Let-7a Downregulation Accompanied by KRAS Mutation Is Predictive of Lung Cancer Onset in Cigarette Smoke-Exposed Mice.

BACKGROUND: Let-7 is a tumor suppressor microRNA targeting the KRAS lung oncogene. Let-7a downregulation is reversible during the early stages of lung carcinogenesis but is irreversible in cancer cells. The aim of this study is to shed light on the relationship between oncogene (KRAS) mutation and let-7a downregulation in cigarette smoke (CS)-induced lung carcinogenesis. METHODS: A total of 184 strain H Swiss albino mice were either unexposed (control) or exposed to CS for 2 weeks (short CS) or 8 months (long CS). After 8 months, the lungs were individually collected. The following end points have been evaluated: (a) DNA methylation of the let-7a gene promoter by bisulphite-PCR and pyrosequencing; (b) let-7a expression by qPCR; (c) KRAS mutation by DNA pyrosequencing; (d) cancer incidence by histopathological examination. RESULTS: let-7a expression decreased by 8.3% in the mice exposed to CS for two weeks (CS short) and by 33.4% (p ≤ 0.01) in the mice exposed to CS for 8 months (CS long). No significant difference was detected in the rate of let-7a-promoter methylation between the Sham-exposed mice (55.1%) and the CS short-(53%) or CS long (51%)-exposed mice. The percentage of G/T transversions in KRAS codons 12 and 13 increased from 2.3% (Sham) to 6.4% in CS short- and to 11.5% in CS long-exposed mice. Cancer incidence increased significantly in the CS long-exposed mice (11%) as compared to both the Sham (4%) and the CS short-exposed (2%) mice. In the CS long-exposed mice, the correlation between let-7a expression and the number of KRAS mutations was positive (R = +0.5506) in the cancer-free mice and negative (R = -0.5568) in the cancer-bearing mice. CONCLUSIONS: The effects of CS-induced mutations in KRAS are neutralized by the high expression of let-7a in cancer-free mice (positive correlation) but not in cancer-bearing mice where an irreversible let-7a downregulation occurs (negative correlation). This result provides evidence that both genetic (high load of KRAS mutation) and epigenetic alterations (let-7a irreversible downregulation) are required to produce lung cancer in CS-exposed organisms.

Association between night shift work and methylation of a subset of immune-related genes.

Introduction: Night shift (NS) work has been associated with an increased risk of different conditions characterized by altered inflammatory and immune responses, such as cardio-metabolic and infectious diseases, cancer, and obesity. Epigenetic modifications, such as DNA methylation, might mirror alterations in biological processes that are influenced by NS work. Methods: The present study was conducted on 94 healthy female workers with different working schedules and aimed at identifying whether NS was associated with plasmatic concentrations of the inflammatory proteins NLRP3 and TNF-alpha, as well as with DNA methylation levels of ten human endogenous retroviral (HERV) sequences, and nine genes selected for their role in immune and inflammatory processes. We also explored the possible role of the body mass index (BMI) as an additional susceptibility factor that might influence the effects of NS work on the tested epigenetic modifications. Results and discussion: We observed a positive association between NS and NLRP3 levels (p-value 0.0379). Moreover, NS workers retained different methylation levels for ERVFRD-1 (p-value = 0.0274), HERV-L (p-value = 0.0377), and HERV-P (p-value = 0.0140) elements, and for BIRC2 (p-value = 0.0460), FLRT3 (p-value = 0.0422), MIG6 (p-value = 0.0085), and SIRT1 (p-value = 0.0497) genes. We also observed that the BMI modified the relationship between NS and the methylation of ERVE, HERV-L, and ERVW-1 elements. Overall, our results suggest that HERV methylation could pose as a promising biomolecular sensor to monitor not only the effect of NS work but also the cumulative effect of multiple stressors.

Human Endogenous Retroviruses Long Terminal Repeat Methylation, Transcription, and Protein Expression in Human Colon Cancer.

