Selected CpG Sites Research Articles

Variation of selected genotoxic and epigenetic markers due to therapeutic exposure to PAHs and ultraviolet radiation.

Goeckerman therapy (GT) of psoriasis involves dermal application of crude coal tar containing polycyclic aromatic hydrocarbons (PAHs) and exposure to ultraviolet radiation (UVR). Little is known about GT influence on DNA epigenetics. The study aim was to discover epigenetic mechanisms altered by the exposure related to the GT of psoriasis. Observed group of patients with plaque psoriasis (n = 23) was treated by GT with 3 % CCT. Before and after GT, we analyzed the levels of benzo[a]pyrene-7,8-diol-9,10-epoxide-DNA adducts (BPDE-DNA), p53 protein in serum, 5-methylcytosine (5-mC, global DNA methylation), and methylation in selected CpG sites of p53 gene. We found a significant increase in the levels of BPDE-DNA (p < 0.01) and serum levels of p53 protein (p < 0.01) after GT, and an insignificant decrease in the percentage of 5-mC in peripheral blood DNA. Methylation of p53 CpG sites was affected neither by psoriasis nor by GT. The study confirmed good effectiveness of GT (significantly reduced psoriasis area and severity index; p < 0.001). Our findings indicate that there is a significantly increased genotoxic hazard related to the exposure of PAHs and UV radiation after GT of psoriasis. However, global DNA methylation and p53 gene methylation evade the effect of GT, as they remained unchanged (Tab. 4, Fig. 3, Ref. 50).

Class-Incremental Learning With Deep Generative Feature Replay for DNA Methylation-Based Cancer Classification

Developing lifelong learning algorithms are mandatory for computational systems biology. Recently, many studies have shown how to extract biologically relevant information from high-dimensional data to understand the complexity of cancer by taking the benefit of deep learning (DL). Unfortunately, new cancer growing up into the hundred types that make systems difficult to classify them efficiently. In contrast, the current state-of-the-art continual learning (CL) methods are not designed for the dynamic characteristics of high-dimensional data. And data security and privacy are some of the main issues in the biomedical field. This article addresses three practical challenges for class-incremental learning (Class-IL) such as data privacy, high-dimensionality, and incremental learning problems. To solve this, we propose a novel continual learning approach, called Deep Generative Feature Replay (DGFR), for cancer classification tasks. DGFR consists of an incremental feature selection (IFS) and a scholar network (SN). IFS is used for selecting the most significant CpG sites from high-dimensional data. We investigate different dimensions to find an optimal number of selected CpG sites. SN employs a deep generative model for generating pseudo data without accessing past samples and a neural network classifier for predicting cancer types. We use a variational autoencoder (VAE), which has been successfully applied to this research field in previous works. All networks are sequentially trained on multiple tasks in the Class-IL setting. We evaluated the proposed method on the publicly available DNA methylation data. The experimental results show that the proposed DGFR achieves a significantly superior quality of cancer classification tasks with various state-of-the-art methods in terms of accuracy.

DNA Methylation Profiles Suggest Intergenerational Transfer of Maternal Effects.

The view of maternal effects (nongenetic maternal environmental influence on offspring phenotype) has changed from one of distracting complications in evolutionary genetics to an important evolutionary mechanism for improving offspring fitness. Recent studies have shown that maternal effects act as an adaptive mechanism to prepare offspring for stressful environments. Although research into the magnitude of maternal effects is abundant, the molecular mechanisms of maternal influences on offspring phenotypic variation are not fully understood. Despite recent work identifying DNA methylation as a potential mechanism of nongenetic inheritance, currently proposed links between DNA methylation and parental effects are indirect and primarily involve genomic imprinting. We combined a factorial breeding design and gene-targeted sequencing methods to assess inheritance of methylation during early life stages at 14 genes involved in growth, development, metabolism, stress response, and immune function of Chinook salmon (Oncorhynchus tshawytscha). We found little evidence for additive or nonadditive genetic effects acting on methylation levels during early development; however, we detected significant maternal effects. Consistent with conventional maternal effect data, maternal effects on methylation declined through development and were replaced with nonadditive effects when offspring began exogenous feeding. We mapped methylation at individual CpG sites across the selected candidate genes to test for variation in site-specific methylation profiles and found significant maternal effects at selected CpG sites that also declined with development stage. While intergenerational inheritance of methylated DNA is controversial, we show that CpG-specific methylation may function as an underlying molecular mechanism for maternal effects, with important implications for offspring fitness.

