Rationale: Exposure to particulate air pollution has been related to increased hospitalization and death, particularly from cardiovascular disease. Lower blood DNA methylation content is found in processes related to cardiovascular outcomes, such as oxidative stress, aging, and atherosclerosis.
Objectives: We evaluated whether particulate pollution modifies DNA methylation in heavily methylated sequences with high representation throughout the human genome.
Methods: We measured DNA methylation of long interspersed nucleotide element (LINE)-1 and Alu repetitive elements by quantitative polymerase chain reaction–pyrosequencing of 1,097 blood samples from 718 elderly participants in the Boston area Normative Aging Study. We used covariate-adjusted mixed models to account for within-subject correlation in repeated measures. We estimated the effects on DNA methylation of ambient particulate pollutants (black carbon, particulate matter with aerodynamic diameter ≤ 2.5 μm [PM2.5], or sulfate) in multiple time windows (4 h to 7 d) before the examination. We estimated standardized regression coefficients (β) expressing the fraction of a standard deviation change in DNA methylation associated with a standard deviation increase in exposure.
Measurements and Main Results: Repetitive element DNA methylation varied in association with time-related variables, such as day of the week and season. LINE-1 methylation decreased after recent exposure to higher black carbon (β = −0.11; 95% confidence interval [CI], −0.18 to −0.04; P = 0.002) and PM2.5 (β = −0.13; 95% CI, −0.19 to −0.06; P < 0.001 for the 7-d moving average). In two-pollutant models, only black carbon, a tracer of traffic particles, was significantly associated with LINE-1 methylation (β = −0.09; 95% CI, −0.17 to −0.01; P = 0.03). No association was found with Alu methylation (P > 0.12).
Conclusions: We found decreased repeated-element methylation after exposure to traffic particles. Whether decreased methylation mediates exposure-related health effects remains to be determined.
epigenetic processes; air pollution; inhalation exposure; interspersed repetitive sequences
Background: DNA methylation is a potential pathway linking environmental exposures to disease. Exposure to particulate air pollution has been associated with increased cardiovascular morbidity and mortality, and lower blood DNA methylation has been found in processes related to cardiovascular morbidity.
Objective: We hypothesized that prolonged exposure to particulate pollution would be associated with hypomethylation of repetitive DNA elements and that this association would be modified by genes involved in glutathione metabolism and other host characteristics.
Methods: DNA methylation of the long interspersed nucleotide element–1 (LINE-1) and the short interspersed nucleotide element Alu were measured by quantitative polymerase chain reaction pyrosequencing in 1,406 blood samples from 706 elderly participants in the Normative Aging Study. We estimated changes in repetitive element DNA methylation associated with ambient particles (particulate matter ≤ 2.5 µm in aerodynamic diameter), black carbon (BC), and sulfates (SO4), with mixed models. We examined multiple exposure windows (1–6 months) before DNA methylation measurement. We investigated whether this association was modified by genotype and phenotype.
Results: An interquartile range (IQR) increase in BC over a 90-day period was associated with a decrease of 0.31% 5-methylcytosine (5mC) (95% confidence interval, 0.12–0.50%) in Alu. An IQR increase in SO4 over a 90-day period was associated with a decrease of 0.27% 5mC (0.02–0.52%) in LINE-1. The glutathione S-transferase mu-1–null genotype strengthened the association between BC and Alu hypomethylation.
Conclusion: Prolonged exposure to BC and SO4 particles was associated with hypomethylation of two types of repetitive elements.
air pollution; DNA methylation; epigenetics; gene–environment. Environ Health Perspect 119:977–982 (2011). doi:10.1289/ehp.1002773 [Online 8 March 2011]
Recent data suggest a link between blood leukocyte DNA methylation, and cancer risk. However, reports on DNA methylation from a prospective study are unavailable for gastric cancer.
We explored the association between methylation in pre-diagnostic blood leukocyte DNA and gastric cancer risk in a case–control study nested in the prospective Shanghai Women's Health Study cohort. Incident gastric cancer cases (n=192) and matched controls (n=384) were included in the study. Methylation of Alu and long interspersed nucleotide elements (LINE)-1 were evaluated using bisulphite pyrosequencing. Odds ratios (ORs) and 95% confidence intervals (CI) were calculated from logistic regression adjusting for potential confounders.
Alu methylation was inversely associated with gastric cancer risk, mainly among cases diagnosed one or more years after blood collection. After excluding cases diagnosed during the first year of follow-up, the ORs for the third, second, and first quartiles of Alu methylation compared with the highest quartile were 2.43 (1.43–4.13), 1.47(0.85–2.57), and 2.22 (1.28–3.84), respectively. This association appeared to be modified by dietary intake, particularly isoflavone. In contrast, LINE-1 methylation levels were not associated with gastric cancer risk.
