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1.  White blood cell global methylation and IL-6 promoter methylation in association with diet and lifestyle risk factors in a cancer-free population 
Epigenetics  2012;7(6):606-614.
Altered levels of global DNA methylation and gene silencing through methylation of promoter regions can impact cancer risk, but little is known about their environmental determinants. We examined the association between lifestyle factors and levels of global genomic methylation and IL-6 promoter methylation in white blood cell DNA of 165 cancer-free subjects, 18–78 years old, enrolled in the COMIR (Commuting Mode and Inflammatory Response) study, New York, 2009–2010. Besides self-administrated questionnaires on diet and physical activity, we measured weight and height, white blood cell (WBC) counts, plasma levels of high sensitivity C-reactive protein (hs-CRP), and genomic (LINE-1) and gene-specific methylation (IL-6) by pyrosequencing in peripheral blood WBC. Mean levels of LINE-1 and IL-6 promoter methylation were 78.2% and 57.1%, respectively. In multivariate linear regression models adjusting for age, gender, race/ethnicity, body mass index, diet, physical activity, WBC counts and CRP, only dietary folate intake from fortified foods was positively associated with LINE-1 methylation. Levels of IL-6 promoter methylation were not significantly correlated with age, gender, race/ethnicity, body mass index, physical activity or diet, including overall dietary patterns and individual food groups and nutrients. There were no apparent associations between levels of methylation and inflammation markers such as WBC counts and hs-CRP. Overall, among several lifestyle factors examined in association with DNA methylation, only dietary folate intake from fortification was associated with LINE-1 methylation. The long-term consequence of folate fortification on DNA methylation needs to be further evaluated in longitudinal settings.
doi:10.4161/epi.20236
PMCID: PMC3398989  PMID: 22531363
DNA Methylation; cancer; diet; lifestyle factors
2.  Recreational and household physical activity at different time points and DNA global methylation 
Background
DNA methylation patterns are heritable but can change over time and in response to exposures. Lower global DNA methylation, which may result in increased genomic and chromosomal instability, has been associated with increased cancer risk. Physical activity is a modifiable factor that has been inversely related to the risk of cancer. Changes in DNA methylation may be a mechanism by which lifestyle and environment factors influence disease. We investigated the relationship between DNA methylation and physical activity in a sample of women enrolled in The Sister Study, a large U.S. cohort study of women aged 35–74 years with a family history of breast cancer.
Methods
Global DNA methylation was measured using bisulfite converted DNA and pyrosequencing of a LINE-1 repetitive sequence in the peripheral blood of 647 non-Hispanic white women. Physical activity (average hours per week) was retrospectively assessed for three time periods: childhood (ages 5–12), teenage years (ages 13–19) and the previous twelve months.
Findings
Compared with women with physical activity levels below the median for all three time periods, those at or above the median physical activity for one (β= 0.20, 95% CI: −0.10, 0.49), two (β= 0.22, 95% CI: −0.08, 0.52) or all three (β= 0.33, 95% CI: 0.01, 0.66) time periods had increased global methylation.
Interpretation
Maintaining higher levels of physical activity over these three time periods was associated with increased global DNA methylation, consistent with reported associations between exercise and decreased cancer risk.
doi:10.1016/j.ejca.2013.02.013
PMCID: PMC3686968  PMID: 23473616
DNA methylation; LINE-1; physical activity; pyrosequencing; Sister Study
3.  The Prognostic Significance of Whole Blood Global and Specific DNA Methylation Levels in Gastric Adenocarcinoma 
PLoS ONE  2010;5(12):e15585.
Background
Epigenetics, particularly DNA methylation, has recently been elucidated as important in gastric cancer (GC) initiation and progression. We investigated the clinical and prognostic importance of whole blood global and site-specific DNA methylation in GC.
Methods
Genomic DNA was extracted from the peripheral blood of 105 Omani GC patients at diagnosis. DNA methylation was quantified by pyrosequencing of global DNA and specific gene promoter regions at 5 CpG sites for CDH1, 7 CpG sites for p16, 4 CpG sites for p53, and 3 CpG sites for RUNX3. DNA methylation levels in patients were categorized into low, medium, and high tertiles. Associations between methylation level category and clinicopathological features were evaluated using χ2 tests. Survival analyses were carried out using the Kaplan-Meier method and log rank test. A backward conditional Cox proportional hazards regression model was used to identify independent predictors of survival.
Results
Older GC patients had increased methylation levels at specific CpG sites within the CDH1, p53, and RUNX-3 promoters. Male gender was significantly associated with reduced global and increased site-specific DNA methylation levels in CDH1, p16, and p53 promoters. Global DNA low methylation level was associated with better survival on univariate analysis. Patients with high and medium methylation vs. low methylation levels across p16 promoter CpG sites, site 2 in particular, had better survival. Multivariate analysis showed that global DNA hypermethylation was a significant independent predictor of worse survival (hazard ratio (HR) = 2.0, 95% CI: 1.1–3.8; p = 0.02) and high methylation mean values across p16 promoter sites 1–7 were associated with better survival with HR of 0.3 (95% CI, 0.1–0.8; p = 0.02) respectively.
Conclusions
Analysis of global and site-specific DNA methylation in peripheral blood by pyrosequencing provides quantitative DNA methylation values that may serve as important prognostic indicators.
doi:10.1371/journal.pone.0015585
PMCID: PMC3009731  PMID: 21203466
4.  Role of DNA Methylation and Epigenetic Silencing of HAND2 in Endometrial Cancer Development 
PLoS Medicine  2013;10(11):e1001551.
TB filled in by Laureen
Please see later in the article for the Editors' Summary
Background
Endometrial cancer incidence is continuing to rise in the wake of the current ageing and obesity epidemics. Much of the risk for endometrial cancer development is influenced by the environment and lifestyle. Accumulating evidence suggests that the epigenome serves as the interface between the genome and the environment and that hypermethylation of stem cell polycomb group target genes is an epigenetic hallmark of cancer. The objective of this study was to determine the functional role of epigenetic factors in endometrial cancer development.
Methods and Findings
Epigenome-wide methylation analysis of >27,000 CpG sites in endometrial cancer tissue samples (n = 64) and control samples (n = 23) revealed that HAND2 (a gene encoding a transcription factor expressed in the endometrial stroma) is one of the most commonly hypermethylated and silenced genes in endometrial cancer. A novel integrative epigenome-transcriptome-interactome analysis further revealed that HAND2 is the hub of the most highly ranked differential methylation hotspot in endometrial cancer. These findings were validated using candidate gene methylation analysis in multiple clinical sample sets of tissue samples from a total of 272 additional women. Increased HAND2 methylation was a feature of premalignant endometrial lesions and was seen to parallel a decrease in RNA and protein levels. Furthermore, women with high endometrial HAND2 methylation in their premalignant lesions were less likely to respond to progesterone treatment. HAND2 methylation analysis of endometrial secretions collected using high vaginal swabs taken from women with postmenopausal bleeding specifically identified those patients with early stage endometrial cancer with both high sensitivity and high specificity (receiver operating characteristics area under the curve = 0.91 for stage 1A and 0.97 for higher than stage 1A). Finally, mice harbouring a Hand2 knock-out specifically in their endometrium were shown to develop precancerous endometrial lesions with increasing age, and these lesions also demonstrated a lack of PTEN expression.