Development of novel in vitro diagnostics to predict the efficacy of anti-EGFR treatment based on DNA methylation status

Abstract Background Anti-EGFR antibody is one of the options for treatment of RAS wild-type metastatic colorectal cancer (mCRC), especially when the primary tumor site locates in the left sided large intestine. We have shown that methylation status evaluated by genome-wide DNA methylation test correlates with clinical outcomes of anti-EGFR treatment in RAS wild-type mCRC. Here, we report about the development of novel in vitro diagnostics (IVD) based on DNA methylation status of selected CpG sites. Method We retrospectively collected KRAS wild-type mCRC cases who received anti-EGFR treatment in salvage line. DNA methylation status was measured for 16 CpG sites by our novel IVD using real-time PCR method for each case. Based on DNA methylation status, they were classified into 2 groups. Highly-methylated colorectal cancer (HMCC) was defined when 8 or more sites among 16 sites were methylated and low-methylated colorectal cancer (LMCC) was classified when it was less than 8 sites. We compared the clinical outcomes between the 2 groups, and the predictive accuracy of the DNA methylation status by the new diagnostic method was examined. Results A total of 83 cases were collected, with 20 cases classified as HMCC and 63 cases classified as LMCC. The response rate of the anti-EGFR treatment was 5.0% (1/20 cases) in HMCC and 30.2% (19/63 cases) in LMCC, respectively (p = 0.032). The median progression-free survival were 2.8 months and 5.3 months, respectively (p Conclusion DNA methylation status of the 16 CpG sites measured by our novel IVD was significantly correlated with the therapeutic effect of anti-EGFR treatment for KRAS wild-type mCRC. Currently, extended analysis is in progress using the data of more than 200 cases. We will report the results including the analysis of RAS status and primary tumor site.

Preclinical and Clinical Evidence for a Distinct Regulation of Mu Opioid and Type 1 Cannabinoid Receptor Genes Expression in Obesity.

Among endogenous signaling networks involved in both rewarding and homeostatic mechanisms of obesity, a relevant role is played by the endocannabinoid (ECS) and the opioid (EOS) systems. We here studied the transcriptional regulation of ECS and EOS genes in the hypothalamus of Diet-induced obesity rats, a preclinical model of obesity, as well as in humans with obesity and healthy controls. A significant and selective increase in type 1 cannabinoid receptor gene (Cnr1) expression was observed at the beginning of obesity development (5 weeks on high fat diet) as well as after 21 weeks of high diet exposure. After 5 weeks on high fat diet, selective up-regulation of mu opioid receptor gene (Oprm1) expression was also observed. Consistently, epigenetic studies showed a selective and significant decrease in DNA methylation at specific CpG sites at both gene promoters in overweight rats, but only after 5 weeks on high fat diet. Moreover, significantly lower levels of DNA methylation were observed at selected CpG sites of both receptor gene promoters, analyzed in peripheral blood mononuclear cells from younger (<30 years old) humans with obesity, as well as in those with shorter time length from disease onset. Taken together, we here provide evidence of selective, synergistic and time-dependent transcriptional regulation of CNR1 and OPRM1 genes in overweight rats, as well as in human subjects. These alterations in genes regulation could contribute to the development of the obese phenotype, and we thus suggest CNR1 and OPRM1 epigenetic modulation as possible biomarkers of obesity development. Due to the reversible nature of the epigenetic hallmark, our data might also open new avenue to early environmental strategies of intervention.

Impaired CpG Demethylation in Common Variable Immunodeficiency Associates With B Cell Phenotype and Proliferation Rate.

