The DNA methylome of human peripheral blood mononuclear cells
ABSTRACT: DNA methylation plays a vital role in genome dynamics and, in the human genome, occurs predominantly at cytosine guanine dinucleotide (CpG) sites. The diploid haploid human genome analysed here contains around 2060 million CpG sites (methylome) where DNA methylation can vary, affecting many biological processes in health and disease. Using whole-genome bisulfite sequencing, we report the essentially complete (92.6282%) methylome of human peripheral blood mononuclear cells (PBMC) which constitute an important source for clinical blood tests world-wide. We find the majority of CpG sites (68.4% at false positive rate of 0.46%) and only <0.2% of non-CpG sites to be methylated, demonstrating that non-CpG cytosine methylation is negligible in human PBMC. Analysis of the PBMC methylome revealed a rich landscape of epigenomic data for 20 distinct features including regulatory, protein-coding, RNA gene coding, non-coding and repeat sequences. Alu element mobility, for instance, was found to negatively correlate with their methylation levels, emphasizing the critical role of DNA methylation in genome stability. Integration of our methylome data with the previously determined genome sequence of the same Asian individual analysed here, enabled a first assessment of allele-specific methylation (ASM) differences between the two haploid methylomes of any individual. Using a conservative cut-off (p <0.001), we identified 599 haploid differentially methylated regions (hDMRs) covering 287 genes. Of these, 76 genes had hDMRs within 2kb of their transcriptional start sites of which >80% displayed allele-specific expression (ASE) after random testing using TA clone sequencing of the same PBMC sample. These data show, that ASM is a recurrent phenomenon and highly correlated with ASE, suggesting that imprinting may be more common than previously thought. Our study not only provides a comprehensive resource for future epigenomic research but also demonstrates a paradigm of large-scale epigenomics studies through new sequencing technology. Overall design: We report the essentially complete (92.6282%) methylome of human peripheral blood mononuclear cells (PBMC) which constitute an important source for clinical blood tests world-wide.
Project description:DNA methylation plays a vital role in genome dynamics and, in the human genome, occurs predominantly at cytosine guanine dinucleotide (CpG) sites. The diploid haploid human genome analysed here contains around 2060 million CpG sites (methylome) where DNA methylation can vary, affecting many biological processes in health and disease. Using whole-genome bisulfite sequencing, we report the essentially complete (92.6282%) methylome of human peripheral blood mononuclear cells (PBMC) which constitute an important source for clinical blood tests world-wide. We find the majority of CpG sites (68.4% at false positive rate of 0.46%) and only <0.2% of non-CpG sites to be methylated, demonstrating that non-CpG cytosine methylation is negligible in human PBMC. Analysis of the PBMC methylome revealed a rich landscape of epigenomic data for 20 distinct features including regulatory, protein-coding, RNA gene coding, non-coding and repeat sequences. Alu element mobility, for instance, was found to negatively correlate with their methylation levels, emphasizing the critical role of DNA methylation in genome stability. Integration of our methylome data with the previously determined genome sequence of the same Asian individual analysed here, enabled a first assessment of allele-specific methylation (ASM) differences between the two haploid methylomes of any individual. Using a conservative cut-off (p <0.001), we identified 599 haploid differentially methylated regions (hDMRs) covering 287 genes. Of these, 76 genes had hDMRs within 2kb of their transcriptional start sites of which >80% displayed allele-specific expression (ASE) after random testing using TA clone sequencing of the same PBMC sample. These data show, that ASM is a recurrent phenomenon and highly correlated with ASE, suggesting that imprinting may be more common than previously thought. Our study not only provides a comprehensive resource for future epigenomic research but also demonstrates a paradigm of large-scale epigenomics studies through new sequencing technology. We report the essentially complete (92.6282%) methylome of human peripheral blood mononuclear cells (PBMC) which constitute an important source for clinical blood tests world-wide.
Project description:In this study, we investigated the interaction between CpG methylation and genetic polymorphisms by taking the advantage of the family structure in 22 nuclear pedigrees. We have identified CpG sites that exhibit heritable methylation patterns, among which the majority are SNPs ditrectly disrupting CpG dinucleotides. We also identified 27.2% of the heritable non-SNP CpGs were associated with cis-regulatory SNPs. Additionally, we have identified hundreds of CpG clusters whose the degree of DNA methylation variation is associated with genetic polymorphism. Investigate the influence of genetic variances on blood DNA methylation patterns in the human genome of 96 subjects from 22 pedigrees by using different approaches, including mid-parent offspring analysis (MPO), methylation quantitative trait loci (mQTL) analysis and allele-specific DNA methylation (ASM) Raw data not provided since the files contain genetic information of the human subject that should be protected.