Conclusions
HAND2 methylation is a common and crucial molecular alteration in endometrial cancer that could potentially be employed as a biomarker for early detection of endometrial cancer and as a predictor of treatment response. The true clinical utility of HAND2 DNA methylation, however, requires further validation in prospective studies.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Cancer, which is responsible for 13% of global deaths, can develop anywhere in the body, but all cancers are characterized by uncontrolled cell growth and reduced cellular differentiation (the process by which unspecialized cells such as “stem” cells become specialized during development, tissue repair, and normal cell turnover). Genetic alterations—changes in the sequence of nucleotides (DNA's building blocks) in specific genes—are required for this cellular transformation and subsequent cancer development (carcinogenesis). However, recent evidence suggests that epigenetic modifications—reversible, heritable changes in gene function that occur in the absence of nucleotide sequence changes—may also be involved in carcinogenesis. For example, the addition of methyl groups to a set of genes called stem cell polycomb group target genes (PCGTs; polycomb genes control the expression of their target genes by modifying their DNA or associated proteins) is one of the earliest molecular changes in human cancer development, and increasing evidence suggests that hypermethylation of PCGTs is an epigenetic hallmark of cancer.
Why Was This Study Done?
The methylation of PCGTs, which is triggered by age and by environmental factors that are associated with cancer development, reduces cellular differentiation and leads to the accumulation of undifferentiated cells that are susceptible to cancer development. It is unclear, however, whether epigenetic modifications have a causal role in carcinogenesis. Here, the researchers investigate the involvement of epigenetic factors in the development of endometrial (womb) cancer. The risk of endometrial cancer (which affects nearly 50,000 women annually in the United States) is largely determined by environmental and lifestyle factors. Specifically, the risk of this cancer is increased in women in whom estrogen (a hormone that drives cell proliferation in the endometrium) is functionally dominant over progesterone (a hormone that inhibits endometrial proliferation and causes cell differentiation); obese women and women who have taken estrogen-only hormone replacement therapies fall into this category. Thus, endometrial cancer is an ideal model in which to study whether epigenetic mechanisms underlie carcinogenesis.
What Did the Researchers Do and Find?
The researchers collected data on genome-wide DNA methylation at cytosine- and guanine-rich sites in endometrial cancers and normal endometrium and integrated this information with the human interactome and transcriptome (all the physical interactions between proteins and all the genes expressed, respectively, in a cell) using an algorithm called Functional Epigenetic Modules (FEM). This analysis identified HAND2 as the hub of the most highly ranked differential methylation hotspot in endometrial cancer. HAND2 is a progesterone-regulated stem cell PCGT. It encodes a transcription factor that is expressed in the endometrial stroma (the connective tissue that lies below the epithelial cells in which most endometrial cancers develop) and that suppresses the production of the growth factors that mediate the growth-inducing effects of estrogen on the endometrial epithelium. The researchers hypothesized, therefore, that epigenetic deregulation of HAND2 could be a key step in endometrial cancer development. In support of this hypothesis, the researchers report that HAND2 methylation was increased in premalignant endometrial lesions (cancer-prone, abnormal-looking tissue) compared to normal endometrium, and was associated with suppression of HAND2 expression. Moreover, a high level of endometrial HAND2 methylation in premalignant lesions predicted a poor response to progesterone treatment (which stops the growth of some endometrial cancers), and analysis of HAND2 methylation in endometrial secretions collected from women with postmenopausal bleeding (a symptom of endometrial cancer) accurately identified individuals with early stage endometrial cancer. Finally, mice in which the Hand2 gene was specifically deleted in the endometrium developed precancerous endometrial lesions with age.
What Do These Findings Mean?
These and other findings identify HAND2 methylation as a common, key molecular alteration in endometrial cancer. These findings need to be confirmed in more women, and studies are needed to determine the immediate molecular and cellular consequences of HAND2 silencing in endometrial stromal cells. Nevertheless, these results suggest that HAND2 methylation could potentially be used as a biomarker for the early detection of endometrial cancer and for predicting treatment response. More generally, these findings support the idea that methylation of HAND2 (and, by extension, the methylation of other PCGTs) is not a passive epigenetic feature of cancer but is functionally involved in cancer development, and provide a framework for identifying other genes that are epigenetically regulated and functionally important in carcinogenesis.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001551
The US National Cancer Institute provides information on all aspects of cancer and has detailed information about endometrial cancer for patients and professionals (in English and Spanish)
The not-for-profit organization American Cancer Society provides information on cancer and how it develops and specific information on endometrial cancer (in several languages)
The UK National Health Service Choices website includes an introduction to cancer, a page on endometrial cancer, and a personal story about endometrial cancer
The not-for-profit organization Cancer Research UK provides general information about cancer and specific information about endometrial cancer
Wikipedia has a page on cancer epigenetics (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
The Eve Appeal charity that supported this research provides useful information on gynecological cancers
doi:10.1371/journal.pmed.1001551
PMCID: PMC3825654  PMID: 24265601
5.  Age-Related Changes in the Global DNA Methylation Profile of Leukocytes Are Linked to Nutrition but Are Not Associated with the MTHFR C677T Genotype or to Functional Capacities 
PLoS ONE  2012;7(12):e52570.
Global DNA methylation of peripheral blood leukocytes has been recently proposed as a potential biomarker for disease risk. However, the amplitude of the changes in DNA methylation associated with normal aging and the impacts of environmental changes on this variation are still unclear. In this context, we evaluated the association of global DNA methylation with nutritional habits, tobacco smoking, body mass index (BMI), clinical laboratory parameters, polymorphism C677T MTHFR, functional cognition and the daily practice of physical activity in a cancer-free older population. Leukocyte global DNA methylation from 126 older individuals was quantified using a high-throughput ELISA-based method. Global DNA hypomethylation was observed in older individuals when compared to a younger population (p = 0.0469), confirming changes in DNA methylation in the aging process. Furthermore, the methylation profile of elders was correlated with the daily ingestion of carbohydrates (p = 0.0494), lipids (p = 0.0494), vitamin B6 (p = 0.0421), magnesium (p = 0.0302), and also to the serum levels of total protein (p = 0.0004), alpha 2 globulin (p = 0.0013) and albumin (p = 0.0015). No statistically significant difference was observed when global DNA methylation were stratified according to C677T MTHFR genotypes (p = 0.7200), BMI (p = 0.1170), smoking habit (p = 0.4382), physical activity in daily life (p = 0.8492), scored cognitive function (p = 0.7229) or depression state (p = 0.8301). Our data indicate that age-related variations in the global DNA methylation profile of leukocytes might be modulated by the daily intake of carbohydrates, lipids, vitamin B6, and magnesium and be associated with serum protein levels, however it is independent of C677T MTHFR genotype and not correlated with BMI, smoking habit, cognitive function or the routine physical activities.
doi:10.1371/journal.pone.0052570
PMCID: PMC3527598  PMID: 23285094
6.  Global DNA Hypomethylation (LINE-1) in the Normal Colon and Lifestyle Characteristics, Dietary and Genetic Factors 
Background
Global loss of methylated cytosines in DNA, thought to predispose to chromosomal instability and aneuploidy, has been associated with an increased risk of colorectal neoplasia. Little is known about the relationships between global hypomethylation and lifestyle, demographics, dietary measures and genetic factors.
