Quality control and statistical modeling for environmental epigenetics: A study on in utero lead exposure and DNA methylation at birth

DNA methylation data assayed using pyrosequencing techniques are increasingly being used in human cohort studies to investigate associations between epigenetic modifications at candidate genes and exposures to environmental toxicants and to examine environmentally-induced epigenetic alterations as a mechanism underlying observed toxicant-health outcome associations. For instance, in utero lead (Pb) exposure is a neurodevelopmental toxicant of global concern that has also been linked to altered growth in human epidemiological cohorts; a potential mechanism of this association is through alteration of DNA methylation (e.g., at growth-related genes). However, because the associations between toxicants and DNA methylation might be weak, using appropriate quality control and statistical methods is important to increase reliability and power of such studies. Using a simulation study, we compared potential approaches to estimate toxicant-DNA methylation associations that varied by how methylation data were analyzed (repeated measures vs. averaging all CpG sites) and by method to adjust for batch effects (batch controls vs. random effects). We demonstrate that correcting for batch effects using plate controls yields unbiased associations, and that explicitly modeling the CpG site-specific variances and correlations among CpG sites increases statistical power. Using the recommended approaches, we examined the association between DNA methylation (in LINE-1 and growth related genes IGF2, H19 and HSD11B2) and 3 biomarkers of Pb exposure (Pb concentrations in umbilical cord blood, maternal tibia, and maternal patella), among mother-infant pairs of the Early Life Exposures in Mexico to Environmental Toxicants (ELEMENT) cohort (n = 247). Those with 10 μg/g higher patella Pb had, on average, 0.61% higher IGF2 methylation (P = 0.05). Sex-specific trends between Pb and DNA methylation (P < 0.1) were observed among girls including a 0.23% increase in HSD11B2 methylation with 10 μg/g higher patella Pb.     

2型糖尿病环境因素与DNA甲基化的研究进展

2 型糖尿病(Type 2 diabetes mellitus, T2DM)是由于遗传与环境因素共同作用而引起葡萄糖代谢紊乱的疾病。DNA甲基化修饰的研究发现环境因素可以通过影响DNA甲基化修饰, 显著地增加T2DM的患病风险。目前, T2DM环境相关基因的DNA甲基化修饰研究已在人及动物的不同组织中取得进展。此外, T2DM相关基因的甲基化研究主要集中在糖代谢、能量代谢、炎症等。文章系统地综述了目前T2DM致病环境因素与DNA甲基化研究进展。     

Global hypomethylation and promoter methylation in small intestinal neuroendocrine tumors: An in vivo and in vitro study

Aberrant DNA methylation is a feature of human cancer affecting gene expression and tumor phenotype. Here, we quantified promoter methylation of candidate genes and global methylation in 44 small intestinal-neuroendocrine tumors (SI-NETs) from 33 patients by pyrosequencing. Findings were compared with gene expression, patient outcome and known tumor copy number alterations. Promoter methylation was observed for WIF1, RASSF1A, CTNNB1, CXCL14, NKX2–3, P16, LAMA1, and CDH1. By contrast APC, CDH3, HIC1, P14, SMAD2, and SMAD4 only had low levels of methylation. WIF1 methylation was significantly increased (P = 0.001) and WIF1 expression was reduced in SI-NETs vs. normal references (P = 0.003). WIF1, NKX2–3, and CXCL14 expression was reduced in metastases vs. primary tumors (P < 0.02). Low expression of RASSF1A and P16 were associated with poor overall survival (P = 0.045 and P = 0.011, respectively). Global methylation determined by pyrosequencing of LINE1 repeats was reduced in tumors vs. normal references, and was associated with loss in chromosome 18. The tumors fell into three clusters with enrichment of WIF1 methylation and LINE1 hypomethylation in Cluster I and RASSF1A and CTNNB1 methylation and loss in 16q in Cluster II. In Cluster III, these alterations were low-abundant and NKX2-3 methylation was low. Similar analyses in the SI-NET cell lines HC45 and CNDT2 showed methylation for CDH1 and WIF1 and/or P16, CXCL14, NKX2-3, LAMA1, and CTNNB1. Treatment with the demethylating agent 5-azacytidine reduced DNA methylation and increased expression of these genes in vitro. In conclusion, promoter methylation of tumor suppressor genes is associated with suppressed gene expression and DNA copy number alterations in SI-NETs, and may be restored in vitro.     

