Cord blood buffy coat DNA methylation is comparable to whole cord blood methylation

Cord blood DNA methylation is associated with numerous health outcomes and environmental exposures. Whole cord blood DNA reflects all nucleated blood cell types, while centrifuging whole blood separates red blood cells, generating a white blood cell buffy coat. Both sample types are used in DNA methylation studies. Cell types have unique methylation patterns and processing can impact cell distributions, which may influence comparability. We evaluated differences in cell composition and DNA methylation between cord blood buffy coat and whole cord blood samples. Cord blood DNA methylation was measured with the Infinium EPIC BeadChip (Illumina) in eight individuals, each contributing buffy coat and whole blood samples. We analyzed principal components (PC) of methylation, performed hierarchical clustering, and computed correlations of mean-centered methylation between pairs. We conducted moderated t-tests on single sites and estimated cell composition. DNA methylation PCs were associated with individual (P_PC1 = 1.4 × 10^?9; P_PC2 = 2.9 × 10^?5; P_PC3 = 3.8 × 10^-5; P_PC4 = 4.2 × 10^-6; P_PC5 = 9.9 × 10^-13, P_PC6 = 1.3 × 10^?11) and not with sample type (P_PC1-6>0.7). Samples hierarchically clustered by individual. Pearson correlations of mean-centered methylation between paired samples ranged from r = 0.66 to r = 0.87. No individual site significantly differed between buffy coat and whole cord blood when adjusting for multiple comparisons (five sites had unadjusted P<10^?5). Estimated cell type proportions did not differ by sample type (P = 0.46), and estimated proportions were highly correlated between paired samples (r = 0.99). Differences in methylation and cell composition between buffy coat and whole cord blood are much lower than inter-individual variation, demonstrating that both sample preparation types can be analytically combined and compared.     

Familial resemblances in blood leukocyte DNA methylation levels

Epigenetic factors such as DNA methylation are DNA alterations affecting gene expression that can convey environmental information through generations. Only a few studies have demonstrated epigenetic inheritance in humans. Our objective is to quantify genetic and common environmental determinants of familial resemblances in DNA methylation levels, using a family based sample. DNA methylation was measured in 48 French Canadians from 16 families as part of the GENERATION Study. We used the Illumina HumanMethylation450 BeadChip array to measure DNA methylation levels in blood leukocytes on 485,577 CpG sites. Heritability was assessed using the variance components method implemented in the QTDT software, which partitions the variance into polygenic (G), common environmental (C), and non-shared environmental (E) effects. We computed maximal heritability, genetic heritability, and common environmental effect for all probes (12.7%, 8.2%, and 4.5%, respectively) and for statistically significant probes (81.8%, 26.9%, and 54.9%, respectively). Higher maximal heritability was observed in the Major Histocompatibility Complex region on chromosome 6. In conclusion, familial resemblances in DNA methylation levels are mainly attributable to genetic factors when considering the average across the genome, but common environmental effect plays an important role when considering statistically significant probes. Further epigenome-wide studies on larger samples combined with genome-wide genotyping studies are needed to better understand the underlying mechanisms of DNA methylation heritability.     

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.     

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.     

Age-related epigenome-wide DNA methylation and hydroxymethylation in longitudinal mouse blood

DNA methylation at cytosine-phosphate-guanine (CpG) dinucleotides changes as a function of age in humans and animal models, a process that may contribute to chronic disease development. Recent studies have investigated the role of an oxidized form of DNA methylation – 5-hydroxymethylcytosine (5hmC) – in the epigenome, but its contribution to age-related DNA methylation remains unclear. We tested the hypothesis that 5hmC changes with age, but in a direction opposite to 5-methylcytosine (5mC), potentially playing a distinct role in aging. To characterize epigenetic aging, genome-wide 5mC and 5hmC were measured in longitudinal blood samples (2, 4, and 10 months of age) from isogenic mice using two sequencing methods – enhanced reduced representation bisulfite sequencing and hydroxymethylated DNA immunoprecipitation sequencing. Examining the epigenome by age, we identified 28,196 unique differentially methylated CpGs (DMCs) and 8,613 differentially hydroxymethylated regions (DHMRs). Mouse blood showed a general pattern of epigenome-wide hypermethylation and hypo-hydroxymethylation with age. Comparing age-related DMCs and DHMRs, 1,854 annotated genes showed both differential 5mC and 5hmC, including one gene – Nfic – at five CpGs in the same 250 bp chromosomal region. At this region, 5mC and 5hmC levels both decreased with age. Reflecting these age-related epigenetic changes, Nfic RNA expression in blood decreased with age, suggesting that age-related regulation of this gene may be driven by 5hmC, not canonical DNA methylation. Combined, our genome-wide results show age-related differential 5mC and 5hmC, as well as some evidence that changes in 5hmC may drive age-related DNA methylation and gene expression.     

