Tissue-specific patterns of allelically-skewed DNA methylation.
ABSTRACT: While DNA methylation is usually thought to be symmetrical across both alleles, there are some notable exceptions. Genomic imprinting and X chromosome inactivation are two well-studied sources of allele-specific methylation (ASM), but recent research has indicated a more complex pattern in which genotypic variation can be associated with allelically-skewed DNA methylation in cis. Given the known heterogeneity of DNA methylation across tissues and cell types we explored inter- and intra-individual variation in ASM across several regions of the human brain and whole blood from multiple individuals. Consistent with previous studies, we find widespread ASM with > 4% of the ?220,000 loci interrogated showing evidence of allelically-skewed DNA methylation. We identify ASM flanking known imprinted regions, and show that ASM sites are enriched in DNase I hypersensitivity sites and often located in an extended genomic context of intermediate DNA methylation. We also detect examples of genotype-driven ASM, some of which are tissue-specific. These findings contribute to our understanding of the nature of differential DNA methylation across tissues and have important implications for genetic studies of complex disease. As a resource to the community, ASM patterns across each of the tissues studied are available in a searchable online database: http://epigenetics.essex.ac.uk/ASMBrainBlood.
Project description:Numerous recent studies have suggested that phenotypic effects of DNA sequence variants can be mediated or modulated by their epigenetic marks, such as allele-skewed DNA modification (ASM). Using Affymetrix SNP microarrays, we performed a comprehensive search of ASM effects in human post-mortem brain and sperm samples (total n = 256) from individuals with major psychosis and control individuals. Depending on the phenotypic category of the brain samples, 1.4%-7.5% of interrogated SNPs exhibited ASM effects. Next, we investigated ASM in the context of genetic studies of schizophrenia and detected that brain ASM SNPs were significantly overrepresented among sub-threshold SNPs from a schizophrenia genome-wide association study (GWAS). Brain ASM SNPs showed a much stronger enrichment in a schizophrenia GWAS than in 17 large GWASs of non-psychiatric diseases and traits, arguing that ASM effects are at least partially tissue specific. Studies of germline and control brain ASM SNPs supported a causal association between ASM and schizophrenia. Finally, significantly higher proportions of ASM SNPs than of non-ASM SNPs were detected at loci exhibiting epigenetic signatures of enhancers and promoters, and they were overrepresented within transcription factor binding regions and DNase I hypersensitive sites. All of these findings collectively indicate that ASM SNPs should be prioritized in follow-up GWASs.
Project description:DNA methylation is assumed to be complementary on both alleles across the genome, although there are exceptions, notably in regions subject to genomic imprinting. We present a genome-wide survey of the degree of allelic skewing of DNA methylation with the aim of identifying previously unreported differentially methylated regions (DMRs) associated primarily with genomic imprinting or DNA sequence variation acting in cis. We used SNP microarrays to quantitatively assess allele-specific DNA methylation (ASM) in amplicons covering 7.6% of the human genome following cleavage with a cocktail of methylation-sensitive restriction enzymes (MSREs). Selected findings were verified using bisulfite-mapping and gene-expression analyses, subsequently tested in a second tissue from the same individuals, and replicated in DNA obtained from 30 parent-child trios. Our approach detected clear examples of ASM in the vicinity of known imprinted loci, highlighting the validity of the method. In total, 2,704 (1.5%) of our 183,605 informative and stringently filtered SNPs demonstrate an average relative allele score (RAS) change > or =0.10 following MSRE digestion. In agreement with previous reports, the majority of ASM ( approximately 90%) appears to be cis in nature, and several examples of tissue-specific ASM were identified. Our data show that ASM is a widespread phenomenon, with >35,000 such sites potentially occurring across the genome, and that a spectrum of ASM is likely, with heterogeneity between individuals and across tissues. These findings impact our understanding about the origin of individual phenotypic differences and have implications for genetic studies of complex disease.
