Modification of Epigenetic Patterns in Low Birth Weight Children: Importance of Hypomethylation of the ACE Gene Promoter

There is a growing body of evidence that epigenetic alterations are involved in the pathological mechanisms of many chronic disorders linked to fetal programming. Angiotensin-converting enzyme (ACE) appears as one candidate gene that brings new insights into the epigenetic control and later development of diseases. In this view, we have postulated that epigenetic modifications in the ACE gene might show different interactions between birth weight (BW), blood pressure levels, plasma ACE activity and ACE I/D polymorphism. To explore this hypothesis, we performed a cross-sectional study to evaluate the DNA methylation of 3 CpG sites using pyrosequencing within the ACE gene promoter of peripheral blood leukocytes from 45 LBW children compared with 70 NBW children. Our results have revealed that LBW children have lower methylation levels (P<0.001) in parallel with a higher ACE activity (P = 0.001). Adjusting for prematurity, gender, age, body mass index, and family history of cardiovascular disease did not alter these findings. We have also performed analyses of individual CpG sites. The frequency of DNA methylation was significantly different at two CpG sites (site 1: nucleotide position +555; and site 3: nucleotide position +563). In addition, we have found a significant inverse correlation between degree of DNA methylation and both ACE activity (P<0.001) and systolic blood pressure levels (P<0.001). We also observed that the methylation level was significantly lower in LBW children who are carriers of the DD genotype compared to NBW children with DD genotype (P<0.024). In conclusion, we are able to demonstrate that the hypomethylation in the 3 CpG sites of ACE gene promoter is associated with LBW in 6 to 12 year-old children. The magnitude of these epigenetic changes appears to be clinically important, which is supported by the observation that discrete changes in DNA methylation can affect systolic blood pressure and ACE protein activity levels.     

Fetal DNA Methylation Associates with Early Spontaneous Preterm Birth and Gestational Age

Spontaneous preterm birth (PTB, <37 weeks gestation) is a major public health concern, and children born preterm have a higher risk of morbidity and mortality throughout their lives. Recent studies suggest that fetal DNA methylation of several genes varies across a range of gestational ages (GA), but it is not yet clear if fetal epigenetic changes associate with PTB. The objective of this study is to interrogate methylation patterns across the genome in fetal leukocyte DNA from African Americans with early PTB (24^1/7–34^0/7 weeks; N = 22) or term births (39^0/7–40^6/7weeks; N = 28) and to evaluate the association of each CpG site with PTB and GA. DNA methylation was assessed across the genome with the HumanMethylation450 BeadChip. For each individual sample and CpG site, the proportion of DNA methylation was estimated. The associations between methylation and PTB or GA were evaluated by fitting a separate linear model for each CpG site, adjusting for relevant covariates. Overall, 29 CpG sites associated with PTB (FDR<.05; 5.7×10⁻¹⁰<p<2.9×10⁻⁶) independent of GA. Also, 9637 sites associated with GA (FDR<.05; 9.5×10⁻¹⁶<p<1.0×10⁻³), with 61.8% decreasing in methylation with shorter GA. GA-associated CpG sites were depleted in the CpG islands of their respective genes (p<2.2×10⁻¹⁶). Gene set enrichment analysis (GSEA) supported enrichment of GA-associated CpG sites in genes that play a role in embryonic development as well as the extracellular matrix. Additionally, this study replicated the association of several CpG sites associated with gestational age in other studies (CRHBP, PIK3CD and AVP). Dramatic differences in fetal DNA methylation are evident in fetuses born preterm versus at term, and the patterns established at birth may provide insight into the long-term consequences associated with PTB.     