Project description:Haplotype-dependent allele-specific methylation (hap-ASM) can impact disease susceptibility, but maps of this phenomenon using stringent criteria in disease-relevant tissues remain sparse. Here we apply array-based and Methyl-seq approaches to multiple human tissues and cell types, including brain, purified neurons and glia, T lymphocytes, and placenta, and identify 795 hap-ASM differentially methylated regions (DMRs) and 3,082 strong methylation quantitative trait loci (mQTLs), most not previously reported. More than half of these DMRs have cell type-restricted ASM, and among them are 188 hap-ASM DMRs and 933 mQTLs located near GWAS signals for immune and neurological disorders. Targeted bis-seq confirmed hap-ASM in 12/13 loci tested, including CCDC155, CD69, FRMD1, IRF1, KBTBD11, and S100A*-ILF2, associated with immune phenotypes, MYT1L, PTPRN2,CMTM8 and CELF2, associated with neurological disorders, NGFR and HLA-DRB6, associated with both immunological and brain disorders, and ZFP57, a trans-acting regulator of genomic imprinting. Polymorphic CTCF and transcription factor (TF) binding sites are over-represented among hap-ASM DMRs and mQTLs, and analysis of the human data, supplemented by cross-species comparisons to Macaca mulattamacaques, indicates that CTCF and TF binding likelihood predicts the strength and direction of the allelic methylation asymmetry. These results show that hap-ASM is highly tissue-specific; an important trans-acting regulator of genomic imprinting is regulated by this phenomenon; variation in CTCF and TF binding sites is an underlying mechanism in primary tissues, and maps of hap-ASM and mQTLs reveal regulatory sequences underlying supera- and sub-threshold GWAS peaks in immunological and neurological disorders. Overall design: We used the Agilent SureSelect Methyl-seq DNA hybrid capture kit, followed by deep Nextgen bis-seq. In this protocol, targeted regions (total of 3.7M CpGs) include RefGenes, promoter regions, CpG islands, CpG island shores, shelves, and DNAse I hypersensitive sites. DNA was sheared to an average size of 250 bp and bisulfite converted with the EZ DNA methylation kit (Zymo). Paired-end reads (150 bp) were generated with an Illumina HiSeq2000 sequencer. One of the brain samples (5984) was relatively under-represented in the library, so additional sequences were generated on a MiSeq sequencer to improve the coverage for this sample. After trimming for low-quality bases (Phred score<30) and reads with a length <40 bp with TrimGalore, the reads were aligned to the human genome (GRCh37) using Bismark and duplicate reads were removed using Samtools. Methylation calling was performed using Bismark and and SNP calling was performed with BisSNP with default settings and dbSNP137 as references. ASM calling was performed with Bismark, after separating the valid SNP-containing reads by allele.
Project description:To determine whether differences between background strains or housing conditions altered the hepatic methylome, We report the generation and analysis of genome-wide DNA methylation profiles at nucleotide resolution in mouse liver from two male mice on a mixed background (mixed-1, mixed-2) and two males on a pure Black-6 (B6-1, B62) background. Using Enhanced high-throughput Reduced Representation Bisulfite Sequencing (ERRBS), we enriched CpG islands in mouse liver, and covered a representative sampling of conserved non-coding elements, transposons and other genomic features, for mouse liver. We found that the total CpG methylation of each methylome was strikingly similar among the 4 mouse liver samples from two different genetic backgrounds. Analysis of all CpG sites with at least 10x coverage showed a bimodal distribution of methylation, with all samples having 25% of hypermethylated CpG sites and 60% as hypomethylated CpG sites. Given the high percent of genome coverage and robust depth at single nucleotide level, these datasets provide a resource for investigation into changes in DNA methylation patterns in liver disease, tumorigenesis and regeneration. Ehanced reduced representation bisulfite sequencing (MspI 70~320bp size fraction) of liver tissue
Project description:Genome wide DNA methylation profiling of cord blood in Faroe Island cohort. The Illumina Infinium 450k Human DNA methylation Beadchip was used to obtain DNA methylation profiles across approximately 485,000 CpGs in cord blood samples. Background: 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. Objective: We aimed to identify key environmental chemicals in cord blood associated with DNA methylation changes in a population with elevated exposure to chemical mixtures. Method: 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 Methylation 450K 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. Results: 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. Conclusion: In this multiple-pollutant and genome-wide study, we identified key epigenetic toxicants. The significant enrichment of specific X-chromosome sites in males implies potentially sex-specific epigenome responses to prenatal chemical exposures. Overall design: Bisulphite converted DNA from the 72 samples were hybridised to the Illumina Infinium 450k Human Methylation Beadchip (samples 2, 3, 4, 6, 12, 13, 15, 17, 18, 24, 30, 32, 33, 34, 40, 42, 43, 44, 46, 68 and 71 were removed from the final analysis due to poor quality).
