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Background DNA methylation in the human genome is established and maintained by DNA methyltransferases (DNMTs). DNMT isoforms show differential expression by cell lineage and during development, but much remains to be elucidated about their shared and unique genomic targets. Results We examined changes in the epigenome following overexpression of 13 DNMT isoforms in HEK293T cells. We observed increased methylation (Δβ > 0.2) at 43,405 CpG sites, with expression of DNMT3A2, DNMTΔ3B4 and DNMTΔ3B2 associated with the greatest impact. De novo methylation occurred primarily within open sea regions and at loci with intermediate methylation levels (β: 0.2–0.6). 53% of differentially methylated loci showed specificity towards a single DNMT subfamily, primarily DNMTΔ3B and DNMT3A and 39% towards a single isoform. These loci were significantly enriched for pathways related to neuronal development (DNMTΔ3B4), calcium homeostasis (DNMTΔ3B3) and ion transport (DNMT3L). Repetitive elements did not display differential sensitivity to overexpressed DNMTs, but hypermethylation of Alu elements was associated with their evolutionary age following overexpression of DNMT3A2, DNMT3B1, DNMT3B2 and DNMT3L. Differential methylation (Δβ > 0.1) was observed at 121 of the 353 loci associated with the Horvath ‘epigenetic clock’ model of ageing, with 51 showing isoform specificity, and was associated with reduction of epigenetic age by 5–15 years following overexpression of seven isoforms. Finally, we demonstrate the potential for dietary constituents to modify epigenetic marks through isoform-specific inhibition of methylation activity. Conclusions Our results provide insight into regions of the genome methylated uniquely by specific DNMT isoforms and demonstrate the potential for dietary intervention to modify the epigenome.

The identification of cancer-specific vulnerability genes is one of the most promising approaches for developing more effective and less toxic cancer treatments. Cancer genomes exhibit thousands of changes in DNA methylation and gene expression, with the vast majority likely to be passenger changes. We hypothesised that, through integration of genome-wide DNA methylation/expression data, we could exploit this inherent variability to identify cancer subtype-specific vulnerability genes that would represent novel therapeutic targets that could allow cancer-specific cell killing. We developed a bioinformatics pipeline integrating genome-wide DNA methylation/gene expression data to identify candidate subtype-specific vulnerability partner genes for the genetic drivers of individual genetic/molecular subtypes. Using acute lymphoblastic leukaemia as an initial model, 21 candidate subtype-specific vulnerability genes were identified across the five common genetic subtypes, with at least one per subtype. To confirm the approach was applicable across cancer types, we also assessed medulloblastoma, identifying 15 candidate subtype-specific vulnerability genes across three of four established subtypes. Almost all identified genes had not previously been implicated in these diseases. Functional analysis of seven candidate subtype-specific vulnerability genes across the two tumour types confirmed that siRNA-mediated knockdown induced significant inhibition of proliferation/induction of apoptosis, which was specific to the cancer subtype in which the gene was predicted to be specifically lethal. Thus, we present a novel approach that integrates genome-wide DNA methylation/expression data to identify cancer subtype-specific vulnerability genes as novel therapeutic targets. We demonstrate this approach is applicable to multiple cancer types and identifies true functional subtype-specific vulnerability genes with high efficiency.

Retrotransposons such as LINE-1 and Alu comprise >25% of the human genome. While global hypomethylation of these elements has been widely reported in solid tumours, their epigenetic dysregulation is yet to be characterised in chronic lymphocytic leukaemia, and there has been scant consideration of their evolutionary history that mediates sensitivity to hypomethylation. Here, we developed an approach for locus- and evolutionary subfamily-specific analysis of retrotransposons using the Illumina Infinium Human Methylation 450K microarray platform, which we applied to publicly-available datasets from chronic lymphocytic leukaemia and other haematological malignancies. We identified 9,797 microarray probes mapping to 117 LINE-1 subfamilies and 13,130 mapping to 37 Alu subfamilies. Of these, 10,782 were differentially methylated (PFDR90% patients) but not observed in healthy individuals or other leukaemias, and was detectable in blood samples taken prior to chronic lymphocytic leukaemia diagnosis in 9 of 82 individuals from the Melbourne Collaborative Cohort Study. Our results demonstrate differential methylation of retrotransposons in chronic lymphocytic leukaemia by their evolutionary heritage that modulates expression of proximal genes.

Background Chronic lymphocytic leukaemia (CLL) patients display a highly variable clinical course, with progressive acquisition of drug resistance. We sought to identify aberrant epigenetic traits that are enriched following exposure to treatment that could impact patient response to therapy. Methods Epigenome-wide analysis of DNA methylation was performed for 20 patients at two timepoints during treatment. The prognostic significance of differentially methylated regions (DMRs) was assessed in independent cohorts of 139 and 163 patients. Their functional role in drug sensitivity was assessed in vitro. Results We identified 490 DMRs following exposure to therapy, of which 31 were CLL-specific and independent of changes occurring in normal B-cell development. Seventeen DMR-associated genes were identified as differentially expressed following treatment in an independent cohort. Methylation of the HOXA4, MAFB and SLCO3A1 DMRs was associated with post-treatment patient survival, with HOXA4 displaying the strongest association. Re-expression of HOXA4 in cell lines and primary CLL cells significantly increased apoptosis in response to treatment with fludarabine, ibrutinib and idelalisib. Conclusion Our study demonstrates enrichment for multiple CLL-specific epigenetic traits in response to chemotherapy that predict patient outcomes, and particularly implicate epigenetic silencing of HOXA4 in reducing the sensitivity of CLL cells to therapy.