DNA methylation in neonates born to women receiving psychiatric care

Prenatal exposure both to maternal psychiatric illness and psychiatric medication has been linked with adverse child outcomes that affect physiological, emotional and psychiatric development. Studies suggest that epigenetic mechanisms, such as DNA methylation, may facilitate these effects. In this report, we explore the association between maternal psychiatric illness and treatment during pregnancy and neonatal DNA methylation patterns in a prospectively-characterized clinical cohort of 201 dyads. Associations between the percent of umbilical cord blood DNA methylated at 27,578 CpG sites and maternal psychiatric diagnosis, symptoms and antidepressant use were evaluated by fitting a separate linear mixed effects model for each CpG site. There were no significant changes in neonatal DNA methylation attributable to maternal psychiatric diagnosis or depressive symptoms during pregnancy. Exposure to an antidepressant medication was associated with differential methylation of CpG sites in TNFRSF21 and CHRNA2 (false discovery rate < 0.05), but the average difference in methylation for both CpG sites was less than 3% between each group. The results were not specific to type of antidepressant or duration of the exposure. This study suggests that there are no large effects of maternal psychiatric illness, depressive symptoms or prenatal exposure to antidepressants on neonatal DNA methylation. Delineation of the influence of maternal psychiatric illness and pharmacological exposures on the developing fetuses has critical implications for clinical care during pregnancy.     

Genome-Wide Investigation of DNA Methylation Marks Associated with FV Leiden Mutation

In order to investigate whether DNA methylation marks could contribute to the incomplete penetrance of the FV Leiden mutation, a major genetic risk factor for venous thrombosis (VT), we measured genome-wide DNA methylation levels in peripheral blood samples of 98 VT patients carrying the mutation and 251 VT patients without the mutation using the dedicated Illumina HumanMethylation450 array. The genome-wide analysis of 388,120 CpG probes identified three sites mapping to the SLC19A2 locus whose DNA methylation levels differed significantly (p<3 10⁻⁸) between carriers and non-carriers. The three sites replicated (p<2 10⁻⁷) in an independent sample of 214 individuals from five large families ascertained on VT and FV Leiden mutation among which 53 were carriers and 161 were non-carriers of the mutation. In both studies, these three CpG sites were also associated (2.33 10⁻¹¹<p<3.02 10⁻⁴) with biomarkers of the Protein C pathway known to be influenced by the FV Leiden mutation. A comprehensive linkage disequilibrium (LD) analysis of the whole locus revealed that the original associations were due to LD between the FV Leiden mutation and a block of single nucleotide polymorphisms (SNP) located in SLC19A2. After adjusting for this block of SNPs, the FV Leiden mutation was no longer associated with any CpG site (p>0.05). In conclusion, our work clearly illustrates some promises and pitfalls of DNA methylation investigations on peripheral blood DNA in large epidemiological cohorts. DNA methylation levels at SLC19A2 are influenced by SNPs in LD with FV Leiden, but these DNA methylation marks do not explain the incomplete penetrance of the FV Leiden mutation.     

Maternal genome-wide DNA methylation patterns and congenital heart defects

The majority of congenital heart defects (CHDs) are thought to result from the interaction between multiple genetic, epigenetic, environmental, and lifestyle factors. Epigenetic mechanisms are attractive targets in the study of complex diseases because they may be altered by environmental factors and dietary interventions. We conducted a population based, case-control study of genome-wide maternal DNA methylation to determine if alterations in gene-specific methylation were associated with CHDs. Using the Illumina Infinium Human Methylation27 BeadChip, we assessed maternal gene-specific methylation in over 27,000 CpG sites from DNA isolated from peripheral blood lymphocytes. Our study sample included 180 mothers with non-syndromic CHD-affected pregnancies (cases) and 187 mothers with unaffected pregnancies (controls). Using a multi-factorial statistical model, we observed differential methylation between cases and controls at multiple CpG sites, although no CpG site reached the most stringent level of genome-wide statistical significance. The majority of differentially methylated CpG sites were hypermethylated in cases and located within CpG islands. Gene Set Enrichment Analysis (GSEA) revealed that the genes of interest were enriched in multiple biological processes involved in fetal development. Associations with canonical pathways previously shown to be involved in fetal organogenesis were also observed. We present preliminary evidence that alterations in maternal DNA methylation may be associated with CHDs. Our results suggest that further studies involving maternal epigenetic patterns and CHDs are warranted. Multiple candidate processes and pathways for future study have been identified.     