Evidence from this prospective study is consistent with the hypothesis that DNA hypomethylation in blood leukocytes may be related to cancer risk, including risk of gastric cancer.
gastric cancer; DNA methylation; leukocyte
Loss of genomic DNA methylation has been found in a variety of common human age-related diseases. Whether DNA methylation decreases over time as individuals age is unresolved. We measured DNA methylation in 1,097 blood DNA samples from 718 elderly subjects between 55–92 years of age (1–3 samples/subjects), who have been repeatedly evaluated over an 8-year time span in the Boston area Normative Aging Study. DNA methylation was measured using quantitative PCR-Pyrosequencing analysis in Alu and LINE-1 repetitive elements, heavily methylated sequences with high representation throughout the human genome. Age at the visit was negatively associated with Alu element methylation (β=−.12 %5mC/year, p=0.0005). A weaker association was observed with LINE-1 elements (β=−.06 %5mC/year, p=0.049). We observed a significant decrease in average Alu methylation over time, with a −0.2 %5mc change (p=0.012) compared to blood samples collected up to 8 years earlier. The longitudinal decline in Alu methylation was linear and highly correlated with time since the first measurement (β=−.089 %5mC/year, p<0.0001). In contrast, average LINE-1 methylation did not vary over time [p=0.51]. Our results demonstrate a progressive loss of DNA methylation in repetitive elements dispersed throughout the genome.
The highly repetitive Alu retroelements are regarded as methylation centres in the genome. Methylation in the gene promoters could be spreading from them. Promoter methylation of MLH1 is frequently detected in cancers, but the underlying mechanism is unclear. The aim of this study is to understand whether the methylation in the Alu elements is associated with promoter methylation in the MLH1 gene. Bisulfite genomic sequencing was used to analyse the CpG sites of the 5′ end (promoter, exon 1 and Alu-containing intron 1) of the MLH1 gene in colorectal cancer cells and tissues, and gastric cancer tissues. Hypomethylation in the Alu elements and hypermethylation in the promoters and the regions between the promoters and the Alu elements were detected in two cancer cell lines and seven cancer tissues. However, demethylation or hypomethylation of the MLH1 promoter and regions between promoter and the Alu elements, and hypermethylation in the Alu elements, were identified in the normal tissues. MLH1 promoter methylation may spread from Alu elements that are located in intron 1 of the MLH1 gene. The trans-acting elements binding to the mutation sites could play a role in the methylation spreading.
We report a method for studying global DNA methylation based on using bisulfite treatment of DNA and simultaneous PCR of multiple DNA repetitive elements, such as Alu elements and long interspersed nucleotide elements (LINE). The PCR product, which represents a pool of approximately 15 000 genomic loci, could be used for direct sequencing, selective restriction digestion or pyrosequencing, in order to quantitate DNA methylation. By restriction digestion or pyrosequencing, the assay was reproducible with a standard deviation of only 2% between assays. Using this method we found that almost two-thirds of the CpG methylation sites in Alu elements are mutated, but of the remaining methylation target sites, 87% were methylated. Due to the heavy methylation of repetitive elements, this assay was especially useful in detecting decreases in DNA methylation, and this assay was validated by examining cell lines treated with the methylation inhibitor 5-aza-2′deoxycytidine (DAC), where we found a 1–16% decrease in Alu element and 18–60% LINE methylation within 3 days of treatment. This method can be used as a surrogate marker of genome-wide methylation changes. In addition, it is less labor intensive and requires less DNA than previous methods of assessing global DNA methylation.
Tobacco smoking and reduced methylation of long interspersed element-1 (LINE-1) are crucial in oral carcinogenesis. 5′UTR of human LINE-1 sequence contains several CpG dinucleotides which are methylated in various proportions (0–100%). Methylation levels of many LINE-1s in cancer were reduced, hypomethylated. The hypomethylation of each LINE-1 locus can promote instability of genome and repress expression of a gene located on that same chromosome. This study investigated if cigarette smoking influences LINE-1 methylation of oral mucosal cells. The methylation of human LINE-1 in clinically normal oral mucosa of current smokers was compared to non-smokers. By using the combined bisulphite restriction analysis, each LINE-1 sequence was categorised into 4 patterns depending on the methylation status and location of the two 18-bp successive CpG from 5′ to 3′ including mCmC, uCuC, mCuC and uCmC. Of these, mC and uC represent methylated and unmethylated CpG, respectively. The DNA bisulphite sequence demonstrated that most CpGs of mCmC and uCuC were methylated and unmethylated, respectively. Nevertheless, some CpGs of each mCuC or uCmC allele were methylated. Imaging of the digestion products was used to generate %methylation value. No significant difference in the overall LINE-1 methylation level but the differences in percentages of some methylation patterns were discovered. The %mCmC and %uCuC increased, while the %mCuC decreased in current smokers (p = 0.002, 0.015, and <0.0001, respectively). Additionally, the lower %mCuC still persisted in persons who had stopped smoking for over 1 year (p = 0.001). The %mCuC also decreased in the higher pack-year smokers (p = 0.028). Smoking possibly altered mCuC to mCmC and uCuC forms, and changes uCmC to uCuC forms. In conclusion, smoking changes methylation levels of partial methylated LINE-1s and increased the number of hypo- and hypermethylated loci. These hypomethylated LINE-1s may possess carcinogenesis potential. Moreover, LINE-1 methylation patterns may be useful for monitoring oral carcinogenesis in smokers.
