Accounting for cellular heterogeneity is critical in epigenome-wide association studies

Background

Epigenome-wide association studies of human disease and other quantitative traits are becoming increasingly common. A series of papers reporting age-related changes in DNA methylation profiles in peripheral blood have already been published. However, blood is a heterogeneous collection of different cell types, each with a very different DNA methylation profile.

Results

Using a statistical method that permits estimating the relative proportion of cell types from DNA methylation profiles, we examine data from five previously published studies, and find strong evidence of cell composition change across age in blood. We also demonstrate that, in these studies, cellular composition explains much of the observed variability in DNA methylation. Furthermore, we find high levels of confounding between age-related variability and cellular composition at the CpG level.

Conclusions

Our findings underscore the importance of considering cell composition variability in epigenetic studies based on whole blood and other heterogeneous tissue sources. We also provide software for estimating and exploring this composition confounding for the Illumina 450k microarray.     

Global DNA hypomethylation in peripheral blood leukocytes as a biomarker for cancer risk: a meta-analysis

Background

Good biomarkers for early detection of cancer lead to better prognosis. However, harvesting tumor tissue is invasive and cannot be routinely performed. Global DNA methylation of peripheral blood leukocyte DNA was evaluated as a biomarker for cancer risk.

Methods

We performed a meta-analysis to estimate overall cancer risk according to global DNA hypomethylation levels among studies with various cancer types and analytical methods used to measure DNA methylation. Studies were systemically searched via PubMed with no language limitation up to July 2011. Summary estimates were calculated using a fixed effects model.

Results

The subgroup analyses by experimental methods to determine DNA methylation level were performed due to heterogeneity within the selected studies (p<0.001, I²: 80%). Heterogeneity was not found in the subgroup of %5-mC (p = 0.393, I²: 0%) and LINE-1 used same target sequence (p = 0.097, I²: 49%), whereas considerable variance remained in LINE-1 (p<0.001, I²: 80%) and bladder cancer studies (p = 0.016, I²: 76%). These results suggest that experimental methods used to quantify global DNA methylation levels are important factors in the association study between hypomethylation levels and cancer risk. Overall, cancer risks of the group with the lowest DNA methylation levels were significantly higher compared to the group with the highest methylation levels [OR (95% CI): 1.48 (1.28–1.70)].

Conclusions

Global DNA hypomethylation in peripheral blood leukocytes may be a suitable biomarker for cancer risk. However, the association between global DNA methylation and cancer risk may be different based on experimental methods, and region of DNA targeted for measuring global hypomethylation levels as well as the cancer type. Therefore, it is important to select a precise and accurate surrogate marker for global DNA methylation levels in the association studies between global DNA methylation levels in peripheral leukocyte and cancer risk.     

Dietary Flavanols Modulate the Transcription of Genes Associated with Cardiovascular Pathology without Changes in Their DNA Methylation State

Background

In a recent intervention study, the daily supplementation with 200 mg monomeric and oligomeric flavanols (MOF) from grape seeds for 8 weeks revealed a vascular health benefit in male smokers. The objective of the present study was to determine the impact of MOF consumption on the gene expression profile of leukocytes and to assess changes in DNA methylation.

Methodology/Principal Findings

Gene expression profiles were determined using whole genome microarrays (Agilent) and DNA methylation was assessed using HumanMethylation450 BeadChips (Illumina). MOF significantly modulated the expression of 864 genes. The majority of the affected genes are involved in chemotaxis, cell adhesion, cell infiltration or cytoskeleton organisation, suggesting lower immune cell adhesion to endothelial cells. This was corroborated by in vitro experiments showing that MOF exposure of monocytes attenuates their adhesion to TNF-α-stimulated endothelial cells. Nuclear factor kappa B (NF-κB) reporter gene assays confirmed that MOF decrease the activity of NF-κB. Strong inter-individual variability in the leukocytes'' DNA methylation was observed. As a consequence, on group level, changes due to MOF supplementation could not be found.

Conclusion

Our study revealed that an 8 week daily supplementation with 200 mg MOF modulates the expression of genes associated with cardiovascular disease pathways without major changes of their DNA methylation state. However, strong inter-individual variation in leukocyte DNA methylation may obscure the subtle epigenetic response to dietary flavanols. Despite the lack of significant changes in DNA methylation, the modulation of gene expression appears to contribute to the observed vascular health effect of MOF in humans.     

