Single CpG Research Articles

TWIST1 DNA methylation is a cell marker of airway and parenchymal lung fibroblasts that are differentially methylated in asthma

BackgroundMesenchymal fibroblasts are ubiquitous cells that maintain the extracellular matrix of organs. Within the lung, airway and parenchymal fibroblasts are crucial for lung development and are altered with disease, but it has been difficult to understand their roles due to the lack of distinct molecular markers. We studied genome-wide DNA methylation and gene expression in airway and parenchymal lung fibroblasts from healthy and asthmatic donors, to identify a robust cell marker and to determine if these cells are molecularly distinct in asthma.ResultsAirway (N = 8) and parenchymal (N = 15) lung fibroblasts from healthy individuals differed in the expression of 158 genes, and DNA methylation of 3936 CpGs (Bonferroni adjusted p value < 0.05). Differential DNA methylation between cell types was associated with differential expression of 42 genes, but no single DNA methylation CpG feature (location, effect size, number) defined the interaction. Replication of gene expression and DNA methylation in a second cohort identified TWIST1 gene expression, DNA methylation and protein expression as a cell marker of airway and parenchymal lung fibroblasts, with DNA methylation having 100% predictive discriminatory power. DNA methylation was differentially altered in parenchymal (112 regions) and airway fibroblasts (17 regions) with asthmatic status, with no overlap between regions.ConclusionsDifferential methylation of TWIST1 is a robust cell marker of airway and parenchymal lung fibroblasts. Airway and parenchymal fibroblast DNA methylation are differentially altered in individuals with asthma, and the role of both cell types should be considered in the pathogenesis of asthma.

Association between NPPA promoter methylation and hypertension: results from Gusu cohort and replication in an independent sample

BackgroundAtrial natriuretic peptide (ANP), one of the main members of the natriuretic peptides system, has been associated with hypertension and related complications, but the underlying molecular mechanisms are not very clear. Here, we aimed to examine whether DNA methylation, a molecular modification to the genome, of the natriuretic peptide A gene (NPPA), the coding gene of ANP, was associated with hypertension.MethodsPeripheral blood DNA methylation of NPPA promoter was quantified by target bisulfite sequencing in 2498 community members (mean aged 53 years, 38% men) as a discovery sample and 1771 independent participants (mean aged 62 years, 54% men) as a replication sample. In both samples, we conducted a single CpG association analysis, followed by a gene-based association analysis, to examine the association between NPPA promoter methylation and hypertension, adjusting for age, sex, education level, cigarette smoking, alcohol consumption, obesity, fasting glucose, and lipids. Multiple testing was controlled by the false discovery rate approach.ResultsOf the 9 CpG loci assayed, hypermethylation at 5 CpGs (CpG1, CpG3, CpG6, CpG8, and CpG9) was significantly associated with a lower odds of prevalent hypertension in the discovery sample, and one CpG methylation (CpG1 located at Chr1:11908353) was successfully replicated in the replication sample (OR = 0.82, 95%CI 0.74–0.91, q = 0.002) after adjusting for covariates and multiple testing. The gene-based analysis found that DNA methylation of the 9 CpGs at NPPA promoter as a whole was significantly associated with blood pressure and prevalent hypertension in both samples (all P < 0.05).ConclusionsDNA methylation levels at NPPA promoter were decreased in Chinese adults with hypertension. Aberrant DNA methylation of the NPPA gene may participate in the mechanisms of hypertension.

Analysis of transcript and methylation levels of INSL3 and RXFP2 in undescended and descended dog testes suggested promising biomarkers associated with cryptorchidism