Colon cancer is the fourth most common malignancy in both incidence and mortality in developed countries. Infectious agents are among the risk factors for colon cancer. Variations in human endogenous retrovirus (HERV) transcript and protein levels are associated with several types of cancers, but few studies address HERV expression in colon cancer. Fifty-eight patients with advanced-stage colon cancer were enrolled in this study. HERV-H, -K (HML-2), -P LTRs, Alu, and LINE-1 methylation levels and transcription of HERV-H, -K (HML-2), and -P env and HERV-K pol genes in normal adjacent and tumor tissues were investigated by pyrosequencing and RT-qPCR, respectively. Expression of the HERV-K (HML-2) Pol and Env proteins in selected tissues was examined by Western blotting. Associations between HERV transcript expression and methylation levels and between clinical characteristics and HERV expression were evaluated. Compared to adjacent normal tissues, LINE-1 was hypomethylated in tumor tissues (p < 0.05), whereas Alu, HERV-K (HML-2), and -H LTRs showed a decreasing trend in tumor tissue compared to normal tissue, though without a significant difference. The transcription levels of HERV env and pol genes were similar. However, the HERV-K (HML-2) Pol protein was more highly expressed (p < 0.01) in surrounding normal tissues, but the HERV-K (HML-2) Env protein was only expressed in tumor tissues. Although HERV LTR methylation and gene expression did not show significant differences between tumor and normal tissues, HERV protein expression differed greatly. Pol protein expression in normal cells may induce reverse transcription and subsequent integration into the host genome, likely favoring cell transformation; in contrast, the Env protein in tumor tissue may contribute to cancer progression through cell-to-cell fusion.

DNA methylation level in blood and relations to breast cancer, risk factors and environmental exposure in Greenlandic Inuit women.

Several studies have found aberrant DNA methylation levels in breast cancer cases, but factors influencing DNA methylation patterns and the mechanisms are not well understood. This case-control study evaluated blood methylation level of two repetitive elements and selected breast cancer-related genes in relation to breast cancer risk, and the associations with serum level of persistent organic pollutants (POPs) and breast cancer risk factors in Greenlandic Inuit. DNA methylation was determined using bisulphite pyrosequencing in blood from 74 breast cancer cases and 80 controls. Using first tertile as reference, the following was observed. Positive associations for ATM in second tertile (OR: 2.33, 95% CI: 1.04; 5.23) and ESR2 in third tertile (OR: 2.22, 95% CI: 0.97; 5.05) suggest an increased breast cancer risk with high DNA methylation. LINE-1 methylation was lower in cases than controls. In third tertile (OR: 0.42, 95% CI: 0.18; 0.98), associations suggest in accordance with the literature an increased risk of breast cancer with LINE-1 hypomethylation. Among controls, significant associations between methylation levels and serum level of POPs and breast cancer risk factors (age, body mass index, cotinine level) were found. Thus, breast cancer risk factors and POPs may alter the risk through changes in methylation levels; further studies are needed to elucidate the mechanisms.

Human endogenous retroviruses env gene expression and long terminal repeat methylation in colorectal cancer patients.

Human endogenous retroviruses (HERV) are remnants of exogenous retroviral infections, representing 8% of the human genome. Their regulation is based on the DNA methylation of promoters, the long terminal repeats (LTRs). Transcripts from HERV have been associated with cancers, but reports concerning HERV expression in colorectal cancer remain sporadic. Sixty-three patients with advanced stages of colorectal cancer were enrolled in this study. The expressions of HERV env gene, and HERV-H, -K, -R and -P LTRs and Alu, LINE-1 methylation levels, were investigated in the tumor, normal adjacent tissues, and, where possible, blood and plasmatic extracellular vesicles (EVs). Associations among HERV env expression, methylation status and clinical characteristics were evaluated. No differences were observed in HERV env gene expression levels among the clinical specimens, while Alu, LINE-1, HERV-H and -K LTRs were demethylated in the tumor compared to the normal adjacent tissues (p < 0.05).The HERV env gene was expressed in the EVs at of 54% (-H), 38% (-K), 31% (-R) patients. Association was not found between HERV env expression and LTR methylation, but significant higher expression of HERV-P and -R env was found in tumor tissues arising from the right colon. Our findings do not demonstrate significant overexpression of the studied HERV in colorectal cancer, but their association with tumor localization and specificity of the changes in DNA methylation of retroelements are shown. HERV sequences were packaged in the EVs and might be transferred from one cell to another.