Common Variable Immunodeficiency (CVID) is characterized by impaired antibody production and poor terminal differentiation of the B cell compartment, yet its pathogenesis is still poorly understood. We first reported the occurrence of epigenetic alterations in CVID by high-throughput methylation analysis in CVID-discordant monozygotic twins. Data from a recent whole DNA methylome analysis throughout different stages of normal B cell differentiation allowed us to design a new experimental approach. We selected CpG sites for analysis based on two criteria: one, CpGs with potential association with the transcriptional status of relevant genes for B cell activation and differentiation; and two, CpGs that undergo significant demethylation from naïve to memory B cells in healthy individuals. DNA methylation was analyzed by bisulfite pyrosequencing of specific CpG sites in sorted naïve and memory B cell subsets from CVID patients and healthy donors. We observed impaired demethylation in two thirds of the selected CpGs in CVID memory B cells, in genes that govern B cell-specific processes or participate in B cell signaling. The degree of demethylation impairment associated with the extent of the memory B cell reduction. The impaired demethylation in such functionally relevant genes as AICDA in switched memory B cells correlated with a lower proliferative rate. Our new results reinforce the hypothesis of altered demethylation during B cell differentiation as a contributing pathogenic mechanism to the impairment of B cell function and maturation in CVID. In particular, deregulated epigenetic control of AICDA could play a role in the defective establishment of a post-germinal center B cell compartment in CVID.

Abstract 5316: DNA methylation index of lifetime estrogen exposure in breast cancer

Abstract Breast cancer is the most common female cancer worldwide and makes up 25% of all cancers diagnosed in women. There is a need for improved risk assessment methods to identify women at high risk before the disease develops. It is well established that estrogens are involved in estrogen receptor positive (ER+) breast cancer carcinogenesis, however the mechanisms are not fully understood. We hypothesize that lifetime estrogen exposure accumulates changes in DNA methylation detectable in blood as a surrogate for other tissues. Here, we have identified a methylation signature of estrogen exposure measured in blood DNA that could improve risk assessment for breast cancer. Our Estimated Lifetime Estrogen Exposure (ELEE) model, taking into account reproductive time (age at menopause minus age at menarche), number of pregnancies and breastfeeding duration, shows a 5% increased risk of developing breast cancer per unit increase in ELEE (range 18 – 44) in the European Prospective into Cancer and Nutrition (EPIC)-Italy study cohort (30671 healthy controls, 1193 cases, age at diagnosis ≥ 50). An Epigenome-Wide Association Study (EWAS) of ELEE was conducted on 450K methylation data in EPIC-Italy (199 controls, 97 cases) and the Generations Study (GS) cohort (n=65 controls). For validation targeted bisulfite sequencing using the Fluidigm 48.48 Access Array was performed on independent DNA samples from the GS cohort (440 matched ER+ case-control pairs). The Methylation Index (MI) of ELEE was developed on 450K data using ridge regression and includes DNA methylation levels at selected CpG sites. DNA methylation levels at 694 CpG sites show significant (false discovery rate q &amp;lt; 0.05) association with ELEE in the EWAS and of these 42 CpG sites from the top-probes were selected for validation. The MI was developed on 28 CpG sites passing quality control and shows high correlation with ELEE in 450K training data (r=0.69) and significant association with breast cancer risk in EPIC-Italy with an OR of 1.38 per unit MI (range 24 – 43, 95% CI: 1.23 – 1.57, P=2.0E-07). Validation step is yet to be finalized; initial analysis on the first 318 case-control pairs suggests a modest but significant association with ER+ breast cancer risk (unadjusted OR=1.04 per unit MI, 95% CI: 1.00 – 1.07, P=0.030, MI range 20 – 53). In summary, we show that changes in DNA methylation following estrogen exposure are detectable in blood and we have developed a Methylation Index of ELEE that is significantly increased in breast cancer cases compared to controls in EPIC-Italy, and modestly increased in the GS validation cohort. This molecular measure of estrogen exposure could potentially improve risk assessment methods and be used to identify women at high risk of developing breast cancer. Citation Format: Annelie Johansson, Domenico Palli, Giovanna Masala, Sara Grioni, Claudia Agnoli, Rosario Tumino, Maria Concetta Giurdanella, Francesca Fasanelli, Carlotta Sacerdote, Salvatore Panico, Amalia Mattiello, Anthony Swerdlow, Minouk Schoemaker, Michael Jones, Nick Orr, Olivia Fletcher, Nichola Johnson, Katarzyna Tomczyk, Paolo Vineis, James M. Flanagan. DNA methylation index of lifetime estrogen exposure in breast cancer [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 5316.