Cryptorchidism is the most common disorder of sex development (DSD) in dogs. This malformation is associated with reduced fertility and with a higher risk of gonadal cancer. Testicular descent is a complex process, and the functions of many environmental and genetic factors are crucial for the proper migration of fetal gonads into the scrotum. Among these, the hormone INSL3 (insulin-like peptide 3) and its receptor RXFP2 (relaxin family peptide receptor 2) play crucial roles in the transabdominal migration of the testes. The genetic background of canine cryptorchidism is poorly elucidated. The aim of this study was to compare the transcript and methylation levels of INSL3 and RXFP2 genes in undescended and descended testes of isolated unilateral cryptorchids, and in gonads of control male dogs with scrotal testes. Next, we searched for polymorphic variants in the 5′-regulatory regions of both genes associated with predispositions to cryptorchidism. The INSL3 transcript level was significantly higher in the undescended testes than in the descended testes of both the affected and control dogs. On the other hand, the mRNA level of RXFP2 was significantly lower in the retained gonads of cryptorchids than in the scrotal testes. The methylation level of a single CpG site located 15 bp upstream of the translation start codon in INSL3 was significantly higher in the testes of the control dogs than in both gonads of cryptorchids. The methylation level of 14 CpG sites in the coding region of INSL3 was significantly higher in undescended testes than in the scrotal testes, which may be associated with the higher mRNA levels of INSL3 observed in these samples. The methylation pattern of two CpG sites in the 5′-flanking region of RXFP2 was similar in both descended and undescended testes. We detected three and seven single nucleotide polymorphisms (SNPs) in the 5′-regulatory regions of INSL3 and RXFP2, respectively. Among these, the frequency of A > C substitution (ss7093349755) located 495 bp upstream of the transcription start site of RXFP2 differed significantly between cryptorchids and control dogs. Our study showed two possible genetic biomarkers associated with canine cryptorchidism: a hypomethylation of a single CpG site in the 5′-flanking region of INSL3, and the ss7093349755 SNP in the 5′-flanking region of RXFP2.

Epigenetic effect of folic acid (FA) on the gene proximal promoter area and mRNA expression of chicken B cell as antigen presenting cells

ABSTRACT 1. This study evaluated and characterised the effect of folic acid (FA) on chromosomal DNA methylation and the epigenetic result on gene expression control mechanisms in chicken B cells as a model of antigen presenting cells. 2. After FA supplementation, the methylation pattern on the proximal promoter area and mRNA expression of toll-like receptor (TLR) 2b, TLR4, B cell receptor (BCR) immunoglobulin (Ig) β and major histocompatibility complex (MHC) II β chain genes in chicken B cells was observed 3. Chicken B cell line (DT40) cultures were incubated with 0, 1.72 or 3.96 mM of FA for 4 and 8 h and samples were taken at specific time points. After 4 h of incubation, cells were challenged with 0, 1 or 10 µg/ml of lipopolysaccharide (LPS) and samples were collected 4 h post-challenge. 4. FA supplementation modified the methylation patterns of the proximal promoter regions of TLR4, Igß, and MHCII ß chain at 4 and 8 hours of incubation; however, the single CpG dinucleotide of TLR2b remained methylated regardless of the treatment. 5. A positive association was found between FA concentration and percentage DNA methylation on the promoter area of Igβ and TLR2b. However, there was a negative association between FA and MHCII β chain. 6. There were downregulatory effects in TLR4, Igß and MHCII ß chain gene expression after 8 h of incubation, nut not at 4 h. Although incubation time did not affect TLR2b gene expression, FA concentration did, whereby it increased TLR2b expression at 1.72 mM FA (P < 0.05). 7. LPS significant downregulated TLR2b expression, while an interaction between FA and LPS concentration affected TLR4 and Igβ gene expression. 8. In conclusion, the results showed that FA can have an immunomodulatory effect on chicken B cells, possibly affecting their ability to both recognise antigens through the TLR and BCR pathways, and to present it via the MHCII presentation pathway.

Novel candidate genes for ECT response prediction\u2014a pilot study analyzing the DNA methylome of depressed patients receiving electroconvulsive therapy