Evaluation of allelic expression of imprinted genes in adult human blood

Background

Imprinted genes are expressed from only one allele in a parent-of-origin dependent manner. Loss of imprinted (LOI) expression can result in a variety of human disorders and is frequently reported in cancer. Biallelic expression of imprinted genes in adult blood has been suggested as a useful biomarker and is currently being investigated in colorectal cancer. In general, the expression profiles of imprinted genes are well characterised during human and mouse fetal development, but not in human adults.

Methodology/Principal Findings

We investigated quantitative expression of 36 imprinted genes in adult human peripheral blood leukocytes obtained from healthy individuals. Allelic expression was also investigated in B and T lymphocytes and myeloid cells. We found that 21 genes were essentially undetectable in adult blood. Only six genes were demonstrably monoallelic, and most importantly, we found that nine genes were either biallelic or showed variable expression in different individuals. Separated leukocyte populations showed the same expression patterns as whole blood. Differential methylation at each of the imprinting control loci analysed was maintained, including regions that contained biallelically expressed genes. This suggests in some cases methylation has become uncoupled from its role in regulating gene expression.

Conclusions/Significance

We conclude that only a limited set of imprinted genes, including IGF2 and SNRPN, may be useful for LOI cancer biomarker studies. In addition, blood is not a good tissue to use for the discovery of new imprinted genes. Finally, lymphocyte DNA methylation status in the adult may not always be a reliable indicator of monoallelic gene expression.     

Genome-Wide DNA Methylation Profiles Indicate CD8+ T Cell Hypermethylation in Multiple Sclerosis

Objective

Determine whether MS-specific DNA methylation profiles can be identified in whole blood or purified immune cells from untreated MS patients.

Methods

Whole blood, CD4+ and CD8+ T cell DNA from 16 female, treatment naïve MS patients and 14 matched controls was profiled using the HumanMethylation450K BeadChip. Genotype data were used to assess genetic homogeneity of our sample and to exclude potential SNP-induced DNA methylation measurement errors.

Results

As expected, significant differences between CD4+ T cells, CD8+ T cells and whole blood DNA methylation profiles were observed, regardless of disease status. Strong evidence for hypermethylation of CD8+ T cell, but not CD4+ T cell or whole blood DNA in MS patients compared to controls was observed. Genome-wide significant individual CpG-site DNA methylation differences were not identified. Furthermore, significant differences in gene DNA methylation of 148 established MS-associated risk genes were not observed.

Conclusion

While genome-wide significant DNA methylation differences were not detected for individual CpG-sites, strong evidence for DNA hypermethylation of CD8+ T cells for MS patients was observed, indicating a role for DNA methylation in MS. Further, our results suggest that large DNA methylation differences for CpG-sites tested here do not contribute to MS susceptibility. In particular, large DNA methylation differences for CpG-sites within 148 established MS candidate genes tested in our study cannot explain missing heritability. Larger studies of homogenous MS patients and matched controls are warranted to further elucidate the impact of CD8+ T cell and more subtle DNA methylation changes in MS development and pathogenesis.     

DNA Methylation Signatures Triggered by Prenatal Maternal Stress Exposure to a Natural Disaster: Project Ice Storm

Background

Prenatal maternal stress (PNMS) predicts a wide variety of behavioral and physical outcomes in the offspring. Although epigenetic processes may be responsible for PNMS effects, human research is hampered by the lack of experimental methods that parallel controlled animal studies. Disasters, however, provide natural experiments that can provide models of prenatal stress.

Methods

Five months after the 1998 Quebec ice storm we recruited women who had been pregnant during the disaster and assessed their degrees of objective hardship and subjective distress. Thirteen years later, we investigated DNA methylation profiling in T cells obtained from 36 of the children, and compared selected results with those from saliva samples obtained from the same children at age 8.

Results

Prenatal maternal objective hardship was correlated with DNA methylation levels in 1675 CGs affiliated with 957 genes predominantly related to immune function; maternal subjective distress was uncorrelated. DNA methylation changes in SCG5 and LTA, both highly correlated with maternal objective stress, were comparable in T cells, peripheral blood mononuclear cells (PBMCs) and saliva cells.

Conclusions

These data provide first evidence in humans supporting the conclusion that PNMS results in a lasting, broad, and functionally organized DNA methylation signature in several tissues in offspring. By using a natural disaster model, we can infer that the epigenetic effects found in Project Ice Storm are due to objective levels of hardship experienced by the pregnant woman rather than to her level of sustained distress.     