Extracellular Vesicles Released by Colorectal Cancer Cell Lines Modulate Innate Immune Response in Zebrafish Model: The Possible Role of Human Endogenous Retroviruses.

Extracellular vesicles (EVs) are important components of the metastatic niche and are crucial in infiltration, metastasis, and immune tolerance processes during tumorigenesis. We hypothesized that human endogenous retroviruses (HERV) positive EVs derived from tumor cellsmay have a role in modulating the innate immune response. The study was conducted in two different colorectal cancer cell lines, representing different stages of cancer development: Caco-2, derived from a non-metastatic colorectal adenocarcinoma, and SK-CO-1, derived from metastatic colorectal adenocarcinoma (ascites). Both cell lines were treated with decitabine to induce global hypomethylation and to reactivate HERV expression. EVs were quantified by nanoparticle tracking analysis, and HERV-positive EV concentrations were measured by flow cytometry. The effect of EVs isolated from both untreated and decitabine-treated cells on the innate immune response was evaluated by injecting them in zebrafish embryos and then assessing Interleukin 1ß (IL1-ß), Interleukin 10 (IL-10), and the myeloperoxidase (mpx) expression levels by real-time qPCR. Interestingly, HERV-K positive EVs concentrations were significantly associated with a reduced expression of IL1-ß and mpx, supporting our hypothesis that HERV-positive EVs may act as immunomodulators in tumor progression. The obtained results open new perspectives about the modulation of the immune response in cancer therapy.

Effects of Physical Exercise on Endothelial Function and DNA Methylation.

Essential hypertension is the leading preventable cause of death in the world. Epidemiological studies have shown that physical training can reduce blood pressure (BP), both in hypertensive and healthy individuals. Increasing evidence is emerging that DNA methylation is involved in alteration of the phenotype and of vascular function in response to environmental stimuli. We evaluated repetitive element and gene-specific DNA methylation in peripheral blood leukocytes of 68 volunteers, taken before (T0) and after (T1) a three-month intervention protocol of continuative aerobic physical exercise. DNA methylation was assessed by bisulfite-PCR and pyrosequencing. Comparing T0 and T1 measurements, we found an increase in oxygen consumption at peak of exercise (VO2peak) and a decrease in diastolic BP at rest. Exercise increased the levels of ALU and Long Interspersed Nuclear Element 1 (LINE-1) repetitive elements methylation, and of Endothelin-1 (EDN1), Inducible Nitric Oxide Synthase (NOS2), and Tumour Necrosis Factor Alpha (TNF) gene-specific methylation. VO2peak was positively associated with methylation of ALU, EDN1, NOS2, and TNF; systolic BP at rest was inversely associated with LINE-1, EDN1, and NOS2 methylation; diastolic BP was inversely associated with EDN1 and NOS2 methylation. Our findings suggest a possible role of DNA methylation for lowering systemic BP induced by the continuative aerobic physical training program.

Night Shift Work, DNA Methylation and Telomere Length: An Investigation on Hospital Female Nurses.

Increased breast cancer risk has been reported in some night shift (NS) workers but underlying biological mechanisms are still unclear. We assessed the association between NS work and DNA methylation of tumor suppressor (TP53, CDKN2A, BRCA1, BRCA2) and estrogen receptor (ESR1, ESR2) genes, methylation of repetitive elements (LINE-1, Alu), and telomere length (TL). Forty six female nurses employed in NS for at least two years were matched by age (30-45 years) and length of service (≥1 year) with 51 female colleagues not working in NS. Each subject underwent a semi-structured interview and gave a blood sample. We applied linear regression and spline models adjusted for age, BMI, smoking habit, oral contraceptive use, parity and marital status/age at marriage. Currently working in NS was associated with ESR1 hypomethylation (ß: -1.85 (95%CI: -3.03; -0.67), p = 0.003). In current and former NS workers we observed TP53 (-0.93 (-1.73; -0.12), p = 0.03) and BRCA1 (-1.14 (-1.71; -0.58), p <0.001) hypomethylation. We found an increase between TL and number of years in NS in subjects employed in NS <12 years (0.06 (0.03; 0.09), p <0.001), while a decrease if employed in NS ≥12 years (-0.07 -0.10; -0.04), p <0.001). Our findings show NS-associated markers potentially involved in cellular aging, genomic instability, and cancer development.