Background: Uterine leiomyoma (UL) is a benign tumour with a prevalence of 4-21% in Western populations and UC occurrence is associate with impaired quality of life. Few studies have studied on psychological stress with the prevalence of UL in non-Western populations. We sought to identify the association of psychological stress with the prevalence of UL and mediation by BMI. Methods: Analysis was performed in a cross-sectional, population-based Project Young ELEFANT study. We analysed clinical data collected from young Chinese women (age 20-40; n=178205) who were residents of Tianjin, China. Work-related, social and financial stress were evaluated by questionnaire and categorised by intensity (none, low, intermediate, and high). Odds ratios of UL were determined by binary logistic regression models adjusted for age at enrolment, smoking, passive smoking, alcohol consumption, BMI, education, occupation, residence, age at menarche, parity, oral contraceptive use, and diagnosis of type 2 diabetes and hypertension. Results: UL prevalence in young adult women was associated with psychosocial stress by cause and intensity. High levels of work-related, social and financial stress were associated with ORs (95% CI) of 1.72 (1.41, 2.10), 1.48 (1.18, 1.84) and 1.44 (1.11, 1.88) respectively. The risk of UL with psychosocial stress showed interaction with BMI, with underweight women at greatest risk. Conclusions: In young adult Chinese women, psychological stress is associated with UL prevalence. While BMI was positively associated with UL incidence, underweight and healthy weight women with psychosocial stress showed highest risk of UL.

Background\ud Studies investigating the impact of fine particulate matter (PM2.5) exposure during pregnancy upon adverse birth outcomes have primarily been performed in Western nations with low ambient PM2.5 levels. We examined associations between high levels of PM2.5 exposure during pregnancy and risk of adverse birth outcomes by timing and level of exposure in a Chinese population.\ud \ud Methods\ud We analysed data from 10,738 live births within the Project ELEFANT study based in Tianjin, China. Personal mean daily PM2.5 exposures were estimated using data from 25 local monitoring sites across the city, used to compute the days exceeding 50, 100, 150, 200 and 250 μg/m3. Relative risk of pre-term birth (50, >150 and >250 μg/m3 PM2.5 in the third trimester were 1.09 (95%CI: 1.03-1.16), 1.30 (1.09-1.54) and 2.73 (2.03-3.66) respectively. For low birthweight, exposures of >50, >150 and >250 μg/m3 PM2.5 in the third trimester were associated with RRs of 0.99 (0.88-1.11), 1.37 (1.04-1.81) and 3.03 (1.75-5.23) respectively.\ud \ud Conclusions\ud Exposure to high levels of PM2.5 from the second trimester onwards were most strongly associated with increased risk of pre-term birth and low birthweight, with a dose-response relationship. Our data demonstrates the need to account for both level and timing of exposure in analysis of PM2.5-associated birth outcomes.

The identification of early markers of dementia is important for higher-risk populations such as those with type 2 diabetes (T2D). Retrotransposons, including long interspersed nuclear element 1 (LINE-1) and Alu, comprise ~40% of the human genome. Although dysregulation of these retrotransposons can induce aberrant gene regulation and genomic instability, their role in the development of pre-symptomatic dementia (PSD) among T2D patients is unknown. Here, we examined locus-specific changes in LINE-1 and Alu methylation in PSD and the potential to offset these changes via supplementation with folate and vitamin B12. We interrogated DNA methylation patterns corresponding to 22,352 probes for LINE-1 and Alu elements using publicly-available Illumina Infinium 450K methylation datasets from i) an 18-month prospective study in 28 T2D patients (GSE62003) and ii) an intervention study in which 44 individuals were supplemented with folic acid (400 μg/day) and vitamin B12 (500 μg/day) over two years (GSE74548). We identified 714 differentially methylated positions (DMP) mapping to retrotransposons in T2D patients who developed PSD in comparison to those who did not (PFDR < 0.05), comprised of 2.4% (228 probes) of all LINE-1 probes and 3.8% (486 probes) of all Alu probes. These loci were enriched in genes with functions related to Alzheimer's disease and cognitive decline, including GNB5, GNG7 and PKN3 (p < 0.05). In older individuals supplemented with folate/vitamin B12, 85 (11.9%) PSD retrotransposon loci showed significant changes in methylation (p < 0.05): participants with the MTHFR CC genotype predominantly showed hypermethylation at these loci, while hypomethylation was observed more frequently in those with the TT genotype. In T2D patients, LINE-1 and Alu elements are differentially methylated in PSD in a locus-specific manner and may offer clinical utility in monitoring risk of dementia. Further work is required to examine the potential for dietary supplementation in lowering the risk of PSD.