BackgroundMajor depressive disorder (MDD) represents a serious global health concern. The urge for efficient MDD treatment strategies is presently hindered by the incomplete knowledge of its underlying pathomechanism. Despite recent progress (highlighting both genetics and the environment, and thus DNA methylation, to be relevant for its development), 30–50% of MDD patients still fail to reach remission with standard treatment approaches. Electroconvulsive therapy (ECT) is one of the most powerful options for the treatment of pharmacoresistant depression; nevertheless, ECT remission rates barely reach 50% in large-scale naturalistic population-based studies. To optimize MDD treatment strategies and enable personalized medicine in the long- term, prospective indicators of ECT response are thus in great need. Because recent target-driven analyses revealed DNA methylation baseline differences between ECT responder groups, we analyzed the DNA methylome of depressed ECT patients using next-generation sequencing. In this pilot study, we did not only aim to find novel targets for ECT response prediction but also to get a deeper insight into its possible mechanism of action.ResultsLongitudinal DNA methylation analysis of peripheral blood mononuclear cells isolated from a cohort of treatment-resistant MDD patients (n = 12; time points: before and after 1st and last ECT, respectively) using a TruSeq-Methyl Capture EPIC Kit for library preparation, led to the following results: (1) The global DNA methylation differed neither between the four measured time points nor between ECT responders (n = 8) and non-responders (n = 4). (2) Analyzing the DNA methylation variance for every probe (=1476812 single CpG sites) revealed eight novel candidate genes to be implicated in ECT response (protein-coding genes: RNF175, RNF213, TBC1D14, TMC5, WSCD1; genes encoding for putative long non-coding RNA transcripts: AC018685.2, AC098617.1, CLCN3P1). (3) In addition, DNA methylation of two CpG sites (located within AQP10 and TRERF1) was found to change during the treatment course.ConclusionsWe suggest ten novel candidate genes to be implicated in either ECT response or its possible mechanism. Because of the small sample size of our pilot study, our findings must be regarded as preliminary.

Altered expression of CYP17A1 and CYP19A1 in undescended testes of dogs with unilateral cryptorchidism.

Cryptorchidism is the most common disorder of sex development in dogs and testosterone plays a crucial role in the inguinal phase of the testes descending into the scrotum. The molecular background of impaired testosterone synthesis in the testes of cryptorchid dogs is poorly elucidated. In this study, we analyzed the expression of four genes involved in testicular steroidogenesis (CYP17A1, CYP19A1, HSD3B2 and HSD17B3) in undescended and contralateral scrotal testes from inguinal unilateral cryptorchid dogs (n=13) and from the scrotal gonads of normal males (n=15). We found that transcript level of CYP17A1 was significantly increased in inguinal gonads, while the level of CYP19A1 was decreased. For these two genes, we analyzed the methylation level of single CpG sites in the promoter region localized within putative target sites for testicular transcription factors (NUR77, CREB, CAR and HSF2). A correlation between decreased methylation in the promoter of CYP17A1 and its increased transcript level in undescended gonads was observed, but the change in protein level was not significant. We also resequenced the 5'-flanking region of both genes and two known polymorphic sites, SNP in CYP17A1 and an indel in CYP19A1, were found. However, the distribution of the variants in affected (n=80) and control (n=75) dogs was not associated with cryptorchidism. We tentatively conclude that the altered expression of CYP17A1 and CYP19A1 in undescended testes could be caused by their exposure to increased temperature in the body. Furthermore, we showed that the identified polymorphisms cannot be considered markers associated with a predisposition to cryptorchidism.

Ethnicity, age and disease-associated variation in body fluid-specific CpG sites in a diverse South African cohort

Tissue-specific differential DNA methylation has been an attractive target for the development of markers for discrimination of body fluids found at crime scenes. Though mostly stable, DNA methylation patterns have been shown to vary between different ethnic groups, in different age groups as well as between healthy and diseased individuals. To the best of our knowledge, none of the markers for body fluid identification have been applied to different ethnic groups to ascertain if variability exists. In the present study, saliva and blood were collected to determine the effects of ethnicity (Blacks, Whites, Coloureds and Indians), age (20–30 years, 40–50years and above 60 years) and diabetes on methylation profiles of potential saliva- and blood-specific DMSs. Both DMSs were previously shown to exhibit hypermethylation in their target body fluids at single CpG sites, however in the present study, additional CpG sites flanking the reported sites were also screened. Bisulfite sequencing revealed that Coloureds showed highest methylation levels for both body fluids, and blacks displayed significant differences between other ethnic groups in the blood-specific CpG sites. A decline in methylation for both potential DMRs was observed with increasing age. Heavily methylated CpG sites in different ethnic groups and previously reported DMSs displayed hypomethylation with increasing age and disease status. Diabetic status did not show any significant difference in methylation when compared to healthy counterparts. Thus, the use of methylation markers for forensics needs thorough investigation of influence of external factors and ideally, several CpG sites should be co-analysed instead of a single DMS.