Background: Fetal programming describes the theory linking environmental conditions during embryonic and fetal development with risk of diseases later in life. Environmental insults in utero may lead to changes in epigenetic mechanisms potentially affecting fetal development.
Objectives: We examined associations between in utero exposures, infant growth, and methylation of repetitive elements and gene-associated DNA in human term placenta tissue samples.
Methods: Placental tissues and associated demographic and clinical data were obtained from subjects delivering at Women and Infants Hospital in Providence, Rhode Island (USA). Methylation levels of long interspersed nuclear element-1 (LINE-1) and the Alu element AluYb8 were determined in 380 placental samples from term deliveries using bisulfite pyrosequencing. Genomewide DNA methylation profiles were obtained in a subset of 184 samples using the Illumina Infinium HumanMethylation27 BeadArray. Multiple linear regression, model-based clustering methods, and gene set enrichment analysis examined the association between birth weight percentile, demographic variables, and repetitive element methylation and gene-associated CpG locus methylation.
Results: LINE-1 and AluYb8 methylation levels were found to be significantly positively associated with birth weight percentile (p = 0.01 and p < 0.0001, respectively) and were found to differ significantly among infants exposed to tobacco smoke and alcohol. Increased placental AluYb8 methylation was positively associated with average methylation among CpG loci found in polycomb group target genes; developmentally related transcription factor binding sites were overrepresented for differentially methylated loci associated with both elements.
Conclusions: Our results suggest that repetitive element methylation markers, most notably AluYb8 methylation, may be susceptible to epigenetic alterations resulting from the intrauterine environment and play a critical role in mediating placenta function, and may ultimately inform on the developmental basis of health and disease.
birth weight; epigenetics; fetal programming; in utero exposures; placenta; retrotransposon
A significant fraction of Alu repeats in human sperm DNA, previously found to be unmethylated, is nearly completely methylated in DNA from many somatic tissues. A similar fraction of unmethylated Alus is observed here in sperm DNA from rhesus monkey. However, Alus are almost completely methylated at the restriction sites tested in monkey follicular oocyte DNA. The Alu methylation patterns in mature male and female monkey germ cells are consistent with Alu methylation in human germ cell tumors. Alu sequences are hypomethylated in seminoma DNAs and more methylated in a human ovarian dysgerminoma. These results contrast with methylation patterns reported for germ cell single-copy, CpG island, satellite, and L1 sequences. The function of Alu repeats is not known, but differential methylation of Alu repeats in the male and female germ lines suggests that they may serve as markers for genomic imprinting or in maintaining differences in male and female meiosis.
Global hypomethylation has been shown to increase genome instability potentially leading to increased cancer risk. We determined whether global methylation in blood leukocyte DNA was associated with gastric cancer in a population-based study on 302 gastric cancer cases and 421 age- and sex-matched controls in Warsaw, Poland, between 1994 and 1996. Using PCR-pyrosequencing, we analyzed methylation levels of Alu and LINE-1, 2 CG-rich repetitive elements, to measure global methylation levels. Gastric cancer risk was highest among those with lowest level of methylation in either Alu (OR = 1.3, 95% CI = 0.9–1.9) or LINE-1 (OR = 1.4, 95% CI = 0.9–2.0) relative to those with the highest levels, although the trends were not statistically significant. For Alu, the association was stronger among those aged 70 or older (OR = 2.6, 95% CI = 1.3–5.5, p for interaction = 0.02). We did not observe meaningful differences in the associations by other risk factors and polymorphisms examined. For LINE-1, the association tended to be stronger among individuals with a family history of cancer (OR = 3.1, 95% CI = 1.4–7.0, p for interaction = 0.01), current alcohol drinkers (OR = 1.9, 95% CI = 1.0–3.6, p for interaction = 0.05), current smokers (OR = 2.3, 95% CI = 1.1–4.6, p for interaction = 0.02), those who rarely or never consumed fruit (OR = 3.1, 95% CI = 1.2–8.1, p for interaction = 0.03), CC carriers for the MTRR Ex5+123C>T polymorphism (OR = 2.3, 95% CI = 1.2–4.4, p for interaction = 0.01) and TT carriers for the MTRR Ex15+572T>C polymorphism (OR = 1.7, 95% CI = 1.0–2.8, p for interaction = 0.06). The association was not different by sex, Helicobacter pylori infection, intake of folate, vitamin B6 and total protein and the remaining polymorphisms examined. Our results indicate that interactions between blood leukocyte DNA hypomethylation and host characteristics may determine gastric cancer risk.