50-Hz MF does not affect global DNA methylation of SH-SY5Y cells treated with the neurotoxin MPP.

Exposure to extremely low frequency magnetic fields (ELF-MFs) has been associated with an increased risk of neurodegenerative disorders. The underlying mechanisms, however, are still debated. Since epigenetics play a key role in the neurodegenerative process, we investigated whether exposure to ELF-MF (50 Hz, 1 mT) might affect global DNA methylation of SH-SY5Y dopaminergic-like neuroblastoma cells. We assessed the percentage of 5-methylcytosine (5-mC) of three repetitive interspersed sequences (ALU, LINE-1, or SATα), through pyrosequencing analysis. We demonstrated that ELF exposure (up to 72 h) does not induce any change in the methylation pattern of ALU, LINE-1, and SATα in both proliferating and differentiated SH-SY5Y cells. Furthermore, when administered in combination with 1-methyl-4-phenylpyridinium (MPP+ ), a neurotoxin mimicking the Parkinson's Disease (PD) phenotype, ELF-MF exposure does not trigger any modulation in the percentage of 5-mC of the repetitive elements. Our findings demonstrate that exposure to 50-Hz MF does not affect global DNA methylation in proliferating and dopaminergic differentiated SH-SY5Y cells, either under basal culture conditions or under neurotoxic stress. Bioelectromagnetics. 40:33-41, 2019. © 2018 Bioelectromagnetics Society.

Evaluation of DNA methylation of inflammatory genes following treatment of chronic periodontitis: A pilot case-control study.

OBJECTIVE: To evaluate the influence of periodontal therapy on DNA methylation in patients with chronic periodontitis as compared to healthy individuals. MATERIAL AND METHODS: Twenty patients were enrolled into two groups: (i) 10 diagnosed as clinically healthy; and (ii) 10 diagnosed with chronic periodontitis. Clinical measures were recorded and gingival biopsies were harvested at baseline (both patient groups) and at 2 and 8 weeks post-baseline for diseased individuals. Molecular DNA methylation analysis was performed by pyrosequencing for the putative inflammation-associated genes LINE-1, COX-2, IFN-γ and TNF-α. Random-intercept linear regression models were applied to evaluate methylation levels across groups at baseline and the methylation changes over time in the diseased and normal tissues. RESULTS: Periodontal therapy did not influence gene expression methylation of TNF-α, IFN-γ and LINE-1 levels at normal and periodontitis sites over time. However, it significantly reduced COX-2 methylation levels comparable to healthy individuals at both 2 and 8 weeks post-treatment (p < .05). CONCLUSIONS: Periodontal therapy resets the DNA methylation status of inflammatory gene for COX-2 in patients with periodontal disease. DNA methylation levels of TNF-α, IFN-γ and LINE-1 were sustained in periodontitis sites despite therapy. Future studies should consider an expanded panel of inflammatory genes over time. (ClinicalTrials.gov NCT02835898).

DNA methylation changes in Mexican children exposed to arsenic from two historic mining areas in San Luis potosí.

Arsenic is a carcinogen and epimutagen that threatens the health of exposed populations worldwide. In this study, we examined the methylation status of Alu and long interspersed nucleotide elements (LINE-1) and their association with levels of urinary arsenic in 84 Mexican children between 6 and 12 years old from two historic mining areas in the State of San Luis Potosí, Mexico. Urinary arsenic levels were determined by atomic absorption spectrophotometry and DNA methylation analysis was performed in peripheral blood leukocytes by bisulfite-pyrosequencing. The geometric mean of urinary arsenic was 26.44 µg/g Cr (range 1.93-139.35). No significant differences in urinary arsenic or methylation patterns due to gender were observed. A positive correlation was found between urinary arsenic and the mean percentage of methylated cytosines in Alu sequences (Spearman correlation coefficient r = 0.532, P < 0.001), and a trend of LINE-1 hypomethylation was also observed (Spearman correlation coefficient r = -0.232, P = 0.038) after adjustment for sex and age. A linear regression model showed an association with log-normalized urinary arsenic for Alu (ß = 1.05, 95% CI: 0.67; 1.43, P < 0.001) and LINE-1 (ß = -0.703, 95% CI: -1.36; -0.38, P = 0.038). Despite the low-level arsenic exposure, a subtle epigenetic imbalance measured as DNA methylation was detected in the leukocytes of Mexican children living in two historic mining areas. Environ. Mol. Mutagen. 57:717-723, 2016. © 2016 Wiley Periodicals, Inc.