Epigenetic regulation of TLR2‐mediated periapical inflammation

To determinethe methylation pattern of TLR2 gene promoter and its association with the transcriptional regulation of periapical inflammatory and angiogenic responses in symptomatic and asymptomatic forms of apical periodontitis. In this cross-sectional study, apical lesions were obtained from volunteers with asymptomatic apical periodontitis (AAP) (n=17) and symptomatic apical periodontitis (SAP) (n=17) scheduled for tooth extraction, and both total RNA and DNA were extracted. DNA was bisulfite-treated, a region of CpG island within the TLR2 gene was amplified by qPCR and the products were sequenced. Additionally, the mRNA expression of TLR2, TLR4, IL-6, IL-12, TNFalpha, IL-23, IL-10, TGFbeta, VEGFA and CDH5 was analysed by qPCR. The data were analysed with chi-square tests, Mann-Whitney or unpaired t-tests, and Spearman´s correlation; variable adjustments were performed using multiple linear regression (P<0.05). TLR2 depicted a hypomethylated DNA profile at the CpG island in SAPwhen compared with AAP, along with upregulated expression of TLR2, with pro-inflammatory cytokines IL-6 and IL-23, and the angiogenesis marker CDH5 (P<0.05). TLR2 methylation percentage negatively correlated with mRNA levels of IL-23 and CDH5 in apical periodontitis. Lower methylation frequencies of single CpG dinucleotides -8 and -10 localized in close proximity to nuclear factor κB (NFκB) binding within the TLR2 promoter were identified in SAP versus AAP (P<0.05). Finally, unmethylated -10 and -8 single sites demonstrated up-regulation of IL-23, IL-10 and CDH5 transcripts compared to their methylated counterparts (P<0.05). TLR2 gene promoter hypomethylation was linked to transcriptional activity of pro-inflammatory cytokines and angiogenic markers in exacerbated periapical inflammation. Moreover, unmethylated single sites in close proximity to NFκB binding were involved in active transcription of IL-23, IL-10 and CDH5.

Hypermethylation of Single CpG Dinucleotides at the Promoter of CXCL13 Gene Promoting Cell Migration in Cervical Cancer.

Chemokine 13 (CXCL13) and its chemokine receptor 5 (CXCR5) are involved in the onset of various types of cancer. However, their role in cervical cancer (CC) remains unknown. To investigate the role of chemokine 13 (CXCL13) and its receptor in CC. The expression of CXCL13/CXCR5 and the infiltration of CXCR5+CD8+ T cells in CC, cervical intraepithelial neoplasia (CIN), normal cervical epithelial (NCE) tissues, and in CC cell lines were analysed and the associated clinical significance was determined. In vitro, CXCL13 overexpression and DNA methyltransferase inhibition (through S110) were used to investigate the biological function and the underlying mechanism that regulates CXCL13 expression. Tumor growth and liver metastasis were also evaluated in the xenogenous subcutaneously implant model. CXCL13/CXCR5 expression levels and the infiltration of CXCR5+CD8+ T cells were significantly decreased in CC tissues compared with CIN and NCE tissues. CXCL13 downregulation was significantly correlated with the FIGO stages, lymph node metastasis, interstitial infiltration depth, and pathological grade. The overexpression of CXCL13 suppressed CC cell migration. CXCL13 downregulation was associated with hypermethylation in CC cell lines, and primary tumor biopsies. Furthermore, a CpG dinucleotide at the HIF-1a transcription factor motifs in the promoter element of CXCL13 was consistently methylated in CC cells and associated with HIF-1a. CXCL13 overexpression and S110 treatment dramatically repressed tumor growth and liver metastasis in the xenograft model; whereas it's low expression increased the risk of death in CC patients. DNA methylation-dependent CXCL13 downregulation may promote cervical carcinogenesis and progression.

