Genetic Control of Individual Differences in Gene-Specific Methylation in Human Brain

We have observed extensive interindividual differences in DNA methylation of 8590 CpG sites of 6229 genes in 153 human adult cerebellum samples, enriched in CpG island “shores” and at further distances from CpG islands. To search for genetic factors that regulate this variation, we performed a genome-wide association study (GWAS) mapping of methylation quantitative trait loci (mQTLs) for the 8590 testable CpG sites. cis association refers to correlation of methylation with SNPs within 1 Mb of a CpG site. 736 CpG sites showed phenotype-wide significant cis association with 2878 SNPs (after permutation correction for all tested markers and methylation phenotypes). In trans analysis of methylation, which tests for distant regulation effects, associations of 12 CpG sites and 38 SNPs remained significant after phenotype-wide correction. To examine the functional effects of mQTLs, we analyzed 85 genes that were with genetically regulated methylation we observed and for which we had quality gene expression data. Ten genes showed SNP-methylation-expression three-way associations—the same SNP simultaneously showed significant association with both DNA methylation and gene expression, while DNA methylation was significantly correlated with gene expression. Thus, we demonstrated that DNA methylation is frequently a heritable continuous quantitatively variable trait in human brain. Unlike allele-specific methylation, genetic polymorphisms mark both cis- and trans-regulatory genetic sites at measurable distances from their CpG sites. Some of the genetically regulated DNA methylation is directly connected with genetically regulated gene expression variation.     

Heritability of DNA methylation in threespine stickleback (Gasterosteus aculeatus)

Epigenetic mechanisms underlying phenotypic change are hypothesized to contribute to population persistence and adaptation in the face of environmental change. To date, few studies have explored the heritability of intergenerationally stable methylation levels in natural populations, and little is known about the relative contribution of cis- and trans-regulatory changes to methylation variation. Here, we explore the heritability of DNA methylation, and conduct methylation quantitative trait loci (meQTLs) analysis to investigate the genetic architecture underlying methylation variation between marine and freshwater ecotypes of threespine stickleback (Gasterosteus aculeatus). We quantitatively measured genome-wide DNA methylation in fin tissue using reduced representation bisulfite sequencing of F1 and F2 crosses, and their marine and freshwater source populations. We identified cytosines (CpG sites) that exhibited stable methylation levels across generations. We found that additive genetic variance explained an average of 24–35% of the methylation variance, with a number of CpG sites possibly autonomous from genetic control. We also detected both cis- and trans-meQTLs, with only trans-meQTLs overlapping with previously identified genomic regions of high differentiation between marine and freshwater ecotypes. Finally, we identified the genetic architecture underlying two key CpG sites that were differentially methylated between ecotypes. These findings demonstrate a potential role for DNA methylation in facilitating adaptation to divergent environments and improve our understanding of the heritable basis of population epigenomic variation.     

Allelic Skewing of DNA Methylation Is Widespread across the Genome

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 ≥0.10 following MSRE digestion. In agreement with previous reports, the majority of ASM (∼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.     

The impact of genetic variation and cigarette smoke on DNA methylation in current and former smokers from the COPDGene study

DNA methylation can be affected by systemic exposures, such as cigarette smoking and genetic sequence variation; however, the relative impact of each on the epigenome is unknown. We aimed to assess if cigarette smoking and genetic variation are associated with overlapping or distinct sets of DNA methylation marks and pathways. We selected 85 Caucasian current and former smokers with genome-wide single nucleotide polymorphism (SNP) genotyping available from the COPDGene study.  Genome-wide methylation was obtained on DNA from whole blood using the Illumina HumanMethylation27 platform. To determine the impact of local sequence variation on DNA methylation (mQTL), we examined the association between methylation and SNPs within 50 kb of each CpG site.  To examine the impact of cigarette smoking on DNA methylation, we examined the differences in methylation by current cigarette smoking status. We detected 770 CpG sites annotated to 708 genes associated at an FDR < 0.05 in the cis-mQTL analysis and 1,287 CpG sites annotated to 1,242 genes, which were nominally associated in the smoking-CpG association analysis (P_unadjusted < 0.05). Forty-three CpG sites annotated to 40 genes were associated with both SNP variation and current smoking; this overlap was not greater than that expected by chance. Our results suggest that cigarette smoking and genetic variants impact distinct sets of DNA methylation marks, the further elucidation of which may partially explain the variable susceptibility to the health effects of cigarette smoking. Ascertaining how genetic variation and systemic exposures differentially impact the human epigenome has relevance for both biomarker identification and therapeutic target development for smoking-related diseases.     