gastric cancer; methylation; global hypomethylation; gastric cancer risk
Loss of genome-wide methylation is a common feature of cancer, and the degree of hypomethylation has been correlated with genomic instability. Global methylation of repetitive elements possibly arose as a defense mechanism against parasitic DNA elements, including retrotransposons and viral pathogens. Given the alterations of global methylation in both viral infection and cancer, we examined genome-wide methylation levels in head and neck squamous cell carcinoma (HNSCC), a cancer causally associated with human papilloma virus (HPV). We assayed global hypomethylation levels in 26 HNSCC samples, compared with their matched normal adjacent tissue, using Pyrosequencing-based methylation assays for LINE repeats. In addition, we examined cell lines derived from a variety of solid tumors for LINE and SINE (Alu) repeats. The degree of LINE and Alu hypomethylation varied among different cancer cell lines. There was only moderate correlation between LINE and Alu methylation levels, with the range of variation in methylation levels being greater for the LINE elements. LINE hypomethylation was more pronounced in HPV-negative than in HPV-positive tumors. Moreover, genomic instability, as measured by genome-wide loss-of-heterozygosity (LOH) single nucleotide polymorphism (SNP) analysis, was greater in HNSCC samples with more pronounced LINE hypomethylation. Global hypomethylation was variable in HNSCC. Its correlation with both HPV status and degree of LOH as a surrogate for genomic instability may reflect alternative oncogenic pathways in HPV-positive versus HPV-negative tumors.
Fetal lead exposure is associated with adverse pregnancy outcomes and developmental and cognitive deficits; however, the mechanism(s) by which lead-induced toxicity occurs remains unknown. Epigenetic fetal programming via DNA methylation may provide a pathway by which environmental lead exposure can influence disease susceptibility.
This study was designed to determine whether prenatal lead exposure is associated with alterations in genomic methylation of leukocyte DNA levels from umbilical cord samples.
We measured genomic DNA methylation, as assessed by Alu and LINE-1 (long interspersed nuclear element-1) methylation via pyrosequencing, on 103 umbilical cord blood samples from the biorepository of the Early Life Exposures in Mexico to Environmental Toxicants (ELEMENT) study group. Prenatal lead exposure had been assessed by measuring maternal bone lead levels at the mid-tibial shaft and the patella using a spot-source 109Cd K-shell X-ray fluorescence instrument.
We found an inverse dose–response relationship in which quartiles of patella lead correlated with cord LINE-1 methylation (p for trend = 0.01) and and tibia lead correlated with Alu methylation (p for trend = 0.05). In mixed effects regression models, maternal tibia lead was negatively associated with umbilical cord genomic DNA methylation of Alu (β= −0.027; p = 0.01). We found no associations between cord blood lead and cord genomic DNA methylation.
Prenatal lead exposure is inversely associated with genomic DNA methylation in cord blood. These data suggest that the epigenome of the developing fetus can be influenced by maternal cumulative lead burden, which may influence long-term epigenetic programming and disease susceptibility throughout the life course.
blood lead; bone lead; DNA methylation; early life; epigenetics; fetal programming; genomic DNA methylation; intergenerational; lead exposure; life course; Mexico
Alu repeats are especially rich in CpG dinucleotides, the principal target sites for DNA methylation in eukaryotes. The methylation state of Alus in different human tissues is investigated by simple, direct genomic blot analysis exploiting recent theoretical and practical advances concerning Alu sequence evolution. Whereas Alus are almost completely methylated in somatic tissues such as spleen, they are hypomethylated in the male germ line and tissues which depend on the differential expression of the paternal genome complement for development. In particular, we have identified a subset enriched in young Alus whose CpGs appear to be almost completely unmethylated in sperm DNA. The existence of this subset potentially explains the conservation of CpG dinucleotides in active Alu source genes. These profound, sequence-specific developmental changes in the methylation state of Alu repeats suggest a function for Alu sequences at the DNA level, such as a role in genomic imprinting.