Studies investigating the impact of fine particulate matter (PMWe analysed data from 10,738 live births within the Project ELEFANT study based in Tianjin, China. Personal mean daily PMA dose-response was exhibited for PMExposure to high levels of PM

Chemical modifications of RNA molecules have gained increasing attention since evidence emerged for their substantive roles in a range of biological processes, such as the stability and translation of mRNA transcripts. More than 150 modifications have been identified in different organisms to date, collectively known as the 'epitranscriptome', with 6-methyladenosine (m(6)A), 5-methylcytidine (m(5)C), pseudouridine and N1-methyladenosine (m(1)A) the most extensively investigated. Although we are just beginning to elucidate the roles of these modifications in cellular functions, there is already evidence for their dysregulation in diseases such as cancer and neumdevelopmental disorders. There is currently more limited knowledge regarding how environmental exposures affect the epitranscriptome and how this may mediate disease risk, but evidence is beginning to emerge. Here, we review the current evidence for the impact of environmental exposures such as benzo[a]pyrene, bisphenol A, pesticides, metals and nanoparticles upon RNA modifications and the expression of their 'writers' (methyl transferases), 'erasers' (demethylases) and `readers'. We discuss future directions of the field and identify areas of particular promise and consider the technical challenges that are faced.

Purpose:\ud Psychosocial stress, including bereavement and work-related stress, is associated with the risk of breast cancer. However, it is unknown whether it may also be linked with increased risk of benign breast disease (BBD). \ud \ud Methods: \ud Our study leveraged 61,907 women aged 17-55 years-old from the Project ELEFANT study. BBD was diagnosed by clinician. Self-reported data on psychosocial stress over a ten-year period was retrospectively collected from questionnaires and categorised by cause (work, social and economic) and severity (none, low, and high). Odd ratios (ORs) for the development of BBD were estimated using logistic regression. The model was adjusted for age, BMI, TSH levels, smoking, alcohol consumption, family history, age of menarche, oral contraceptive usage, education and occupation.\ud \ud Results: \ud Within our study, 8% (4,914) of participants were diagnosed with BBD. Work-related stress (OR = 1.57, 95% confidence interval [CI] = 1.46 - 1.69) and financial stress (OR = 1.34, 95% CI = 1.24 - 1.44) were significantly associated with BBD incidence, with a smaller but still significant association with social stress (OR = 1.11, 95% CI = 1.01 - 1.21). The associations remained significant after exclusion of participants with first- and second-degree family history of breast disease. The presence of multiple forms of stress did not synergistically increase risk. The neutrophil lymphocyte ratio (NLR), a marker of systemic inflammation and prognostic marker for breast cancer, was not associated with BBD.\ud \ud Conclusions:\ud Psychosocial stress, particularly work-related and financial stress, is associated with increased risk of benign breast disease among young Chinese women.

Smoking tobacco is a known risk factor for the development of colorectal cancer, and for mortality associated with the disease. Smoking has been reported to be associated with changes in DNA methylation in blood and in lung tumour tissues, although there has been scant investigation of how epigenetic factors may be implicated in the increased risk of developing colorectal cancer. To identify epigenetic changes associated with smoking behaviours, we performed epigenome-wide analysis of DNA methylation in colorectal tumours from 36 never smokers, 47 former smokers and 13 active smokers, and adjacent mucosa from 49 never smokers, 64 former smokers and 18 active smokers. Our analyses identified 15 CpG sites within the APC 1A promoter that were significantly hypermethylated and 14 CpG loci within the NFATC1 gene body that were significantly hypomethylated (pLIS

Prognostication in patients with chronic lymphocytic leukemia (CLL) is challenging due to heterogeneity in clinical course. We hypothesize that constitutional genetic variation affects disease progression and could aid prognostication. Pooling data from seven studies incorporating 842 cases identifies two genomic locations associated with time from diagnosis to treatment, including 10q26.13 (rs736456, hazard ratio (HR) = 1.78, 95% confidence interval (CI) = 1.47–2.15; P = 2.71 × 10−9) and 6p (rs3778076, HR = 1.99, 95% CI = 1.55–2.55; P = 5.08 × 10−8), which are particularly powerful prognostic markers in patients with early stage CLL otherwise characterized by low-risk features. Expression quantitative trait loci analysis identifies putative functional genes implicated in modulating B-cell receptor or innate immune responses, key pathways in CLL pathogenesis. In this work we identify rs736456 and rs3778076 as prognostic in CLL, demonstrating that disease progression is determined by constitutional genetic variation as well as known somatic drivers., The clinical course of chronic lymphocytic leukaemia (CLL) is variable and difficult to predict. Here, the authors conduct a genome wide association study meta-analysis for time to first treatment in CLL patients and report two loci associating with progressive disease.