Pre-Diagnostic Leukocyte Genomic DNA Methylation and the Risk of Colorectal Cancer in Women

Background

Abnormal one-carbon metabolism may lead to general genomic (global) hypomethylation, which may predispose an individual to the development of colorectal neoplasia.

Methods

We evaluated the association between pre-diagnostic leukocyte genomic DNA methylation level and the risk of colorectal cancer in a nested case-control study of 358 colorectal cancer cases and 661 matched controls within the all-female cohort of the Nurses’ Health Study (NHS). Among control subjects, we further examined major plasma components in the one-carbon metabolism pathway in relation to genomic DNA methylation level. Liquid chromatography/tandem mass spectrometry was used to examine leukocyte genomic DNA methylation level. We calculated odds ratios (ORs) and 95% confidence intervals (95% CIs) using logistic regression.

Results

Overall genomic DNA methylation level was not associated with the risk of colorectal cancer (p for trend, 0.45). Compared with women in the lowest quintile of methylation, the multivariate OR of colorectal cancer risk was 1.32 (95% CI, 0.82–2.13) for those in the highest quintile. We did not find significant associations between major plasma components of one-carbon metabolism or risk factors for colorectal cancer and genomic DNA methylation level (all p for trend >0.05). Also, neither one-carbon metabolism-related plasma components nor well-known risk factors for colorectal cancer modified the association between genomic DNA methylation level and the risk of colorectal cancer (all p for interaction >0.05).

Conclusions

We found no evidence that hypomethylation of leukocyte genomic DNA increases risk of colorectal cancer among women. Additional studies are needed to investigate the association between pre-diagnostic genomic DNA methylation level and colorectal cancer risk among diverse populations.     

Multi-Platform Whole-Genome Microarray Analyses Refine the Epigenetic Signature of Breast Cancer Metastasis with Gene Expression and Copy Number

Background

We have previously identified genome-wide DNA methylation changes in a cell line model of breast cancer metastasis. These complex epigenetic changes that we observed, along with concurrent karyotype analyses, have led us to hypothesize that complex genomic alterations in cancer cells (deletions, translocations and ploidy) are superimposed over promoter-specific methylation events that are responsible for gene-specific expression changes observed in breast cancer metastasis.

Methodology/Principal Findings

We undertook simultaneous high-resolution, whole-genome analyses of MDA-MB-468GFP and MDA-MB-468GFP-LN human breast cancer cell lines (an isogenic, paired lymphatic metastasis cell line model) using Affymetrix gene expression (U133), promoter (1.0R), and SNP/CNV (SNP 6.0) microarray platforms to correlate data from gene expression, epigenetic (DNA methylation), and combination copy number variant/single nucleotide polymorphism microarrays. Using Partek Software and Ingenuity Pathway Analysis we integrated datasets from these three platforms and detected multiple hypomethylation and hypermethylation events. Many of these epigenetic alterations correlated with gene expression changes. In addition, gene dosage events correlated with the karyotypic differences observed between the cell lines and were reflected in specific promoter methylation patterns. Gene subsets were identified that correlated hyper (and hypo) methylation with the loss (or gain) of gene expression and in parallel, with gene dosage losses and gains, respectively. Individual gene targets from these subsets were also validated for their methylation, expression and copy number status, and susceptible gene pathways were identified that may indicate how selective advantage drives the processes of tumourigenesis and metastasis.

Conclusions/Significance

Our approach allows more precisely profiling of functionally relevant epigenetic signatures that are associated with cancer progression and metastasis.     

5-Hydroxymethylcytosine is an essential intermediate of active DNA demethylation processes in primary human monocytes

Background

Cytosine methylation is a frequent epigenetic modification restricting the activity of gene regulatory elements. Whereas DNA methylation patterns are generally inherited during replication, both embryonic and somatic differentiation processes require the removal of cytosine methylation at specific gene loci to activate lineage-restricted elements. However, the exact mechanisms facilitating the erasure of DNA methylation remain unclear in many cases.

