DNA methylation of cord blood cell types: Applications for mixed cell birth studies

Epigenome-wide association studies of disease widely use DNA methylation measured in blood as a surrogate tissue. Cell proportions can vary between people and confound associations of exposure or outcome. An adequate reference panel for estimating cell proportions from adult whole blood for DNA methylation studies is available, but an analogous cord blood cell reference panel is not yet available. Cord blood has unique cell types and the epigenetic signatures of standard cell types may not be consistent throughout the life course. Using magnetic bead sorting, we isolated cord blood cell types (nucleated red blood cells, granulocytes, monocytes, natural killer cells, B cells, CD4⁺T cells, and CD8⁺T cells) from 17 live births at Johns Hopkins Hospital. We confirmed enrichment of the cell types using fluorescence assisted cell sorting and ran DNA from the separated cell types on the Illumina Infinium HumanMethylation450 BeadChip array. After filtering, the final analysis was on 104 samples at 429,794 probes. We compared cell type specific signatures in cord to each other and methylation at 49.2% of CpG sites on the array differed by cell type (F-test P < 10^?8). Differences between nucleated red blood cells and the remainder of the cell types were most pronounced (36.9% of CpG sites at P < 10^?8) and 99.5% of these sites were hypomethylated relative to the other cell types. We also compared the mean-centered sorted cord profiles to the available adult reference panel and observed high correlation between the overlapping cell types for granulocytes and monocytes (both r=0.74), and poor correlation for CD8⁺T cells and NK cells (both r=0.08). We further provide an algorithm for estimating cell proportions in cord blood using the newly developed cord reference panel, which estimates biologically plausible cell proportions in whole cord blood samples.     

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.     

An epigenome-wide association meta-analysis of prenatal maternal stress in neonates: A model approach for replication

Prenatal maternal stress exposure has been associated with neonatal differential DNA methylation. However, the available evidence in humans is largely based on candidate gene methylation studies, where only a few CpG sites were evaluated. The aim of this study was to examine the association between prenatal exposure to maternal stress and offspring genome-wide cord blood methylation using different methods. First, we conducted a meta-analysis and follow-up pathway analyses. Second, we used novel region discovery methods [i.e., differentially methylated regions (DMRs) analyses]. To this end, we used data from two independent population-based studies, the Generation R Study (n = 912) and the Avon Longitudinal Study of Parents and Children (ALSPAC, n = 828), to (i) measure genome-wide DNA methylation in cord blood and (ii) extract a prenatal maternal stress composite. The meta-analysis (n_total = 1,740) revealed no epigenome-wide (meta P <1.00e-07) associations of prenatal maternal stress exposure with neonatal differential DNA methylation. Follow-up analyses of the top hits derived from our epigenome-wide meta-analysis (meta P <1.00e-04) indicated an over-representation of the methyltransferase activity pathway. We identified no Bonferroni-corrected (P <1.00e-06) DMRs associated with prenatal maternal stress exposure. Combining data from two independent population-based samples in an epigenome-wide meta-analysis, the current study indicates that there are no large effects of prenatal maternal stress exposure on neonatal DNA methylation. Such replication efforts are essential in the search for robust associations, whether derived from candidate gene methylation or epigenome-wide studies.     

Cell type specific DNA methylation in cord blood: A 450K-reference data set and cell count-based validation of estimated cell type composition

Epigenome-wide association studies of prenatal exposure to different environmental factors are becoming increasingly common. These studies are usually performed in umbilical cord blood. Since blood comprises multiple cell types with specific DNA methylation patterns, confounding caused by cellular heterogeneity is a major concern. This can be adjusted for using reference data consisting of DNA methylation signatures in cell types isolated from blood. However, the most commonly used reference data set is based on blood samples from adult males and is not representative of the cell type composition in neonatal cord blood. The aim of this study was to generate a reference data set from cord blood to enable correct adjustment of the cell type composition in samples collected at birth. The purity of the isolated cell types was very high for all samples (>97.1%), and clustering analyses showed distinct grouping of the cell types according to hematopoietic lineage. We explored whether this cord blood and the adult peripheral blood reference data sets impact the estimation of cell type composition in cord blood samples from an independent birth cohort (MoBa, n = 1092). This revealed significant differences for all cell types. Importantly, comparison of the cell type estimates against matched cell counts both in the cord blood reference samples (n = 11) and in another independent birth cohort (Generation R, n = 195), demonstrated moderate to high correlation of the data. This is the first cord blood reference data set with a comprehensive examination of the downstream application of the data through validation of estimated cell types against matched cell counts.     