Methods
Our data were collected as part of a randomized clinical trial testing the efficacy of aspirin and folic acid for the prevention of colorectal adenomas. At a surveillance colonoscopy approximately three years after the qualifying exam, we obtained two biopsies of the normal-appearing mucosa from the right colon and two from the left colon. Specimens were assayed for global hypomethylation using a pyrosequencing assay for LINE-1 (long interspersed nucleotide elements) repeats.
Results
The analysis included data from 388 subjects. There was relatively little variability in LINE methylation overall. Mean LINE-1 methylation levels in normal mucosa from the right bowel were significantly lower than those on the left side (p<0.0001). No significant associations were found between LINE-1 methylation and folate treatment, age, sex, body-mass-index, smoking status, alcohol use, dietary intake or circulating levels of B-vitamins, homocysteine, or selected genotypes. Race, dietary folic acid and plasma B6 showed associations with global methylation that differed between the right and left bowel. The effect of folic acid on risk of adenomas did not differ according to extent of LINE-1 methylation and we found no association between LINE-1 methylation and risk of adenomas.
Conclusions
LINE-1 methylation is not influenced by folic acid supplementation, but differs by colon subsite.
doi:10.1158/1055-9965.EPI-08-0926
PMCID: PMC2712652  PMID: 19336559
Global LINE-1 methylation; Folate; Folic Acid; Clinical Trial; Colon; Lifestyle; Dietary; Polymorphisms; Adenomas
7.  DNA Methylation as a Biomarker for Cardiovascular Disease Risk 
PLoS ONE  2010;5(3):e9692.
Background
Elevated serum homocysteine is associated with an increased risk of cardiovascular disease (CVD). This may reflect a reduced systemic remethylation capacity, which would be expected to cause decreased genomic DNA methylation in peripheral blood leukocytes (PBL).
Methodology/Principal Findings
We examined the association between prevalence of CVD (myocardial infarction, stroke) and its predisposing conditions (hypertension, diabetes) and PBL global genomic DNA methylation as represented by ALU and Satellite 2 (AS) repetitive element DNA methylation in 286 participants of the Singapore Chinese Health Study, a population-based prospective investigation of 63,257 men and women aged 45–74 years recruited during 1993–1998. Men exhibited significantly higher global DNA methylation [geometric mean (95% confidence interval (CI)): 159 (143, 178)] than women [133 (121, 147)] (P = 0·01). Global DNA methylation was significantly elevated in men with a history of CVD or its predisposing conditions at baseline (P = 0·03) but not in women (P = 0·53). Fifty-two subjects (22 men, 30 women) who were negative for these CVD/predisposing conditions at baseline acquired one or more of these conditions by the time of their follow-up I interviews, which took place on average about 5·8 years post-enrollment. Global DNA methylation levels of the 22 incident cases in men were intermediate (AS, 177) relative to the 56 male subjects who remained free of CVD/predisposing conditions at follow-up (lowest AS, 132) and the 51 male subjects with a diagnosis of CVD or predisposing conditions reported at baseline (highest AS 184) (P for trend = 0.0008) No such association was observed in women (P = 0.91). Baseline body mass index was positively associated with AS in both men and women (P = 0·007).
Conclusions/Significance
Our findings indicate that elevated, not decreased, PBL DNA methylation is positively associated with prevalence of CVD/predisposing conditions and obesity in Singapore Chinese.
doi:10.1371/journal.pone.0009692
PMCID: PMC2837739  PMID: 20300621
8.  Predictors of global methylation levels in blood DNA of healthy subjects: a combined analysis 
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.
doi:10.1093/ije/dyq154
PMCID: PMC3304518  PMID: 20846947
Blood; DNA methylation; epigenetics; meta-analysis; repetitive elements
9.  A Genome-Wide Screen for Promoter Methylation in Lung Cancer Identifies Novel Methylation Markers for Multiple Malignancies  
PLoS Medicine  2006;3(12):e486.
Background
Promoter hypermethylation coupled with loss of heterozygosity at the same locus results in loss of gene function in many tumor cells. The “rules” governing which genes are methylated during the pathogenesis of individual cancers, how specific methylation profiles are initially established, or what determines tumor type-specific methylation are unknown. However, DNA methylation markers that are highly specific and sensitive for common tumors would be useful for the early detection of cancer, and those required for the malignant phenotype would identify pathways important as therapeutic targets.
Methods and Findings
In an effort to identify new cancer-specific methylation markers, we employed a high-throughput global expression profiling approach in lung cancer cells. We identified 132 genes that have 5′ CpG islands, are induced from undetectable levels by 5-aza-2′-deoxycytidine in multiple non-small cell lung cancer cell lines, and are expressed in immortalized human bronchial epithelial cells. As expected, these genes were also expressed in normal lung, but often not in companion primary lung cancers. Methylation analysis of a subset (45/132) of these promoter regions in primary lung cancer (n = 20) and adjacent nonmalignant tissue (n = 20) showed that 31 genes had acquired methylation in the tumors, but did not show methylation in normal lung or peripheral blood cells. We studied the eight most frequently and specifically methylated genes from our lung cancer dataset in breast cancer (n = 37), colon cancer (n = 24), and prostate cancer (n = 24) along with counterpart nonmalignant tissues. We found that seven loci were frequently methylated in both breast and lung cancers, with four showing extensive methylation in all four epithelial tumors.
Conclusions
By using a systematic biological screen we identified multiple genes that are methylated with high penetrance in primary lung, breast, colon, and prostate cancers. The cross-tumor methylation pattern we observed for these novel markers suggests that we have identified a partial promoter hypermethylation signature for these common malignancies. These data suggest that while tumors in different tissues vary substantially with respect to gene expression, there may be commonalities in their promoter methylation profiles that represent targets for early detection screening or therapeutic intervention.
John Minna and colleagues report that a group of genes are commonly methylated in primary lung, breast, colon, and prostate cancer.
Editors' Summary
Background.
Tumors or cancers contain cells that have lost many of the control mechanisms that normally regulate their behavior. Unlike normal cells, which only divide to repair damaged tissues, cancer cells divide uncontrollably. They also gain the ability to move round the body and start metastases in secondary locations. These changes in behavior result from alterations in their genetic material. For example, mutations (permanent changes in the sequence of nucleotides in the cell's DNA) in genes known as oncogenes stimulate cells to divide constantly. Mutations in another group of genes—tumor suppressor genes—disable their ability to restrain cell growth. Key tumor suppressor genes are often completely lost in cancer cells. But not all the genetic changes in cancer cells are mutations. Some are “epigenetic” changes—chemical modifications of genes that affect the amount of protein made from them. In cancer cells, methyl groups are often added to CG-rich regions—this is called hypermethylation. These “CpG islands” lie near gene promoters—sequences that control the transcription of DNA into RNA, the template for protein production—and their methylation switches off the promoter. Methylation of the promoter of one copy of a tumor suppressor gene, which often coincides with the loss of the other copy of the gene, is thought to be involved in cancer development.