Project description:Autoimmune thyroid diseases (AITDs) predominantly develop in females. One of two X chromosomes is randomly inactivated by methylation in each female cell, but it has been reported that skewed X chromosome inactivation (XCI) may be associated with the development of autoimmune diseases. To clarify the significance of skewed XCI in the prognosis and development of AITD, we investigated the proportion of skewed XCI in female patients with AITD.We analyzed the degree of XCI skewing in 120 female patients with AITD (77 patients with Graves' disease [GD] and 43 patients with Hashimoto's disease [HD]) and 49 female controls in DNA from peripheral blood mononuclear cells (PBMC). We performed XCI analysis by digesting inactive DNA with a methylation-sensitive restriction enzyme (HpaII) followed by a polymerase chain reaction (PCR) assay for the polymorphic CAG repeat of the androgen receptor gene and electrophoresis of the PCR products.The proportion of skewed XCI (≥65% skewing) was not significantly different between AITD patients and control subjects but was higher in patients with intractable GD (66.7%) than those with GD in remission (25.0%, p=0.0033) and control subjects (32.6%, p=0.0038). When the cutoff value for XCI skewing was relaxed, the proportion of skewed XCI (≥60% skewing) was higher in patients with severe HD (76.5%) than in those with mild HD (41.2%, p=0.0342).Skewed XCI is related to the prognosis of AITD, particularly the intractability of GD.
Project description:In diploid mammalian genomes, parental alleles can exhibit different methylation patterns (allele-specific DNA methylation, ASM), which have been documented in a small number of cases except for the imprinted regions and X chromosomes in females. We carried out a chromosome-wide survey of ASM across 16 human pluripotent and adult cell lines using Illumina bisulfite sequencing. We applied the principle of linkage disequilibrium (LD) analysis to characterize the correlation of methylation between adjacent CpG sites on single DNA molecules, and also investigated the correlation between CpG methylation and single nucleotide polymorphisms (SNPs). We observed ASM on 23% approximately 37% heterozygous SNPs in any given cell line. ASM is often cell-type-specific. Furthermore, we found that a significant fraction (38%-88%) of ASM regions is dependent on the presence of heterozygous SNPs in CpG dinucleotides that disrupt their methylation potential. This study identified distinct types of ASM across many cell types and suggests a potential role for CpG-SNP in connecting genetic variation with the epigenome.
Project description:Background:Genetic variants within the fatty acid desaturase (FADS) gene cluster (human Chr11) are important regulators of long-chain (LC) polyunsaturated fatty acid (PUFA) biosynthesis in the liver and consequently have been associated with circulating LC-PUFA levels. More recently, epigenetic modifications such as DNA methylation, particularly within the FADS cluster, have been shown to affect LC-PUFA levels. Our lab previously demonstrated strong associations of allele-specific methylation (ASM) between a single nucleotide polymorphism (SNP) rs174537 and CpG sites across the FADS region in human liver tissues. Given that epigenetic signatures are tissue-specific, we aimed to evaluate the methylation status and ASM associations between rs174537 and DNA methylation obtained from human saliva, CD4+ cells and total leukocytes derived from whole blood. The goals were to (1) determine if DNA methylation from these peripheral samples would display similar ASM trends as previously observed in human liver tissues and (2) evaluate the associations between DNA methylation and circulating LC-PUFAs. Results:DNA methylation at six CpG sites spanning FADS1 and FADS2 promoter regions and a putative FADS enhancer region were determined in two Caucasian cohorts of healthy volunteers: leukocytes in cohort 1 (n?=?89, median age?=?43, 35% male) and saliva and CD4+ cells in cohort 2 (n?=?32, median age?=?41, 41% male). Significant ASM between rs174537 and DNA methylation at three CpG sites located in the FADS2 promoter region (i.e., chr11:61594865, chr11:61594876, chr11:61594907) and one CpG site in the putative enhancer region (chr11:61587979) were observed with leukocytes. In CD4+ cells, significant ASM was observed at CpG sites chr11:61594876 and chr11:61584894. Genotype at rs174537 was significantly associated with DNA methylation from leukocytes. Similar trends were observed with CD4+ cells, but not with saliva. DNA methylation from leukocytes and CD4+ cells also significantly correlated with circulating omega-6 LC-PUFAs. Conclusions:We observed significant ASM between rs174537 and DNA methylation at key regulatory regions in the FADS region from leukocyte and CD4+ cells. DNA methylation from leukocytes also correlated with circulating omega-6 LC-PUFAs. These results support the use of peripheral whole blood samples, with leukocytes showing the most promise for future nutrigenomic studies evaluating epigenetic modifications affecting LC-PUFA biosynthesis in humans.