Methylation of a CGATA element inhibits binding and regulation by GATA-1

Alterations in DNA methylation occur during development, but the mechanisms by which they influence gene expression remain uncertain. There are few examples where modification of a single CpG dinucleotide directly affects transcription factor binding and regulation of a target gene in vivo. Here, we show that the erythroid transcription factor GATA-1 — that typically binds T/AGATA sites — can also recognise CGATA elements, but only if the CpG dinucleotide is unmethylated. We focus on a single CGATA site in the c-Kit gene which progressively becomes unmethylated during haematopoiesis. We observe that methylation attenuates GATA-1 binding and gene regulation in cell lines. In mice, converting the CGATA element to a TGATA site that cannot be methylated leads to accumulation of megakaryocyte-erythroid progenitors. Thus, the CpG dinucleotide is essential for normal erythropoiesis and this study illustrates how a single methylated CpG can directly affect transcription factor binding and cellular regulation.

PretiMeth: precise prediction models for DNA methylation based on single methylation mark

BackgroundThe computational prediction of methylation levels at single CpG resolution is promising to explore the methylation levels of CpGs uncovered by existing array techniques, especially for the 450 K beadchip array data with huge reserves. General prediction models concentrate on improving the overall prediction accuracy for the bulk of CpG loci while neglecting whether each locus is precisely predicted. This leads to the limited application of the prediction results, especially when performing downstream analysis with high precision requirements.ResultsHere we reported PretiMeth, a method for constructing precise prediction models for each single CpG locus. PretiMeth used a logistic regression algorithm to build a prediction model for each interested locus. Only one DNA methylation feature that shared the most similar methylation pattern with the CpG locus to be predicted was applied in the model. We found that PretiMeth outperformed other algorithms in the prediction accuracy, and kept robust across platforms and cell types. Furthermore, PretiMeth was applied to The Cancer Genome Atlas data (TCGA), the intensive analysis based on precise prediction results showed that several CpG loci and genes (differentially methylated between the tumor and normal samples) were worthy for further biological validation.ConclusionThe precise prediction of single CpG locus is important for both methylation array data expansion and downstream analysis of prediction results. PretiMeth achieved precise modeling for each CpG locus by using only one significant feature, which also suggested that our precise prediction models could be probably used for reference in the probe set design when the DNA methylation beadchip update. PretiMeth is provided as an open source tool via https://github.com/JxTang-bioinformatics/PretiMeth.

The sperm epigenome does not display recurrent epimutations in patients with severely impaired spermatogenesis

BackgroundIn the past 15 years, numerous studies have described aberrant DNA methylation of imprinted genes (e.g. MEST and H19) in sperm of oligozoospermic men, but the prevalence and genomic extent of abnormal methylation patterns have remained unknown.ResultsUsing deep bisulfite sequencing (DBS), we screened swim-up sperm samples from 40 normozoospermic and 93 patients diagnosed as oligoasthenoteratozoospermic, oligoteratozoospermic or oligozoospermic, which are termed OATs throughout the manuscript, for H19 and MEST methylation. Based on this screening, we defined three patient groups: normal controls (NC), abnormally methylated oligozoospermic (AMO; n = 7) and normally methylated oligozoospermic (NMO; n = 86). Whole-genome bisulfite sequencing (WGBS) of five NC and five AMO samples revealed abnormal methylation levels of all 50 imprinting control regions in each AMO sample. To investigate whether this finding reflected epigenetic germline mosaicism or the presence of residual somatic DNA, we made a genome-wide inventory of soma-germ cell-specific DNA methylation. We found that > 2000 germ cell-specific genes are promoter-methylated in blood and that AMO samples had abnormal methylation levels at these genes, consistent with the presence of somatic cell DNA. The comparison between the five NC and six NMO samples revealed 19 differentially methylated regions (DMRs), none of which could be validated in an independent cohort of 40 men. Previous studies reported a higher incidence of epimutations at single CpG sites in the CTCF-binding region 6 of H19 in infertile patients. DBS analysis of this locus, however, revealed an association between DNA methylation levels and genotype (rs2071094), but not fertility phenotype.ConclusionsOur results suggest that somatic DNA contamination and genetic variation confound methylation studies in sperm of infertile men. While we cannot exclude the existence of rare patients with slightly abnormal sperm methylation at non-recurrent CpG sites, the prevalence of aberrant methylation in swim-up purified sperm of infertile men has likely been overestimated, which is reassuring for patients undergoing assisted reproduction.