Psychological factors and DNA methylation of genes related to immune/inflammatory system markers: the VA Normative Aging Study.

OBJECTIVES: Although psychological factors have been associated with chronic diseases such as coronary heart disease (CHD), the underlying pathways for these associations have yet to be elucidated. DNA methylation has been posited as a mechanism linking psychological factors to CHD risk. In a cohort of community-dwelling elderly men, we explored the associations between positive and negative psychological factors with DNA methylation in promoter regions of multiple genes involved in immune/inflammatory processes related to atherosclerosis. DESIGN: Prospective cohort study. SETTING: Greater Boston, Massachusetts area. PARTICIPANTS: Samples of 538 to 669 men participating in the Normative Aging Study cohort with psychological measures and DNA methylation measures, collected on 1-4 visits between 1999 and 2006 (mean age=72.7 years at first visit). OUTCOME MEASURES: We examined anxiety, depression, hostility and life satisfaction as predictors of leucocyte gene-specific DNA methylation. We estimated repeated measures linear mixed models, controlling for age, smoking, education, history of heart disease, stroke or diabetes, % lymphocytes, % monocytes and plasma folate. RESULTS: Psychological distress measured by anxiety, depression and hostility was positively associated, and happiness and life satisfaction were inversely associated with average Intercellular Adhesion Molecule-1 (ICAM-1) and coagulation factor III (F3) promoter methylation levels. There was some evidence that hostility was positively associated with toll-like receptor 2 (TLR-2) promoter methylation, and that life satisfaction was inversely associated with TLR-2 and inducible nitric oxide synthase (iNOS) promoter methylation. We observed less consistent and significant associations between psychological factors and average methylation for promoters of the genes for glucocorticoid receptor (NR3C1), interferon-γ (IFN-γ) and interleukin 6 (IL-6). CONCLUSIONS: These findings suggest that positive and negative psychological factors affect DNA methylation of selected genes involved in chronic immune/inflammatory processes and inflammation-related endothelial dysfunction. Such epigenetic changes may represent biological pathways that mediate the effects of psychological factors on CHD.

Nutrients intake is associated with DNA methylation of candidate inflammatory genes in a population of obese subjects.

The aim of the present study was to evaluate the potential association between dietary nutrients and alterations in DNA methylation in a set of five candidate genes, including CD14, Et-1, iNOS, HERV-w and TNFα, in a population of overweight/obese subjects. We evaluated possible associations between gene methylation and clinical blood parameters, including total cholesterol (TC), low- and high-density lipoprotein cholesterol (LDL-C and HDL-C), triglyceride and homocysteine levels. We employed validated methods to assess anthropometric, clinical and dietary data, as well as pyrosequencing to evaluate DNA methylation of the five candidate genes in 165 overweight/obese subjects. There was no association between body mass index and DNA methylation of the five candidate genes in this group of subjects. Positive associations were observed between TNFα methylation and blood levels of LDL-C (ß = 0.447, p = 0.002), TC/HDL-C (ß = 0.467, p = 0.001) and LDL-C/HDL-C (ß = 0.445, p = 0.002), as well as between HERV-w methylation and dietary intakes of ß-carotene (ß = 0.088, p = 0.051) and carotenoids (ß = 0.083, p = 0.029). TNFα methylation showed negative associations with dietary intakes of cholesterol (ß = -0.278, p = 0.048), folic acid (ß = -0.339, p = 0.012), ß-carotene (ß = -0.332, p = 0.045), carotenoids (ß = -0.331, p = 0.015) and retinol (ß = -0.360, p = 0.008). These results suggest a complex relationship among nutrient intake, oxidative stress and DNA methylation.