Project description:Given the tissue-specific nature of epigenetic processes, the assessment of disease-relevant tissue is an important consideration for epigenome-wide association studies (EWAS). Little is known about whether easily accessible tissues, such as whole blood, can be used to address questions about interindividual epigenomic variation in inaccessible tissues, such as the brain. We quantified DNA methylation in matched DNA samples isolated from whole blood and 4 brain regions (prefrontal cortex, entorhinal cortex, superior temporal gyrus, and cerebellum) from 122 individuals. We explored co-variation between tissues and the extent to which methylomic variation in blood is predictive of interindividual variation identified in the brain. For the majority of DNA methylation sites, interindividual variation in whole blood is not a strong predictor of interindividual variation in the brain, although the relationship with cortical regions is stronger than with the cerebellum. Variation at a subset of probes is strongly correlated across tissues, even in instances when the actual level of DNA methylation is significantly different between them. A substantial proportion of this co-variation, however, is likely to result from genetic influences. Our data suggest that for the majority of the genome, a blood-based EWAS for disorders where brain is presumed to be the primary tissue of interest will give limited information relating to underlying pathological processes. These results do not, however, discount the utility of using a blood-based EWAS to identify biomarkers of disease phenotypes manifest in the brain. We have generated a searchable database for the interpretation of data from blood-based EWAS analyses ( http://epigenetics.essex.ac.uk/bloodbrain/).
Project description:Genetic polymorphisms can shape the global landscape of DNA methylation, by either changing substrates for DNA methyltransferases or altering the DNA binding affinity of cis-regulatory proteins. The interactions between CpG methylation and genetic polymorphisms have been previously investigated by methylation quantitative trait loci (mQTL) and allele-specific methylation (ASM) analysis. However, it remains unclear whether these approaches can effectively and comprehensively identify all genetic variants that contribute to the inter-individual variation of DNA methylation levels. Here we used three independent approaches to systematically investigate the influence of genetic polymorphisms on variability in DNA methylation by characterizing the methylation state of 96 whole blood samples in 52 parent-child trios from 22 nuclear pedigrees. We performed targeted bisulfite sequencing with padlock probes to quantify the absolute DNA methylation levels at a set of 411,800 CpG sites in the human genome. With mid-parent offspring analysis (MPO), we identified 10,593 CpG sites that exhibited heritable methylation patterns, among which 70.1% were SNPs directly present in methylated CpG dinucleotides. We determined the mQTL analysis identified 49.9% of heritable CpG sites for which regulation occurred in a distal cis-regulatory manner, and that ASM analysis was only able to identify 5%. Finally, we identified hundreds of clusters in the human genome for which the degree of variation of CpG methylation, as opposed to whether or not CpG sites were methylated, was associated with genetic polymorphisms, supporting a recent hypothesis on the genetic influence of phenotypic plasticity. These results show that cis-regulatory SNPs identified by mQTL do not comprise the full extent of heritable CpG methylation, and that ASM appears overall unreliable. Overall, the extent of genome-methylome interactions is well beyond what is detectible with the commonly used mQTL and ASM approaches, and is likely to include effects on plasticity.