Placental mitochondrial methylation and exposure to airborne particulate matter in the early life environment: An ENVIRONAGE birth cohort study

Most research to date has focused on epigenetic modifications in the nuclear genome, with little attention devoted to mitochondrial DNA (mtDNA). Placental mtDNA content has been shown to respond to environmental exposures that induce oxidative stress, including airborne particulate matter (PM). Damaged or non-functioning mitochondria are specifically degraded through mitophagy, exemplified by lower mtDNA content, and could be primed by epigenetic modifications in the mtDNA. We studied placental mtDNA methylation in the context of the early life exposome. We investigated placental tissue from 381 mother-newborn pairs that were enrolled in the ENVIRONAGE birth cohort. We determined mtDNA methylation by bisulfite-pyrosequencing in 2 regions, i.e., the D-loop control region and 12S rRNA (MT-RNR1), and measured mtDNA content by qPCR. PM_2.5 exposure was calculated for each participant''s home address using a dispersion model. An interquartile range (IQR) increment in PM_2.5 exposure over the entire pregnancy was positively associated with mtDNA methylation (MT-RNR1: +0.91%, P = 0.01 and D-loop: +0.21%, P = 0.05) and inversely associated with mtDNA content (relative change of −15.60%, P = 0.001) in placental tissue. mtDNA methylation was estimated to mediate 54% [P = 0.01 (MT-RNR1)] and 27% [P = 0.06 (D-loop)] of the inverse association between PM_2.5 exposure and mtDNA content. This study provides new insight into the mechanisms of altered mitochondrial function in the early life environment. Epigenetic modifications in the mitochondrial genome, especially in the MT-RNR1 region, substantially mediate the association between PM_2.5 exposure during gestation and placental mtDNA content, which could reflect signs of mitophagy and mitochondrial death.     

Correlation of DNA methylation levels in blood and saliva DNA in young girls of the LEGACY Girls study

Many epidemiologic studies of environmental exposures and disease susceptibility measure DNA methylation in white blood cells (WBC). Some studies are also starting to use saliva DNA as it is usually more readily available in large epidemiologic studies. However, little is known about the correlation of methylation between WBC and saliva DNA. We examined DNA methylation in three repetitive elements, Sat2, Alu, and LINE-1, and in four CpG sites, including AHRR (cg23576855, cg05575921), cg05951221 at 2q37.1, and cg11924019 at CYP1A1, in 57 girls aged 6–15 years with blood and saliva collected on the same day. We measured all DNA methylation markers by bisulfite-pyrosequencing, except for Sat2 and Alu, which were measured by the MethyLight assay. Methylation levels measured in saliva DNA were lower than those in WBC DNA, with differences ranging from 2.8% for Alu to 14.1% for cg05575921. Methylation levels for the three repetitive elements measured in saliva DNA were all positively correlated with those in WBC DNA. However, there was a wide range in the Spearman correlations, with the smallest correlation found for Alu (0.24) and the strongest correlation found for LINE-1 (0.73). Spearman correlations for cg05575921, cg05951221, and cg11924019 were 0.33, 0.42, and 0.79, respectively. If these findings are replicated in larger studies, they suggest that, for selected methylation markers (e.g., LINE-1), methylation levels may be highly correlated between blood and saliva, while for others methylation markers, the levels may be more tissue specific. Thus, in studies that differ by DNA source, each interrogated site should be separately examined in order to evaluate the correlation in DNA methylation levels across DNA sources.     

Correlation of DNA methylation levels in blood and saliva DNA in young girls of the LEGACY Girls study