Results

We previously established human post-proliferative monocytes as a model to study active DNA demethylation. We now show, for several previously identified genomic sites, that the loss of DNA methylation during the differentiation of primary, post-proliferative human monocytes into dendritic cells is preceded by the local appearance of 5-hydroxymethylcytosine. Monocytes were found to express the methylcytosine dioxygenase Ten-Eleven Translocation (TET) 2, which is frequently mutated in myeloid malignancies. The siRNA-mediated knockdown of this enzyme in primary monocytes prevented active DNA demethylation, suggesting that TET2 is essential for the proper execution of this process in human monocytes.

Conclusions

The work described here provides definite evidence that TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine initiates targeted, active DNA demethylation in a mature postmitotic myeloid cell type.     

Quantification of epigenetic biomarkers: an evaluation of established and emerging methods for DNA methylation analysis

Background

DNA methylation is an important epigenetic mechanism in several human diseases, most notably cancer. The quantitative analysis of DNA methylation patterns has the potential to serve as diagnostic and prognostic biomarkers, however, there is currently a lack of consensus regarding the optimal methodologies to quantify methylation status. To address this issue we compared five analytical methods: (i) MethyLight qPCR, (ii) MethyLight digital PCR (dPCR), methylation-sensitive and -dependent restriction enzyme (MSRE/MDRE) digestion followed by (iii) qPCR or (iv) dPCR, and (v) bisulfite amplicon next generation sequencing (NGS). The techniques were evaluated for linearity, accuracy and precision.

Results

MethyLight qPCR displayed the best linearity across the range of tested samples. Observed methylation measured by MethyLight- and MSRE/MDRE-qPCR and -dPCR were not significantly different to expected values whilst bisulfite amplicon NGS analysis over-estimated methylation content. Bisulfite amplicon NGS showed good precision, whilst the lower precision of qPCR and dPCR analysis precluded discrimination of differences of < 25% in methylation status. A novel dPCR MethyLight assay is also described as a potential method for absolute quantification that simultaneously measures both sense and antisense DNA strands following bisulfite treatment.

Conclusions

Our findings comprise a comprehensive benchmark for the quantitative accuracy of key methods for methylation analysis and demonstrate their applicability to the quantification of circulating tumour DNA biomarkers by using sample concentrations that are representative of typical clinical isolates.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1174) contains supplementary material, which is available to authorized users.     

Genome-Wide Divergence of DNA Methylation Marks in Cerebral and Cerebellar Cortices

Background

Emerging evidence suggests that DNA methylation plays an expansive role in the central nervous system (CNS). Large-scale whole genome DNA methylation profiling of the normal human brain offers tremendous potential in understanding the role of DNA methylation in brain development and function.

Methodology/Significant Findings

Using methylation-sensitive SNP chip analysis (MSNP), we performed whole genome DNA methylation profiling of the prefrontal, occipital, and temporal regions of cerebral cortex, as well as cerebellum. These data provide an unbiased representation of CpG sites comprising 377,509 CpG dinucleotides within both the genic and intergenic euchromatic region of the genome. Our large-scale genome DNA methylation profiling reveals that the prefrontal, occipital, and temporal regions of the cerebral cortex compared to cerebellum have markedly different DNA methylation signatures, with the cerebral cortex being hypermethylated and cerebellum being hypomethylated. Such differences were observed in distinct genomic regions, including genes involved in CNS function. The MSNP data were validated for a subset of these genes, by performing bisulfite cloning and sequencing and confirming that prefrontal, occipital, and temporal cortices are significantly more methylated as compared to the cerebellum.

Conclusions

These findings are consistent with known developmental differences in nucleosome repeat lengths in cerebral and cerebellar cortices, with cerebrum exhibiting shorter repeat lengths than cerebellum. Our observed differences in DNA methylation profiles in these regions underscores the potential role of DNA methylation in chromatin structure and organization in CNS, reflecting functional specialization within cortical regions.     

Genome-wide signatures of differential DNA methylation in pediatric acute lymphoblastic leukemia

Background

Although aberrant DNA methylation has been observed previously in acute lymphoblastic leukemia (ALL), the patterns of differential methylation have not been comprehensively determined in all subtypes of ALL on a genome-wide scale. The relationship between DNA methylation, cytogenetic background, drug resistance and relapse in ALL is poorly understood.