Why Was This Study Done?
The rules that govern which genes are hypermethylated during the development of different cancer types are not known, but it would be useful to identify any DNA methylation events that occur regularly in common cancers for two reasons. First, specific DNA methylation markers might be useful for the early detection of cancer. Second, identifying these epigenetic changes might reveal cellular pathways that are changed during cancer development and so identify new therapeutic targets. In this study, the researchers have used a systematic biological screen to identify genes that are methylated in many lung, breast, colon, and prostate cancers—all cancers that form in “epithelial” tissues.
What Did the Researchers Do and Find?
The researchers used microarray expression profiling to examine gene expression patterns in several lung cancer and normal lung cell lines. In this technique, labeled RNA molecules isolated from cells are applied to a “chip” carrying an array of gene fragments. Here, they stick to the fragment that represents the gene from which they were made, which allows the genes that the cells express to be catalogued. By comparing the expression profiles of lung cancer cells and normal lung cells before and after treatment with a chemical that inhibits DNA methylation, the researchers identified genes that were methylated in the cancer cells—that is, genes that were expressed in normal cells but not in cancer cells unless methylation was inhibited. 132 of these genes contained CpG islands. The researchers examined the promoters of 45 of these genes in lung cancer cells taken straight from patients and found that 31 of the promoters were methylated in tumor tissues but not in adjacent normal tissues. Finally, the researchers looked at promoter methylation of the eight genes most frequently and specifically methylated in the lung cancer samples in breast, colon, and prostate cancers. Seven of the genes were frequently methylated in both lung and breast cancers; four were extensively methylated in all the tumor types.
What Do These Findings Mean?
These results identify several new genes that are often methylated in four types of epithelial tumor. The observation that these genes are methylated in multiple independent tumors strongly suggests, but does not prove, that loss of expression of the proteins that they encode helps to convert normal cells into cancer cells. The frequency and diverse patterning of promoter methylation in different tumor types also indicates that methylation is not a random event, although what controls the patterns of methylation is not yet known. The identification of these genes is a step toward building a promoter hypermethylation profile for the early detection of human cancer. Furthermore, although tumors in different tissues vary greatly with respect to gene expression patterns, the similarities seen in this study in promoter methylation profiles might help to identify new therapeutic targets common to several cancer types.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030486.
US National Cancer Institute, information for patients on understanding cancer
CancerQuest, information provided by Emory University about how cancer develops
Cancer Research UK, information for patients on cancer biology
Wikipedia pages on epigenetics (note that Wikipedia is a free online encyclopedia that anyone can edit)
The Epigenome Network of Excellence, background information and latest news about epigenetics
doi:10.1371/journal.pmed.0030486
PMCID: PMC1716188  PMID: 17194187
10.  Genomic DNA methylation among Women in a Multi-ethnic New York City Birth Cohort 
One plausible mechanism for the environment to alter cancer susceptibility is through DNA methylation. Alterations in DNA methylation can lead to genomic instability and altered gene transcription. Genomic DNA methylation levels have been inversely associated with age suggesting that factors throughout life may be associated with declines in DNA methylation. Using information from a multi-ethnic New York City birth cohort (born between 1959 and 1963), we examined whether genomic DNA methylation, measured in peripheral blood mononuclear cells, was associated with smoking exposure and other epidemiologic risk factors across the lifecourse. Information on prenatal and childhood exposures was collected prospectively through 1971; and information on adult exposures and blood specimens were collected in adulthood from 2001-2007. Methylation levels of leukocyte DNA were determined using a [3H]-methyl acceptance assay where higher values of DPM/μg DNA indicate less DNA methylation. Genomic methylation of leukocyte DNA differed by ethnicity (66% of blacks, 48% of whites, and 29% of Hispanics were above the median level of DPM/μg DNA) (p = 0.03). In multivariable modeling, DNA methylation was statistically significantly associated with maternal smoking during pregnancy, longer birth length, later age at menarche, nulliparity, and later age at first birth. These data, if replicated in larger samples, suggest that risk factors across the lifecourse may be associated with DNA methylation in adulthood. Larger studies and studies that measure within-individual changes in DNA methylation over time are a necessary next step.
doi:10.1158/1055-9965.EPI-08-0312
PMCID: PMC3832298  PMID: 18768498
11.  Associations between antibiotic exposure during pregnancy, birth weight and aberrant methylation at imprinted genes among offspring 
Objectives:
Low birth weight (LBW) has been associated with common adult-onset chronic diseases, including obesity, cardiovascular disease, type II diabetes and some cancers. The etiology of LBW is multi-factorial. However, recent evidence suggests exposure to antibiotics may also increase the risk of LBW. The mechanisms underlying this association are unknown, although epigenetic mechanisms are hypothesized. In this study, we evaluated the association between maternal antibiotic use and LBW and examined the potential role of altered DNA methylation that controls growth regulatory imprinted genes in these associations.
Methods:
Between 2009–2011, 397 pregnant women were enrolled and followed until delivery. Prenatal antibiotic use was ascertained through maternal self-report. Imprinted genes methylation levels were measured at differentially methylated regions (DMRs) using bisulfite pyrosequencing. Generalized linear models were used to examine associations among antibiotic use, birth weight and DMR methylation fractions.
Results:
After adjusting for infant gender, race/ethnicity, maternal body mass index, delivery route, gestational weight gain, gestational age at delivery, folic acid intake, physical activity, maternal smoking and parity, antibiotic use during pregnancy was associated with 138 g lower birth weight compared with non-antibiotic use (β-coefficient=−132.99, s.e.=50.70, P=0.008). These associations were strongest in newborns of women who reported antibiotic use other than penicillins (β-coefficient=−135.57, s.e.=57.38, P=0.02). Methylation at five DMRs, IGF2 (P=0.05), H19 (P=0.15), PLAGL1 (P=0.01), MEG3 (P=0.006) and PEG3 (P=0.08), was associated with maternal antibiotic use; among these, only methylation at the PLAGL1 DMR was also associated with birth weight.
Conclusion:
We report an inverse association between in utero exposure to antibiotics and lower infant birth weight and provide the first empirical evidence supporting imprinted gene plasticity in these associations.
doi:10.1038/ijo.2013.47
PMCID: PMC3705584  PMID: 23609933
newborns; antibiotics; pregnancy; birth weight; epigenetics; race
12.  LINE-1 Methylation Levels in Leukocyte DNA and Risk of Renal Cell Cancer 
PLoS ONE  2011;6(11):e27361.
Purpose
Leukocyte global DNA methylation levels are currently being considered as biomarkers of cancer susceptibility and have been associated with risk of several cancers. In this study, we aimed to examine the association between long interspersed nuclear elements (LINE-1) methylation levels, as a biomarker of global DNA methylation in blood cell DNA, and renal cell cancer risk.
Experimental Design
LINE-1 methylation of bisulfite-converted genomic DNA isolated from leukocytes was quantified by pyrosequencing measured in triplicate, and averaged across 4 CpG sites. A total of 328 RCC cases and 654 controls frequency-matched(2∶1) on age(±5years), sex and study center, from a large case-control study conducted in Central and Eastern Europe were evaluated.