The landscape of CD28, CD80, CD86, CTLA4, and ICOS DNA methylation in head and neck squamous cell carcinomas

ABSTRACT CTLA-4 blocking therapeutic antibodies are currently under investigation in head and neck squamous cell carcinoma (HNSCC). A better understanding of the epigenetic regulation of the CD28 superfamily members CD28, CTLA-4, and ICOS and their B7 ligands, CD80 and CD86, could support the development of biomarkers for response prediction to anti-CTLA-4 immunotherapy. We investigated methylation of the encoding genes CD28, CTLA4, ICOS, CD80, and CD86 at single CpG resolution (51 CpG sites) in a cohort of HNSCC (N = 528) and normal adjacent tissue samples (N = 50) provided by The Cancer Genome Research Atlas, in isolated blood leukocytes from healthy individuals (N = 28), and HNSCC cell lines (N = 39). We analysed methylation levels with regard to mRNA expression, overall survival, mutational load, interferon-γ signature, and signatures of immune cell infiltrates. Depending on the location of the CpG sites (promoter, promoter flank, gene body, and intergenic sites), we found significant differences in methylation levels among isolated leukocytes, between leukocytes and HNSCC cell lines, and among HNSCCs. Methylation of all analysed genes correlated inversely or positively with mRNA expression, depending on the CpG site. CD28, CTLA4, and ICOS revealed almost identical correlation patterns. Furthermore, we found significant correlations with survival and features of response to immunotherapy, i.e. interferon-γ signature, signatures of tumour infiltrating immune cells, and mutational load. Our results suggest CD28, CTLA4, ICOS, CD80, and CD86 expression levels are epigenetically co-regulated by DNA methylation. This study provides rationale to test their DNA methylation as potential biomarker for prediction of response to CTLA-4 immune checkpoint inhibitors.

DNA methylation profiling identifies a high effect genetic variant for lipoprotein(a) levels

ABSTRACT Changes in whole blood DNA methylation levels at several CpG sites have been associated with circulating blood lipids, specifically high-density lipoprotein and triglycerides. This study performs a discovery and validation epigenome-wide association study (EWAS) for circulating lipoprotein(a) [Lp(a)], an independent risk factor for cardiovascular diseases. Whole-blood DNA methylation profiles were assessed in a cohort of 1020 elderly individuals using the Illumina EPIC array and independent validation in 359 elderly males using the Illumina 450 k array. Plasma Lp(a) was measured using an apolipoprotein(a)-size-independent ELISA. Epigenome-wide rank regression analysis identified and validated a single CpG site, cg17028067 located in intron 1 of the LPA gene, that was significantly associated with plasma Lp(a) levels after correction for multiple testing. Genotyping of the site identified a relatively uncommon SNP (rs76735376, MAF <0.02) at the CpG site that largely explained the observed methylation effect. Rs76735376 is an expression quantitative trait loci for the LPA gene and could affect expression by altering enhancer activity. This EWAS for plasma Lp(a) identified a single CpG site within LPA. This association is due to an uncommon, but highly effective genetic variant, which was not in significant linkage disequilibrium with other variants known to influence Lp(a) levels or apo(a) isoform size. This study highlights the utility of CpG site methylation to identify potentially important genetic associations that would not be readily apparent in a comparable size genetic association study.