The epitranscriptome comprises more than 100 forms of RNA modifications. Of these, N6-methyladenosine (m(6)A) is the most abundant form of RNA methylation, with roles in modulating mRNA transcript processing and regulation. The aims of the study were to examine changes inm(6)A RNA methylation in A549 lung epithelial cells in response to environmental toxicants, and differential gene expression of m(6)A modulator genes ('readers', 'writers' and 'erasers') in human subjects exposed to particulate matter (PM) and in lung cancer tissue using publicly-available microarray datasets. Global m(6)A methylation levels were measured in total RNA after exposure to two carcinogens (PM and sodium arsenite) for 24- and 48-h, and to two endocrine disruptors (bisphenol A and vinclozolin)for 24-h. Global m(6)A methylation level significantly decreased with exposure to > 62 mu g/mlPM, > 1 mu M sodium arsenite, > 1 mu M bisphenol A (BPA), and 0.1 mu M vinclozolin. In an analysis of a published dataset derived from a population study, we observed that m(6)A writers (METTL3 and WTAP), erasers (FTO and ALKBH5) and readers (HNRPC) showed significantly higher expression among participants in the high-PM2.5 exposure group compared to those in the low-exposure control group (all p < 0.05). Further, the m(6)A writer METTL3 shows reduced expression in lung tumors in comparison to normal lung epithelia (p < 0.0001). Our findings reveal that m(6)A RNA methylation can be modified by exposure to environmental toxicants, and exposure to particulate matter is associated with differential expression level of m(6)A RNA methylation modification machinery.

Introduction Genomic level data has significantly increased the depth of our understanding of genetic and epigenetic changes in cancer. However, using this complex data to improve patient management is challenging. Synthetic lethal (SL) genes (which are only required for growth/survival of cancer cells and not normal cells), represent ideal targets for the development of more targeted, less toxic and more effective cancer treatments. Here we report a novel bioinformatics approach that exploits the complexity of DNA methylation/expression changes to identify cancer subtype specific SL genes. Materials and Method Publicly available genome wide DNA methylation and expression data was obtained for ALL (n=517), medulloblastoma (n=763) and neuroblastoma (n=213). For neuroblastoma, which lacks established subgroups, novel subgroups were defined based on differential methylation (using the t-SNE/dbSCAN method) SL gene candidates were derived from genomic methylation/expression data using our in-house developed bioinformatic pipeline. Functional validation of identified candidate SL genes was performed using siRNA-mediated knockdown, coupled with analysis of cell proliferation (MTT assay) and apoptosis (annexin-V staining/caspase activity). Results 22 candidate SL genes were identified across six ALL genetic subgroups, ranging from nine in the TCF3-PBX1 to one in the iAMP21 subgroup. The identified genes lack any apparent genetic/epigenetic alterations, and thus most of the candidates have not previously been implicated in cancer. siRNA-mediated partial knockdown of ETV6/RUNX1 and TCF3-PBX1-specific candidate SL genes TUSC3 and FAT1 respectively resulted in a 40-60% reduction in cell proliferation (p Conclusion We have demonstrated that SL genes, specific for defined molecular subtypes of cancer, can be identified utilising a novel approach combining methylation/expression data in multiple cancer types. Citation Format: Lalchung Nunga, Ed Schwalbe, Fadhel Lafta, Deborah Tweddle, John Maris, Timothy Barrow, Gordon Strathdee. Identification of cancer-specific synthetic lethal genes as novel therapeutic targets [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1275.

Background Early and late age at menarche are associated with risk of hypertension, but little is known whether modifiable lifestyle can reduce this risk. Methods Our study leverages 60,135 healthy young Chinese women from the Environmental and LifEstyle FActors iN metabolic health throughout life-course Trajectories (ELEFANT) study. Menarche age and lifestyle factors were assessed by self-reported questionnaires and hypertension was diagnosed by physicians. We estimated the odds ratios (ORs) of hypertension associated with menarche age using multivariable logistic regression. We further investigated whether modifiable lifestyles (body mass index, BMI; psychological stress; passive smoking; and imbalanced diet) increased risk in joint analyses. Results The association between age at menarche and hypertension was U-shaped, with age ≤ 12 at menarche giving the highest OR (1.46, 95% confidence interval [CI], 1.27–1.69) and ≥ 16 the second highest (OR = 1.36, 95% CI = 1.15–1.62). Simultaneous analysis of lifestyle risk factors and age of menarche showed that having one or more modifiable risk factors increased the menarche age-hypertension association. The risk of hypertension among participants with menarche age ≤ 12 decreased from OR 13.21 (95% CI = 5.17–29.36) with four high-risk lifestyle factors to 12.36 (95% CI = 9.51–16.05) with three high-risk factors, 5.24 (95% CI = 4.11–6.69) with two, and 2.76 (95% CI = 2.09–3.60) with one, in comparison to individuals with no high-risk lifestyle factors and menarche age 14. Conclusions Our results suggest that modification of lifestyle, including maintenance of normal weight and a balanced diet, are associated with substantially reduce the risk of hypertension in high-risk individuals. Plain English summary Early and late age at menarche are risk factors for the development of hypertension in Western populations, and there is limited evidence that this is also true of Chinese populations. Targeted prevention of hypertension in vulnerable populations would be highly beneficial in efforts to reduce the incidence of cardiovascular disease, but it is not currently known whether lifestyle intervention could reduce hypertension risk. In this study, we analysed the risk of hypertension by age at menarche and four modifiable lifestyle factors (BMI, diet, psychological stress, and smoking tobacco) in a cohort of 60,135 young adult Chinese women (mean age 29). We identified that early and late age at menarche are associated with increased risk of hypertension in young Chinese women. There was joint effects between age at menarche and lifestyles on hypertension only participants with age at menarche ≤12 and being overweight or obese. Modification of lifestyle, including maintenance of normal weight and a balanced diet, can substantially reduce the risk of hypertension in high-risk individuals. In conclusion, our study has revealed that early and late menarche age are associated with the development of hypertension in young Chinese women, and that this risk is modified by healthy lifestyle traits. Electronic supplementary material The online version of this article (10.1186/s12905-018-0677-y) contains supplementary material, which is available to authorized users.

