Pharmacoepigenomic effects of anti-hypertensive drugs on DNA methylation and its implication to drug response and side effects.

DNA methylation profile at a satellite region is associated with aberrant placentation in cloned calves

Introduction Environmental factors, such as nutrition and occupational exposure can influence epigenetic marks like DNA methylation, which play a role in the development of chronic diseases. Methods Data of the MAternal Nutrition and Offspring’s Epigenome (MANOE) study was used to assess the effect of maternal occupation on maternal and infant DNA (hydroxy)methylation levels. Mothers were categorised in job categories according to the International Standard Classification of Occupations (ISCO). Maternal global DNA (hydroxy)methylation levels during each trimester of pregnancy and at delivery (n=122) was measured in whole blood via LC-MS/MS. Data were analysed with a one-Way ANOVA. Results We found statistically significant differences in maternal global DNA methylation (p=0.008) and global DNA hydroxymethylation (p=0.004) at 20 weeks of pregnancy. Post hoc tests revealed that global DNA methylation and global DNA hydroxymethylation level was significantly lower when the mother had an intellectual/scientific/artistic profession (6.36% and 0.13%) as opposed to being a manager (7.77%, p=0.007% and 0.22%, p=0.002) or administrative staff (7.71%, p=0.003% and 0.2%, p=0.005). No significant differences between different working groups were found for global DNA (hydroxy)methylation in the first and third trimester of pregnancy and at delivery. Conclusion The mother’s occupation was associated with maternal global DNA (hydroxy)methylation levels only in the second trimester of pregnancy. The change in maternal global DNA (hydroxy)methylation in the second trimester of pregnancy could be due to hormonal changes during pregnancy, a shift in the one-carbon metabolism in the middle of pregnancy, but based on these results we also have to take into account maternal occupational exposure.

Background: The association between human blood DNA global methylation and global hydroxymethylation has not been evaluated in population-based studies. No studies have evaluated environmental determinants of global DNA hydroxymethylation, including exposure to metals. Aims: To evaluate the association between global DNA methylation and global DNA hydroxymethylation in a subsample of 48 Strong Heart Study participants who had selected metals measured in urine at baseline and DNA available in 1989-1991 and 1998-1999. Methods: Global 5-methylcytosine (5-mC) and 5-hydroxymethyl-cytosine (5-hmC) levels in DNA were measured by capture and detection antibodies followed by colorimetric quantification. We used linear regression models to evaluate trends and compare relative differences in methylation and hydroxymethylation levels by participant characteristics (age, sex, education, adiposity, smoking, alcohol intake, metal exposure and arsenic metabolism). Results: The Spearman’s correlation coefficient for 5-mC and 5-hmC levels was 0.32 (p –value = 0.03) at visit 1 and 0.54 (p – value < 0.001) at visit 3, with consistent trends for both epigenetic markers across characteristics. The associations were significant for urine cadmium concentrations and global methylation and for arsenic metabolism and global hydroxymethylation. Conclusions: Our findings support that both epigenetic measures are related at the population level. The consistent trends in the associations between these epigenetic modifications and the characteristics evaluated suggest the need for understanding which of the two measures is a better biomarker for environmental epigenetic effects in future large-scale epidemiologic studies.

Influence of smoking on methylation and hydroxymethylation levels in global DNA and specific sites of KRT14, KRT19, MIR-9-3 and MIR-137 genes of oral mucosa

This study was designed to analyze the effect of global and gene-specific DNA methylation patterns on the outcome of patients with acute myeloid leukemia (AML). Methylation of CDKN2B (p15), E-cadherin (CDH) and hypermethylated in cancer 1 (HIC1) promoters and global DNA methylation by luminometric methylation assay (LUMA) was analyzed in 107 AML patients and cytogenetic and molecular mutational analysis was performed. In addition, genome-wide promoter-associated methylation was assessed using the Illumina HumanMethylation27 array in a proportion of the patients. Promoter methylation was discovered in 66, 66 and 51% of the patients for p15, CDH and HIC1, respectively. In multivariate analysis, low global DNA methylation was associated with higher complete remission rate (hazard ratio (HR) 5.9, P=0.005) and p15 methylation was associated with better overall (HR 0.4, P=0.001) and disease-free survival (HR 0.4, P=0.016). CDH and HIC1 methylation were not associated with clinical outcome. Mutational status and karyotype were not significantly associated with gene-specific methylation or global methylation. Increased genome-wide promoter-associated methylation was associated with better overall and disease-free survival as well as with LUMA hypomethylation. We conclude that global and gene-specific methylation patterns are independently associated with the clinical outcome in AML patients.