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.     

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.     

The circadecadal rhythm of oscillation of umbilical cord blood parameters correlates with geomagnetic activity – An analysis of long-term measurements (1999–2011)

Recently, we have shown that the contents of total nucleated cells (TNCs) and CD34⁺ hematopoietic stem and progenitor cells (CD34⁺ HSPCs) as well as the cord blood volume (CBV) in umbilical cord blood (UCB) show a circadecadal (~10 years) rhythm of oscillation. This observation was based on an analysis of 17,936 cord blood donations collected during 1999–2011. The aim of the present study was to investigate whether this circadecadal rhythm of oscillation in TNCs, CD34⁺ HSPCs and CBV is related to geomagnetic activity. For the analysis, the yearly averages of TNCs, CD34⁺ HSPCs and CBV in UCB were correlated with geomagnetic activity (Dcx index). Our analysis revealed that (i) all three UCB parameters were statistically significantly correlated with the level of geomagnetic activity, (ii) CBV showed a linear correlation with the Dcx index (r = 0.5290), (iii) the number of TNCs and CD34⁺ HSPCs were quadratic inversely correlated with the Dcx index (r = ?0.5343 and r = ?0.7749, respectively). Furthermore, (iv) CBV and the number of TNCs were not statistically significantly correlated with the number of either modest or intense geomagnetic storms per year, but (v) the number of CD34⁺ HSPCs was statistically significantly correlated with the number of modest (r = 0.9253) as well as intense (r = 0.8683) geomagnetic storms per year. In conclusion, our study suggests that UCB parameters correlate with the state of the geomagnetic field (GMF) modulated by solar activity. Possible biophysical mechanisms underlying this observation, as well as the outcome of these findings, are discussed.     

Epigenome-wide differential DNA methylation between HIV-infected and uninfected individuals

Epigenetic control of human immunodeficiency virus-1 (HIV-1) genes is critical for viral integration and latency. However, epigenetic changes in the HIV-1-infected host genome have not been well characterized. Here, we report the first large-scale epigenome-wide association study of DNA methylation for HIV-1 infection. We recruited HIV-infected (n = 261) and uninfected (n = 117) patients from the Veteran Aging Cohort Study (VACS) and all samples were profiled for 485,521 CpG sites in DNA extracted from the blood. After adjusting for cell type and clinical confounders, we identified 20 epigenome-wide significant CpGs for HIV-1 infection. Importantly, 2 CpGs in the promoter of the NLR family, CARD domain containing gene 5 (NLRC5), a key regulator of major histocompatibility complex class I gene expression, showed significantly lower methylation in HIV-infected subjects than in uninfected subjects (cg07839457: t = ?6.03, P_nominal = 4.96 × 10^?9; cg16411857: t = ?7.63, P_nominal = 3.07 × 10^?13). Hypomethylation of these 2 CpGs was replicated in an independent sample (GSE67705: cg07839457: t = ?4.44, P_nominal = 1.61 × 10^?5; cg16411857: t = ?5.90; P = 1.99 × 10^?8). Methylation of these 2 CpGs in NLRC5 was negatively correlated with viral load in the 2 HIV-infected samples (cg07839457: P = 1.8 × 10^?4; cg16411857: P = 0.03 in the VACS; and cg07839457: P = 0.04; cg164111857: P = 0.01 in GSE53840). Our findings demonstrate that differential DNA methylation is associated with HIV infection and suggest the involvement of a novel host gene, NLRC5, in HIV pathogenesis.     