Many epidemiologic studies of environmental exposures and disease susceptibility measure DNA methylation in white blood cells (WBC). Some studies are also starting to use saliva DNA as it is usually more readily available in large epidemiologic studies. However, little is known about the correlation of methylation between WBC and saliva DNA. We examined DNA methylation in three repetitive elements, Sat2, Alu, and LINE-1, and in four CpG sites, including AHRR (cg23576855, cg05575921), cg05951221 at 2q37.1, and cg11924019 at CYP1A1, in 57 girls aged 6–15 years with blood and saliva collected on the same day. We measured all DNA methylation markers by bisulfite-pyrosequencing, except for Sat2 and Alu, which were measured by the MethyLight assay. Methylation levels measured in saliva DNA were lower than those in WBC DNA, with differences ranging from 2.8% for Alu to 14.1% for cg05575921. Methylation levels for the three repetitive elements measured in saliva DNA were all positively correlated with those in WBC DNA. However, there was a wide range in the Spearman correlations, with the smallest correlation found for Alu (0.24) and the strongest correlation found for LINE-1 (0.73). Spearman correlations for cg05575921, cg05951221, and cg11924019 were 0.33, 0.42, and 0.79, respectively. If these findings are replicated in larger studies, they suggest that, for selected methylation markers (e.g., LINE-1), methylation levels may be highly correlated between blood and saliva, while for others methylation markers, the levels may be more tissue specific. Thus, in studies that differ by DNA source, each interrogated site should be separately examined in order to evaluate the correlation in DNA methylation levels across DNA sources.     

DNA methylation levels and long-term trihalomethane exposure in drinking water: an epigenome-wide association study

Trihalomethanes (THM) are undesired disinfection byproducts (DBPs) formed during water treatment. Mice exposed to DBPs showed global DNA hypomethylation and c-myc and c-jun gene-specific hypomethylation, while evidence of epigenetic effects in humans is scarce. We explored the association between lifetime THM exposure and DNA methylation through an epigenome-wide association study. We selected 138 population-based controls from a case-control study of colorectal cancer conducted in Barcelona, Spain, exposed to average lifetime THM levels ≤85 μg/L vs. >85 μg/L (N = 68 and N = 70, respectively). Mean age of participants was 70 years, and 54% were male. Average lifetime THM level in the exposure groups was 64 and 130 µg/L, respectively. DNA was extracted from whole blood and was bisulphite converted to measure DNA methylation levels using the Illumina HumanMethylation450 BeadChip. Data preprocessing was performed using RnBeads. Methylation was compared between exposure groups using empirical Bayes moderated linear regression for CpG sites and Gaussian kernel for CpG regions. ConsensusPathDB was used for gene set enrichment. Statistically significant differences in methylation between exposure groups was found in 140 CpG sites and 30 gene-related regions, after false discovery rate <0.05 and adjustment for age, sex, methylation first principal component, and blood cell proportion. The annotated genes were localized to several cancer pathways. Among them, 29 CpGs had methylation levels associated with THM levels (|Δβ|≥0.05) located in 11 genes associated with cancer in other studies. Our results suggest that THM exposure may affect DNA methylation in genes related to tumors, including colorectal and bladder cancers. Future confirmation studies are required.     

Differential DNA methylation in umbilical cord blood of infants exposed to mercury and arsenic in utero

Mercury and arsenic are known developmental toxicants. Prenatal exposures are associated with adverse childhood health outcomes that could be in part mediated by epigenetic alterations that may also contribute to altered immune profiles. In this study, we examined the association between prenatal mercury exposure on both DNA methylation and white blood cell composition of cord blood, and evaluated the interaction with prenatal arsenic exposure. A total of 138 mother-infant pairs with postpartum maternal toenail mercury, prenatal urinary arsenic concentrations, and newborn cord blood were assessed using the Illumina Infinium Methylation450 array. White blood cell composition was inferred from DNA methylation measurements. A doubling in toenail mercury concentration was associated with a 2.5% decrease (95% CI: 5.0%, 1.0%) in the estimated monocyte proportion. An increase of 3.5% (95% CI: 1.0, 7.0) in B-cell proportion was observed for females only. Among the top 100 CpGs associated with toenail mercury levels (ranked on P-value), there was a significant enrichment of loci located in North shore regions of CpG islands (P = 0.049), and the majority of these loci were hypermethylated (85%). Among the top 100 CpGs for the interaction between arsenic and mercury, there was a greater than expected proportion of loci located in CpG islands (P = 0.045) and in South shore regions (P = 0.009) and all of these loci were hypermethylated. This work supports the hypothesis that mercury may be contributing to epigenetic variability and immune cell proportion changes, and suggests that in utero exposure to mercury and arsenic, even at low levels, may interact to impact the epigenome.     