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 were over-represented among hap-ASM DMRs and mQTLs, and analysis of the human data, supplemented by cross-species comparisons to macaques, indicated 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; and variation in CTCF and TF binding sites is an underlying mechanism, and maps of hap-ASM and mQTLs reveal regulatory sequences underlying supra- and sub-threshold GWAS peaks in immunological and neurological disorders.
Project description:Genome-wide analysis of gene expression or protein binding patterns using different array or sequencing based technologies is now routinely performed to compare different populations, such as treatment and reference groups. It is often necessary to normalize the data obtained to remove technical variation introduced in the course of conducting experimental work, but standard normalization techniques are not capable of eliminating technical bias in cases where the distribution of the truly altered variables is skewed, i.e. when a large fraction of the variables are either positively or negatively affected by the treatment. However, several experiments are likely to generate such skewed distributions, including ChIP-chip experiments for the study of chromatin, gene expression experiments for the study of apoptosis, and SNP-studies of copy number variation in normal and tumour tissues. A preliminary study using spike-in array data established that the capacity of an experiment to identify altered variables and generate unbiased estimates of the fold change decreases as the fraction of altered variables and the skewness increases. We propose the following work-flow for analyzing high-dimensional experiments with regions of altered variables: (1) Pre-process raw data using one of the standard normalization techniques. (2) Investigate if the distribution of the altered variables is skewed. (3) If the distribution is not believed to be skewed, no additional normalization is needed. Otherwise, re-normalize the data using a novel HMM-assisted normalization procedure. (4) Perform downstream analysis. Here, ChIP-chip data and simulated data were used to evaluate the performance of the work-flow. It was found that skewed distributions can be detected by using the novel DSE-test (Detection of Skewed Experiments). Furthermore, applying the HMM-assisted normalization to experiments where the distribution of the truly altered variables is skewed results in considerably higher sensitivity and lower bias than can be attained using standard and invariant normalization methods.
Project description:BACKGROUND: Genes subject to genomic imprinting are mono-allelically expressed in a parent-of-origin dependent manner. Each imprinted locus has at least one differentially methylated region (DMR) which has allele specific DNA methylation and contributes to imprinted gene expression. Once DMRs are established, they are potentially able to withstand normal genome reprogramming events that occur during cell differentiation and germ-line DMRs are stably maintained throughout development. These DMRs, in addition to being either maternally or paternally methylated, have differences in whether methylation was acquired in the germ-line or post fertilization and are present in a variety of genomic locations with different Cytosine-phosphate guanine (CpG) densities and CTCF binding capacities. We therefore examined the stability of maintenance of DNA methylation imprints and determined the normal baseline DNA methylation levels in several adult tissues for all imprinted genes. In order to do this, we first developed and validated 50 highly specific, quantitative DNA methylation pyrosequencing assays for the known DMRs associated with human imprinted genes. RESULTS: Remarkable stability of the DNA methylation imprint was observed in all germ-line DMRs and paternally methylated somatic DMRs (which maintained average methylation levels of between 35% - 65% in all somatic tissues, independent of gene expression). Maternally methylated somatic DMRs were found to have more variation with tissue specific methylation patterns. Most DMRs, however, showed some intra-individual variability for DNA methylation levels in peripheral blood, suggesting that more than one DMR needs to be examined in order to get an overall impression of the epigenetic stability in a tissue. The plasticity of DNA methylation at imprinted genes was examined in a panel of normal and cancer cell lines. All cell lines showed changes in DNA methylation, especially at the paternal germ-line and the somatic DMRs. CONCLUSIONS: Our validated pyrosequencing methylation assays can be widely used as a tool to investigate DNA methylation levels of imprinted genes in clinical samples. This first comprehensive analysis of normal methylation levels in adult somatic tissues at human imprinted regions confirm that, despite intra-individual variability and tissue specific expression, imprinted genes faithfully maintain their DNA methylation in healthy adult tissue. DNA methylation levels of a selection of imprinted genes are, therefore, a valuable indicator for epigenetic stability.