Assessment of RainDrop BS-seq as a method for large-scale,targeted bisulfite sequencing

We present a systematic assessment of RainDrop BS-seq, a novel method for large-scale, targeted bisulfite sequencing using microdroplet-based PCR amplification coupled with next-generation sequencing. We compared DNA methylation levels at 498 target loci (1001 PCR amplicons) in human whole blood, osteosarcoma cells and an archived tumor tissue sample. We assessed the ability of RainDrop BS-seq to accurately measure DNA methylation over a range of DNA quantities (from 10 to 1500 ng), both with and without whole-genome amplification (WGA) following bisulfite conversion. DNA methylation profiles generated using at least 100 ng correlated well (median R = 0.92) with those generated on Illumina Infinium HumanMethylation450 BeadChips, currently the platform of choice for epigenome-wide association studies (EWAS). WGA allowed for testing of samples with a starting DNA amount of 10 and 50 ng, although a reduced correlation was observed (median R = 0.79). We conclude that RainDrop BS-seq is suitable for measuring DNA methylation levels using nanogram quantities of DNA, and can be used to study candidate epigenetic biomarker loci in an accurate and high-throughput manner, paving the way for its application to routine clinical diagnostics.     

Regions of variable DNA methylation in human placenta associated with newborn neurobehavior

The placenta regulates the in utero environment and functionally impacts fetal development. Candidate gene studies identified variation in placental DNA methylation is associated with newborn neurologic and behavioral outcomes including movement quality, lethargic behavior, attention, and arousal. We sought to identify novel regions of variable DNA methylation associated with newborn attention, lethargy, quality of movement, and arousal by performing an epigenome-wide association study in 335 infants from a US birth cohort. Methylation status was quantified using the Illumina HumanMethylation450 BeadChip array and associations to newborn outcomes assessed by the NICU Network Neurobehavioral Scales (NNNS) were identified while incorporating established bioinformatics algorithms to control for confounding by cell type composition. Methylation of CpGs within FHIT (cg15970800) and ANKRD11 (cg16710656) demonstrated genome-wide significance (P < 1.8 × 10^?7) in specific associations with infant attention. CpGs whose differential methylation was associated with all 4 neurobehavioral outcomes were common to 50 genes involved in biological processes relating to cellular adhesion and nervous system development. Comprehensive methylation profiling identified relationships between methylation of FHIT and ANKRD11, which have been previously linked to neurodevelopment and behavioral outcomes in genetic association studies. Subtle changes in DNA methylation of these genes within the placenta may impact normal variation of a newborn's ability to alter and track visual and auditory stimuli. Gene ontology analysis suggested that those genes with variable methylation related to these outcomes are over-represented in biological pathways involved in brain development and placental physiology, supportive of our hypothesis for a key role of the placenta in neurobehavioral outcomes.     

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.     

Concentrations of heavy metals in maternal and umbilical cord blood

Concentrations of lead, cadmium, methylmercury and total mercury were measured in maternal and umbilical cord blood using graphite atomic absorption spectrometry. Two essential metals, copper and zinc, were also determined using ion chromatography. Lead, copper and zinc were found to be lower in the cord blood, whereas methylmercury and total mercury were higher in cord blood than in maternal blood. Little differences were noted for cadmium in maternal and cord blood. Significant positive correlations were observed between the concentrations in maternal and cord blood with regard to lead (correlation coefficient, r = 0.44), copper (r = 0.34), zinc (r = 0.29), methylmercury (r = 0.44) and total mercury (r = 0.58). These results suggest that, like essential metals, most heavy metals can move rather freely across the human placenta. The potential health effects of heavy metal transfer from mothers to young infants cannot be discounted.     

Association between DNA Methylation in Whole Blood and Measures of Glucose Metabolism: KORA F4 Study