Application of the LUminometric Methylation Assay to ecological species: tissue quality requirements and a survey of DNA methylation levels in animals

The LUminometric Methylation Assay (LUMA) measures global DNA methylation. LUMA depends on digestion of DNA with methyl‐sensitive and methyl‐insensitive restriction enzymes, followed by pyrosequencing. Until recently, LUMA has been principally used for biomedical research. Here, we use chickens as a model to investigate sample quality issues relating to LUMA and then apply the method to ecological species. First, we assessed the effect of tissue storage conditions on DNA methylation values. This is an important consideration for ecological species because samples are not always ideally preserved and LUMA is sensitive to poor DNA quality. We found that good quality LUMA data could be obtained from chicken liver and brain tissues stored at 21 °C for at least 2 and 12 h, respectively. Longer storage times introduced nonspecific peaks to pyrograms which were associated with reduced DNA methylation. Repeatedly, freezing and thawing the tissues did not affect LUMA data. Second, we measured DNA methylation in 12 species representing five animal classes: amphibians (African and Western clawed frog), reptiles (green anole lizard), fish (yellow perch, goldfish, lake trout), mammals (American mink, polar bear, short‐beaked common dolphin, Atlantic white‐sided dolphin) and birds (chicken, Japanese quail). We saw a pattern of high DNA methylation in fish (84–87%), and intermediate levels in mammals (68–72%) and birds (52–71%). This pattern corresponds well with previous measures of DNA methylation generated by HPLC. Our data represent the first CpG methylation values to be reported in several species and provide a basis for studying patterns of epigenetic inheritance in an ecological context.     

In utero arsenic exposure and epigenome-wide associations in placenta,umbilical artery,and human umbilical vein endothelial cells

Exposure to arsenic early in life has been associated with increased risk of several chronic diseases and is believed to alter epigenetic programming in utero. In the present study, we evaluate the epigenome-wide association of arsenic exposure in utero and DNA methylation in placenta (n = 37), umbilical artery (n = 45) and human umbilical vein endothelial cells (HUVEC) (n = 52) in a birth cohort using the Infinium HumanMethylation450 BeadChip array. Unadjusted and cell mixture adjusted associations for each tissue were examined along with enrichment analyses relative to CpG island location and omnibus permutation tests of association among biological pathways. One CpG in artery (cg26587014) and 4 CpGs in placenta (cg12825509; cg20554753; cg23439277; cg21055948) reached a Bonferroni adjusted level of significance. Several CpGs were differentially methylated in artery and placenta when controlling the false discovery rate (q-value<0.05), but none in HUVEC. Enrichment of hypomethylated CpG islands was observed for artery while hypermethylation of open sea regions were present in placenta relative to prenatal arsenic exposure. The melanogenesis pathway was differentially methylated in artery (Max F P < 0.001), placenta (Max F P < 0.001), and HUVEC (Max F P = 0.02). Similarly, the insulin-signaling pathway was differentially methylated in artery (Max F P = 0.02), placenta (Max F P = 0.02), and HUVEC (Max F P = 0.02). Our results show that prenatal arsenic exposure can alter DNA methylation in artery and placenta but not in HUVEC. Further studies are needed to determine if these alterations in DNA methylation mediate the effect of prenatal arsenic exposure and health outcomes later in life.     

DNA methylation in mycobacteria: Absence of methylation at GATC (Dam) and CCA/TGG (Dcm) sequences

Abstract The presence of 6-methyladenine and 5-methylcytosine at Dam (GATC) and Dcm (CCA/TGG) sites in DNA of mycobacterial species was investigated using isoschizomer restriction enzymes. In all species examined, Dam and Dcm recognition sequences were not methylated indicating the absence of these methyltransferases. On the other hand, high performance liquid chromatographic analysis of genomic DNA from Mycobacterium smegmatis and Mycobacterium tuberculosis showed significant levels of 6-methyladenine and 5-methylcytosine suggesting the presence of DNA methyltransferases other than Dam and Dcm. Occurrence of methylation was also established by a sensitive genetic assay.     

