 83% female, 46% African American, 8% Hispanic/Latino American). In each subpopulation, we estimated daily mean ambient pollutant concentrations at geocoded participant addresses using national-scale, lognormal ordinary kriging. We averaged the concentrations over the 2, 28, and 365 days before phlebotomy, the source of leukocyte DNA used in estimating DNAm at 485,000 Cytosine-phosphate-Guanine (CpG) sites on the Illumina Infinium HumanMethylation450 BeadChip. We estimated pollutant-DNAm associations using multi-level, linear mixed-effects models adjusted for socio-demographic, behavioral, and meteorological factors; estimated leukocyte proportions; and technical covariates. Then we combined estimates from the 12 subpopulations in inverse variance-weighted meta-analyses and characterized CpG sites associated with significant associations and little evidence of heterogeneity among subpopulations ( p &lt;1.0x10 -7 ; p Cochran’s Q &gt;0.05). Results: Although we found no significant or suggestive CO-, O 3 -, or SO 2 -DNAm associations, we found a significant 28-day mean NO 2 -DNAm association at cg01885635 near ZNF621 on chr. 3 ( p =5.01x10 -8 ) and suggestive 365-day mean NO 2 -DNAm associations at cg12684684 near ARPC2 on chr. 2 ( p =9.3x10 -7 ), cg19693031 near TXNIP on chr. 1 ( p =7.3x10 -7 ), and cg01885635 near ZNF621 on chr. 3 ( p =6.7x10 -7 ). We also found 2- and 365-day mean NOx-DNAm associations at cg27032760 near KCTD16 on chr. 5 ( p =4.1x10 -7 ) and cg15008743 near ZNF83 on chr. 19 ( p =2.8x10 -7 ), respectively. ZNF621 and ZNF83 encode zinc finger proteins that possess transcription factor activity. ARPC2 encodes an actin-related protein complex subunit that has been associated with mean platelet volume, a known risk factor for myocardial infarction (MI) and post-MI mortality. TXNIP encodes a thioredoxin-binding protein that protects cells from oxidative stress, is highly glucose responsive, and has been associated with triglycerides, blood pressure, diabetes, and CAD. KCTD16 encodes a potassium channel tetramerization domain-containing protein. Conclusions: Pending replication, the preliminary findings suggest that biologically plausible epigenetic mechanisms may underlie associations between nitrogen oxide exposures and CAD in multi-racial/ethnic populations.

Abstract P101: Methylome-wide Association Study Provides Evidence of Particulate Matter Air Pollution-associated Dna Methylation at Cardiovascular Disease-related Genes

Background: DNA methylation (DNAm), a heritable but dynamic epigenetic modification that can influence gene expression without altering the genome, may underlie the associations between air pollution and cardiovascular disease risk. Therefore, our objective was to evaluate associations between DNAm and ambient concentrations of particulate matter (PM) ≤ 2.5 and ≤ 10 micrometers in diameter (PM 2.5; PM 10 ). Methods: We conducted a methylome-wide association study among twelve cohort- and race/ethnicity- stratified subpopulations from the Women’s Health Initiative and the Atherosclerosis Risk in Communities study (discovery n = 6,720; replication n = 1,936; mean age: 61.3 yrs; 83% female; 46% African American; 9% Hispanic/Latino American). We averaged geocoded address-specific estimates of daily and monthly mean PM concentrations over 2, 7, 28, and 365 days and 1 and 12 months before exams at which we measured leukocyte DNAm in whole blood. In each subpopulation, we estimated PM-DNAm associations at approximately 485,000 Cytosine-phosphate-Guanine (CpG) sites in multi-level, linear mixed-effects models adjusting for sociodemographic, behavioral, and meteorological characteristics; estimated leukocyte proportions; and technical covariates. We combined subpopulation-specific PM-DNAm associations in fixed-effects, inverse variance-weighted meta-analyses of the discovery, replication, and overall populations. Then we conducted in silico characterization of CpG sites at which PM-DNAm associations exceeded methylome-wide significance and were not heterogeneous ( P &lt; 1.0 x 10 -7 ; P Cochran’s Q &gt; 0.10) to assess their putative function and biological plausibility. Results: Discovery analyses identified significant PM 2.5 - and PM 10 -DNAm associations at four CpG sites, but none survived Bonferroni correction. Overall analyses identified significant associations at two CpG sites. On chromosome 20 near MATN4 , 28-day mean PM 10 was associated with increased DNAm at cg19004594 ( P all = 2.8 x 10 -8 ; P Cochran’s Q = 0.61). MATN4 is expressed in heart and lung tissues. It encodes Matrilin 4, a von Willebrand factor A domain-containing protein linked to cardiac remodeling. On chromosome 10 near ARPP21 , 1-month mean PM 10 was inversely associated with DNAm at cg24102420 ( P all = 4.8 x 10 -8 ; P Cochran’s Q = 0.51). ARPP21 is expressed in the brain/spinal cord and neutrophils. It encodes cAMP-regulated phosphoprotein 21, a regulator of calmodulin/calcium signaling. Conclusions: Findings from this methylome-wide association study suggest that ambient PM 10 concentrations affect DNA methylation at regions of the genome potentially related to cardiovascular disease among racially, ethnically and environmentally diverse populations of U.S. men and women. Further investigation is warranted to uncover epigenetic mechanisms of PM-associated cardiovascular traits.

DNA methylation analyses of the candidate genes identified by a methylome-wide association study revealed common epigenetic alterations in schizophrenia and bipolar disorder.

Schizophrenia (SZ) and bipolar disorder (BD) have been known to share genetic and environmental risk factors, and complex gene-environmental interactions may contribute to their pathophysiology. In contrast to high genetic overlap between SZ and BD, as revealed by genome-wide association studies, the extent of epigenetic overlap remains largely unknown. In the present study, we explored whether SZ and BD share epigenetic risk factors in the same manner as they share genetic components. We performed DNA methylation analyses of the CpG sites in the top five candidate regions (FAM63B, ARHGAP26, CTAGE11P, TBC1D22A, and intergenic region [IR] on chromosome 16) reported in a previous methylome-wide association study (MWAS) of SZ, using whole blood samples from subjects with BD and controls. Among the five candidate regions, the CpG sites in FAM63B and IR on chromosome 16 were significantly hypomethylated in the samples from subjects with BD as well as those from subjects with SZ. On the other hand, the CpG sites in TBC1D22A were hypermethylated in the samples from subjects with BD, in contrast to hypomethylation in the samples from subjects with SZ. Hypomethylation of FAM63B and IR on chromosome 16 could be common epigenetic risk factors for SZ and BD. Further comprehensive epigenetic studies for BD, such as MWAS, will uncover the extent of similarity and uniqueness of epigenetic alterations.