Results
LINE-1 methylation levels were significantly higher in RCC cases with a median of 81.97% (interquartile range[IQR]: 80.84–83.47) compared to 81.67% (IQR: 80.35–83.03) among controls (p = 0.003, Wilcoxon). Compared to the lowest LINE-1 methylation quartile(Q1), the adjusted ORs for increasing methylation quartiles were as follows: OR(Q2) = 1.84(1.20−2.81), OR(Q3) = 1.72(1.11−2.65) and OR(Q4) = 2.06(1.34−3.17), with a p-trend = 0.004. The association was stronger among current smokers (p-trend<0.001) than former or never smokers (p-interaction = 0.03). To eliminate the possibility of selection bias among controls, the relationship between LINE-1 methylation and smoking was evaluated and confirmed in a case-only analysis, as well.
Conclusions
Higher levels of LINE-1 methylation appear to be positively associated with RCC risk, particularly among current smokers. Further investigations using both post- and pre-diagnostic genomic DNA is warranted to confirm findings and will be necessary to determine whether the observed differences occur prior to, or as a result of carcinogenesis.
doi:10.1371/journal.pone.0027361
PMCID: PMC3208631  PMID: 22076155
13.  Association of postmenopausal endogenous sex hormones with global methylation level of leukocyte DNA among Japanese women 
BMC Cancer  2012;12:323.
Background
Although global hypomethylation of leukocyte DNA has been associated with an increased risk of several sites of cancer, including breast cancer, determinants of global methylation level among healthy individuals remain largely unexplored. Here, we examined whether postmenopausal endogenous sex hormones were associated with the global methylation level of leukocyte DNA.
Methods
A cross-sectional study was conducted using the control group of a breast cancer case–control study in Nagano, Japan. Subjects were postmenopausal women aged 55 years or over who provided blood samples. We measured global methylation level of peripheral blood leukocyte DNA by luminometric methylation assay; estradiol, estrone, androstenedione, dehydroepiandrosterone sulfate, testosterone and free testosterone by radioimmunoassay; bioavailable estradiol by the ammonium sulfate precipitation method; and sex-hormone binding globulin by immunoradiometric assay. A linear trend of association between methylation and hormone levels was evaluated by regression coefficients in a multivariable liner regression model. A total of 185 women were included in the analyses.
Results
Mean global methylation level (standard deviation) was 70.3% (3.1) and range was from 60.3% to 79.2%. Global methylation level decreased 0.27% per quartile category for estradiol and 0.39% per quartile category for estrone while it increased 0.41% per quartile category for bioavailable estradiol. However, we found no statistically significant association of any sex hormone level measured in the present study with global methylation level of leukocyte DNA.
Conclusions
Our findings suggest that endogenous sex hormones are not major determinants of the global methylation level of leukocyte DNA.
doi:10.1186/1471-2407-12-323
PMCID: PMC3481397  PMID: 22839213
Cross-sectional study; Endogenous sex hormones; Global methylation level; Luminometric methylation assay; Peripheral blood leukocyte DNA
14.  DNA methylation in white blood cells 
Epigenetics  2011;6(7):828-837.
Alterations in DNA methylation patterns, both at specific loci and overall in the genome, have been associated with many different health outcomes. In cancer and other diseases, most of these changes have been observed at the tissue level. Data on whether DNA methylation changes in white blood cells (WBC) can serve as a useful biomarker for different health outcomes are much more limited, but rapidly emerging. Epidemiologic studies have reported associations between global WBC methylation and several different cancers including cancers of the colon, bladder, stomach, breast, and head and neck, as well as schizophrenia and myelodysplastic syndrome. Evidence for WBC methylation at specific loci and disease risk is more limited, but increasing. Differences in WBC DNA methylation by selected risk factors including demographic (age, gender, race), environmental exposures (benzene, persistent organic pollutants, lead, arsenic and air pollution), and other risk factors (cigarette smoke, alcohol drinking, body size, physical activity and diet) have been observed in epidemiologic studies though the patterns are far from consistent. Challenges in inferences from the existing data are primarily due to the cross-sectional fashion and small size of most studies performed to date, as well as to the differences in results across assay type and source of DNA. Large, prospective studies will be needed to understand whether changes in risk factors are associated with changes in DNA methylation patterns, and if changes in DNA methylation patterns are associated with changes in disease endpoints.
doi:10.4161/epi.6.7.16500
PMCID: PMC3154425  PMID: 21636973
white blood cells; DNA methylation; global; gene-specific; risk factors; epidemiology
15.  Micronutrient status and global DNA methylation in school-age children 
Epigenetics  2012;7(10):1133-1141.
Aberrations in global LINE-1 DNA methylation have been related to risk of cancer and cardiovascular disease. Micronutrients including methyl-donors and retinoids are involved in DNA methylation pathways. We investigated associations of micronutrient status and LINE-1 methylation in a cross-sectional study of school-age children from Bogotá, Colombia. Methylation of LINE-1 repetitive elements was quantified in 568 children 5–12 years of age using pyrosequencing technology. We examined the association of LINE-1 methylation with erythrocyte folate, plasma vitamin B12, vitamin A ferritin (an indicator of iron status) and serum zinc concentrations using multivariable linear regression. We also considered associations of LINE-1 methylation with socio-demographic and anthropometric characteristics. Mean (± SD) LINE-1 methylation was 80.25 (± 0.65) percentage of 5-mC (%5-mC). LINE-1 methylation was inversely related to plasma vitamin A. After adjustment for potential confounders, children with retinol levels higher than or equal to 1.05 µmol/L showed 0.19% 5-mC lower LINE-1 methylation than children with retinol levels lower than 0.70 µmol/L. LINE-1 methylation was also inversely associated with C-reactive protein, a marker of chronic inflammation, and female sex. We identified positive associations of maternal body mass index and socioeconomic status with LINE-1 methylation. These associations were not significantly different by sex. Whether modification of these exposures during school-age years leads to changes in global DNA methylation warrants further investigation.
doi:10.4161/epi.21915
PMCID: PMC3469455  PMID: 22918385
C-reactive protein; LINE-1; children; global DNA methylation; inflammation; maternal BMI; methyl-donor nutrients; socioeconomic status; vitamin A
16.  Physical activity and global genomic DNA methylation in a cancer-free population 
Epigenetics  2011;6(3):293-299.