Scope: Alterations in DNA methylation patterns are correlated with aging, environmental exposures and disease pathophysiology; the possibility of reverting or preventing these processes through dietary intervention is gaining momentum. In particular, methyl donors that provide S-adenosyl-methionine for one-carbon metabolism and polyphenols such as flavanols that inhibit the activity of DNA methyltransferases (DNMTs) could be key modifiers of epigenetic patterns. \ud Methods and results: We assessed DNA methylation patterns in publicly available Illumina Infinium 450K methylation datasets from intervention studies with either folic acid + vitamin B12 (GSE74548) or monomeric and oligomeric flavanols (MOF) (GSE54690) in 44 and 13 participants, respectively. Global DNA methylation levels increased in unmethylated regions such as CpG islands and shores following folic acid + vitamin B12 supplementation and decreased in highly methylated regions, including shelves and open-seas following intervention with MOF. After supplementation with folic acid + vitamin B12, epigenetic age, estimated by the Horvath ‘epigenetic clock’ model, was reduced in women with the MTHFR 677CC genotype. \ud Conclusions: The effects of supplementation with folic acid + vitamin B12 and MOF on DNA methylation age are dependent upon gender and MTHFR genotype. Additionally, our findings demonstrate the potential for these dietary factors to modulate global DNA methylation profiles.

Background/Aims: Exposure to fine particulate matter of

Background: Thyroid disease is one of the leading endocrine disorders, with disruption of thyroid stimulating hormone (TSH) levels commonly implicated in disease aetiology.Aims: We sought to examin...

OBJECTIVES:\ud We investigated the association between occupational noise exposure and the risk of elevated blood pressure and hypertension by stage in young adults.\ud \ud METHODS:\ud We utilized 124,286 young adults (18-40 years) from the Project ELEFANT study. We categorized occupational noise exposure as high (75 dBA noise exposure for more than 4 hours per day) or low, and measured blood pressure (mmHg) and categorized participants by hypertension stage (normal, elevated, Stage 1, Stage 2). We applied adjusted logistic regression models to identify associations with hypertension risk, and we further examined the noise-BMI, noise-gender, and noise-residence interactions on hypertension risk in separate models.\ud \ud RESULTS:\ud High occupational noise exposure was associated with increases in blood pressure among participants with elevated blood pressure (Estimate = 0.23, 95% CI: 1.09, 1.46, p = 0.0009), in Stage 1 hypertension (Estimate = 0.15, 95% CI: 1.06, 1.25, p = 0.0008), and in Stage 2 hypertension (Estimate = 0.41 95% CI: 1.31, 1.73, p

Epigenetic epidemiology is the study of epigenetic traits in populations and their influence upon disease risk. The epigenome is modifiable through environmental exposures, such as air pollution, tobacco smoke, and diet, and epigenetic dysregulation of tumor-suppressor genes such as mutL homolog 1 ( MLH1 ) and breast cancer 1 ( BRCA1 ) have been identified in healthy individuals that are associated with increased cancer risk. Through the study of these aberrant epigenetic marks and the effect of environmental exposures, epigenetic epidemiology can bring significant insights into the mechanisms of carcinogenesis and the risk of disease. In this chapter, the design and selection of appropriate tissues for studies will be addressed. Evidence for epigenetic changes in response to environmental exposures and for the presence of “epimutations” in oncogenes and tumor-suppressor genes will be reviewed. In addition, novel approaches to integrated analysis of the epigenome, such as the “epigenetic clock” model of aging, will be discussed and the future directions of the field will be commented upon.

Epigenetic regulation of imprinted genes enables monoallelic expression according to parental origin, and its disruption is implicated in many cancers and developmental disorders. The expression of hormone receptors is significant in breast cancer because they are indicators of cancer cell growth rate and determine response to endocrine therapies. We investigated the frequency of aberrant events and variation in DNA methylation at nine imprinted sites in invasive breast cancer and examined the association with estrogen and progesterone receptor status. Breast tissue and blood from patients with invasive breast cancer (n = 38) and benign breast disease (n = 30) were compared with those from healthy individuals (n = 36), matched with the cancer patients by age at diagnosis, ethnicity, body mass index, menopausal status and familial history of cancer. DNA methylation and allele-specific expression were analyzed by pyrosequencing. Tumor-specific methylation changes at IGF2 DMR2 were observed in 59% of cancer patients, IGF2 DMR0 in 38%, DIRAS3 DMR in 36%, GRB10 ICR in 23%, PEG3 DMR in 21%, MEST ICR in 19%, H19 ICR in 18%, KvDMR in 8% and SNRPN/SNURF ICR in 4%. Variation in methylation was significantly greater in breast tissue from cancer patients compared with that in healthy individuals and benign breast disease. Aberrant methylation of three or more sites was significantly associated with negative estrogen-alpha (Fisher's exact test, p = 0.02) and progesterone-A (p = 0.02) receptor status. Aberrant events and increased variation in imprinted gene DNA methylation, therefore, seem to be frequent in invasive breast cancer and are associated with negative estrogen and progesterone receptor status, without loss of monoallelic expression.