Project description:Methylation of cytosines at CpG sites is a common epigenetic DNA modification that can be measured by a large number of methods, now even in a genome-wide manner for hundreds of thousands of sites. The application of DNA methylation analysis is becoming widely popular in complex disorders, for example, to understand part of the “missing inheritance”. The DNA samples most readily available for methylation studies are derived from whole blood. However, blood consists of many functionally and developmentally distinct cell populations in varying proportions. We studied whether such variation might affect the interpretation of methylation studies based on whole blood DNA. We found in healthy male blood donors there is important variation in the methylation profiles of whole blood, mononuclear cells, granulocytes, and cells from seven selected purified lineages. CpG methylation between mononuclear cells and granulocytes differed for 22% of the 8252 probes covering the selected 343 genes implicated in immune-related disorders by genome-wide association studies, and at least one probe was differentially methylated for 85% of the genes, indicating that whole blood methylation results might be unintelligible. For individual genes, even if the overall methylation patterns might appear similar, a few CpG sites in the regulatory regions may have opposite methylation patterns (i.e., hypo/hyper) in the main blood cell types. We conclude that interpretation of whole blood methylation profiles should be performed with great caution and for any differences implicated in a disorder, the differences resulting from varying proportions of white blood cell types should be considered. Six healthy male blood donors, age 38 ± 13.6 years, were included in the study. From each individual, global DNA methylation levels were analyzed in whole blood, peripheral blood mononuclear cells (PBMC) and granulocytes as well as for seven isolated cell populations (CD4+ T cells, CD8+ T cells, CD56+ NK cells, CD19+ B cells, CD14+ monocytes, neutrophils, and eosinophils), n=60 samples analyzed in total.
Project description:Elevated plasma homocysteine is an independent risk factor for cardiovascular disease and stroke, however the etiology remains poorly understood. Elevated homocysteine is known to inhibit methyltransferases including DNA methyltransferases, but no methylome-wide analysis of elevated homocysteine has been reported. Peripheral blood genomic DNA methylation in 8 Singaporean-Chinese ischemic stroke patients (4 male, 4 female) with varying homocysteine titer and hypertensive status were profiled using methyl-CpG binding domain (MBD) protein-enriched genome sequencing (MBD-seq) on Illumina Genome Analyzer IIx. A methylome wide screen was undertaken for gender, total plasma homocysteine, hypertension and age. The data show considerable variability within the small cohort, including at genes which are related to one carbon metabolism and cardiovascular disease. Peripheral blood genomic DNA methylation in 8 Singaporean-Chinese ischemic stroke patients (4 male, 4 female) was profiled using methyl-CpG binding domain (MBD) protein-enriched genome sequencing (MBD-seq) on Illumina Genome Analyzer IIx. Methylation parrterns were correlated with homocysteine levels, lypertensive status, gender and age.
Project description:Genome-scale measurements of DNA methylation levels are necessary to decipher the epigenetic events involved in glioblastoma aggressive phenotype, and to guide new therapeutic strategies. In that purpose, we performed a whole genome integrative analysis of the methylation and expression profiles for 40 newly diagnosed glioblastoma patients. We have also screened for associations between CpG sites methylation levels and overall survival in a cohort of 50 patients uniformly treated with radiotherapy and chemotherapy with concomitant and adjuvant temozolomide (STUPP protocol). The methylation analysis identified 616 CpG sites differentially methylated between glioblastoma and control brain, a quarter being differentially expressed in a concordant way. Among these concordant CpG sites, 13 genes displayed, within our glioblastoma cohort, an inverse correlation between promoter methylation and expression levels: B3GNT5, FABP7, ZNF217, BST2, OAS1, SLC13A5, GSTM5, ME1, UBXD3, TSPYL5, FAAH, C7orf13, and C3orf14. The expression of these genes may be tightly regulated by epigenetic mechanisms. The survival analysis identified six CpG sites associated with overall survival. The SOX10 promoter methylation status (two CpG sites) stratifies the patients in a way similar to MGMT with improved performance based on Area Under the Curve criteria (0.78 vs. 0.71, p-value < 5.10-4). The methylation status of FNDC3B, TBX3, DGKI, and FSD1 promoters identify patients with MGMT methylated tumors non-responding to STUPP treatment (p-value < 1.10-4). These markers have a potential impact on therapeutic decision. 40 glioblastoma samples and 6 control brain samples were analysed. 2 distinct series of hybridizations were carried out, each containing GBMs and control brains.