Mixed connective tissue disease (MCTD) is a very rare disorder that belongs in the rare and clinically multifactorial groups of diseases. The pathogenesis of MCTD is still unclear. The best understood epigenetic alteration is DNA methylation whose role is to regulate gene expression. In the literature, there are ever-increasing assumptions that DNA methylation can be one of the possible reasons for the development of Autoimmune Connective Tissue Diseases (ACTDs) such as systemic sclerosis (SSc) and systemic lupus erythematosus (SLE). The aim of this study was to define the global DNA methylation changes between MCTD and other ACTDs patients in whole blood samples. The study included 54 MCTD patients, 43 SSc patients, 45 SLE patients, and 43 healthy donors (HC). The global DNA methylation level was measured by ELISA. Although the global DNA methylation was not significantly different between MCTD and control, we observed that hypomethylation distinguishes the MCTD patients from the SSc and SLE patients. The present analysis revealed a statistically significant difference of global methylation between SLE and MCTD (p < 0.001), SLE and HC (p = 0.008), SSc and MCTD (p ≤ 0.001), and SSc and HC (p < 0.001), but neither between MCTD and HC (p = 0.09) nor SSc and SLE (p = 0.08). The highest % of global methylation (median, IQR) has been observed in the group of patients with SLE [0.73 (0.43, 1.22] and SSc [0,91 (0.59, 1.50)], whereas in the MCTD [0.29 (0.20, 0.54)], patients and healthy subjects [0.51 (0.24, 0.70)] were comparable. In addition, our study provided evidence of different levels of global DNA methylation between the SSc subtypes (p = 0.01). Our study showed that patients with limited SSc had a significantly higher global methylation level when compared to diffuse SSc. Our data has shown that the level of global DNA methylation may not be a good diagnostic marker to distinguish MCTD from other ACTDs. Our research provides the groundwork for a more detailed examination of the significance of global DNA methylation as a distinguishing factor in patients with MCTD compared to other ACTDs patients.

B45 Polycyclic aromatic hydrocarbons (PAHs) are widespread organic carcinogenic pollutants. When metabolized in vivo, PAHs form reactive diol epoxides that bind to DNA at guanine residues forming adducts. PAH exposure has also been associated with alterations in genomic cytosine methylation. Both PAH-DNA adducts and altered DNA methylation have been associated with increased cancer risk. We sought to explore the relationship between prenatal PAH exposure, global DNA methylation and PAH-DNA adducts in a New York City birth cohort. Cord blood was collected from deliveries of non-smoking, African Americans and Dominicans, ages 18-35, residing in N. Manhattan and S. Bronx. Personal air monitors measured PAH during their 8th month of pregnancy. We measured benzo[a]pyrene (B[a]P)-DNA adducts (which serve as a proxy for PAH-DNA adducts) in cord blood using high-performance liquid chromatography-fluorescence. We measured genomic DNA methylation in cord blood leukocytes using the MethylampTM global DNA methylation quantification kit. Demographic and epidemiologic risk factors were collected prospectively, including maternal age, ethnicity, parity, and prenatal tobacco smoke exposure, and child’s gender We found that prenatal PAH exposure was associated global DNA hypomethylation such that average methylation among those in the highest quartile of prenatal PAH exposure was 1.17 ng/100 ng total DNA as compared 1.83 ng/100 ng total DNA in the lowest quartile (p &amp;lt; 0.01). Conversely, those with detectable PAH-DNA adducts had higher levels of DNA methylation (1.43 ng/100 ng total DNA) as compared to those with non-detectable adducts (1.16 ng/100 ng total DNA) (p = 0.02). Using multiple variable linear regression, PAH exposure was independently associated with hypomethylation (average difference in methylation among those in the highest vs. lowest quartile of exposure = -0.42, 95% CI: -0.75, -0.09) and the presence of DNA adducts was associated with hypermethyaltion (average difference in methylation among those with detectable vs. non-detectable adducts = 0.28, 95% CI: 0.05, 0.49). Adjusting for potential confounders did not significantly alter these independent associations, indicating that the lowest level of global DNA methylation was found among those with non-detectable adducts and high PAH exposure, and the highest level of DNA methylation was found among those with detectable adducts and low PAH exposure. While the mechanism by which prenatal PAH exposure results in DNA adducts is well understood, the way exposure may impact methylation is not known. These data suggest that in the presence of DNA adducts, PAH exposure is associated with a reduction in methylation, but when adducts are non-detectable, the same PAH exposure is associated with an even larger methylation reduction. It is possible that the presence of adducts may be partially preventing PAH compounds (or metabolites) from accessing the DNA, leading to less of a change in methylation. It is also possible that the shift in global methylation from PAH exposure may prevent adduct formation. These speculations as well as the associations observed in this analysis require confirmation in other settings/populations. Because methylation changes are potentially reversible, it is possible that if these associations are confirmed, methylation may serve as a target for intervention. Citation Information: Cancer Prev Res 2008;1(7 Suppl):B45.