Modeling methylation dynamics with simultaneous changes in CpG islands

BackgroundIn vertebrate genomes, CpG sites can be clustered into CpG islands, and the amount of methylation in a CpG island can change due to gene regulation processes. Thus, single regulatory events can simultaneously change the methylation states of many CpG sites within a CpG island. This should be taken into account when quantifying the amount of change in methylation, for example in form of a branch length in a phylogeny of cell types.ResultsWe propose a probabilistic model (the IWE-SSE model) of methylation dynamics that accounts for simultaneous methylation changes in multiple CpG sites belonging to the same CpG island. We further propose a Markov-chain Monte-Carlo (MCMC) method to fit this model to methylation data from cell type phylogenies and apply this method to available data from murine haematopoietic cells and from human cell lines. Combined with simulation studies, these analyses show that accounting for CpG island wide methylation changes has a strong effect on the inferred branch lengths and leads to a significantly better model fit for the methylation data from murine haematopoietic cells and human cell lines.ConclusionThe MCMC based parameter estimation method for the IWE-SSE model in combination with our MCMC based inference method allows to quantify the amount of methylation changes at single CpG sites as well as on entire CpG islands. Accounting for changes affecting entire islands can lead to more accurate branch length estimation in the presence of simultaneous methylation change.

Profiling haplotype specific CpG and CpH methylation within a schizophrenia GWAS locus on chromosome 14 in schizophrenia and healthy subjects

Interrogating DNA methylation within schizophrenia risk loci holds promise to identify mechanisms by which genes influence the disease. Based on the hypothesis that allele specific methylation (ASM) of a single CpG, or perhaps CpH, might mediate or mark the effects of genetic variants on disease risk and phenotypes, we explored haplotype specific methylation levels of individual cytosines within a genomic region harbouring the BAG5, APOPT1 and KLC1 genes in peripheral blood of schizophrenia patients and healthy controls. Three DNA fragments located in promoter, intronic and intergenic areas were studied by single-molecule real-time bisulfite sequencing enabling the analysis of long reads of DNA with base-pair resolution and the determination of haplotypes directly from sequencing data. Among 1,012 cytosines studied, we did not find any site where methylation correlated with the disease or cognitive deficits after correction for multiple testing. At the same time, we determined the methylation profile associated with the schizophrenia risk haplotype within the KLC1 fourth intron and confirmed ASM for cytosines located in the vicinity of rs67899457. These genetically associated DNA methylation variations may be related to the pathophysiological mechanism differentiating the risk and non-risk haplotypes and merit further investigation.

Collective effects of long-range DNA methylations predict gene expressions and estimate phenotypes in cancer

DNA methylation of various genomic regions has been found to be associated with gene expression in diverse biological contexts. However, most genome-wide studies have focused on the effect of (1) methylation in cis, not in trans and (2) a single CpG, not the collective effects of multiple CpGs, on gene expression. In this study, we developed a statistical machine learning model, geneEXPLORE (geneexpression prediction by long-range epigenetics), that quantifies the collective effects of both cis- and trans- methylations on gene expression. By applying geneEXPLORE to The Cancer Genome Atlas (TCGA) breast and 10 other types of cancer data, we found that most genes are associated with methylations of as much as 10 Mb from the promoters or more, and the long-range methylation explains 50% of the variation in gene expression on average, far greater than cis-methylation. geneEXPLORE outperforms competing methods such as BioMethyl and MethylXcan. Further, the predicted gene expressions could predict clinical phenotypes such as breast tumor status and estrogen receptor status (AUC = 0.999, 0.94 respectively) as accurately as the measured gene expression levels. These results suggest that geneEXPLORE provides a means for accurate imputation of gene expression, which can be further used to predict clinical phenotypes.

Identification of methylation states of DNA regions for Illumina methylation BeadChip

BackgroundMethylation of cytosine bases in DNA is a critical epigenetic mark in many eukaryotes and has also been implicated in the development and progression of normal and diseased cells. Therefore, profiling DNA methylation across the genome is vital to understanding the effects of epigenetic. In recent years the Illumina HumanMethylation450 (HM450K) and MethylationEPIC (EPIC) BeadChip have been widely used to profile DNA methylation in human samples. The methods to predict the methylation states of DNA regions based on microarray methylation datasets are critical to enable genome-wide analyses.ResultWe report a computational approach based on the two layers two-state hidden Markov model (HMM) to identify methylation states of single CpG site and DNA regions in HM450K and EPIC BeadChip. Using this mothed, all CpGs detected by HM450K and EPIC in H1-hESC and GM12878 cell lines are identified as un-methylated, middle-methylated and full-methylated states. A large number of DNA regions are segmented into three methylation states as well. Comparing the identified regions with the result from the whole genome bisulfite sequencing (WGBS) datasets segmented by MethySeekR, our method is verified. Genome-wide maps of chromatin states show that methylation state is inversely correlated with active histone marks. Genes regulated by un-methylated regions are expressed and regulated by full-methylated regions are repressed. Our method is illustrated to be useful and robust.ConclusionOur method is valuable for DNA methylation genome-wide analyses. It is focusing on identification of DNA methylation states on microarray methylation datasets. For the features of array datasets, using two layers two-state HMM to identify to methylation states on CpG sites and regions creatively, our method which takes into account the distribution of genome-wide methylation levels is more reasonable than segmentation with a fixed threshold.