The Relationship of DNA Methylation with Age,Gender and Genotype in Twins and Healthy Controls

Cytosine-5 methylation within CpG dinucleotides is a potentially important mechanism of epigenetic influence on human traits and disease. In addition to influences of age and gender, genetic control of DNA methylation levels has recently been described. We used whole blood genomic DNA in a twin set (23 MZ twin-pairs and 23 DZ twin-pairs, N = 92) as well as healthy controls (N = 96) to investigate heritability and relationship with age and gender of selected DNA methylation profiles using readily commercially available GoldenGate bead array technology. Despite the inability to detect meaningful methylation differences in the majority of CpG loci due to tissue type and locus selection issues, we found replicable significant associations of DNA methylation with age and gender. We identified associations of genetically heritable single nucleotide polymorphisms with large differences in DNA methylation levels near the polymorphism (cis effects) as well as associations with much smaller differences in DNA methylation levels elsewhere in the human genome (trans effects). Our results demonstrate the feasibility of array-based approaches in studies of DNA methylation and highlight the vast differences between individual loci. The identification of CpG loci of which DNA methylation levels are under genetic control or are related to age or gender will facilitate further studies into the role of DNA methylation and disease.     

Identification of a new locus and validation of previously reported loci showing differential methylation associated with smoking. The REGICOR study

Smoking increases the risk of many diseases and could act through changes in DNA methylation patterns. The aims of this study were to determine the association between smoking and DNA methylation throughout the genome at cytosine-phosphate-guanine (CpG) site level and genomic regions. A discovery cross-sectional epigenome-wide association study nested in the follow-up of the REGICOR cohort was designed and included 645 individuals. Blood DNA methylation was assessed using the Illumina HumanMethylation450 BeadChip. Smoking status was self-reported using a standardized questionnaire. We identified 66 differentially methylated CpG sites associated with smoking, located in 38 genes. In most of these CpG sites, we observed a trend among those quitting smoking to recover methylation levels typical of never smokers. A CpG site located in a novel smoking-associated gene (cg06394460 in LNX2) was hypomethylated in current smokers. Moreover, we validated two previously reported CpG sites (cg05886626 in THBS1, and cg24838345 in MTSS1) for their potential relation to atherosclerosis and cancer diseases, using several different approaches: CpG site methylation, gene expression, and plasma protein level determinations. Smoking was also associated with higher THBS1 gene expression but with lower levels of thrombospondin-1 in plasma. Finally, we identified differential methylation regions in 13 genes and in four non-coding RNAs. In summary, this study replicated previous findings and identified and validated a new CpG site located in LNX2 associated with smoking.     

Epigenetic natural variation in Arabidopsis thaliana

Cytosine methylation of repetitive sequences is widespread in plant genomes, occurring in both symmetric (CpG and CpNpG) as well as asymmetric sequence contexts. We used the methylation-dependent restriction enzyme McrBC to profile methylated DNA using tiling microarrays of Arabidopsis Chromosome 4 in two distinct ecotypes, Columbia and Landsberg erecta. We also used comparative genome hybridization to profile copy number polymorphisms. Repeated sequences and transposable elements (TEs), especially long terminal repeat retrotransposons, are densely methylated, but one third of genes also have low but detectable methylation in their transcribed regions. While TEs are almost always methylated, genic methylation is highly polymorphic, with half of all methylated genes being methylated in only one of the two ecotypes. A survey of loci in 96 Arabidopsis accessions revealed a similar degree of methylation polymorphism. Within-gene methylation is heritable, but is lost at a high frequency in segregating F₂ families. Promoter methylation is rare, and gene expression is not generally affected by differences in DNA methylation. Small interfering RNA are preferentially associated with methylated TEs, but not with methylated genes, indicating that most genic methylation is not guided by small interfering RNA. This may account for the instability of gene methylation, if occasional failure of maintenance methylation cannot be restored by other means.     