Abstract 2209: Lung cancer risk prediction using DNA methylation markers

Abstract There is an urgent need to improve lung cancer risk assessment as current screening criteria miss a large proportion of cases and result in a high rate of false positives on low-dose CT screening. A fixed effect meta-analyses of 4 epigenome-wide association studies of lung cancer revealed differential DNA methylation at 16 CpG sites (FDR&amp;lt;0.05). The current study aimed to evaluate the extent to which such methylation markers can improve upon smoking-based risk-discrimination among ever smokers. We used data on 662 ever smoking lung cancer case-control pairs from 4 individual prospective cohorts that measured DNA methylation using the Illumina Infinium HumanMethylation450 BeadChip in peripheral blood samples before diagnosis: The Italian part of the European Prospective Investigation into Cancer (EPIC) cohort, the Melbourne Collaborative Cohort (MCCS), the Norwegian Women and Cancer cohort (NOWAC) and the Northern Sweden Health and Disease Study (NSHDS). We adopted a training-testing design where the training was performed on MCCS and NSHDS (N=511 case-control pairs), and the testing on EPIC-Italy and NOWAC (N=151 case-control pairs). Logistic regressions with lasso penalties were performed in the training set to select the best set of CpGs jointly predicting lung cancer. A methylation score was trained by fitting a logistic regression model including the selected CpGs in the training set. A baseline score based on self-reported smoking information (duration, cigarettes/day for current smokers and time since smoking cessation for former smokers) was also developed. The discriminative performances of both scores, as well as integrated model where both smoking and methylation info was incorporated, were assessed by the AUC under the ROC curves in the validation set. The methylation score based on the 9 selected CpG sites yielded an AUC of 0.78 [0.73-0.84] compared to 0.73 [0.68-0.79] for the baseline-smoking score. The model integrating both scores yielded an AUC of 0.79 [0.73-0.84], a notable 0.06-increase in AUC from using the smoking score alone (P=0.008 for difference in AUC). In conclusion, specific methylation biomarkers have a strong potential to improve lung cancer risk assessment and current USPSTF criteria for CT-screening. During the conference, we will present absolute risk estimates based on the integrated risk prediction model. Citation Format: Florence Guida, Therese H. Nøst, Caroline Relton, Paul Brennan, Torkjel M. Sandanger, Marc Chadeau-Hyam, Mattias Johansson. Lung cancer risk prediction using DNA methylation markers [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 2209.

Novel Epigenomic Biomarkers of Male Infertility Identified by Methylation Patterns of CpG Sites Within Imprinting Control Regions of H19 and SNRPN Genes.

Male infertility is an important global health burden that can benefit from novel biomarkers and diagnostics innovation. Aberrant methylation of the imprinted genes H19 and SNRPN (small nuclear ribonucleoprotein polypeptide N) in sperm DNA has been implicated in abnormal sperm parameters and male infertility. However, whether certain methylation patterns of one or multiple CpG sites within an imprinted gene are pathological for multiple sperm defects remains poorly understood. To examine the diagnostic potential of certain methylation patterns of CpG sites for multiphenotype defects in human sperm, the sperm DNA methylation patterns of individual CpG sites within imprinting control regions (ICRs) of imprinted genes H19 and SNRPN were measured by bisulfite pyrosequencing in a Han Chinese population sample: 39 oligoasthenozoospermia (OA) patients, 36 asthenoteratozoospermia (AT) patients, and 50 normozoospermia (N) controls. A partial least squares discriminant analysis model was built with the CpG sites as independent variables. Among the 16 CpG sites screened, the methylation patterns of eight CpG sites within H19-ICR (CpG sites 1, 6-9, 12 and 15-16), and eight CpG sites within SNRPN-ICR (CpG sites 2, 5-6, 8-10, 13, and 16) correctly classified 74.4% and 72.0% of the samples in terms of male fertile status, respectively. Furthermore, by combination of these 16 selected CpG sites within ICRs of H19 and SNRPN, 88.0% of the samples could be successfully classified. Our study demonstrates that methylation profiles of CpG sites within ICRs of imprinted genes H19 and SNRPN may potentially serve as epigenomic biomarkers for assessment of infertility in men with multiple sperm defects. Further studies in independent population samples are called for diagnostic significance of methylation patterns of CpG sites within imprinted genes.

Whole-genome methylation profiling of peripheral blood mononuclear cell for acute exacerbations of chronic obstructive pulmonary disease treated with corticosteroid.