Alu methylation is correlated with the overall level of DNA methylation and recombination activity of the genome. However, the maintenance and methylation status of each CpG site within Alu elements (Alu) and its methylation status have not well characterized. This information is useful for understanding natural status of Alu in the genome and helpful for developing an optimal assay to quantify Alu hypomethylation.
Bisulfite clone sequencing was carried out in 14 human gastric samples initially. A Cac8I COBRA-DHPLC assay was developed to detect methylated-Alu proportion in cell lines and 48 paired gastric carcinomas and 55 gastritis samples. DHPLC data were statistically interpreted using SPSS version 16.0.
From the results of 427 Alu bisulfite clone sequences, we found that only 27.2% of CpG sites within Alu elements were preserved (4.6 of 17 analyzed CpGs, A ~ Q) and that 86.6% of remaining-CpGs were methylated. Deamination was the main reason for low preservation of methylation targets. A high correlation coefficient of methylation was observed between Alu clones and CpG site J (0.963), A (0.950), H (0.946), D (0.945). Comethylation of the sites H and J were used as an indicator of the proportion of methylated-Alu in a Cac8I COBRA-DHPLC assay. Validation studies showed that hypermethylation or hypomethylation of Alu elements in human cell lines could be detected sensitively by the assay after treatment with 5-aza-dC and M.SssI, respectively. The proportion of methylated-Alu copies in gastric carcinomas (3.01%) was significantly lower than that in the corresponding normal samples (3.19%) and gastritis biopsies (3.23%).
Most Alu CpG sites are deaminated in the genome. 27% of Alu CpG sites represented in our amplification products. 87% of the remaining CpG sites are methylated. Alu hypomethylation in primary gastric carcinomas could be detected with the Cac8I COBRA-DHPLC assay quantitatively.
Background Estimates of global DNA methylation from repetitive DNA elements, such as Alu and LINE-1, have been increasingly used in epidemiological investigations because of their relative low-cost, high-throughput and quantitative results. Nevertheless, determinants of these methylation measures in healthy individuals are still largely unknown. The aim of this study was to examine whether age, gender, smoking habits, alcohol drinking and body mass index (BMI) are associated with Alu or LINE-1 methylation levels in blood leucocyte DNA of healthy individuals.
Methods Individual data from five studies including a total of 1465 healthy subjects were combined. DNA methylation was quantified by PCR-pyrosequencing.
Results Age [β = −0.011% of 5-methyl-cytosine (%5mC)/year, 95% confidence interval (CI) −0.020 to −0.001%5mC/year] and alcohol drinking (β = −0.214, 95% CI −0.415 to −0.013) were inversely associated with Alu methylation. Compared with females, males had lower Alu methylation (β = −0.385, 95% CI −0.665 to −0.104) and higher LINE-1 methylation (β = 0.796, 95% CI 0.261 to 1.330). No associations were found with smoking or BMI. Percent neutrophils and lymphocytes in blood counts exhibited a positive (β = 0.036, 95% CI 0.010 to 0.061) and negative (β = −0.038, 95% CI −0.065 to −0.012) association with LINE-1 methylation, respectively.
Conclusions Global methylation measures in blood DNA vary in relation with certain host and lifestyle characteristics, including age, gender, alcohol drinking and white blood cell counts. These findings need to be considered in designing epidemiological investigations aimed at identifying associations between DNA methylation and health outcomes.
Blood; DNA methylation; epigenetics; meta-analysis; repetitive elements
Shorter telomere length (TL) in peripheral blood lymphocytes (PBLs) is predictive of lung cancer risk. Polycyclic aromatic hydrocarbons (PAHs) are established lung carcinogens that cause chromosome instability. Whether PAH exposure and its molecular effects are linked with shorter TL has never been evaluated. In the present study, we investigated the effect of chronic exposure to PAHs on TL measured in PBLs of Polish male non-current smoking cokeoven workers and matched controls. PAH exposure and molecular effects were characterized using measures of internal dose (urinary 1-pyrenol), effective dose [anti-benzo[a]pyrene diolepoxide (anti-BPDE)–DNA adduct], genetic instability (micronuclei, MN) and DNA methylation [p53 promoter and Alu and long interspersed nuclear element-1 (LINE-1) repetitive elements, as surrogate measures of global methylation] in PBLs. TL was measured by real-time polymerase chain reaction. Cokeoven workers were heavily exposed to PAHs (79% exceeded the urinary 1-pyrenol biological exposure index) and exhibited lower TL (P = 0.038) than controls, as well as higher levels of genetic and chromosomal alterations [i.e. anti-BPDE–DNA adduct and MN (P < 0.0001)] and epigenetic changes [i.e. p53 gene-specific promoter and global methylation (P ≤ 0.001)]. TL decreased with longer duration of work as cokeoven worker (P = 0.039) and in all subjects with higher levels of anti-BPDE–DNA adduct (P = 0.042), p53 hypomethylation (P = 0.005) and MN (P = 0.009). In multivariate analysis, years of work in cokery (P = 0.008) and p53 hypomethylation (P = 0.001) were the principal determinants of shorter TL. Our results indicate that shorter TL is associated with chronic PAH exposure. The interrelations with other genetic and epigenetic mechanisms in our data suggest that shorter TL could be a central event in PAH carcinogenesis.