Array-based profiling of reference-independent methylation status (aPRIMES) identifies frequent promoter methylation and consecutive downregulation of ZIC2 in pediatric medulloblastoma

Existing microarray-based approaches for screening of DNA methylation are hampered by a number of shortcomings, such as the introduction of bias by DNA copy-number imbalances in the test genome and negligence of tissue-specific methylation patterns. We developed a method designated array-based profiling of reference-independent methylation status (aPRIMES) that allows the detection of direct methylation status rather than relative methylation. Array-PRIMES is based on the differential restriction and competitive hybridization of methylated and unmethylated DNA by methylation-specific and methylation-sensitive restriction enzymes, respectively. We demonstrate the accuracy of aPRIMES in detecting the methylation status of CpG islands for different states of methylation. Application of aPRIMES to the DNA from desmoplastic medulloblastomas of monozygotic twins showed strikingly similar methylation profiles. Additional analysis of 18 sporadic medulloblastomas revealed an overall correlation between highly methylated tumors and poor clinical outcome and identified ZIC2 as a frequently methylated gene in pediatric medulloblastoma.     

Characterization of the level,target sites and inheritance of cytosine methylation in tomato nuclear DNA

The tomato nuclear genome was determined to have a G+C content of 37% which is among the lowest reported for any plant species. Non-coding regions have a G+C content even lower (32% average) whereas coding regions are considerably richer in G+C (46%).5-methyl cytosine was the only modified base detected and on average 23% of the cytosine residues are methylated. Immature tissues and protoplasts have significantly lower levels of cytosine methylation (average 20%) than mature tissues (average 25%). Mature pollen has an intermediate level of methylation (22%). Seeds gave the highest value (27%), suggesting de novo methylation after pollination and during seed development.Based on isoschizomer studies we estimate 55% of the CpG target sites (detected by Msp I/Hpa II) and 85% of the CpNpG target sites (detected by Bst NI/Eco RI)are methylated. Unmethylated target sites (both CpG and CpNpG) are not randomly distributed throughout the genome, but frequently occur in clusters. These clusters resemble CpG islands recently reported in maize and tobacco.The low G+C content and high levels of cytosine methylation in tomato may be due to previous transitions of 5mCT. This is supported by the fact that G+C levels are lowest in non-coding portions of the genome in which selection is relaxed and thus transitions are more likely to be tolerated. This hypothesis is also supported by the general deficiency of methylation target sites in the tomato genome, especially in non-coding regions.Using methylation isoschizomers and RFLP analysis we have also determined that polymorphism between plants, for cytosine methylation at allelic sites, is common in tomato. Comparing DNA from two tomato species, 20% of the polymorphisms detected by Bst NI/Eco RII could be attributed to differential methylation at the CpNpG target sites. With Msp I/Hpa II, 50% of the polymorphisms were attributable to methylation (CpG and CpNpG sites). Moreover, these polymorphisms were demonstrated to be inherited in a mendelian fashion and to co-segregate with the methylation target site and thus do not represent variation for transacting factors that might be involved in methylation of DNA. The potential role of heritable methylation polymorphism in evolution of gene regulation and in RFLP studies is discussed.     