Results

We surveyed the DNA methylation levels of 435,941 CpG sites in samples from 764 children at diagnosis of ALL and from 27 children at relapse. This survey uncovered four characteristic methylation signatures. First, compared with control blood cells, the methylomes of ALL cells shared 9,406 predominantly hypermethylated CpG sites, independent of cytogenetic background. Second, each cytogenetic subtype of ALL displayed a unique set of hyper- and hypomethylated CpG sites. The CpG sites that constituted these two signatures differed in their functional genomic enrichment to regions with marks of active or repressed chromatin. Third, we identified subtype-specific differential methylation in promoter and enhancer regions that were strongly correlated with gene expression. Fourth, a set of 6,612 CpG sites was predominantly hypermethylated in ALL cells at relapse, compared with matched samples at diagnosis. Analysis of relapse-free survival identified CpG sites with subtype-specific differential methylation that divided the patients into different risk groups, depending on their methylation status.

Conclusions

Our results suggest an important biological role for DNA methylation in the differences between ALL subtypes and in their clinical outcome after treatment.     

Deciphering the heterogeneity in DNA methylation patterns during stem cell differentiation and reprogramming

Background

Human induced pluripotent stem cells (iPSCs) have a wide range of applications throughout the fields of basic research, disease modeling and drug screening. Epigenetic instable iPSCs with aberrant DNA methylation may divide and differentiate into cancer cells. Unfortunately, little effort has been taken to compare the epigenetic variation in iPSCs with that in differentiated cells. Here, we developed an analytical procedure to decipher the DNA methylation heterogeneity of mixed cells and further exploited it to quantitatively assess the DNA methylation variation in the methylomes of adipose-derived stem cells (ADS), mature adipocytes differentiated from ADS cells (ADS-adipose) and iPSCs reprogrammed from ADS cells (ADS-iPSCs).

Results

We observed that the degree of DNA methylation variation varies across distinct genomic regions with promoter and 5’UTR regions exhibiting low methylation variation and Satellite showing high methylation variation. Compared with differentiated cells, ADS-iPSCs possess globally decreased methylation variation, in particular in repetitive elements. Interestingly, DNA methylation variation decreases in promoter regions during differentiation but increases during reprogramming. Methylation variation in promoter regions is negatively correlated with gene expression. In addition, genes showing a bipolar methylation pattern, with both completely methylated and completely unmethylated reads, are related to the carbohydrate metabolic process, cellular development, cellular growth, proliferation, etc.

Conclusions

This study delivers a way to detect cell-subset specific methylation genes in a mixed cell population and provides a better understanding of methylation dynamics during stem cell differentiation and reprogramming.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-978) contains supplementary material, which is available to authorized users.     

DNA Methylation Profiles of Airway Epithelial Cells and PBMCs from Healthy, Atopic and Asthmatic Children

Background

Allergic inflammation is commonly observed in a number of conditions that are associated with atopy including asthma, eczema and rhinitis. However, the genetic, environmental or epigenetic factors involved in these conditions are likely to be different. Epigenetic modifications, such as DNA methylation, can be influenced by the environment and result in changes to gene expression.

Objectives

To characterize the DNA methylation pattern of airway epithelial cells (AECs) compared to peripheral blood mononuclear cells (PBMCs) and to discern differences in methylation within each cell type amongst healthy, atopic and asthmatic subjects.

Methods

PBMCs and AECs from bronchial brushings were obtained from children undergoing elective surgery for non-respiratory conditions. The children were categorized as atopic, atopic asthmatic, non-atopic asthmatic or healthy controls. Extracted DNA was bisulfite treated and 1505 CpG loci across 807 genes were analyzed using the Illumina GoldenGate Methylation Cancer Panel I. Gene expression for a subset of genes was performed using RT-PCR.

Results

We demonstrate a signature set of CpG sites that are differentially methylated in AECs as compared to PBMCs regardless of disease phenotype. Of these, 13 CpG sites were specific to healthy controls, 8 sites were only found in atopics, and 6 CpGs were unique to asthmatics. We found no differences in the methylation status of PBMCs between disease phenotypes. In AECs derived from asthmatics compared to atopics, 8 differentially methylated sites were identified including CpGs in STAT5A and CRIP1. We demonstrate STAT5A gene expression is decreased whereas CRIP1 gene expression is elevated in the AECs from asthmatic compared to both healthy and atopic subjects.

Discussion

We characterized a cell specific DNA methylation signature for AECs compared to PBMCs regardless of asthmatic or atopic status. Our data highlight the importance of understanding DNA methylation in the epithelium when studying the epithelial contribution to asthma.