DNA methylation of CpGs located in two types of repetitive elements—LINE1 (L1) and Alu—is used to assess “global” changes in DNA methylation in studies of human disease and environmental exposure. L1 and Alu contribute close to 30% of all base pairs in the human genome and transposition of repetitive elements is repressed through DNA methylation. Few studies have investigated whether repetitive element DNA methylation is associated with DNA methylation at other genomic regions, or the biological and technical factors that influence potential associations. Here, we assess L1 and Alu DNA methylation by Pyrosequencing of consensus sequences and using subsets of probes included in the Illumina Infinium HumanMethylation27 BeadChip array. We show that evolutionary age and assay method affect the assessment of repetitive element DNA methylation. Additionally, we compare Pyrosequencing results for repetitive elements to average DNA methylation of CpG islands, as assessed by array probes classified into strong, weak and non-islands. We demonstrate that each of these dispersed sequences exhibits different patterns of tissue-specific DNA methylation. Correlation of DNA methylation suggests an association between L1 and weak CpG island DNA methylation in some of the tissues examined. We caution, however, that L1, Alu and CpG island DNA methylation are distinct measures of dispersed DNA methylation and one should not be used in lieu of another. Analysis of DNA methylation data is complex and assays may be influenced by environment and pathology in different or complementary ways.
Alu; CpG island; DNA Methylation; L1; epigenetics; placenta; repetitive elements
Repetitive elements represent a large portion of the human genome and contain much of the CpG methylation found in normal human postnatal somatic tissues. Loss of DNA methylation in these sequences might account for most of the global hypomethylation that characterizes a large percentage of human cancers that have been studied. There is widespread interest in correlating the genomic 5-methylcytosine content with clinical outcome, dietary history, lifestyle, etc. However, a high-throughput, accurate and easily accessible technique that can be applied even to paraffin-embedded tissue DNA is not yet available. Here, we report the development of quantitative MethyLight assays to determine the levels of methylated and unmethylated repeats, namely, Alu and LINE-1 sequences and the centromeric satellite alpha (Satα) and juxtacentromeric satellite 2 (Sat2) DNA sequences. Methylation levels of Alu, Sat2 and LINE-1 repeats were significantly associated with global DNA methylation, as measured by high performance liquid chromatography, and the combined measurements of Alu and Sat2 methylation were highly correlative with global DNA methylation measurements. These MethyLight assays rely only on real-time PCR and provide surrogate markers for global DNA methylation analysis. We also describe a novel design strategy for the development of methylation-independent MethyLight control reactions based on Alu sequences depleted of CpG dinucleotides by evolutionary deamination on one strand. We show that one such Alu-based reaction provides a greatly improved detection of DNA for normalization in MethyLight applications and is less susceptible to normalization errors caused by cancer-associated aneuploidy and copy number changes.
Rationale: Prenatal exposure to tobacco smoke increases the risk for diseases later in the child's life that may be mediated through alterations in DNA methylation.
Objectives: To demonstrate that differences in DNA methylation patterns occur in children exposed to tobacco smoke and that variation in detoxification genes may alter these associations.
Methods: Methylation of DNA repetitive elements, LINE1 and AluYb8, was measured using bisulfite conversion and pyrosequencing in buccal cells of 348 children participating in the Children's Health Study. Gene-specific CpG methylation differences associated with smoke exposure were screened in 272 participants in the Children's Health Study children using an Illumina GoldenGate panel. CpG loci that demonstrated a statistically significant difference in methylation were validated by pyrosequencing. Estimates were standardized across loci using a Z score to enable cross-comparison of results.
Measurements and Main Results: DNA methylation patterns were associated with in utero exposure to maternal smoking. Exposed children had significantly lower methylation of AluYb8 (β, −0.31; P = 0.03). Differences in smoking-related effects on LINE1 methylation were observed in children with the common GSTM1 null genotype. Differential methylation of CpG loci in eight genes was identified through the screen. Two genes, AXL and PTPRO, were validated by pyrosequencing and showed significant increases in methylation of 0.37 (P = 0.005) and 0.34 (P = 0.02) in exposed children. The associations with maternal smoking varied by a common GSTP1 haplotype.