Although association studies in the general population may be relevant for determining susceptibility to chronic obstructive pulmonary disease (COPD), they may be less applicable for pharmacogenetics research in participants who have already acquired the disease. A genome-wide methylation profiling (generated by HumanMethylation450 BeadChips study was performed on peripheral blood mononuclear cells of 24 patients with AECOPD (acute exacerbation COPD), with good and poor responsiveness to standard corticosteroid treatment. Pyrosequencing was used to replicate the selected CpG sites in 50 patients with AECOPD with standard corticosteroid treatment. The results showed the patients with AECOPD with good and poor response to standard corticosteroid treatment have a distinct DNA methylation pattern. A total of 23 CpG loci located in 19 known gene regions, including gene-body and promoter, appeared to be significantly differentially methylated. Replication by pyrosequencing revealed that one CpG site in PSMD8 showed the same trend of differential methylation and reached to statistical significance as the microarray result. Our preliminary findings provide evidence for molecular heterogeneity in patients with AECOPD, which may contribute to significant differences in their response to COPD treatment.

Colorectal adenoma and cancer detection based on altered methylation pattern of SFRP1, SFRP2, SDC2, and PRIMA1 in plasma samples

ABSTRACTAberrant methylation is one of the most frequent epigenetic alterations that can contribute to tumor formation. Cell-free DNA can originate from tumor tissue; therefore, the evaluation of methylation markers in cell-free DNA can be a promising method for cancer screening. Our aim was to develop a panel of biomarkers with altered methylation along the colorectal adenoma-carcinoma sequence in both colonic tissue and plasma. Methylation of selected CpG sites in healthy colonic (n = 15), adenoma (n = 15), and colorectal cancer (n = 15) tissues was analyzed by pyrosequencing. MethyLight PCR was applied to study the DNA methylation of SFRP1, SFRP2, SDC2, and PRIMA1 gene promoters in 121 plasma and 32 biopsy samples. The effect of altered promoter methylation on protein expression was examined by immunohistochemistry. Significantly higher (P < 0.05) DNA methylation levels were detected in the promoter regions of all 4 markers, both in CRC and adenoma tissues compared with healthy controls. Methylation of SFRP1, SFRP2, SDC2, and PRIMA1 promoter sequences was observed in 85.1%, 72.3%, 89.4%, and 80.9% of plasma samples from patients with CRC and 89.2%, 83.8%, 81.1% and 70.3% from adenoma patients, respectively. When applied as a panel, CRC patients could be distinguished from controls with 91.5% sensitivity and 97.3% specificity [area under the curve (AUC) = 0.978], while adenoma samples could be differentiated with 89.2% sensitivity and 86.5% specificity (AUC = 0.937). Immunohistochemical analysis indicated decreasing protein levels of all 4 markers along the colorectal adenoma-carcinoma sequence. Our findings suggest that this methylation biomarker panel allows non-invasive detection of colorectal adenoma and cancer from plasma samples.

Epigenetic changes in myelofibrosis: Distinct methylation changes in the myeloid compartments and in cases with ASXL1 mutations

This is the first study to compare genome-wide DNA methylation profiles of sorted blood cells from myelofibrosis (MF) patients and healthy controls. We found that differentially methylated CpG sites located to genes involved in ‘cancer’ and ‘embryonic development’ in MF CD34+ cells, in ‘inflammatory disease’ in MF mononuclear cells, and in ‘immunological diseases’ in MF granulocytes. Only few differentially methylated CpG sites were common among the three cell populations. Mutations in the epigenetic regulators ASXL1 (47%) and TET2 (20%) were not associated with a specific DNA methylation pattern using an unsupervised approach. However, in a supervised analysis of ASXL1 mutated versus wild-type cases, differentially methylated CpG sites were enriched in regions marked by histone H3K4me1, histone H3K27me3, and the bivalent histone mark H3K27me3 + H3K4me3 in human CD34+ cells. Hypermethylation of selected CpG sites was confirmed in a separate validation cohort of 30 MF patients by pyrosequencing. Altogether, we show that individual MF cell populations have distinct differentially methylated genes relative to their normal counterparts, which likely contribute to the phenotypic characteristics of MF. Furthermore, differentially methylated CpG sites in ASXL1 mutated MF cases are found in regulatory regions that could be associated with aberrant gene expression of ASXL1 target genes.

Effects of DNA Methylation on Progression to Interstitial Fibrosis and Tubular Atrophy in Renal Allograft Biopsies: A Multi-Omics Approach.