Changes in DNA methylation may represent an intermediate step between the environment and human diseases. Little is known on whether behavioral risk factors may modify gene expression through DNA methylation. To assess whether DNA methylation is associated with different levels of physical activity, we measured global genomic DNA methylation using bisulfite-converted DNA and real-time PCR (MethyLight) for LINE-1 in peripheral blood of 161 participants aged 45–75 years enrolled in the North Texas Healthy Heart Study and levels of physical activity using an accelerometer (Actigraph GT1M Monitor). We found that individuals with physical activity 26–30 min/day had a significantly higher level of global genomic DNA methylation compared to those with physical activity ≤10 min/day (β = 2.52, 95% CI: 0.70, 4.35). However, the association was attenuated and became statistically insignificant after multivariate adjustment (β = 1.24, 95% CI: −0.93, 3.40). There were some suggestions of a positive association between physical activity and global genomic DNA methylation in non-Hispanics (β = 1.50, 95% CI: −0.08, 3.08) that warrants further investigation.
doi:10.4161/epi.6.3.14378
PMCID: PMC3092677  PMID: 21178401
DNA methylation; physical activity; peripheral blood
17.  LINE-1 hypomethylation is associated with bladder cancer risk among non-smoking Chinese 
Reduced levels of global DNA methylation, assessed in peripheral blood, have been associated with bladder cancer risk in European and American populations. Similar data are lacking in Asian populations where genetic differences, lifestyle factors, and different environmental exposures may affect DNA methylation and its risk relationship with bladder cancer. The association between global DNA methylation measured at long interspersed nuclear element (LINE-1) repeat regions through bisulfite pyrosequencing in lymphocyte DNA and bladder cancer risk was examined in a case-control study of 510 bladder cancer patients and 528 healthy control subjects in Shanghai, China. In an initial analysis restricted to control subjects, LINE-1 methylation was elevated among men, those who frequently consumed cruciferous vegetables, and those with a null genotype for either glutathione S-transferase M1 (GSTM1) or GSTT1. In contrast, reduced LINE-1 methylation was found in current smokers with a high cytochrome P450 1A2 (CYP1A2) phenotype index. In a case-control analysis, there was no significant association of LINE-1 methylation with case status, although reduced LINE-1 methylation was associated with increased risk of bladder cancer among never smokers (P for trend = 0.03); analysis by tertile revealed odds ratios (ORs) of 1.91 (lowest tertile; 95% CI = 1.17–3.13) and 1.34 (middle tertile; 95% CI = 0.79–2.28) when compared to the highest tertile. This association was strongest among nonsmokers null for either the GSTM1 or GSTT1 genotype (P for trend = 0.006). Further research is needed to understand the relationships between methyl group availability and LINE-1 methylation in relation to bladder cancer risk.
doi:10.1002/ijc.26098
PMCID: PMC3208798  PMID: 21445976
18.  Methylome Analysis and Epigenetic Changes Associated with Menarcheal Age 
PLoS ONE  2013;8(11):e79391.
Reproductive factors have been linked to both breast cancer and DNA methylation, suggesting methylation as an important mechanism by which reproductive factors impact on disease risk. However, few studies have investigated the link between reproductive factors and DNA methylation in humans. Genome-wide methylation in peripheral blood lymphocytes of 376 healthy women from the prospective EPIC study was investigated using LUminometric Methylation Assay (LUMA). Also, methylation of 458877 CpG sites was additionally investigated in an independent group of 332 participants of the EPIC-Italy sub-cohort, using the Infinium HumanMethylation 450 BeadChip. Multivariate logistic regression and linear models were used to investigate the association between reproductive risk factors and genome wide and CpG-specific DNA methylation, respectively. Menarcheal age was inversely associated with global DNA methylation as measured with LUMA. For each yearly increase in age at menarche, the risk of having genome wide methylation below median level was increased by 32% (OR:1.32, 95%CI:1.14–1.53). When age at menarche was treated as a categorical variable, there was an inverse dose-response relationship with LUMA methylation levels (OR12–14vs.≤11 yrs:1.78, 95%CI:1.01–3.17 and OR≥15vs.≤11 yrs:4.59, 95%CI:2.04–10.33; P for trend<0.0001). However, average levels of global methylation as measured by the Illumina technology were not significantly associated with menarcheal age. In locus by locus comparative analyses, only one CpG site had significantly different methylation depending on the menarcheal age category examined, but this finding was not replicated by pyrosequencing in an independent data set. This study suggests a link between age at menarche and genome wide DNA methylation, and the difference in results between the two arrays suggests that repetitive element methylation has a role in the association. Epigenetic changes may be modulated by menarcheal age, or the association may be a mirror of other important changes in early life that have a detectable effect on both methylation levels and menarcheal age.
doi:10.1371/journal.pone.0079391
PMCID: PMC3835804  PMID: 24278132
19.  Aberrant DNA Methylation of OLIG1, a Novel Prognostic Factor in Non-Small Cell Lung Cancer 
PLoS Medicine  2007;4(3):e108.
Background
Lung cancer is the leading cause of cancer-related death worldwide. Currently, tumor, node, metastasis (TNM) staging provides the most accurate prognostic parameter for patients with non-small cell lung cancer (NSCLC). However, the overall survival of patients with resectable tumors varies significantly, indicating the need for additional prognostic factors to better predict the outcome of the disease, particularly within a given TNM subset.
Methods and Findings
In this study, we investigated whether adenocarcinomas and squamous cell carcinomas could be differentiated based on their global aberrant DNA methylation patterns. We performed restriction landmark genomic scanning on 40 patient samples and identified 47 DNA methylation targets that together could distinguish the two lung cancer subgroups. The protein expression of one of those targets, oligodendrocyte transcription factor 1 (OLIG1), significantly correlated with survival in NSCLC patients, as shown by univariate and multivariate analyses. Furthermore, the hazard ratio for patients negative for OLIG1 protein was significantly higher than the one for those patients expressing the protein, even at low levels.
Conclusions
Multivariate analyses of our data confirmed that OLIG1 protein expression significantly correlates with overall survival in NSCLC patients, with a relative risk of 0.84 (95% confidence interval 0.77–0.91, p < 0.001) along with T and N stages, as indicated by a Cox proportional hazard model. Taken together, our results suggests that OLIG1 protein expression could be utilized as a novel prognostic factor, which could aid in deciding which NSCLC patients might benefit from more aggressive therapy. This is potentially of great significance, as the addition of postoperative adjuvant chemotherapy in T2N0 NSCLC patients is still controversial.
Christopher Plass and colleagues find thatOLIG1 expression correlates with survival in lung cancer patients and suggest that it could be used in deciding which patients are likely to benefit from more aggressive therapy.
Editors' Summary
Background.
Lung cancer is the commonest cause of cancer-related death worldwide. Most cases are of a type called non-small cell lung cancer (NSCLC). Like other cancers, treatment of NCSLC depends on the “TNM stage” at which the cancer is detected. Staging takes into account the size and local spread of the tumor (its T classification), whether nearby lymph nodes contain tumor cells (its N classification), and whether tumor cells have spread (metastasized) throughout the body (its M classification). Stage I tumors are confined to the lung and are removed surgically. Stage II tumors have spread to nearby lymph nodes and are treated with a combination of surgery and chemotherapy. Stage III tumors have spread throughout the chest, and stage IV tumors have metastasized around the body; patients with both of these stages are treated with chemotherapy alone. About 70% of patients with stage I or II lung cancer, but only 2% of patients with stage IV lung cancer, survive for five years after diagnosis.
Why Was This Study Done?