A Genome-Wide,Fine-Scale Map of Natural Pigmentation Variation in Drosophila melanogaster

Various approaches can be applied to uncover the genetic basis of natural phenotypic variation, each with their specific strengths and limitations. Here, we use a replicated genome-wide association approach (Pool-GWAS) to fine-scale map genomic regions contributing to natural variation in female abdominal pigmentation in Drosophila melanogaster, a trait that is highly variable in natural populations and highly heritable in the laboratory. We examined abdominal pigmentation phenotypes in approximately 8000 female European D. melanogaster, isolating 1000 individuals with extreme phenotypes. We then used whole-genome Illumina sequencing to identify single nucleotide polymorphisms (SNPs) segregating in our sample, and tested these for associations with pigmentation by contrasting allele frequencies between replicate pools of light and dark individuals. We identify two small regions near the pigmentation genes tan and bric-à-brac 1, both corresponding to known cis-regulatory regions, which contain SNPs showing significant associations with pigmentation variation. While the Pool-GWAS approach suffers some limitations, its cost advantage facilitates replication and it can be applied to any non-model system with an available reference genome.     

Genome-Wide Investigation of DNA Methylation Marks Associated with FV Leiden Mutation

In order to investigate whether DNA methylation marks could contribute to the incomplete penetrance of the FV Leiden mutation, a major genetic risk factor for venous thrombosis (VT), we measured genome-wide DNA methylation levels in peripheral blood samples of 98 VT patients carrying the mutation and 251 VT patients without the mutation using the dedicated Illumina HumanMethylation450 array. The genome-wide analysis of 388,120 CpG probes identified three sites mapping to the SLC19A2 locus whose DNA methylation levels differed significantly (p<3 10⁻⁸) between carriers and non-carriers. The three sites replicated (p<2 10⁻⁷) in an independent sample of 214 individuals from five large families ascertained on VT and FV Leiden mutation among which 53 were carriers and 161 were non-carriers of the mutation. In both studies, these three CpG sites were also associated (2.33 10⁻¹¹<p<3.02 10⁻⁴) with biomarkers of the Protein C pathway known to be influenced by the FV Leiden mutation. A comprehensive linkage disequilibrium (LD) analysis of the whole locus revealed that the original associations were due to LD between the FV Leiden mutation and a block of single nucleotide polymorphisms (SNP) located in SLC19A2. After adjusting for this block of SNPs, the FV Leiden mutation was no longer associated with any CpG site (p>0.05). In conclusion, our work clearly illustrates some promises and pitfalls of DNA methylation investigations on peripheral blood DNA in large epidemiological cohorts. DNA methylation levels at SLC19A2 are influenced by SNPs in LD with FV Leiden, but these DNA methylation marks do not explain the incomplete penetrance of the FV Leiden mutation.     

Epigenome-wide association study reveals longitudinally stable DNA methylation differences in CD4+ T cells from children with IgE-mediated food allergy

Food allergy is mediated by a combination of genetic and environmental risk factors, potentially mediated by epigenetic mechanisms. CD4+ T-cells are key drivers of the allergic response, and may therefore harbor epigenetic variation in association with the disease phenotype. Here we retrospectively examined genome-wide DNA methylation profiles (~450 000 CpGs) from CD4+ T-cells on a birth cohort of 12 children with IgE-mediated food allergy diagnosed at 12-months, and 12 non-allergic controls. DNA samples were available at two time points, birth and 12-months. Case:control comparisons of CD4+ methylation profiles identified 179 differentially methylated probes (DMP) at 12-months and 136 DMP at birth (FDR-adjusted P value < 0.05, delta β > 0.1). Approximately 30% of DMPs were coincident with previously annotated SNPs. A total of 96 allergy-associated non-SNP DMPs were present at birth when individuals were initially disease-free, potentially implicating these loci in the causal pathway. Pathway analysis of differentially methylated genes identified several MAP kinase signaling molecules. Mass spectrometry was used to validate 15 CpG sites at 3 candidate genes. Combined analysis of differential methylation with gene expression profiles revealed gene expression differences at some but not all allergy associated differentially methylated genes. Thus, dysregulation of DNA methylation at MAPK signaling-associated genes during early CD4+ T-cell development may contribute to suboptimal T-lymphocyte responses in early childhood associated with the development of food allergy.