Distinctive patterns of DNA methylation associated with Parkinson disease

Parkinson disease (PD) is a multifactorial neurodegenerative disorder with high incidence in the elderly, where environmental and genetic factors are involved in etiology. In addition, epigenetic mechanisms, including deregulation of DNA methylation have been recently associated to PD. As accurate diagnosis cannot be achieved pre-mortem, identification of early pathological changes is crucial to enable therapeutic interventions before major neuropathological damage occurs. Here we investigated genome-wide DNA methylation in brain and blood samples from PD patients and observed a distinctive pattern of methylation involving many genes previously associated to PD, therefore supporting the role of epigenetic alterations as a molecular mechanism in neurodegeneration. Importantly, we identified concordant methylation alterations in brain and blood, suggesting that blood might hold promise as a surrogate for brain tissue to detect DNA methylation in PD and as a source for biomarker discovery.     

Genome-wide DNA methylation analysis in precursor B-cells

DNA methylation is responsible for regulating gene expression and cellular differentiation and for maintaining genomic stability during normal human development. Furthermore, it plays a significant role in the regulation of hematopoiesis. In order to elucidate the influence of DNA methylation during B-cell development, genome-wide DNA methylation status of pro-B, pre-BI, pre-BII, and naïve-B-cells isolated from human umbilical cord blood was determined using the methylated CpG island recovery assay followed by next generation sequencing. On average, 182–200 million sequences were generated for each precursor B-cell subset in 10 biological replicates. An overall decrease in methylation was observed during the transition from pro-B to pre-BI, whereas no differential methylation was observed in the pre-BI to pre-BII transition or in the pre-BII to naïve B-cell transition. Most of the methylated regions were located within intergenic and intronic regions not present in a CpG island context. Putative novel enhancers were identified in these regions that were differentially methylated between pro-B and pre-BI cells. The genome-wide methylation profiles are publically available and may be used to gain a better understanding of the involvement of atypical DNA methylation in the pathogenesis of malignancies associated with precursor B-cells.     

Genome-wide DNA methylation analysis in precursor B-cells

DNA methylation is responsible for regulating gene expression and cellular differentiation and for maintaining genomic stability during normal human development. Furthermore, it plays a significant role in the regulation of hematopoiesis. In order to elucidate the influence of DNA methylation during B-cell development, genome-wide DNA methylation status of pro-B, pre-BI, pre-BII, and naïve-B-cells isolated from human umbilical cord blood was determined using the methylated CpG island recovery assay followed by next generation sequencing. On average, 182–200 million sequences were generated for each precursor B-cell subset in 10 biological replicates. An overall decrease in methylation was observed during the transition from pro-B to pre-BI, whereas no differential methylation was observed in the pre-BI to pre-BII transition or in the pre-BII to naïve B-cell transition. Most of the methylated regions were located within intergenic and intronic regions not present in a CpG island context. Putative novel enhancers were identified in these regions that were differentially methylated between pro-B and pre-BI cells. The genome-wide methylation profiles are publically available and may be used to gain a better understanding of the involvement of atypical DNA methylation in the pathogenesis of malignancies associated with precursor B-cells.     

Validation of a DNA methylation reference panel for the estimation of nucleated cells types in cord blood

Cord blood is widely used as surrogate tissue in epigenome-wide association studies of prenatal conditions. Cell type composition variation across samples can be an important confounder of epigenome-wide association studies in blood that constitute a mixture of cells. We evaluated a newly developed cord blood reference panel to impute cell type composition from DNA methylation levels, including nucleated red blood cells (nRBCs). We estimated cell type composition from 154 unique cord blood samples with available DNA methylation data as well as direct measurements of nucleated cell types. We observed high correlations between the estimated and measured composition for nRBCs (r = 0.92, R² = 0.85), lymphocytes (r = 0.77, R² = 0.58), and granulocytes (r = 0.72, R² = 0.52), and a moderate correlation for monocytes (r = 0.51, R² = 0.25) as well as relatively low root mean square errors from the residuals ranging from 1.4 to 5.4%. These results validate the use of the cord blood reference panel and highlight its utility and limitations for epidemiological studies.     