Progressive fibrosis of the interstitium is the dominant final pathway in renal destruction in native and transplanted kidneys. Over time, the continuum of molecular events following immunological and nonimmunological insults lead to interstitial fibrosis and tubular atrophy and culminate in kidney failure. We hypothesize that these insults trigger changes in DNA methylation (DNAm) patterns, which in turn could exacerbate injury and slow down the regeneration processes, leading to fibrosis development and graft dysfunction. Herein, we analyzed biopsy samples from kidney allografts collected 24 months posttransplantation and used an integrative multi-omics approach to understand the underlying molecular mechanisms. The role of DNAm and microRNAs on the graft gene expression was evaluated. Enrichment analyses of differentially methylated CpG sites were performed using GenomeRunner. CpGs were strongly enriched in regions that were variably methylated among tissues, implying high tissue specificity in their regulatory impact. Corresponding to this methylation pattern, gene expression data were related to immune response (activated state) and nephrogenesis (inhibited state). Preimplantation biopsies showed similar DNAm patterns to normal allograft biopsies at 2 years posttransplantation. Our findings demonstrate for the first time a relationship among epigenetic modifications and development of interstitial fibrosis, graft function, and inter-individual variation on long-term outcomes.

DNA methylation-based age prediction from saliva: High age predictability by combination of 7 CpG markers

DNA methylation is currently one of the most promising age-predictive biomarkers. Many studies have reported DNA methylation-based age predictive models, but most of these are based on DNA methylation patterns from blood. Only a few studies have examined age-predictive DNA patterns in saliva, which is one of the most frequently-encountered body fluids at crime scenes. In this study, we generated genome-wide DNA methylation profiles of saliva from 54 individuals and identified CpG markers that showed a high correlation between methylation and age. Because the age-associated marker candidates from saliva differed from those of blood, we investigated DNA methylation patterns of 6 age-associated CpG marker candidates (cg00481951, cg19671120, cg14361627, cg08928145, cg12757011, and cg07547549 of the SST, CNGA3, KLF14, TSSK6, TBR1, and SLC12A5 genes, respectively) in addition to a cell type-specific CpG marker (cg18384097 of the PTPN7 gene) in an independent set of saliva samples obtained from 226 individuals aged 18 to 65 years. Multiplex methylation SNaPshot reactions were used to generate the data. We then generated a linear regression model with age information and the methylation profile from the 113 training samples. The model exhibited a 94.5% correlation between predicted and chronological age with a mean absolute deviation (MAD) from chronological age of 3.13 years. In subsequent validation using 113 test samples, we also observed a high correlation between predicted and chronological age (Spearman’s rho=0.952, MAD from chronological age=3.15years). The model composed of 7 selected CpG sites enabled age prediction in saliva with high accuracy, which will be useful in saliva analysis for investigative leads.

Tetanus vaccination is associated with differential DNA-methylation: Reduces the risk of asthma in adolescence

BackgroundVaccinations have been suggested to be associated with increased risk of allergic diseases. Tetanus vaccination is one of the most frequently administered vaccines as a part of wound management and was also found to be associated with increased serum IgE levels. We hypothesized that the vaccination modifies the risk of allergic diseases through epigenetic changes such as DNA methylation. MethodData on tetanus vaccination between 10 and 18years of age was collected from a birth cohort established on the Isle of Wight UK in 1989. DNA methylation data were collected from individuals at different ages (at birth [n=30], age 10 [n=34], age 18 [n=245] and during pregnancy [n=121]) using the Illumina Infinium HumanMethylation450K array. Firstly, we performed an epigenome-wide screening to identify cytosine-phosphate-guanine sites (CpGs) associated with tetanus vaccination in 18-year-olds. Secondly, we tested their association with asthma, allergic sensitization, eczema, serum IgE and pulmonary lung function (FVC, FEV1, FEV1/FVC, and FEF25-75%). We then described changes in the methylation of the selected CpG sites over age, and by vaccination status. ResultsTetanus vaccination was found to be associated with decreased methylation of cg14472551 (p value 0.5×10−5, FDR-adjusted p value 2.1×10−4) and increased methylation of cg01669161 (p value 0.0007, FDR-adjusted p value 0.014). Both CpGs, in turn, were associated with decreased risk of asthma at 18years of age. Cg14472551 is located in an intron of KIAA1549L, whose protein binds to a B-cell commitment transcription factor; cg01669161 is located between an antisense regulator of the proteasome assembly chaperone PSMG3, and TFAMP1, a pseudogene. Increased methylation of cg01669161 was also associated with decreased serum IgE levels. ConclusionDNA methylation changes following tetanus vaccination may offer a novel prospect to explain a differential occurrence of asthma in adolescence.