Conclusions: Life-long effects of in utero exposures may be mediated through alterations in DNA methylation. Variants in detoxification genes may modulate the effects of in utero exposure through epigenetic mechanisms.
DNA methylation; epigenetics; prenatal; smoke
Previous studies suggest that air pollution is related to thrombosis, inflammation, and endothelial dysfunction. Mechanisms and sources of susceptibility are still unclear. One possibility is that these associations can be modified by DNA methylation states.
We conducted a cohort study with repeated measurements of fibrinogen, C-reactive protein, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) in 704 elderly men participating in the Veterans Administration Normative Aging Study (2000-2009). We investigated short- and intermediate-term air pollution effects on these blood markers, and epigene-environment interactions by DNA methylation of Alu, LINE-1, tissue factor (F3), Toll-Like Receptor 2 (TLR-2), and ICAM-1.
We found effects of particle number, black carbon, nitrogen dioxide (NO2), and carbon monoxide (CO) on fibrinogen. Ozone was a significant predictor of C-reactive protein and ICAM-1. Particle number, black carbon, NO2, CO, PM2.5, and sulfates were associated with ICAM-1and VCAM-1. An interquartile range increase in 24-hour exposure for NO2; was associated with a 1.7% (95% confidence interval = 0.2% to 3.3%) increase in fibrinogen for ozone a 10.8% (2.2% to 20.0%) increase in C-reactive protein for particle number, a 5.9% (3.6% to 8.3%) increase in ICAM-1; and for PM2.5 a 3.7% (1.7% to 5.8%) increase in VCAM-1. The air pollution effect was stronger among subjects having higher Alu, lower LINE-1, tissue factor, or TLR-2 methylation status.
We observed associations of traffic-related pollutants on fibrinogen, and both traffic and secondary particles on C-reactive protein, ICAM-1, and VCAM-1. There was effect modification by DNA methylation status, indicating that epigenetic states can convey susceptibility to air pollution.
Lower blood DNA methylation has been associated with atherosclerosis and high cardiovascular risk. Mechanisms linking DNA hypomethylation to increased cardiovascular risk are still largely unknown.
In a population of community-dwelling elderly individuals, we evaluated whether DNA methylation in LINE-1 repetitive element, heavily methylated sequences dispersed throughout the human genome, was associated with circulating Vascular Cell Adhesion Molecule-1 (VCAM-1), Inter-Cellular Adhesion Molecule-1 (ICAM-1), and C-reactive protein (CRP).
METHODS AND RESULTS
We measured LINE-1 methylation by bisulfite PCR-Pyrosequencing on 742 blood DNA samples from male participants in the Boston area Normative Aging Study (mean age=74.8 years). Mean serum VCAM-1 increased progressively in association with LINE-1 hypomethylation (from 975.2 to 1063.4 ng/ml in the highest vs. lowest methylation quintiles; p-trend=0.004). The association between VCAM-1 and LINE-1 hypomethylation was significant in individuals without ischemic heart disease or stroke (n=480; p=0.001), but not in those with prevalent disease (n=262; p=0.57). Serum ICAM-1 and CRP were not associated with LINE-1 methylation (p-trend=>0.25). All results were confirmed by multivariable analyses adjusting for age, BMI, smoking, pack-years, and ischemic heart disease/stroke.
LINE-1 element hypomethylation is associated with higher serum VCAM-1. Our data provide new insights into epigenetic events that may accompany the development of cardiovascular disease.
cell adhesion molecules; epidemiology; cardiovascular diseases; risk factors; LINE-1; VCAM-1
Retrotransposons have been extensively studied in plants and animals and have been shown to have an impact on human genome dynamics and evolution. Their ability to move within genomes gives retrotransposons to affect genome instability.
we examined the polymorphic inserted AluYa5, evolutionary young Alu, in the progesterone receptor gene to determine the effects of Alu insertion on molecular environment. We used mono-allelic inserted cell lines which carry both Alu-present and Alu-absent alleles. To determine the epigenetic change and gene expression, we performed restriction enzyme digestion, Pyrosequencing, and Chromatin Immunoprecipitation.
We observed that the polymorphic insertion of evolutionally young Alu causes increasing levels of DNA methylation in the surrounding genomic area and generates inactive histone tail modifications. Consequently the Alu insertion deleteriously inactivates the neighboring gene expression.
The mono-allelic Alu insertion cell line clearly showed that polymorphic inserted repetitive elements cause the inactivation of neighboring gene expression, bringing aberrant epigenetic changes.