Gestational intake of methyl donors and global LINE-1 DNA methylation in maternal and cord blood: Prospective results from a folate-replete population

Maternal diet affects offspring DNA methylation in animal models, but evidence from humans is limited. We investigated the extent to which gestational intake of methyl donor nutrients affects global DNA methylation in maternal and umbilical cord blood. Among mother-infant pairs in Project Viva, a folate-replete US population, we estimated maternal intakes of vitamin B12, betaine, choline, folate, cadmium, zinc and iron periconceptionally and during the second trimester. We examined associations of these nutrients with DNA methylation, measured as %5-methyl cytosines (%5mC) in Long Interspersed Nuclear Element-1 (LINE-1), in first trimester (n = 830) and second trimester (n = 671) maternal blood and in cord blood at delivery (n = 516). Cord blood methylation was higher for male than female infants {mean [standard deviation (SD)] 84.8 [0.6] vs. 84.4 [0.7]%}. In the multivariable-adjusted model, maternal intake of methyl donor nutrients periconceptionally and during the second trimester of pregnancy was not positively associated with first trimester, second trimester or cord blood LINE-1 methylation. Periconceptional betaine intake was inversely associated with cord blood methylation [regression coefficient = -0.08% (95% confidence interval (CI): -0.14,-0.01)] but this association was attenuated after adjustment for dietary cadmium, which itself was directly associated with first trimester methylation and inversely associated with cord blood methylation. We also found an inverse association between periconceptional choline [-0.10%, 95% CI: -0.17,-0.03 for each SD (~63 mg/d)] and cord blood methylation in males only. In this folate-replete population, we did not find positive associations between intake of methyl donor nutrients during pregnancy and DNA methylation overall, but among males, higher early pregnancy intakes of choline were associated with lower cord blood methylation.     

Global DNA methylation in a population with aflatoxin B1 exposure

We previously reported that global DNA hypomethylation, measured as Sat2 methylation in white blood cells (WBC), and aflatoxin B₁ (AFB₁) exposure were associated with increased hepatocellular carcinoma risk. In this study, we assessed the association between AFB₁ exposure and global DNA methylation. We measured LINE-1 and Sat2 methylation in WBC DNA samples from 1140 cancer free participants of the Cancer Screening Program (CSP) cohort. Blood and urine samples were used to determine the level of AFB₁-albumin (AFB₁-Alb) adducts and urinary AFB₁ metabolites. In continuous models, we found reverse associations of urinary AFB₁ with LINE-1 and Sat2 methylation. The odds ratio (OR) per 1 unit decrease were 1.12 (95%CI = 1.03–1.22) for LINE-1 and 1.48 (95%CI = 1.10–2.00) for Sat2 methylation. When compared with subjects in the highest quartile of LINE-1, we found that individuals in the 2nd and 3rd quartiles were less likely to have detectable AFB₁-Alb adducts, with ORs (95%CI) of 0.61 (0.40–0.93), 0.61 (0.40-.94), and 1.09 (0.69–1.72), respectively. The OR for detectable AFB₁-Alb was 1.81 (95%CI = 1.15–2.85) for subjects in the lowest quartile of Sat2 methylation. The OR for detection of urinary AFB₁ for those with LINE-1 methylation in the lowest quartile compared with those in the highest quartile was 1.87 (95%CI = 1.15–3.04). The corresponding OR was 1.75 (95%CI = 1.08–2.82) for subjects in the lowest quartile of Sat2 methylation. The association between AFB₁ exposure and global DNA methylation may have implications for the epigenetic effect of AFB₁ on hepatocellular carcinoma development and also suggests that changes in DNA methylation may represent an epigenetic biomarker of dietary AFB₁ exposure.     

Nightshift work,chronotype, and genome-wide DNA methylation in blood

Molecular mechanisms underlying the negative health effects of shift work are poorly understood, which remains a barrier to developing intervention strategies to protect the long-term health of shift workers. We evaluated genome-wide differences in DNA methylation (measured in blood) between 111 actively employed female nightshift and 86 actively employed female dayshift workers from the Seattle metropolitan area. We also explored the effect of chronotype (i.e., measure of preference for activity earlier or later in the day) on DNA methylation among 110 of the female nightshift workers and an additional group of 131 male nightshift workers. Methylation data were generated using the Illumina Infinium HumanMethylation450 BeadChip (450K) Array. After applying the latest methylation data processing methods, we compared methylation levels at 361,210 CpG loci between the groups using linear regression models adjusted for potential confounders and applied the false-discovery rate (FDR) ≤ 0.05 to account for multiple comparisons. No statistically significant associations at the genome-wide level were observed with shift work or chronotype, though based on raw P values and absolute effect sizes, there were suggestive associations in genes that have been previously linked with cancer (e.g., BACH2, JRK, RPS6KA2) and type-2 diabetes (e.g., KCNQ1). Given that our study was underpowered to detect moderate effects, examining these suggestive results in well-powered independent studies or in pooled data sets may improve our understanding of the pathways underlying the negative health effects of shift work and the influence of personal factors such as chronotype. Such an approach may help identify potential interventions that can be used to protect the long-term health of shift workers.