Effect of prenatal arsenic exposure on DNA methylation and leukocyte subpopulations in cord blood

Prenatal arsenic exposure is associated with increased risk of disease in adulthood. This has led to considerable interest in arsenic’s ability to disrupt fetal programming. Many studies report that arsenic exposure alters DNA methylation in whole blood but these studies did not adjust for cell mixture. In this study, we examined the relationship between arsenic in maternal drinking water collected ≤ 16 weeks gestational age and DNA methylation in cord blood (n = 44) adjusting for leukocyte-tagged differentially methylated regions. DNA methylation was quantified using the Infinium HumanMethylation 450 BeadChip array. Recursively partitioned mixture modeling examined the relationship between arsenic and methylation at 473,844 CpG sites. Median arsenic concentration in water was 12 µg/L (range < 1- 510 µg/L). Log₁₀ arsenic was associated with altered DNA methylation across the epigenome (P = 0.002); however, adjusting for leukocyte distributions attenuated this association (P = 0.013). We also observed that arsenic had a strong effect on the distribution of leukocytes in cord blood. In adjusted models, every log₁₀ increase in maternal drinking water arsenic exposure was estimated to increase CD8+ T cells by 7.4% (P = 0.0004) and decrease in CD4+ T cells by 9.2% (P = 0.0002). These results show that prenatal exposure to arsenic had an exposure-dependent effect on specific T cell subpopulations in cord blood and altered DNA methylation in cord blood. Future research is needed to determine if these small changes in DNA methylation alter gene expression or are associated with adverse health effects.     

Effect of prenatal arsenic exposure on DNA methylation and leukocyte subpopulations in cord blood

Prenatal arsenic exposure is associated with increased risk of disease in adulthood. This has led to considerable interest in arsenic’s ability to disrupt fetal programming. Many studies report that arsenic exposure alters DNA methylation in whole blood but these studies did not adjust for cell mixture. In this study, we examined the relationship between arsenic in maternal drinking water collected ≤ 16 weeks gestational age and DNA methylation in cord blood (n = 44) adjusting for leukocyte-tagged differentially methylated regions. DNA methylation was quantified using the Infinium HumanMethylation 450 BeadChip array. Recursively partitioned mixture modeling examined the relationship between arsenic and methylation at 473,844 CpG sites. Median arsenic concentration in water was 12 µg/L (range < 1- 510 µg/L). Log₁₀ arsenic was associated with altered DNA methylation across the epigenome (P = 0.002); however, adjusting for leukocyte distributions attenuated this association (P = 0.013). We also observed that arsenic had a strong effect on the distribution of leukocytes in cord blood. In adjusted models, every log₁₀ increase in maternal drinking water arsenic exposure was estimated to increase CD8+ T cells by 7.4% (P = 0.0004) and decrease in CD4+ T cells by 9.2% (P = 0.0002). These results show that prenatal exposure to arsenic had an exposure-dependent effect on specific T cell subpopulations in cord blood and altered DNA methylation in cord blood. Future research is needed to determine if these small changes in DNA methylation alter gene expression or are associated with adverse health effects.     

Genome-wide DNA methylation associations with spontaneous preterm birth in US blacks: findings in maternal and cord blood samples

Preterm birth (PTB) affects one in six Black babies in the United States. Epigenetics is believed to play a role in PTB; however, only a limited number of epigenetic studies of PTB have been reported, most of which have focused on cord blood DNA methylation (DNAm) and/or were conducted in white populations. Here we conducted, by far, the largest epigenome-wide DNAm analysis in 300 Black women who delivered early spontaneous preterm (sPTB, n = 150) or full-term babies (n = 150) and replicated the findings in an independent set of Black mother-newborn pairs from the Boston Birth Cohort. DNAm in maternal blood and/or cord blood was measured using the Illumina HumanMethylation450 BeadChip. We identified 45 DNAm loci in maternal blood associated with early sPTB, with a false discovery rate (FDR) <5%. Replication analyses confirmed sPTB associations for cg03915055 and cg06804705, located in the promoter regions of the CYTIP and LINC00114 genes, respectively. Both loci had comparable associations with early sPTB and early medically-indicated PTB, but attenuated associations with late sPTB. These associations could not be explained by cell composition, gestational complications, and/or nearby maternal genetic variants. Analyses in the newborns of the 110 Black women showed that cord blood methylation levels at both loci had no associations with PTB. The findings from this study underscore the role of maternal DNAm in PTB risk, and provide a set of maternal loci that may serve as biomarkers for PTB. Longitudinal studies are needed to clarify temporal relationships between maternal DNAm and PTB risk.     