Epigenetics; Retrotransposons; Long interspersed elements; Alu
To complement next-generation sequencing technologies, there is a pressing need for efficient pre-sequencing capture methods with reduced costs and DNA requirement. The Alu family of short interspersed nucleotide elements is the most abundant type of transposable elements in the human genome and a recognized source of genome instability. With over one million Alu elements distributed throughout the genome, they are well positioned to facilitate genome-wide sequence amplification and capture of regions likely to harbor genetic variation hotspots of biological relevance.
Here we report on the use of inter-Alu PCR with an enhanced range of amplicons in conjunction with next-generation sequencing to generate an Alu-anchored scan, or 'AluScan', of DNA sequences between Alu transposons, where Alu consensus sequence-based 'H-type' PCR primers that elongate outward from the head of an Alu element are combined with 'T-type' primers elongating from the poly-A containing tail to achieve huge amplicon range. To illustrate the method, glioma DNA was compared with white blood cell control DNA of the same patient by means of AluScan. The over 10 Mb sequences obtained, derived from more than 8,000 genes spread over all the chromosomes, revealed a highly reproducible capture of genomic sequences enriched in genic sequences and cancer candidate gene regions. Requiring only sub-micrograms of sample DNA, the power of AluScan as a discovery tool for genetic variations was demonstrated by the identification of 357 instances of loss of heterozygosity, 341 somatic indels, 274 somatic SNVs, and seven potential somatic SNV hotspots between control and glioma DNA.
AluScan, implemented with just a small number of H-type and T-type inter-Alu PCR primers, provides an effective capture of a diversity of genome-wide sequences for analysis. The method, by enabling an examination of gene-enriched regions containing exons, introns, and intergenic sequences with modest capture and sequencing costs, computation workload and DNA sample requirement is particularly well suited for accelerating the discovery of somatic mutations, as well as analysis of disease-predisposing germline polymorphisms, by making possible the comparative genome-wide scanning of DNA sequences from large human cohorts.
The evolution, mobility and deleterious genetic effects of human Alus are fairly well understood. The complexity of regulated transcriptional expression of Alus is becoming apparent and insight into the mechanism of retrotransposition is emerging. Unresolved questions concern why mobile, highly repetitive short interspersed elements (SINEs) have been tolerated throughout evolution and why and how families of such sequences are periodically replaced. Either certain SINEs are more successful genomic parasites or positive selection drives their relative success and genomic maintenance. A complete understanding of the evolutionary dynamics and significance of SINEs requires determining whether or not they have a function(s). Recent evidence suggests two possibilities, one concerning DNA and the other RNA. Dispersed Alus exhibit remarkable tissue-specific differences in the level of their 5-methylcytosine content. Differences in Alu methylation in the male and female germlines suggest that Alu DNA may be involved in either the unique chromatin organization of sperm or signaling events in the early embryo. Alu RNA is increased by cellular insults and stimulates protein synthesis by inhibiting PKR, the eIF2 kinase that is regulated by double-stranded RNA. PKR serves other roles potentially linking Alu RNA to a variety of vital cell functions. Since Alus have appeared only recently within the primate lineage, this proposal provokes the challenging question of how Alu RNA could have possibly assumed a significant role in cell physiology.
Alu elements are short (~300 bp) interspersed elements that amplify in primate genomes through a process termed retroposition. The expansion of these elements has had a significant impact on the structure and function of primate genomes. Approximately 10 % of the mass of the human genome is comprised of Alu elements, making them the most abundant short interspersed element (SINE) in our genome. The majority of Alu amplification occurred early in primate evolution, and the current rate of Alu retroposition is at least 100 fold slower than the peak of amplification that occurred 30–50 million years ago. Alu elements are therefore a rich source of inter- and intra-species primate genomic variation.
A total of 153 Alu elements from the Ye subfamily were extracted from the draft sequence of the human genome. Analysis of these elements resulted in the discovery of two new Alu subfamilies, Ye4 and Ye6, complementing the previously described Ye5 subfamily. DNA sequence analysis of each of the Alu Ye subfamilies yielded average age estimates of ~14, ~13 and ~9.5 million years old for the Alu Ye4, Ye5 and Ye6 subfamilies, respectively. In addition, 120 Alu Ye4, Ye5 and Ye6 loci were screened using polymerase chain reaction (PCR) assays to determine their phylogenetic origin and levels of human genomic diversity.
The Alu Ye lineage appears to have started amplifying relatively early in primate evolution and continued propagating at a low level as many of its members are found in a variety of hominoid (humans, greater and lesser ape) genomes. Detailed sequence analysis of several Alu pre-integration sites indicated that multiple types of events had occurred, including gene conversions, near-parallel independent insertions of different Alu elements and Alu-mediated genomic deletions. A potential hotspot for Alu insertion in the Fer1L3 gene on chromosome 10 was also identified.