Blood DNA methylation, nevi number, and the risk of melanoma.

Germline mutations determining increased cutaneous malignant melanoma (CMM) risk have been identified in familial and sporadic CMM cases, but they account only for a small proportion of CMM cases. Recent evidence suggests that germline epimutations (e.g. DNA methylation alterations), which can be inherited similarly to genomic mutations and can be detected in normal body cells (including blood), might increase susceptibility to cancer. The aim of the study was to identify germline epimutations of genes that were found to be mutated in familial CMM (p16, p14, CDK4, MC1R, hTERT), immune and inflammatory genes (ICAM-1, TNFα), DNA mismatch repair gene (MLH1), and repetitive elements (ALU, LINE-1, HERV-w). We measured DNA methylation using bisulfite pyrosequencing in peripheral blood mononuclear cells from 167 CMM cases and 164 sex-matched and age-matched controls. We used multivariable logistic regression models to evaluate the association between methylation levels and CMM status or presence of dysplastic nevi. We found an association between the risk of CMM and peripheral blood mononuclear cell methylation levels of TNFα [odds ratio (OR)=1.11, 95% confidence interval (CI)=1.03-1.18], CDK4 (OR=0.76, 95% CI=0.64-0.91), and MLH1 (OR=1.12, 95% CI=1.02-1.22). In control participants, the risk of developing dysplastic nevi was associated with methylation levels of TNFα (OR=0.81, 95% CI=0.69-0.95), hTERT (OR=0.90, 95% CI=0.82-0.99), and ALU (OR=1.56, 95% CI=1.02-2.39). Epimutations in CMM susceptibility genes and in genes involved in response to oxidative damage are associated with the risk of developing CMM or dysplastic nevi. Further studies measuring methylation levels of these genes in prospectively collected samples are warranted to further elucidate their role in the development and progression of CMM.

Air pollution and gene-specific methylation in the Normative Aging Study: association, effect modification, and mediation analysis.

The mechanisms by which air pollution has multiple systemic effects in humans are not fully elucidated, but appear to include inflammation and thrombosis. This study examines whether concentrations of ozone and components of fine particle mass are associated with changes in methylation on tissue factor (F3), interferon gamma (IFN-γ), interleukin 6 (IL-6), toll-like receptor 2 (TLR-2), and intercellular adhesion molecule 1 (ICAM-1). We investigated associations between air pollution exposure and gene-specific methylation in 777 elderly men participating in the Normative Aging Study (1999-2009). We repeatedly measured methylation at multiple CpG sites within each gene's promoter region and calculated the mean of the position-specific measurements. We examined intermediate-term associations between primary and secondary air pollutants and mean methylation and methylation at each position with distributed-lag models. Increase in air pollutants concentrations was significantly associated with F3, ICAM-1, and TLR-2 hypomethylation, and IFN-γ and IL-6 hypermethylation. An interquartile range increase in black carbon concentration averaged over the four weeks prior to assessment was associated with a 12% reduction in F3 methylation (95% CI: -17% to -6%). For some genes, the change in methylation was observed only at specific locations within the promoter region. DNA methylation may reflect biological impact of air pollution. We found some significant mediated effects of black carbon on fibrinogen through a decrease in F3 methylation, and of sulfate and ozone on ICAM-1 protein through a decrease in ICAM-1 methylation.

Quantitative analysis of methylation defects and correlation with clinical characteristics in patients with pseudohypoparathyroidism type I and GNAS epigenetic alterations.