Epigenetic regulation has been postulated to affect glucose metabolism, insulin sensitivity and the risk of type 2 diabetes. Therefore, we performed an epigenome-wide association study for measures of glucose metabolism in whole blood samples of the population-based Cooperative Health Research in the Region of Augsburg F4 study using the Illumina HumanMethylation 450 BeadChip. We identified a total of 31 CpG sites where methylation level was associated with measures of glucose metabolism after adjustment for age, sex, smoking, and estimated white blood cell proportions and correction for multiple testing using the Benjamini-Hochberg (B-H) method (four for fasting glucose, seven for fasting insulin, 25 for homeostasis model assessment-insulin resistance [HOMA-IR]; B-H-adjusted p-values between 9.2x10^-5 and 0.047). In addition, DNA methylation at cg06500161 (annotated to ABCG1) was associated with all the aforementioned phenotypes and 2-hour glucose (B-H-adjusted p-values between 9.2x10^-5 and 3.0x10^-3). Methylation status of additional three CpG sites showed an association with fasting insulin only after additional adjustment for body mass index (BMI) (B-H-adjusted p-values = 0.047). Overall, effect strengths were reduced by around 30% after additional adjustment for BMI, suggesting that this variable has an influence on the investigated phenotypes. Furthermore, we found significant associations between methylation status of 21 of the aforementioned CpG sites and 2-hour insulin in a subset of samples with seven significant associations persisting after additional adjustment for BMI. In a subset of 533 participants, methylation of the CpG site cg06500161 (ABCG1) was inversely associated with ABCG1 gene expression (B-H-adjusted p-value = 1.5x10^-9). Additionally, we observed an enrichment of the top 1,000 CpG sites for diabetes-related canonical pathways using Ingenuity Pathway Analysis. In conclusion, our study indicates that DNA methylation and diabetes-related traits are associated and that these associations are partially BMI-dependent. Furthermore, the interaction of ABCG1 with glucose metabolism is modulated by epigenetic processes.     

Carbohydrate-mediated inhibition of ice recrystallization in cryopreserved human umbilical cord blood

Cryopreservation of human umbilical cord blood (UCB) typically involves the cryoprotectant dimethylsulfoxide (DMSO), however, infusional toxicity and reductions in cell viability remain a concern. Ice recrystallization (IR) is an important source of cryopreservation-induced cellular injury and limits the stem cell dose in UCB units. Carbohydrates have wide-ranging intrinsic IR inhibition (IRI) activity related to structural properties. We investigated the impact of carbohydrate IRI on cell viability, induction of apoptosis and hematopoietic progenitor function in cryopreserved UCB. Mononuclear cells (MNCs) from UCB were cryopreserved in storage media containing specific carbohydrates (200 mM) and compared to 5% DMSO. Samples were analyzed under conditions of high IR (‘slow’ thaw) and low IR (‘fast’ thaw). Thawed samples were analyzed for viability and apoptosis by flow cytometry and hematopoietic function using colony-forming unit (CFU) assays. IRI of carbohydrate solutions was determined using the ‘splat cooling’ assay. Greater IRI capacity of carbohydrates correlated with increased yield of viable MNCs (r² = 0.92, p = 0.004) and CD34(+) cells (r² = 0.96, p = 0.019) after thawing under conditions of high IR. The correlations were less apparent under conditions of low IR. Carbohydrates with greater IRI modulate the induction of early apoptosis during thawing, especially in CD34+ cells (r² = 0.96, p = 0.0001) as compared to total mononuclear cells (p = 0.006), and preserve CFU capacity in vitro (r² = 0.92, p = <0.0001). Our results suggest that carbohydrates with potent IRI increase the yield of non-apoptotic and functional hematopoietic progenitors and provide a foundation for the development of novel synthetic carbohydrates with enhanced IRI properties to improve cryopreservation of UCB.     

Assessment of RainDrop BS-seq as a method for large-scale,targeted bisulfite sequencing

We present a systematic assessment of RainDrop BS-seq, a novel method for large-scale, targeted bisulfite sequencing using microdroplet-based PCR amplification coupled with next-generation sequencing. We compared DNA methylation levels at 498 target loci (1001 PCR amplicons) in human whole blood, osteosarcoma cells and an archived tumor tissue sample. We assessed the ability of RainDrop BS-seq to accurately measure DNA methylation over a range of DNA quantities (from 10 to 1500 ng), both with and without whole-genome amplification (WGA) following bisulfite conversion. DNA methylation profiles generated using at least 100 ng correlated well (median R = 0.92) with those generated on Illumina Infinium HumanMethylation450 BeadChips, currently the platform of choice for epigenome-wide association studies (EWAS). WGA allowed for testing of samples with a starting DNA amount of 10 and 50 ng, although a reduced correlation was observed (median R = 0.79). We conclude that RainDrop BS-seq is suitable for measuring DNA methylation levels using nanogram quantities of DNA, and can be used to study candidate epigenetic biomarker loci in an accurate and high-throughput manner, paving the way for its application to routine clinical diagnostics.