Proliferation-Dependent Alterations of the DNA Methylation Landscape Underlie Hematopoietic Stem Cell Aging

Aberrant DNA methylation is an early event in colorectal tumorigenesis and in colorectal cancer (CRC) patients these changes are detected in their normal colonic mucosa. DNA methylation changes may arise from aging or environmental exposures e.g. folate deficiency. We aimed to compare global and repeat-sequence DNA methylation in the colonic mucosa and blood of three groups of individuals – CRC patients, healthy people and patients with folate deficiency. We conducted a multisite cohort study collecting mucosa and blood from 176 patients who had undergone CRC resection and 194 healthy individuals at screening colonoscopy. Blood was also collected from 30 individuals with megaloblastic anaemia secondary to folate deficiency (red cell folate &amp;lt;539nM). Global DNA methylation was measured using liquid chromatography tandem mass spectrometry adapted from Quinlivan &amp; Gregory (2008)1. Alu repeat methylation was determined using a novel, ultra-sensitive real-time PCR assay based upon methylation-sensitive restriction enzyme digestion coupled with a fluorescence-based readout2. Genotyping for a common polymorphism in the folate metabolism enzyme MTHFR (C677T) was performed using pyrosequencing. The mean age of CRC patients (68 ± 14 years) was significantly higher than healthy subjects (55 ± 14 years; p&amp;lt;0.01) but not significantly different from low-folate subjects (72 ± 22 years). The gender distribution was similar in all 3 groups. The prevalence of the variant T allele of MTHFR in CRC subjects (58%) and healthy controls (64%) were comparable to population-based estimates. However, the T allele was over-represented in low folate patients (79%). In the analysis of the first subset of CRC (n=38) and healthy controls (n=38), mean global DNA methylation was significantly lower in the colonic mucosa (CRC n=20: 4.1% ± 0.3; controls n=36: 4.0% ± 0.3) compared to peripheral blood (CRC n=17: 4.4% ± 0.4; controls n=36: 4.4% ± 0.3, p&amp;lt;0.01). No differences in DNA methylation were found in CRC patients compared to healthy subjects. Consistent with global methylation findings, Alu repeats were 2-fold less methylated in colon compared to peripheral blood in healthy (n=38) and CRC cohorts(n=36). Low-folate subjects had a trend towards lower blood global (4.26% ± 0.2) and Alu methylation (1.2-fold hypomethylated) compared to healthy subjects. To date, our results suggest that Alu repeat methylation reflect global DNA methylation and that this holds true across tissue types irrespective of cancer status. Global and Alu repeat demethylation in individuals with folate deficiency and overrepresentation of the T MTHFR allele suggests a possible link between diet and the epigenome that warrants further study. 1. Quinlivan, E.P. &amp; Gregory, J.F., 3rd. (2008) Nucleic acids research 36, e119. 2. Rand, K. &amp; Molloy, P. (2010) BioTechniques 49, xiii-xvii. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 91. doi:10.1158/1538-7445.AM2011-91