CONTEXT: Pseudohypoparathyroidism type I (PHP-I) includes two main subtypes, PHP-Ia and -Ib. About 70% of PHP-Ia patients, who show Albright hereditary osteodystrophy (AHO) associated with resistance toward multiple hormones (PTH/TSH/GHRH/gonadotropins), carry heterozygous mutations in the α-subunit of the stimulatory G protein (Gsα) exons 1-13, encoded by the guanine nucleotide binding-protein α-stimulating activity polypeptide 1 (GNAS), whereas the majority of PHP-Ib patients, who classically display hormone resistance limited to PTH and TSH with no AHO sign, have methylation defects in the imprinted GNAS cluster. Recently methylation defects have been detected also in patients with PHP and different degrees of AHO, indicating a molecular overlap between the two forms. OBJECTIVES: The objectives of the study were to collect patients with the following characteristics: clinical PHP-I (with or without AHO), no mutation in Gsα coding sequence, but the presence of GNAS methylation alterations and to investigate the existence of correlations between the degree of the epigenetic defect and the severity of the disease. PATIENTS AND METHODS: We quantified GNAS methylation alterations by both PCR-pyrosequencing and methylation specific-multiplex ligation-dependent probe amplification assay in genomic DNA from 63 patients with PHP-I and correlated these findings with clinical parameters (age at diagnosis; calcium, phosphorus, PTH, TSH levels; presence or absence of each AHO sign). RESULTS: By both approaches, the degree of the imprinting defect did not correlate with the onset of the disease, the severity of endocrine resistances, or with the presence/absence of specific AHO signs. CONCLUSIONS: Similar molecular alterations may lead to a broad spectrum of diseases, from isolated PTH resistance to complete PHP-Ia, and the degree of methylation alterations does not reflect or anticipate the severity and the type of different PHP/AHO manifestations.

A panel study of occupational exposure to fine particulate matter and changes in DNA methylation over a single workday and years worked in boilermaker welders.

BACKGROUND: Exposure to pollutants including metals and particulate air pollution can alter DNA methylation. Yet little is known about intra-individual changes in DNA methylation over time in relationship to environmental exposures. Therefore, we evaluated the effects of acute- and chronic metal-rich PM2.5 exposures on DNA methylation. METHODS: Thirty-eight male boilermaker welders participated in a panel study for a total of 54 person days. Whole blood was collected prior to any welding activities (pre-shift) and immediately after the exposure period (post-shift). The percentage of methylated cytosines (%mC) in LINE-1, Alu, and inducible nitric oxide synthase gene (iNOS) were quantified using pyrosequencing. Personal PM2.5 (particulate matter with an aerodynamic diameter ≤ 2.5 µm) was measured over the work-shift. A questionnaire assessed job history and years worked as a boilermaker. Linear mixed models with repeated measures evaluated associations between DNA methylation, PM2.5 concentration (acute exposure), and years worked as a boilermaker (chronic exposure). RESULTS: PM2.5 exposure was associated with increased methylation in the promoter region of the iNOS gene (ß = 0.25, SE: 0.11, p-value = 0.04). Additionally, the number of years worked as a boilermaker was associated with increased iNOS methylation (ß = 0.03, SE: 0.01, p-value = 0.03). No associations were observed for Alu or LINE-1. CONCLUSIONS: Acute and chronic exposure to PM2.5 generated from welding activities was associated with a modest change in DNA methylation of the iNOS gene. Future studies are needed to confirm this association and determine if the observed small increase in iNOS methylation are associated with changes in NO production or any adverse health effect.

Allergen sensitization is associated with increased DNA methylation in older men.

BACKGROUND: Variation in epigenetic modifications, arising from either environmental exposures or internal physiological changes, can influence gene expression and may ultimately contribute to complex diseases such as asthma and allergies. We examined the association of asthma and allergic phenotypes with DNA methylation levels of retrotransposon-derived elements. METHODS: We used data from 704 men (mean age 73 years) in the longitudinal Normative Aging Study to assess the relationship between asthma, allergic phenotypes and DNA methylation levels of the retrotransposon-derived elements Alu and long interspersed nuclear element (LINE)-1. Retrotransposons represent a large fraction of the genome (>30%) and are heavily methylated to prevent expression. Percent methylation of Alu and LINE-1 elements in peripheral white blood cells was quantified using PCR pyrosequencing. Data on sensitization to common allergens from skin prick testing, asthma and methacholine responsiveness were gathered approximately 8 years prior to DNA methylation analysis. RESULTS: Prior allergen sensitization was associated with increased methylation of Alu (ß = 0.32 for sensitized vs. nonsensitized patients; p = 0.003) in models adjusted for pack-years of smoking, body mass index, current smoking, air pollutants, percentage of eosinophils, white blood cell count and age. Of the men interviewed, 5% of subjects reported a diagnosis of asthma. Neither Alu nor LINE-1 methylation was associated with asthma. CONCLUSIONS: These data suggest that increased DNA methylation of repetitive elements may be associated with allergen sensitization but does not appear to be associated with asthma. Future work is needed to identify potential underlying mechanisms for these relationships.