Methylation loci associated with body mass index, waist circumference, and waist-to-hip ratio in Chinese adults: an epigenome-wide analysis

BackgroundDNA methylation has been implicated in the pathology of obesity, but little is known about such epigenetic variants in Chinese people. We conducted an epigenome-wide analysis of methylation at CpG sites in relation to body mass index (BMI), waist circumference (WC) and waist-to-hip ratio (WHR) among Chinese adult populations. Methods469 twins from the Chinese National Twin Registry were the discovery cohort. After quality control, methylation levels in whole blood leukocyte DNA were tested for association with BMI, WC, and WHR using mixed linear regressions, adjusting for demographics, lifestyle, and family structure. Methylation sites at a false discovery rate (FDR) q value of 0·05 or less were selected for replication in four independent panels of general populations: Dongfeng-tongji cohort (n=688), Coke Oven Workers (n=137), Wuhan-Zhuhai cohort (n=241), and Shiyan participants (n=144). Association of obesity traits with the selected CpG sites in each replication panel was examined using linear regressions with adjustment of covariates similar to that in the discovery cohort, and results were combined using fixed effect meta-analysis with significance level penalised by Bonferroni correction. All participants gave written informed consent, and the Peking University Biomedical Ethics Committee approved the study protocol. FindingsMethylation levels at 6, 3, and 1 CpG sites were significantly associated with BMI, WC, and WHR, respectively in discovery cohort (FDR q value range 0·0010–0·0443); these findings were replicated by meta-analysis in the four panels. We found associations between obesity traits and methylation at genes CPT1A (meta-analysis p=0·0013 for BMI, p=0·0047 for WC, and p=0·0178 for WHR), ABCG1 (p<0·0001 for BMI and WC) and SREBF1 (p<0·0001 for BMI). Additionally, CpG sites at gene ARID1B (meta-analysis effect size β −0·0221, 95% CI −0·0363 to −0·0079, p=0·0022 for BMI), gene TOP1 (−0·0237, −0·0404 to −0·0071, p=0·0053 for BMI), and methylation site cg17061862 (−0·0379, −0·0575 to −0·018, p=0·0002 for BMI; −0·0356, −0·0608 to −0·0103, p=0·0057 for WC) showed inverse associations with BMI or WC. InterpretationOur findings support the association of DNA methylation at CPT1A, ABCG1, and SREBF1 with obesity traits in Chinese populations. We identified additional associated methylation sites at genes ARID1B and TOP1 and at cg17061862. FundingThis study was supported by the Specific Research Project of Health Public Service, Ministry of Health, China (201002007, 201502006) and Key Grant Project of Chinese Ministry of Education (310006).

Epigenetic control of phospholipase A2 receptor expression in mammary cancer cells.

BackgroundIt has recently been proposed that the M-type phospholipase A2 receptor (PLA2R1) acts as a tumour suppressor in certain malignancies including mammary cancer. Considering that DNA methylation is an important regulator of gene transcription during carcinogenesis, in the current study we analyzed the PLA2R1 expression, PLA2R1 promoter methylation, and selected micro RNA (miRNA) levels in normal human mammary epithelial cells (HMEC) and cancer cell lines.MethodsLevels of PLA2R1 and DNA methyltransferases (DNMT) specific mRNA were determined using real-time RT-PCR. Methylation specific-high resolution melting (MS-HRM) analysis was utilized to quantify the methylation degree of selected CpG sites localized in the promoter region of the PLA2R1 gene. Expression of miRNA was tested using miScript Primer Assay system.ResultsNearly complete methylation of the analyzed PLA2R1 promoter region along with PLA2R1 gene silencing was identified in MDA-MB-453 mammary cancer cells. In MCF-7 and BT-474 mammary cancer cell lines, a higher DNA methylation degree and reduced PLA2R1 expression were found in comparison with those in normal HMEC. Synergistic effects of demethylating agent (5-aza-2′-deoxycytidine) and histone deacetylase inhibitor (trichostatin A) on PLA2R1 transcription in MDA-MB-453 cells confirmed the importance of DNA methylation and histone modification in the regulation of the PLA2R1 gene expression in mammary cells. Furthermore, significant positive correlation between the expression of DNMT1 and PLA2R1 gene methylation and negative correlation between the cellular levels of hsa-mir-141, −181b, and -181d-1 and the expression of PLA2R1 were identified in the analyzed cells. Analysis of combined z-score of miR-23b, −154 and -302d demonstrated a strong and significant positive correlation with PLA2R1 expression.ConclusionsOur data indicate that (i) PLA2R1 expression in breast cancer cells is controlled by DNA methylation and histone modifications, (ii) hypermethylation of the PLA2R1 promoter region is associated with up-regulation of DNMT1, and (iii) hsa-miR-23b, −154, and −302d, as well as hsa-miR-141, −181b, and −181d-1 are potential candidates for post-transcriptional regulation of PLA2R1 expression in mammary cancer cells.