Morphine is one of the most effective analgesics in medicine. However, its use is associated with the development of tolerance and dependence. Recent studies demonstrating epigenetic changes in the brain after exposure to opiates have provided insight into mechanisms possibly underlying addiction. In this study, we sought to identify epigenetic changes in ten regions of the rat brain following acute and chronic morphine exposure. We analyzed DNA methylation of six nuclear-encoded genes implicated in brain function (Bdnf, Comt, Il1b, Il6, Nr3c1, and Tnf) and three mitochondrially-encoded genes (Mtco1, Mtco2, and Mtco3), and measured global 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5 hmC) levels. We observed differential methylation of Bdnf and Il6 in the pons, Nr3c1 in the cerebellum, and Il1b in the hippocampus in response to acute morphine exposure (all P value

Epigenetic epidemiology includes the study of variation in epigenetic traits and the risk of disease in populations. Its application to the field of cancer has provided insight into how lifestyle and environmental factors influence the epigenome and how epigenetic events may be involved in carcinogenesis. Furthermore, it has the potential to bring benefit to patients through the identification of diagnostic markers that enable the early detection of disease and prognostic markers that can inform upon appropriate treatment strategies. However, there are a number of challenges associated with the conduct of such studies, and with the identification of biomarkers that can be applied to the clinical setting. In this review, we delineate the challenges faced in the design of epigenetic epidemiology studies in cancer, including the suitability of blood as a surrogate tissue and the capture of genome-wide DNA methylation. We describe how epigenetic epidemiology has brought insight into risk factors associated with lung, breast, colorectal and bladder cancer and review relevant research. We discuss recent findings on the identification of epigenetic diagnostic and prognostic biomarkers for these cancers.

The increased incidence of chronic lymphocytic leukemia (CLL) in first-degree relatives of affected patients indicates an element of genetic susceptibility to this malignancy, borne out in large scale genome-wide association studies (GWAS), which have identified over 40 constitutional risk alleles. Given the important genetic contribution to CLL susceptibility we hypothesized that constitutional genetic variants also affect disease progression. We employed GWAS methods in a large United Kingdom multi-center cohort study of well-characterized predominantly early-stage CLL cases to identify risk alleles for progressive CLL. We conducted six GWAS for single nucleotide polymorphisms (SNPs) associating with progressive CLL incorporating a total of 774 cases of European ancestry recruited to 6 clinical centers across the United Kingdom. CLL cases were genotyped on the Illumina OmniExpress platform and genotypes were determined using Illumina GenomeStudio software. After imputation, we combined the association test statistic for 5,199,911 autosomal SNPs common to all 6 GWAS after exclusion of those with an imputation quality score of

Environmental noise exposure is associated with adverse effects on human health including hearing loss, heart disease, and changes in stress-related hormone levels. Alteration in DNA methylation in response to environmental exposures is a well-known phenomenon and it is implicated in many human diseases. Understanding how environmental noise exposures affect DNA methylation patterns may help to elucidate the link between noise and adverse effects on health. In this pilot study we examined the effects of environmental noise exposure on DNA methylation of genes related to brain function and investigated whether these changes are related with metabolic health. We exposed four groups of male Wistar rats to moderate intensity noise (70–75 dB with 20–4000 Hz) at night for three days as short-term exposure, and for three weeks as long-term exposure. Noise exposure was limited to 45 dB during the daytime. Control groups were exposed to only 45 dB, day and night. We measured DNA methylation in the Bdnf, Comt, Crhr1, Mc2r, and Snca genes in tissue from four brain regions of the rats (hippocampus, frontal lobe, medulla oblongata, and inferior colliculus). Further, we measured blood pressure and body weight after long-term noise exposure. We found that environmental noise exposure is associated with gene-specific DNA methylation changes in specific regions of the brain. Changes in DNA methylation are significantly associated with changes in body weight (between Bdnf DNA methylation and Δ body weight: r=0.59, p=0.018; and between LINE-1 ORF DNA methylation and Δ body weight: =−0.80, p=0.0004). We also observed that noise exposure decreased blood pressure (p=0.038 for SBP, p=0.017 for DBP and p 0. 017 for MAP) and decreased body weight (β=−26 g, p=0.008). In conclusion, environmental noise exposures can induce changes in DNA methylation in the brain, which may be associated with adverse effects upon metabolic health through modulation of response to stress-related hormones.