Alu and LINE-1 methylation and lung function in the normative ageing study.

OBJECTIVES: To investigate the association between methylation of transposable elements Alu and long-interspersed nuclear elements (LINE-1) and lung function. DESIGN: Cohort study. SETTING: Outpatient Veterans Administration facilities in greater Boston, Massachusetts, USA. PARTICIPANTS: Individuals from the Veterans Administration Normative Aging Study, a longitudinal study of aging in men, evaluated between 1999 and 2007. The majority (97%) were white. PRIMARY AND SECONDARY OUTCOME MEASURES: Primary predictor was methylation, assessed using PCR-pyrosequencing after bisulphite treatment. Primary outcome was lung function as assessed by spirometry, performed according to American Thoracic Society/European Respiratory Society guidelines at the same visit as the blood draws. RESULTS: In multivariable models adjusted for age, height, body mass index (BMI), pack-years of smoking, current smoking and race, Alu hypomethylation was associated with lower forced expiratory volume in 1 s (FEV(1)) (ß=28 ml per 1% change in Alu methylation, p=0.017) and showed a trend towards association with a lower forced vital capacity (FVC) (ß=27 ml, p=0.06) and lower FEV(1)/FVC (ß=0.3%, p=0.058). In multivariable models adjusted for age, height, BMI, pack-years of smoking, current smoking, per cent lymphocytes, race and baseline lung function, LINE-1 hypomethylation was associated with more rapid decline of FEV(1) (ß=6.9 ml/year per 1% change in LINE-1 methylation, p=0.005) and of FVC (ß=9.6 ml/year, p=0.002). CONCLUSIONS: In multiple regression analysis, Alu hypomethylation was associated with lower lung function, and LINE-1 hypomethylation was associated with more rapid lung function decline in a cohort of older and primarily white men from North America. Future studies should aim to replicate these findings and determine if Alu or LINE-1 hypomethylation may be due to specific and modifiable environmental exposures.

Temporal stability of epigenetic markers: sequence characteristics and predictors of short-term DNA methylation variations.

BACKGROUND: DNA methylation is an epigenetic mechanism that has been increasingly investigated in observational human studies, particularly on blood leukocyte DNA. Characterizing the degree and determinants of DNA methylation stability can provide critical information for the design and conduction of human epigenetic studies. METHODS: We measured DNA methylation in 12 gene-promoter regions (APC, p16, p53, RASSF1A, CDH13, eNOS, ET-1, IFNγ, IL-6, TNFα, iNOS, and hTERT) and 2 of non-long terminal repeat elements, i.e., L1 and Alu in blood samples obtained from 63 healthy individuals at baseline (Day 1) and after three days (Day 4). DNA methylation was measured by bisulfite-PCR-Pyrosequencing. We calculated intraclass correlation coefficients (ICCs) to measure the within-individual stability of DNA methylation between Day 1 and 4, subtracted of pyrosequencing error and adjusted for multiple covariates. RESULTS: Methylation markers showed different temporal behaviors ranging from high (IL-6, ICC = 0.89) to low stability (APC, ICC = 0.08) between Day 1 and 4. Multiple sequence and marker characteristics were associated with the degree of variation. Density of CpG dinucleotides nearby the sequence analyzed (measured as CpG(o/e) or G+C content within ±200 bp) was positively associated with DNA methylation stability. The 3' proximity to repeat elements and range of DNA methylation on Day 1 were also positively associated with methylation stability. An inverted U-shaped correlation was observed between mean DNA methylation on Day 1 and stability. CONCLUSIONS: The degree of short-term DNA methylation stability is marker-dependent and associated with sequence characteristics and methylation levels.