Project description:Purpose: We characterized genome-wide DNA methylation profiles (methylome) in purified peripheral blood monocytes (PBMs) from 18 healthy postmenopausal Caucasian females aged 50-56 years. Methods: DNA methylome of Human Peripheral Blood Monocytes were generated by methylated DNA immunoprecipitation combined with high-throughput sequencing (MeDIP-seq), using Illumina GAIIx. The sequence reads that passed quality filters were analyzed using MEDIPS package. Targeted methylation validation analysis was performed by using MassARRAY EpiTYPER assays. Genome-wide gene expression profiles have been obtained for 7 of the 18 subjects by using Affymetrix 1.0 Human Exon ST arrays following the manufacturer's recommended protocols. Results: Using MeDIP-seq,a total of approximately 283 million reads were uniquely aligned to human genome (Build NCBI37, HG19), resulting in average ~16 million uniquely aligned high quality reads per sample. Distinct patterns were revealed at different genomic features. For instance, promoters were commonly (~58%) found to be unmethylated; whereas protein coding regions were largely (~84%) methylated. We found that approximately 24% CpG islands (CGIs) were highly methylated in PBMs. Further characterization of CGIs with respect to their relative locations to RefSeq genes revealed that the highly methylated CGIs were largely enriched (~89%) in CGIs located in gene bodies and intergenic regions. By integration of the methylome data with genome-wide PBM gene expression data, we found negative correlation between promoter methylation levels and gene transcription levels when comparing groups of genes with different expression levels, and this relationship was consistently observed across promoters with high to low CpG densities. Furthermore, we observed a modest but significant excess (permutation p<0.0001) of genes showing negative correlation between inter-individual promoter methylation and transcription levels, particularly for genes associated with CpG-rich promoters. Across the 18 individual PBM methylomes, we also identified genomic regions that were constitutively highly methylated in PBMs as well as regions showing large inter-individual variability. Conclusions: This study represents a comprehensive analysis of the PBM methylome and our data provides a valuable resource for future epigenomic and multi-omic studies exploring biological and disease-related regulatory mechanisms in PBMs. DNA methylome of human peripheral blood monocytes were generated by MeDIP-seq, using Illumina GAIIx.
Project description:Treatment with recombinant human growth hormone (rhGH) has been consistently reported to induce transcriptional changes in various human tissues including peripheral blood. For other hormones it has been shown that the induction of such transcriptional effects is conferred or at least accompanied by DNA-methylation changes. To analyse effects of short term rhGH treatment on the DNA-methylome we investigated a total of 24 patients at baseline and after 4-day rhGH stimulation. We performed array-based DNA-methylation profiling of paired peripheral blood mononuclear cell samples followed by targeted validation using bisulfite pyrosequencing. Unsupervised analysis of DNA-methylation in this short-term treated cohort revealed clustering according to individuals rather than treatment. Supervised analysis identified 239 CpGs as significantly differentially methylated between baseline and rhGH-stimulated samples (p<0.0001, unadjusted paired t-test), which nevertheless did not retain significance after adjustment for multiple testing. An individualised evaluation strategy led to the identification of 2350 CpG and 3 CpH sites showing methylation differences of at least 10% in more than 2 of the 24 analysed sample pairs. To investigate the long term effects of rhGH treatment on the DNA-methylome, we analysed peripheral blood cells from an independent cohort of 36 rhGH treated children born small for gestational age (SGA) as compared to 18 untreated controls. Median treatment interval was 33 months. In line with the groupwise comparison in the short-term treated cohort no differentially methylated targets reached the level of significance in the long-term treated cohort. We identified marked intra-individual responses of DNA-methylation to short-term rhGH treatment. These responses seem to be predominately associated with immunologic functions and show considerable inter-individual heterogeneity. The latter is likely the cause for the lack of a rhGH induced homogeneous DNA-methylation signature after short- and long-term treatment, which nevertheless is well in line with generally assumed safety of rhGH treatment. Bisulfite-converted DNA of PBMC from 24 patients before and after 4 days of rhGH treatment were hybridized to the Illumina Infinium HumanMethylation 450k BeadChip.