The role of environmental stress and DNA methylation in the longitudinal course of bipolar disorder

BackgroundStressful life events influence the course of affective disorders, however, the mechanisms by which they bring about phenotypic change are not entirely known.MethodsWe explored the role of DNA methylation in response to recent stressful life events in a cohort of bipolar patients from the longitudinal PsyCourse study (n = 96). Peripheral blood DNA methylomes were profiled at two time points for over 850,000 methylation sites. The association between impact ratings of stressful life events and DNA methylation was assessed, first by interrogating methylation sites in the vicinity of candidate genes previously implicated in the stress response and, second, by conducting an exploratory epigenome-wide association analysis. Third, the association between epigenetic aging and change in stress and symptom measures over time was investigated.ResultsInvestigation of methylation signatures over time revealed just over half of the CpG sites tested had an absolute difference in methylation of at least 1% over a 1-year period. Although not a single CpG site withstood correction for multiple testing, methylation at one site (cg15212455) was suggestively associated with stressful life events (p < 1.0 × 10−5). Epigenetic aging over a 1-year period was not associated with changes in stress or symptom measures.ConclusionsTo the best of our knowledge, our study is the first to investigate epigenome-wide methylation across time in bipolar patients and in relation to recent, non-traumatic stressful life events. Limited and inconclusive evidence warrants future longitudinal investigations in larger samples of well-characterized bipolar patients to give a complete picture regarding the role of DNA methylation in the course of bipolar disorder.

Genome-Wide DNA Methylation Patterns in Persistent Attention-Deficit/Hyperactivity Disorder and in Association With Impulsive and Callous Traits.

Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder that often persists into adulthood. ADHD and related personality traits, such as impulsivity and callousness, are caused by genetic and environmental factors and their interplay. Epigenetic modifications of DNA, including methylation, are thought to mediate between such factors and behavior and may behave as biomarkers for disorders. Here, we set out to study DNA methylation in persistent ADHD and related traits. We performed epigenome-wide association studies (EWASs) on peripheral whole blood from participants in the NeuroIMAGE study (age range 12–23 years). We compared participants with persistent ADHD (n = 35) with healthy controls (n = 19) and with participants with remittent ADHD (n = 19). Additionally, we performed EWASs of impulsive and callous traits derived from the Conners Parent Rating Scale and the Callous-Unemotional Inventory, respectively, across all participants. For every EWAS, the linear regression model analyzed included covariates for age, sex, smoking scores, and surrogate variables reflecting blood cell type composition and genetic background. We observed no epigenome-wide significant differences in single CpG site methylation between participants with persistent ADHD and healthy controls or participants with remittent ADHD. However, epigenome-wide analysis of differentially methylated regions provided significant findings showing that hypermethylated regions in the APOB and LPAR5 genes were associated with ADHD persistence compared to ADHD remittance (p = 1.68 * 10−24 and p = 9.06 * 10−7, respectively); both genes are involved in cholesterol signaling. Both findings appeared to be linked to genetic variation in cis. We found neither significant epigenome-wide single CpG sites nor regions associated with impulsive and callous traits; the top-hits from these analyses were annotated to genes involved in neurotransmitter release and the regulation of the biological clock. No link to genetic variation was observed for these findings, which thus might reflect environmental influences. In conclusion, in this pilot study with a small sample size, we observed several DNA-methylation–disorder/trait associations of potential significance for ADHD and the related behavioral traits. Although we do not wish to draw conclusions before replication in larger, independent samples, cholesterol signaling and metabolism may be of relevance for the onset and/or persistence of ADHD.