Altered DNA methylation pattern in sperms has been associated with infertility in males demonstrating defective spermatogenesis or low semen quality. Vitamin B-12, by affecting 1-carbon metabolism pathways, might alter the DNA methylation pattern. We aimed to study the correlation of serum vitamin B12 levels with aberrant DNA methylation in infertile male patients. A cross-sectional study was conducted on 17 oligozoospermic infertile males (WHO criteria, 2010) and 10 healthy fertile males. Serum vitamin B12 levels were estimated using the chemiluminescence method. Global methylation was determined using the ELISA system (Imprint Methylated DNA Quantification Kit, Sigma-Aldrich). The levels of global DNA methylation were calculated and compared relative to the methylated (100%) control DNA provided with the kit. Mean serum vitamin B12 concentration in the control group was higher than that of the case group. This difference in serum vitamin B12 concentration in both groups was found statistically significant. Although the results of this study show that oligozoospermic men have relatively lower global DNA methylation as compared to normozoospermic control, the values could not reach a statistically significant level. A small positive correlation was found between serum vitamin B12 levels and percent methylation defect (r = 0.14) but was statistically insignificant. Our study concludes that oligozoospermic infertile males have a significant deficiency of vitamin B12 as compared to normozoospermic fertile males. This study did not find any significant difference in global DNA methylation between the two groups. The present study does not suggest any correlation between serum vitamin B12 level and percent DNA methylation.

Background: Epigenetic markers are often quantified and related to disease in stored samples. While, effects of storage on stability of these markers have not been thoroughly examined. In this longitudinal study, we investigated the influence of storage time, material, temperature, and freeze-thaw cycles on stability of global DNA (hydroxy)methylation. Methods: EDTA blood was collected from 90 individuals. Blood (n = 30, group 1) and extracted DNA (n = 30, group 2) were stored at 4°C, −20°C and −80°C for 0, 1 (endpoint blood 4°C), 6, 12 or 18 months. Additionally, freeze-thaw cycles of blood and DNA samples (n = 30, group 3) were performed over three days. Global DNA methylation and hydroxymethylation (mean ± SD) were quantified using liquid chromatography–electrospray ionization–tandem mass spectrometry (LC-ESI-MS/MS) with between-run precision of 2.8% (methylation) and 6.3% (hydroxymethylation). Effects on stability were assessed using linear mixed models. Results: global DNA methylation was stable over 18 months in blood at −20°C and −80°C and DNA at 4°C and −80°C. However, at 18 months DNA methylation from DNA stored at −20°C relatively decreased −6.1% compared to baseline. Global DNA hydroxymethylation was more stable in DNA samples compared to blood, independent of temperature (p = 0.0131). Stability of global DNA methylation and hydroxymethylation was not affected up to three freeze – thaw cycles. Conclusion: Global DNA methylation and hydroxymethylation stored as blood and DNA can be used for epigenetic studies. The relevance of small differences occuring during storage depend on the expected effect size and research question.

DNA methylation may mediate persistent changes in gene function following chronic stress. To examine this hypothesis, we evaluated African American subjects matched by age and sex, and stratified into four groups by post-traumatic stress disorder (PTSD) diagnosis and history of child abuse. Total Life Stress (TLS) was also assessed in all subjects. We evaluated DNA extracted from peripheral blood using the HumanMethylation27 BeadChip and analyzed both global and site-specific methylation. Methylation levels were examined for association with PTSD, child abuse history, and TLS using a linear mixed model adjusted for age, sex, and chip effects. Global methylation was increased in subjects with PTSD. CpG sites in five genes (TPR, CLEC9A, APC5, ANXA2, and TLR8) were differentially methylated in subjects with PTSD. Additionally, a CpG site in NPFFR2 was associated with TLS after adjustment for multiple testing. Notably, many of these genes have been previously associated with inflammation. Given these results and reports of immune dysregulation associated with trauma history, we compared plasma cytokine levels in these subjects and found IL4, IL2, and TNFα levels associated with PTSD, child abuse, and TLS. Together, these results suggest that psychosocial stress may alter global and gene-specific DNA methylation patterns potentially associated with peripheral immune dysregulation. Our results suggest the need for further research on the role of DNA methylation in stress-related illnesses.