Epigenetic age signatures in the forensically relevant body fluid of semen: a preliminary study

To date, DNA methylation has been regarded as the most promising age-predictive biomarker. In support of this, several researchers have reported age predictive models based on the use of blood or even across a broad spectrum of tissues. However, there have been no publications that report epigenetic age signatures from semen, one of the most forensically relevant body fluids. In genome-wide DNA methylation profiles of 36 body fluids including blood, saliva, and semen, the previous age predictive models showed considerable prediction accuracy in blood and saliva but not in semen. Therefore, we selected CpG sites, whose methylation levels are strongly correlated with age in 12 semen profiles obtained from individuals of different ages, and investigated DNA methylation changes at these CpGs in 68 additional semen samples obtained from individuals aged 20 to 73 years using methylation SNaPshot reaction. Among the selected age-related CpG candidates, outstanding age correlation was obtained at cg06304190 in the TTC7B gene. Interestingly, the region around the TTC7B gene has been reported to show age-related DNA methylation alteration in the sperm methylome of 2 samples collected from individuals at certain time intervals. The age-predictive linear regression model trained with 3 CpGs (cg06304190 in the TTC7B gene, cg06979108 in the NOX4 gene and cg12837463) showed a high correlation between the predicted age and the chronological age, with an average absolute difference of approximately 5 years. These selected epigenetic age signatures are expected to be useful for considerably accurate age estimation in the forensically relevant body fluid of semen. However, because the findings were limited by small sample size, it will be necessary to further evaluate the age correlation of the selected CpGs and to encourage further investigation.

Screening genome-wide DNA methylation CpG sites via training and testing data utilizing surrogate variables

Background Screening Cytosine-phosphate-Guanine dinucleotide (CpG) DNA methylation sites in association with singlenucleotide polymorphisms (SNPs), or covariate of interest, and/or their interactions is desired before performing more complicated analyses due to high dimensionality. It is possible the variation in methylation cannot be fully explained by SNPs and covariates of interest and thus it is important to account for variations introduced by other unknown factors. Furthermore, CpG sites screened from one data set may be inconsistent with those from another data set and it is equally important to improve the reproducibility of the selected CpG sites.

Methylation of BDNF in women with bulimic eating syndromes: associations with childhood abuse and borderline personality disorder.

DNA methylation allows for the environmental regulation of gene expression and is believed to link environmental stressors to such mental-illness phenotypes as eating disorders. Numerous studies have shown an association between bulimia nervosa (BN) and variations in brain-derived neurotrophic factor (BDNF). BDNF has also been linked to borderline personality disorder (BPD) and to such traits as reward dependence. We examined the extent to which BDNF methylation corresponded to bulimic or normal-eater status, and also to the presence of comorbid borderline personality disorder (BPD) and childhood abuse. Our sample consisted of 64 women with BN and 32 normal-eater (NE) control women. Participants were assessed for eating-disorder symptoms, comorbid psychopathology, and childhood trauma, and then they were required to provide blood samples for methylation analyses. We observed a significant site×group (BN vs. NE) interaction indicating that women with BN showed increases in methylation at specific regions of the BDNF promoter. Furthermore, examining effects of childhood abuse and BPD, we observed significant site×group interactions such that groups composed of individuals with childhood abuse or BPD had particularly high levels of methylation at selected CpG sites. Our findings suggest that BN, especially when co-occurring with childhood abuse or BPD, is associated with a propensity towards elevated methylation at specific BDNF promoter region sites. These findings imply that hypermethylation of the BDNF gene may be related to eating disorder status, developmental stress exposure, and comorbid psychopathology.