Population differences in Chinook salmon (Oncorhynchus tshawytscha) DNA methylation: Genetic drift and environmental factors

Local adaptation and phenotypic differences among populations have been reported in many species, though most studies focus on either neutral or adaptive genetic differentiation. With the discovery of DNA methylation, questions have arisen about its contribution to individual variation in and among natural populations. Previous studies have identified differences in methylation among populations of organisms, although most to date have been in plants and model animal species. Here we obtained eyed eggs from eight populations of Chinook salmon (Oncorhynchus tshawytscha) and assayed DNA methylation at 23 genes involved in development, immune function, stress response, and metabolism using a gene‐targeted PCR‐based assay for next‐generation sequencing. Evidence for population differences in methylation was found at eight out of 23 gene loci after controlling for developmental timing in each individual. However, we found no correlation between freshwater environmental parameters and methylation variation among populations at those eight genes. A weak correlation was identified between pairwise DNA methylation dissimilarity among populations and pairwise F _ST based on 15 microsatellite loci, indicating weak effects of genetic drift or geographic distance on methylation. The weak correlation was primarily driven by two genes, GTIIBS and Nkef. However, single‐gene Mantel tests comparing methylation and pairwise F _ST were not significant after Bonferroni correction. Thus, population differences in DNA methylation are more likely related to unmeasured oceanic environmental conditions, local adaptation, and/or genetic drift. DNA methylation is an additional mechanism that contributes to among population variation, with potential influences on organism phenotype, adaptive potential, and population resilience.     

Detection of differential DNA methylation in repetitive DNA of mice and humans perinatally exposed to bisphenol A

Developmental exposure to bisphenol A (BPA) has been shown to induce changes in DNA methylation in both mouse and human genic regions; however, the response in repetitive elements and transposons has not been explored. Here we present novel methodology to combine genomic DNA enrichment with RepeatMasker analysis on next-generation sequencing data to determine the effect of perinatal BPA exposure on repetitive DNA at the class, family, subfamily, and individual insertion level in both mouse and human samples. Mice were treated during gestation and lactation to BPA in chow at 0, 50, or 50,000 ng/g levels and total BPA was measured in stratified human fetal liver tissue samples as low (non-detect to 0.83 ng/g), medium (3.5 to 5.79 ng/g), or high (35.44 to 96.76 ng/g). Transposon methylation changes were evident in human classes, families, and subfamilies, with the medium group exhibiting hypomethylation compared to both high and low BPA groups. Mouse repeat classes, families, and subfamilies did not respond to BPA with significantly detectable differential DNA methylation. In human samples, 1251 individual transposon loci were detected as differentially methylated by BPA exposure, but only 19 were detected in mice. Of note, this approach recapitulated the discovery of a previously known mouse environmentally labile metastable epiallele, Cabp^IAP. Thus, by querying repetitive DNA in both mouse and humans, we report the first known transposons in humans that respond to perinatal BPA exposure.     

Type 2 diabetes mellitus in relation to global LINE-1 DNA methylation in peripheral blood: A cohort study

In the last years, epigenetic processes have emerged as a promising area of complex diseases research. DNA methylation measured in Long Interspersed Nucleotide Element 1 (LINE-1) sequences has been considered a surrogate marker for global genome methylation. New findings have suggested the potential involvement of epigenetic mechanisms in Type 2 diabetes (T2DM) as a crucial interface between the effects of genetic predisposition and environmental influences. Our study evaluated whether global DNA methylation predicted increased risk from T2DM or other carbohydrate metabolism disorders in a cohort study. We used a prospective cohort intervention study and a control group. We collected phenotypic, anthropometric, biochemical, and nutritional information from all subjects. Global LINE-1 DNA methylation was quantified by pyrosequencing technology. Subjects that did not improve their carbohydrate metabolism status showed lower levels of global LINE-1 DNA methylation (63.9 ± 1.7 vs. 64.7 ± 2.4) and they practiced less intense physical activity (5.8% vs. 21.5%). Logistic regression analyses showed a significant association between LINE-1 DNA methylation and metabolic status after adjustment for sex, age, BMI, and physical activity. Our study showed that lower LINE-1 DNA methylation levels were associated with a higher risk metabolic status worsening, independent of other classic risk factors. This finding highlights the potential role for epigenetic biomarkers as predictors of T2DM risk or other related metabolic disorders.