TNM staging is the best way to predict the likely outcome (prognosis) for patients with NSCLC, but survival times for patients with stage I and II tumors vary widely. Another prognostic marker—maybe a “molecular signature”—that could distinguish patients who are likely to respond to treatment from those whose cancer will inevitably progress would be very useful. Unlike normal cells, cancer cells divide uncontrollably and can move around the body. These behavioral changes are caused by alterations in the pattern of proteins expressed by the cells. But what causes these alterations? The answer in some cases is “epigenetic changes” or chemical modifications of genes. In cancer cells, methyl groups are aberrantly added to GC-rich gene regions. These so-called “CpG islands” lie near gene promoters (sequences that control the transcription of DNA into mRNA, the template for protein production), and their methylation stops the promoters working and silences the gene. In this study, the researchers have investigated whether aberrant methylation patterns vary between NSCLC subtypes and whether specific aberrant methylations are associated with survival and can, therefore, be used prognostically.
What Did the Researchers Do and Find?
The researchers used “restriction landmark genomic scanning” (RLGS) to catalog global aberrant DNA methylation patterns in human lung tumor samples. In RLGS, DNA is cut into fragments with a restriction enzyme (a protein that cuts at specific DNA sequences), end-labeled, and separated using two-dimensional gel electrophoresis to give a pattern of spots. Because methylation stops some restriction enzymes cutting their target sequence, normal lung tissue and lung tumor samples yield different patterns of spots. The researchers used these patterns to identify 47 DNA methylation targets (many in CpG islands) that together distinguished between adenocarcinomas and squamous cell carcinomas, two major types of NSCLCs. Next, they measured mRNA production from the genes with the greatest difference in methylation between adenocarcinomas and squamous cell carcinomas. OLIG1 (the gene that encodes a protein involved in nerve cell development) had one of the highest differences in mRNA production between these tumor types. Furthermore, three-quarters of NSCLCs had reduced or no expression of OLIG1 protein and, when the researchers analyzed the association between OLIG1 protein expression and overall survival in patients with NSCLC, reduced OLIG1 protein expression was associated with reduced survival.
What Do These Findings Mean?
These findings indicate that different types of NSCLC can be distinguished by examining their aberrant methylation patterns. This suggests that the establishment of different DNA methylation patterns might be related to the cell type from which the tumors developed. Alternatively, the different aberrant methylation patterns might reflect the different routes that these cells take to becoming tumor cells. This research identifies a potential new prognostic marker for NSCLC by showing that OLIG1 protein expression correlates with overall survival in patients with NSCLC. This correlation needs to be tested in a clinical setting to see if adding OLIG1 expression to the current prognostic parameters can lead to better treatment choices for early-stage lung cancer patients and ultimately improve these patients' overall survival.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040108.
Patient and professional information on lung cancer, including staging (in English and Spanish), is available from the US National Cancer Institute
The MedlinePlus encyclopedia has pages on non-small cell lung cancer (in English and Spanish)
Cancerbackup provides patient information on lung cancer
CancerQuest, provided by Emory University, has information about how cancer develops (in English, Spanish, Chinese and Russian)
Wikipedia pages on epigenetics (note that Wikipedia is a free online encyclopedia that anyone can edit)
The Epigenome Network of Excellence gives background information and the latest news about epigenetics (in several European languages)
doi:10.1371/journal.pmed.0040108
PMCID: PMC1831740  PMID: 17388669
20.  Global DNA Hypomethylation in Peripheral Blood Leukocytes as a Biomarker for Cancer Risk: A Meta-Analysis 
PLoS ONE  2012;7(4):e34615.
Background
Good biomarkers for early detection of cancer lead to better prognosis. However, harvesting tumor tissue is invasive and cannot be routinely performed. Global DNA methylation of peripheral blood leukocyte DNA was evaluated as a biomarker for cancer risk.
Methods
We performed a meta-analysis to estimate overall cancer risk according to global DNA hypomethylation levels among studies with various cancer types and analytical methods used to measure DNA methylation. Studies were systemically searched via PubMed with no language limitation up to July 2011. Summary estimates were calculated using a fixed effects model.
Results
The subgroup analyses by experimental methods to determine DNA methylation level were performed due to heterogeneity within the selected studies (p<0.001, I2: 80%). Heterogeneity was not found in the subgroup of %5-mC (p = 0.393, I2: 0%) and LINE-1 used same target sequence (p = 0.097, I2: 49%), whereas considerable variance remained in LINE-1 (p<0.001, I2: 80%) and bladder cancer studies (p = 0.016, I2: 76%). These results suggest that experimental methods used to quantify global DNA methylation levels are important factors in the association study between hypomethylation levels and cancer risk. Overall, cancer risks of the group with the lowest DNA methylation levels were significantly higher compared to the group with the highest methylation levels [OR (95% CI): 1.48 (1.28–1.70)].
Conclusions
Global DNA hypomethylation in peripheral blood leukocytes may be a suitable biomarker for cancer risk. However, the association between global DNA methylation and cancer risk may be different based on experimental methods, and region of DNA targeted for measuring global hypomethylation levels as well as the cancer type. Therefore, it is important to select a precise and accurate surrogate marker for global DNA methylation levels in the association studies between global DNA methylation levels in peripheral leukocyte and cancer risk.
doi:10.1371/journal.pone.0034615
PMCID: PMC3324531  PMID: 22509334
21.  Implications of LINE1 Methylation for Bladder Cancer Risk in Women 
Purpose
Epigenetic alterations including changes to cellular DNA methylation levels contribute to carcinogenesis and may serve as powerful biomarkers of the disease. This investigation sought to determine whether hypomethylation at the long interspersed nuclear elements (LINE1), reflective of the level of global DNA methylation, in peripheral blood-derived DNA is associated with increased risk of bladder cancer.
Experimental Design
LINE1 methylation was measured from blood-derived DNA obtained from participants of a population-based incident case control study of bladder cancer in New Hampshire. Bisulfite-modified DNA was pyrosequenced to determine LINE1 methylation status; a total of 285 cases and 465 controls were evaluated for methylation.
Results
Being in the lowest LINE1 methylation decile was associated with a 1.8-fold increased risk of bladder cancer (95% CI, 1.12-2.90) in models controlling for gender, age and smoking, and the association was stronger in women than in men (ORs = 2.48, 95% CI 1.19-5.17 in women and 1.47, 95% CI 0.79-2.74 in men). Amongst controls, women were more likely to have lower LINE1 methylation than men (p-value 0.04), and levels of arsenic in the 90th percentile were associated with reduced LINE1 methylation (p-value 0.04).
Conclusions
LINE1 hypomethylation may be an important biomarker of bladder cancer risk, especially amongst women.
doi:10.1158/1078-0432.CCR-09-2983
PMCID: PMC2831156  PMID: 20179218
Bladder Cancer; Epidemiology; Gender Differences
22.  Effect of Body Mass Index on Global DNA Methylation in Healthy Korean Women 
Molecules and Cells  2014;37(6):467-472.