BACKGROUND/OBJECTIVESPrevious studies have indicated that when compared to young mice, old mice have lower global DNA methylation and higher p16 promoter methylation in colonic mucosa, which is a common finding in colon cancer. It is also known that a Western-style diet (WSD) high in fat and calories, and low in calcium, vitamin D, fiber, methionine and choline (based on the AIN 76A diet) is tumorigenic in colons of mice. Because DNA methylation is modifiable by diet, we investigate whether a WSD disrupts DNA methylation patterns, creating a tumorigenic environment.SUBJECTVIES/METHODSWe investigated the effects of a WSD and aging on global and p16 promoter DNA methylation in the colon. Two month old male C57BL/6 mice were fed either a WSD or a control diet (AIN76A) for 6, 12 or 17 months. Global DNA methylation, p16 promoter methylation and p16 expression were determined by LC/MS, methyl-specific PCR and real time RT-PCR, respectively.RESULTSThe WSD group demonstrated significantly decreased global DNA methylation compared with the control at 17 months (4.05 vs 4.31%, P = 0.019). While both diets did not change global DNA methylation over time, mice fed the WSD had lower global methylation relative to controls when comparing all animals (4.13 vs 4.30%, P = 0.0005). There was an increase in p16 promoter methylation from 6 to 17 months in both diet groups (P < 0.05) but no differences were observed between diet groups. Expression of p16 increased with age in both control and WSD groups.CONCLUSIONSIn this model a WSD reduces global DNA methylation, whereas aging itself has no affect. Although the epigenetic effect of aging was not strong enough to alter global DNA methylation, changes in promoter-specific methylation and gene expression occurred with aging regardless of diet, demonstrating the complexity of epigenetic patterns.

Air pollution and DNA methylation in adults: A systematic review and meta-analysis of observational studies

BackgroundDespite DNA methylation being one of the most widely studied epigenetic modifications in eukaryotes, only a few studies have examined the global methylation status of marsupial chromosomes. The emergence of devil facial tumour disease (DFTD), a clonally transmissible cancer spreading through the Tasmanian devil population, makes it a particularly pertinent time to determine the methylation status of marsupial and devil facial tumour chromosomes. DNA methylation perturbations are known to play a role in genome instability in human tumours. One of the interesting features of the devil facial tumour is its remarkable karyotypic stability over time as only four strains with minor karyotypic differences having been reported.The cytogenetic monitoring of devil facial tumour (DFT) samples collected over an eight year period and detailed molecular cytogenetic analysis performed on the different DFT strains enables chromosome rearrangements to be correlated with methylation status as the tumour evolves.ResultsWe used immunofluorescent staining with an antibody to 5-methylcytosine on metaphase chromosomes prepared from fibroblast cells of three distantly related marsupials, including the Tasmanian devil, as well as DFTD chromosomes prepared from samples collected from different years and representing different karyotypic strains. Staining of chromosomes from male and female marsupial cell lines indicate species-specific differences in global methylation patterns but with the most intense staining regions corresponding to telomeric and/or centromeric regions of autosomes. In males, the X chromosome was hypermethylated as was one X in females. Similarly, telomeric regions on DFTD chromosomes and regions corresponding to material from one of the two X chromosomes were hypermethylated. No difference in global methylation in samples of the same strain taken in different years was observed.ConclusionsThe methylation patterns on DFTD chromosomes suggests that the hypermethylated active X was shattered in the formation of the tumour chromosomes, with atypical areas of methylation on DFTD chromosomes corresponding to locations of X chromosome material from the shattered X. The incredibly stable broad methylation patterns observed between strains and over time may reflect the overall genomic stability of the devil facial tumour.Electronic supplementary materialThe online version of this article (doi:10.1186/s13039-015-0176-x) contains supplementary material, which is available to authorized users.