BACKGROUND:\ud The menstrual cycle is regulated by reproductive hormones such as estrogen which has been implicated in the pathogenesis of hypertension and is associated with obesity. However, to date there has scant study of hypertension in relation to menstrual characteristics and abnormalities. We hypothesize that adverse menstrual characteristics are associated with an increase the prevalence of hypertension and that this relationship is exacerbated by obesity.\ud \ud METHODS:\ud Our study leverages 178,205 healthy female participants (mean age = 29) in a population-based cross-sectional study in Tianjin, China. Menstrual characteristics including menstrual cycle length and regularity, menstrual bleeding length, menstrual blood loss and dysmenorrhea were assessed by self-reported questionnaires, and hypertension was diagnosed by physician. Multiple logistic regression models were used to assess the relationships between menstrual characteristics and hypertension.\ud \ud RESULTS:\ud Normal length menstrual cycle (OR = 1.21, 95% CI: 1.03-1.41), oligomenorrhea (OR = 1.54, 95% CI: 1.12-2.07), irregular cycle (OR = 1.54, 95% CI: 1.22-1.93), and light menstrual blood loss (OR = 1.36, 95% CI: 1.06-1.72) were associated with hypertension among women who are overweight or obese, but not among women who are normal weight. Longer menstrual bleeding duration (OR = 1.44, 95% CI: 1.24-1.67) and dysmenorrhea were associated with increased prevalence of hypertension (OR = 1.20, 95% CI: 1.14-1.41) in all young women.\ud \ud CONCLUSIONS:\ud The prevalence of hypertension is higher among women with menstrual abnormalities, and this association is modified by overweight and obesity.

Transposable elements (TE) comprise half of the human genome. LINE-1 and ALU are the most common TE, and they have been used to assess changes in the DNA methylation of repetitive elements in response to intrinsic and extrinsic cellular events. Pyrosequencing(®) is a real-time sequencing technology that enables quantitative assessment of TE methylation at single-base resolution. In Pyrosequencing, a region of interest is first amplified from bisulfite-converted DNA by polymerase chain reaction (PCR), before PCR amplicons are rendered single stranded and annealed with the Pyrosequencing primer prior to sequencing. In this chapter, we provide an overview of the analysis of repetitive element DNA methylation by bisulfite Pyrosequencing, and we describe a protocol that can be used for such purposes.

There is increasing evidence that exposure to air pollutants are associated with human disease and may act through epigenetic modification of the nuclear genome, but there have been few publications describing their impact upon the mitochondrial genome. Mitochondrial DNA may be more susceptible to pollutant-induced changes via increased oxidative stress in the cell, and therefore this field of research is of growing interest. Many techniques employed to study DNA methylation of the nuclear genome are also applicable to mitochondrial epigenetic studies. In this chapter, we describe a protocol for the isolation of mitochondrial DNA from peripheral blood samples and the analysis of 5-methlycytosine content by bisulfite-pyrosequencing.

Transposable elements (TE) comprise half of the human genome. LINE-1 and ALU are the most common TE, and they have been used to assess changes in the DNA methylation of repetitive elements in response to intrinsic and extrinsic cellular events. Pyrosequencing(®) is a real-time sequencing technology that enables quantitative assessment of TE methylation at single-base resolution. In Pyrosequencing, a region of interest is first amplified from bisulfite-converted DNA by polymerase chain reaction (PCR), before PCR amplicons are rendered single stranded and annealed with the Pyrosequencing primer prior to sequencing. In this chapter, we provide an overview of the analysis of repetitive element DNA methylation by bisulfite Pyrosequencing, and we describe a protocol that can be used for such purposes.

Chronic lymphocytic leukaemia (CLL) is the most common form of adult leukaemia worldwide. Patients display a highly variable clinical course, with some requiring immediate therapeutic intervention while others can remain untreated for years. We have previously reported that DNA methylation of the homeobox A4 (HOXA4) promoter can serve as part of a three gene prognostic signature with CD38 and BTG4 to predict time to first treatment (TTT) in Stage A patients. HOXA4 encodes a transcription factor that is expressed in haematopoietic progenitor cells and is involved in embryonic development and B-cell differentiation, and its aberrant epigenetic regulation has been identified in multiple forms of leukaemia. In this study we have sought to elucidate the role of HOXA4in the progression of CLL and determine the functional consequences of its expression. We analysed DNA methylation of the HOXA4 promoter by pyrosequencing in a heterogeneous cohort of 163 CLL patients (median age: 70; median follow-up: 10 years), of whom 60% were Binet Stage A, 16% Stage B and 24% Stage C. Data was collected regarding treatment history, TTT and overall survival, as well as cytogenetic abnormalities and IGVH mutation status. HOXA4 methylation increased with disease progression and was significantly higher in Stage C patients (median 74%) than those with Stage A (62%; p = 0.03) and Stage B disease (65%; p < 0.05). HOXA4 methylation was positively correlated with IGVH sequence homology (r = 0.34, p < 0.0001) and negatively associated with TTT among patients who have started chemotherapy (p = 0.04) and with overall survival (p = 0.04). No associations were observed between HOXA4 methylation and 11q, 13q or 17p deletions, or TP53 and ATMmutations. To investigate the role of HOXA4 in the evolution of the disease, we analysed samples taken at multiple timepoints from 42 patients, of whom 29 were undergoing treatment and 13 remained untreated. HOXA4methylation significantly increased in patients undergoing treatment (p = 0.01), but did not differ in untreated patients (p = 0.19). We hypothesised that silencing of HOXA4 may be selected for during treatment due to its expression conferring increased sensitivity to chemotherapy. Using a lentiviral system, we observed that re-expression of HOXA4increased drug sensitivity in a malignant differentiated B-cell line (Raji). Significantly higher apoptosis was identified after treatment with 3 μM and 10 μM fludarabine (both p < 0.001) and 1 μM and 10 μM ibrutinib (p < 0.01 and p < 0.001), but not 1 μM and 10 μM idelalisib. To confirm the translational relevance our observations, we overexpressed HOXA4 in primary CLL cells derived from four patients and confirmed increased apoptosis in response to 3 μM and 10 μM fludarabine treatment in comparison to control cells (p = 0.02 and p < 0.01). Further work is underway in primary CLL cells to elucidate the pathways under the control of HOXA4 that may confer this drug sensitivity. Our ongoing work may indicate that HOXA4 is also implicated in the progression of CLL through directing malignant cells to the protective bone marrow niche, thereby further reducing sensitivity to antimetabolites. In cell lines HOXA4 up-regulates the expression of RGS2 and RGS16, which are negative regulators of the CXCR4-CXCL12 signalling axis, and we have identified selection for biallelic HOXA4methylation in primary acute lymphoblastic leukaemia cells following engraftment in mice (median in primary cells: 80%; engrafted cells: 92%; p < 0.0001). To determine the origins of HOXA4 dysregulation during the course of the disease, we analysed prospective blood samples from the European Prospective Investigation into Cancer and Nutrition (EPIC) from 20 individuals diagnosed with CLL up to 17 years after blood draw (median: 7 years) and 20 age-matched controls who remained free of cancer. We observed that HOXA4methylation was significantly higher among future CLL patients (median: 49% vs 42%; p = 0.01) and was inversely correlated with time to diagnosis, but did not reach statistical significance (r = -0.39, p = 0.09). Together, our findings suggest that silencing of the HOXA4 gene is an early event in CLL which is selected for during the course of disease through reduced sensitivity to chemotherapeutic agents. Our ongoing work will identify downstream targets that may be implicated in conferring sensitivity, and which may serve as biomarkers to predict prognosis and inform treatment strategies. Disclosures Junge: AstraZeneca: Other: Salary, Research Funding.