In Utero Exposure to Diethylstilbestrol and Blood DNA Methylation in Women Ages 40–59 Years from the Sister Study

In utero exposure to diethylstilbestrol (DES) has been associated with increased risk of adverse health outcomes such as fertility problems and vaginal as well as breast cancer. Animal studies have linked prenatal DES exposure to lasting DNA methylation changes. We investigated genome-wide DNA methylation and in utero DES exposure in a sample of non-Hispanic white women aged 40–59 years from the Sister Study, a large United States cohort study of women with a family history of breast cancer. Using questionnaire information from women and their mothers, we selected 100 women whose mothers reported taking DES while pregnant and 100 control women whose mothers had not taken DES. DNA methylation in blood was measured at 485,577 CpG sites using the Illumina HumanMethylation450 BeadChip. Associations between CpG methylation and DES exposure status were analyzed using robust linear regression with adjustment for blood cell composition and multiple comparisons. Although four CpGs had p<10⁵, after accounting for multiple comparisons using the false discovery rate (FDR), none reached genome-wide significance. In conclusion, adult women exposed to DES in utero had no evidence of large persistent changes in blood DNA methylation.     

Differential DNA methylation identified in the blood and retina of AMD patients

Age-related macular degeneration (AMD) is a major cause of blindness in the western world. While genetic studies have linked both common and rare variants in genes involved in regulation of the complement system to increased risk of development of AMD, environmental factors, such as smoking and nutrition, can also significantly affect the risk of developing the disease and the rate of disease progression. Since epigenetics has been implicated in mediating, in part, the disease risk associated with some environmental factors, we investigated a possible epigenetic contribution to AMD. We performed genome-wide DNA methylation profiling of blood from AMD patients and controls. No differential methylation site reached genome-wide significance; however, when epigenetic changes in and around known GWAS-defined AMD risk loci were explored, we found small but significant DNA methylation differences in the blood of neovascular AMD patients near age-related maculopathy susceptibility 2 (ARMS2), a top-ranked GWAS locus preferentially associated with neovascular AMD. The methylation level of one of the CpG sites significantly correlated with the genotype of the risk SNP rs10490924, suggesting a possible epigenetic mechanism of risk. Integrating genome-wide DNA methylation analysis of retina samples with and without AMD together with blood samples, we further identified a consistent, replicable change in DNA methylation in the promoter region of protease serine 50 (PRSS50). These methylation changes may identify sites in novel genes that are susceptible to non-genetic factors known to contribute to AMD development and progression.     

The impact of genetic variation and cigarette smoke on DNA methylation in current and former smokers from the COPDGene study

DNA methylation can be affected by systemic exposures, such as cigarette smoking and genetic sequence variation; however, the relative impact of each on the epigenome is unknown. We aimed to assess if cigarette smoking and genetic variation are associated with overlapping or distinct sets of DNA methylation marks and pathways. We selected 85 Caucasian current and former smokers with genome-wide single nucleotide polymorphism (SNP) genotyping available from the COPDGene study.  Genome-wide methylation was obtained on DNA from whole blood using the Illumina HumanMethylation27 platform. To determine the impact of local sequence variation on DNA methylation (mQTL), we examined the association between methylation and SNPs within 50 kb of each CpG site.  To examine the impact of cigarette smoking on DNA methylation, we examined the differences in methylation by current cigarette smoking status. We detected 770 CpG sites annotated to 708 genes associated at an FDR < 0.05 in the cis-mQTL analysis and 1,287 CpG sites annotated to 1,242 genes, which were nominally associated in the smoking-CpG association analysis (P_unadjusted < 0.05). Forty-three CpG sites annotated to 40 genes were associated with both SNP variation and current smoking; this overlap was not greater than that expected by chance. Our results suggest that cigarette smoking and genetic variants impact distinct sets of DNA methylation marks, the further elucidation of which may partially explain the variable susceptibility to the health effects of cigarette smoking. Ascertaining how genetic variation and systemic exposures differentially impact the human epigenome has relevance for both biomarker identification and therapeutic target development for smoking-related diseases.     