Obesity is known to be strongly associated with cardiovascular disease and cancer, the leading causes of mortality worldwide, and develops owing to interactions between genes and the environment. DNA methylation can act as a downstream effector of environmental signals, and analysis of this process therefore holds substantial promise for identifying mechanisms through which genetic and environmental factors jointly contribute to disease risk. Global DNA methylation of peripheral blood cells has recently been proposed as a potential biomarker for disease risk. Repetitive element DNA methylation has been shown to be associated with prominent obesity-related chronic diseases, but little is known about its relationship with weight status. In this study, we quantified the methylation of Alu elements in the peripheral blood DNA of 244 healthy women with a range of body mass indexes (BMIs) using pyrosequencing technology. Among the study participants, certain clinical laboratory parameters, including hemoglobin, serum glutamic oxaloacetic transaminase, serum glutamic-pyruvic transaminase, total cholesterol, and triglyceride levels were found to be strongly associated with BMI. Moreover, a U-shaped association between BMI and Alu methylation was observed, with the lowest methylation levels occurring at BMIs of between 23 and 30 kg/m2. However, there was no significant association between Alu methylation and age, smoking status, or alcohol consumption. Overall, we identified a differential influence of BMI on global DNA methylation in healthy Korean women, indicating that BMI-related changes in Alu methylation might play a complex role in the etiology and pathogenesis of obesity. Further studies are required to elucidate the mechanisms underlying this relationship.
doi:10.14348/molcells.2014.0073
PMCID: PMC4086340  PMID: 24938226
Alu; BMI; methylation; pyrosequencing; U-shape
23.  Association between global DNA hypomethylation in leukocytes and risk of breast cancer 
Carcinogenesis  2009;30(11):1889-1897.
Background: Global DNA hypomethylation may result in chromosomal instability and oncogene activation, and as a surrogate of systemic methylation activity, may be associated with breast cancer risk. Methods: Samples and data were obtained from women with incident early-stage breast cancer (I–IIIa) and women who were cancer free, frequency matched on age and race. In preliminary analyses, genomic methylation of leukocyte DNA was determined by measuring 5-methyldeoxycytosine (5-mdC), as well as methylation analysis of the LINE-1-repetitive DNA element. Further analyses used only 5-mdC levels. Logistic regression models were used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for risk of breast cancer in relation to amounts of methylation. Results: In a subset of samples tested (n = 37), 5-mdC level was not correlated with LINE-1 methylation. 5-mdC level in leukocyte DNA was significantly lower in breast cancer cases than healthy controls (P = 0.001), but no significant case–control differences were observed with LINE-1 methylation (P = 0.176). In the entire data set, we noted significant differences in 5-mdC levels in leukocytes between cases (n = 176) and controls (n = 173); P value < 0.001. Compared with women in the highest 5-mdC tertile (T3), women in the second (T2; OR = 1.49, 95% CI = 0.84–2.65) and lowest tertile (T1; OR = 2.86, 95% CI = 1.65–4.94) had higher risk of breast cancer (P for trend ≤0.001). Among controls only and cases and controls combined, only alcohol intake was found to be inversely associated with methylation levels. Conclusion: These findings suggest that leukocyte DNA hypomethylation is independently associated with development of breast cancer.
doi:10.1093/carcin/bgp143
PMCID: PMC2783000  PMID: 19584139
24.  Global DNA Methylation Is Associated With Insulin Resistance 
Diabetes  2012;61(2):542-546.
Insulin resistance (IR), the hallmark of type 2 diabetes, may be under epigenetic control. This study examines the association between global DNA methylation and IR using 84 monozygotic twin pairs. IR was estimated using homeostasis model assessment (HOMA). Global DNA methylation of Alu repeats in peripheral blood leukocytes was quantified by bisulfite pyrosequencing. The association between global DNA methylation and IR was examined using generalized estimating equation (GEE) and within–twin pair analyses, adjusting for potential confounders. Results show that methylation levels at all four CpG sites were individually associated with IR by GEE (all false discovery rate–adjusted P values ≤0.026). A 10% increase in mean Alu methylation was associated with an increase of 4.55 units (95% CI 2.38–6.73) in HOMA. Intrapair difference in IR was significantly associated with intrapair difference in global methylation level. A 10% increase in the difference in mean Alu methylation was associated with an increase of 4.54 units (0.34–8.71; P = 0.036) in the difference in HOMA. Confirmation of the results by intrapair analyses suggests that genetic factors do not confound the association between global DNA methylation and IR. Exclusion of twins taking diabetes medication (n = 17) did not change our results.
doi:10.2337/db11-1048
PMCID: PMC3266395  PMID: 22210312
25.  Function and Evolution of DNA Methylation in Nasonia vitripennis 
PLoS Genetics  2013;9(10):e1003872.
The parasitoid wasp Nasonia vitripennis is an emerging genetic model for functional analysis of DNA methylation. Here, we characterize genome-wide methylation at a base-pair resolution, and compare these results to gene expression across five developmental stages and to methylation patterns reported in other insects. An accurate assessment of DNA methylation across the genome is accomplished using bisulfite sequencing of adult females from a highly inbred line. One-third of genes show extensive methylation over the gene body, yet methylated DNA is not found in non-coding regions and rarely in transposons. Methylated genes occur in small clusters across the genome. Methylation demarcates exon-intron boundaries, with elevated levels over exons, primarily in the 5′ regions of genes. It is also elevated near the sites of translational initiation and termination, with reduced levels in 5′ and 3′ UTRs. Methylated genes have higher median expression levels and lower expression variation across development stages than non-methylated genes. There is no difference in frequency of differential splicing between methylated and non-methylated genes, and as yet no established role for methylation in regulating alternative splicing in Nasonia. Phylogenetic comparisons indicate that many genes maintain methylation status across long evolutionary time scales. Nasonia methylated genes are more likely to be conserved in insects, but even those that are not conserved show broader expression across development than comparable non-methylated genes. Finally, examination of duplicated genes shows that those paralogs that have lost methylation in the Nasonia lineage following gene duplication evolve more rapidly, show decreased median expression levels, and increased specialization in expression across development. Methylation of Nasonia genes signals constitutive transcription across developmental stages, whereas non-methylated genes show more dynamic developmental expression patterns. We speculate that loss of methylation may result in increased developmental specialization in evolution and acquisition of methylation may lead to broader constitutive expression.
Author Summary
Insects use methylation to modulate genome function in a different manner from vertebrates. Here, we quantified the global methylation profile in a parasitic wasp species, Nasonia vitripennis, a model with some advantages over ant and honeybee for functional and genetic analyses of methylation, such as short generation time, inbred lines, and inter-fertile species. Using a highly inbred line permitted us to precisely characterize DNA methylation, which is compared to gene expression variation across developmental stages, and contrasted to other insect species. DNA methylation is almost exclusively on the 5′-most 1 kbp coding exons, and ∼1/3 of protein coding genes are methylated. Methylated genes tend to occur in small clusters in the genome. Unlike many organisms, Nasonia leaves nearly all transposable element genes non-methylated. Methylated genes exhibit more uniform expression across developmental stages for both moderately and highly expressed genes, suggesting that DNA methylation is marking the genes for constitutive expression. Among pairs of differentially methylated duplicated genes, the paralogs that lose DNA methylation after duplication in the Nasonia lineage show lower expression and greater specialization of expression. Finally, by comparative analysis, we show that methylated genes are more conserved at three different time scales during evolution.
doi:10.1371/journal.pgen.1003872
PMCID: PMC3794928  PMID: 24130511

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