Haem in red meat (RM) stimulates the endogenous production of mutagenic nitroso compounds (NOC). Processed (nitrite-preserved red) meat additionally contains high concentrations of preformed NOC. In two studies, of a fresh RM versus a vegetarian (VEG) diet (six males and six females) and of a nitrite-preserved red meat (PM) versus a VEG diet (5 males and 11 females), we investigated whether processing of meat might increase colorectal cancer risk by stimulating nitrosation and DNA damage. Meat diets contained 420 g (males) or 366 g (females) meat/per day. Faecal homogenates from day 10 onwards were analysed for haem and NOC and asso- ciated supernatants for genotoxicity. Means are adjusted for differ- ences in male to female ratios between studies. Faecal NOC concentrations on VEG diets were low (2.6 and 3.5 mmol/g) but significantly higher on meat diets (PM 175 ± 19 nmol/g versus RM 185 ± 22 nmol/g; P 5 0.75). The RM diet resulted in a larger pro- portion of nitrosyl iron (RM 78% versus PM 54%; P < 0.0001) and less nitrosothiols (RM 12% versus PM 19%; P < 0.01) and other NOC (RM 10% versus PM 27%; P < 0.0001). There was no statis- tically significant difference in DNA breaks induced by faecal water (FW) following PM and RM diets (P 5 0.80). However, PM re- sulted in higher levels of oxidized pyrimidines (P < 0.05). Surpris- ingly, VEG diets resulted in significantly more FW-induced DNA strand breaks than the meat diets (P < 0.05), which needs to be clarified in further studies. Meats cured with nitrite have the same effect as fresh RM on endogenous nitrosation but show increased FW-induced oxidative DNA damage.

Genomic imprinting is the epigenetic regulation of genes to enable monoallelic expression according to parental origin. Imprinting is established in the germline and is particularly important in the regulation of fetal development; loss of imprinting (LOI) has been implicated in the development of a variety of childhood disorders and several cancer types. We investigated loss of imprinting in PEG3, MEST and ARHI/DIRAS3 in breast cancer. Blood and tissue samples from women with benign breast disease or no abnormalities (n = 52) and patients with invasive breast cancer (n = 37) were provided by the Clinical Breast Care Project at the Walter Reed Army Medical Center (Washington, USA). DNA extracted from the samples was bisulfite converted and the methylation status of the three genes determined by pyrosequencing. Loss of PEG3 imprinting was observed in 2% of benign tissues and 34% of invasive cancers. LOI in MEST was similarly observed in 4% of benign tissues and 39% of invasive cancers, while in ARHI/DIRAS3 it was observed in 11% of benign samples and 54% of invasive ones. LOI was not observed in blood samples for any of the three genes, suggesting that these epigenetic alterations are highly tissue-specific. There was no correlation between the methylation profiles of the three genes (Spearman's rank correlation coefficient; p = >0.25). Loss of imprinting in PEG3, MEST and ARHI/DIRAS3 appears to be a frequent event in breast cancer, and it is more common in invasive forms than benign proliferative breast disease. The impact of the observed methylation changes upon allele-specific expression is currently investigated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4039. doi:1538-7445.AM2012-4039