Prenatal antidepressant exposure associated with CYP2E1 DNA methylation change in neonates

Some but not all neonates are affected by prenatal exposure to serotonin reuptake inhibitor antidepressants (SRI) and maternal mood disturbances. Distinguishing the impact of these 2 exposures is challenging and raises critical questions about whether pharmacological, genetic, or epigenetic factors can explain the spectrum of reported outcomes. Using unbiased DNA methylation array measurements followed by a detailed candidate gene approach, we examined whether prenatal SRI exposure was associated with neonatal DNA methylation changes and whether such changes were associated with differences in birth outcomes. Prenatal SRI exposure was first associated with increased DNA methylation status primarily at CYP2E1(β_Non-exposed = 0.06, β_SRI-exposed = 0.30, FDR = 0); however, this finding could not be distinguished from the potential impact of prenatal maternal depressed mood. Then, using pyrosequencing of CYP2E1 regulatory regions in an expanded cohort, higher DNA methylation status—both the mean across 16 CpG sites (P < 0.01) and at each specific CpG site (P < 0.05)—was associated with exposure to lower 3rd trimester maternal depressed mood symptoms only in the SRI-exposed neonates, indicating a maternal mood x SRI exposure interaction. In addition, higher DNA methylation levels at CpG2 (P = 0.04), CpG9 (P = 0.04) and CpG10 (P = 0.02), in the interrogated CYP2E1 region, were associated with increased birth weight independently of prenatal maternal mood, SRI drug exposure, or gestational age at birth. Prenatal SRI antidepressant exposure and maternal depressed mood were associated with altered neonatal CYP2E1 DNA methylation status, which, in turn, appeared to be associated with birth weight.     

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.     

DNA hypermethylation and DNA hypomethylation is present at different loci in chronic kidney disease

Genetic risk factors for chronic kidney disease (CKD) are being identified through international collaborations. By comparison, epigenetic risk factors for CKD have only recently been considered using population-based approaches. DNA methylation is a major epigenetic modification that is associated with complex diseases, so we investigated methylome-wide loci for association with CKD. A total of 485,577 unique features were evaluated in 255 individuals with CKD (cases) and 152 individuals without evidence of renal disease (controls). Following stringent quality control, raw data were quantile normalized and β values calculated to reflect the methylation status at each site. The difference in methylation status was evaluated between cases and controls with resultant P values adjusted for multiple testing. Genes with significantly increased and decreased levels of DNA methylation were considered for biological relevance by functional enrichment analysis using KEGG pathways in Partek Genomics Suite. Twenty-three genes, where more than one CpG per loci was identified with P_adjusted < 10⁻⁸, demonstrated significant methylation changes associated with CKD and additional support for these associated loci was sought from published literature. Strong biological candidates for CKD that showed statistically significant differential methylation include CUX1, ELMO1, FKBP5, INHBA-AS1, PTPRN2, and PRKAG2 genes; several genes are differentially methylated in kidney tissue and RNA-seq supports a functional role for differential methylation in ELMO1 and PRKAG2 genes. This study reports the largest, most comprehensive, genome-wide quantitative evaluation of DNA methylation for association with CKD. Evidence confirming methylation sites influence development of CKD would stimulate research to identify epigenetic therapies that might be clinically useful for CKD.     

Identification of sex-specific DNA methylation changes driven by specific chemicals in cord blood in a Faroese birth cohort

Faroe islanders consume marine foods contaminated with methylmercury (MeHg), polychlorinated biphenyls (PCBs), and other toxicants associated with chronic disease risks. Differential DNA methylation at specific CpG sites in cord blood may serve as a surrogate biomarker of health impacts from chemical exposures. We aimed to identify key environmental chemicals in cord blood associated with DNA methylation changes in a population with elevated exposure to chemical mixtures. We studied 72 participants of a Faroese birth cohort recruited between 1986 and 1987 and followed until adulthood. The cord blood DNA methylome was profiled using Infinium HumanMethylation450 BeadChips. We determined the associations of CpG site changes with concentrations of MeHg, major PCBs, other organochlorine compounds [hexachlorobenzene (HCB), p,p’-dichlorodiphenyldichloroethylene (p,p’-DDE) and p,p’-dichlorodiphenyltrichloroethane], and perfluoroalkyl substances. In a combined sex analysis, among the 16 chemicals studied, PCB congener 105 (CB-105) exposure was associated with the majority of differentially methylated CpG sites (214 out of a total of 250). In female-only analysis, only 73 CB-105 associated CpG sites were detected, 44 of which were mapped to genes in the ELAV1-associated cancer network. In males-only, methylation changes were seen for perfluorooctane sulfonate, HCB, and p,p’-DDE in 10,598, 1,238, and 1,473 CpG sites, respectively, 15% of which were enriched in cytobands of the X-chromosome associated with neurological disorders. In this multiple-pollutant and genome-wide study, we identified key epigenetic toxicants. The significant enrichment of specific X-chromosome sites in males implies potential sex-specific epigenome responses to prenatal chemical exposures.     

Association of cigarette smoking and CRP levels with DNA methylation in α-1 antitrypsin deficiency

Alpha-1 antitrypsin (AAT) deficiency and tobacco smoking are confirmed risk factors for Chronic Obstructive Pulmonary Disease. We hypothesized that variable DNA methylation would be associated with smoking and inflammation, as reflected by the level of C-Reactive Protein (CRP) in AAT-deficient subjects. Methylation levels of 1,411 autosomal CpG sites from the Illumina GoldenGate Methylation Cancer Panel I were analyzed in 316 subjects. Associations of five smoking behaviors and CRP levels with individual CpG sites and average methylation levels were assessed using non-parametric testing, linear regression and linear mixed effect models, with and without adjustment for age and gender. Univariate linear regression analysis revealed that methylation levels of 16 CpG sites significantly associated with ever-smoking status. A CpG site in the TGFBI gene was the only site associated with ever-smoking after adjustment for age and gender. No highly significant associations existed between age at smoking initiation, pack-years smoked, duration of smoking, and time since quitting smoking as predictors of individual CpG site methylation levels. However, ever-smoking and younger age at smoking initiation associated with lower methylation level averaged across all sites. DNA methylation at CpG sites in the RUNX3, JAK3 and KRT1 genes associated with CRP levels. The most significantly associated CpG sites with gender and age mapped to the CASP6 and FZD9 genes, respectively. In summary, this study identified multiple potential candidate CpG sites associated with ever-smoking and CRP level in AAT-deficient subjects. Phenotypic variability in Mendelian diseases may be due to epigenetic factors.     

TGF-β induces global changes in DNA methylation during the epithelial-to-mesenchymal transition in ovarian cancer cells

A key step in the process of metastasis is the epithelial-to-mesenchymal transition (EMT). We hypothesized that epigenetic mechanisms play a key role in EMT and to test this hypothesis we analyzed global and gene-specific changes in DNA methylation during TGF-β-induced EMT in ovarian cancer cells. Epigenetic profiling using the Infinium HumanMethylation450 BeadChip (HM450) revealed extensive (P < 0.01) methylation changes after TGF-β stimulation (468 and 390 CpG sites altered at 48 and 120 h post cytokine treatment, respectively). The majority of gene-specific TGF-β-induced methylation changes occurred in CpG islands located in or near promoters (193 and 494 genes hypermethylated at 48 and 120 h after TGF-β stimulation, respectively). Furthermore, methylation changes were sustained for the duration of TGF-β treatment and reversible after the cytokine removal. Pathway analysis of the hypermethylated loci identified functional networks strongly associated with EMT and cancer progression, including cellular movement, cell cycle, organ morphology, cellular development, and cell death and survival. Altered methylation and corresponding expression of specific genes during TGF-β-induced EMT included CDH1 (E-cadherin) and COL1A1 (collagen 1A1). Furthermore, TGF-β induced both expression and activity of DNA methyltransferases (DNMT) -1, -3A, and -3B, and treatment with the DNMT inhibitor SGI-110 prevented TGF-β-induced EMT. These results demonstrate that dynamic changes in the DNA methylome are implicated in TGF-β-induced EMT and metastasis. We suggest that targeting DNMTs may inhibit this process by reversing the EMT genes silenced by DNA methylation in cancer.