Interindividual Variation In DNA Methylation Research Articles

Inter-Individual Variation in DNA Methylation Patterns across Two Tissues and Leukocytes in Mature Brahman Cattle

Quantifying the natural inter-individual variation in DNA methylation patterns is important for identifying its contribution to phenotypic variation, but also for understanding how the environment affects variability, and for incorporation into statistical analyses. The inter-individual variation in DNA methylation patterns in female cattle and the effect that a prenatal stressor has on such variability have yet to be quantified. Thus, the objective of this study was to utilize methylation data from mature Brahman females to quantify the inter-individual variation in DNA methylation. Pregnant Brahman cows were transported for 2 h durations at days 60 ± 5; 80 ± 5; 100 ± 5; 120 ± 5; and 140 ± 5 of gestation. A non-transport group was maintained as a control. Leukocytes, amygdala, and anterior pituitary glands were harvested from eight cows born from the non-transport group (Control) and six from the transport group (PNS) at 5 years of age. The DNA harvested from the anterior pituitary contained the greatest variability in DNA methylation of cytosine-phosphate-guanine (mCpG) sites from both the PNS and Control groups, and the amygdala had the least. Numerous variable mCpG sites were associated with retrotransposable elements and highly repetitive regions of the genome. Some of the genomic features that had high variation in DNA methylation are involved in immune responses, signaling, responses to stimuli, and metabolic processes. The small overlap of highly variable CpG sites and features between tissues and leukocytes supports the role of variable DNA methylation in regulating tissue-specific gene expression. Many of the CpG sites that exhibited high variability in DNA methylation were common between the PNS and Control groups within a tissue, but there was little overlap in genomic features with high variability. The interaction between the prenatal environment and the genome could be responsible for the differences in location of the variable DNA methylation.

A Cautionary Tale of Sexing by Methylation: Hybrid Bisulfite-Conversion Sequencing of Immunoprecipitated Methylated DNA in Chrysemys picta Turtles with Temperature-Dependent Sex Determination Reveals Contrasting Patterns of Somatic and Gonadal Methylation, but No Unobtrusive Sex Diagnostic.

Background: The gonads of Chrysemys picta, a turtle with temperature-dependent sex determination (TSD), exhibit differential DNA methylation between males and females, but whether the same is true in somatic tissues remains unknown. Such differential DNA methylation in the soma would provide a non-lethal sex diagnostic for TSD turtle hatchings who lack visually detectable sexual dimorphism when young. Methods: Here, we tested multiple approaches to study DNA methylation in tail clips of Chrysemys picta hatchlings, to identify differentially methylated candidate regions/sites that could serve as molecular sex markers To detect global differential methylation in the tails we used methylation-sensitive ELISA, and to test for differential local methylation we developed a novel hybrid method by sequencing immunoprecipitated and bisulfite converted DNA (MeDIP-BS-seq) followed by PCR validation of candidate regions/sites after digestion with a methylation-sensitive restriction enzyme. Results: We detected no global differences in methylation between males and females via ELISA. While we detected inter-individual variation in DNA methylation in the tails, this variation was not sexually dimorphic, in contrast with hatchling gonads. Conclusions: Results highlight that differential DNA methylation is tissue-specific and plays a key role in gonadal formation (primary sexual development) and maintenance post-hatching, but not in the somatic tail tissue.

Sequence features of retrotransposons allow for epigenetic variability.

Transposable elements (TEs) are mobile genetic elements that make up a large fraction of mammalian genomes. While select TEs have been co-opted in host genomes to have function, the majority of these elements are epigenetically silenced by DNA methylation in somatic cells. However, some TEs in mice, including the Intracisternal A-particle (IAP) subfamily of retrotransposons, have been shown to display interindividual variation in DNA methylation. Recent work has revealed that IAP sequence differences and strain-specific KRAB zinc finger proteins (KZFPs) may influence the methylation state of these IAPs. However, the mechanisms underlying the establishment and maintenance of interindividual variability in DNA methylation still remain unclear. Here, we report that sequence content and genomic context influence the likelihood that IAPs become variably methylated. IAPs that differ from consensus IAP sequences have altered KZFP recruitment that can lead to decreased KAP1 recruitment when in proximity of constitutively expressed genes. These variably methylated loci have a high CpG density, similar to CpG islands, and can be bound by ZF-CxxC proteins, providing a potential mechanism to maintain this permissive chromatin environment and protect from DNA methylation. These observations indicate that variably methylated IAPs escape silencing through both attenuation of KZFP binding and recognition by ZF-CxxC proteins to maintain a hypomethylated state.

A map of human individuality.

A study in Genome Biology reports an unbiased screen for human genomic regions at which interindividual variation in DNA methylation is consistent across cell types and tissues, providing new targets for studying epigenetic links to disease.

A genomic atlas of systemic interindividual epigenetic variation in humans

BackgroundDNA methylation is thought to be an important determinant of human phenotypic variation, but its inherent cell type specificity has impeded progress on this question. At exceptional genomic regions, interindividual variation in DNA methylation occurs systemically. Like genetic variants, systemic interindividual epigenetic variants are stable, can influence phenotype, and can be assessed in any easily biopsiable DNA sample. We describe an unbiased screen for human genomic regions at which interindividual variation in DNA methylation is not tissue-specific.ResultsFor each of 10 donors from the NIH Genotype-Tissue Expression (GTEx) program, CpG methylation is measured by deep whole-genome bisulfite sequencing of genomic DNA from tissues representing the three germ layer lineages: thyroid (endoderm), heart (mesoderm), and brain (ectoderm). We develop a computational algorithm to identify genomic regions at which interindividual variation in DNA methylation is consistent across all three lineages. This approach identifies 9926 correlated regions of systemic interindividual variation (CoRSIVs). These regions, comprising just 0.1% of the human genome, are inter-correlated over long genomic distances, associated with transposable elements and subtelomeric regions, conserved across diverse human ethnic groups, sensitive to periconceptional environment, and associated with genes implicated in a broad range of human disorders and phenotypes. CoRSIV methylation in one tissue can predict expression of associated genes in other tissues.ConclusionsIn addition to charting a previously unexplored molecular level of human individuality, this atlas of human CoRSIVs provides a resource for future population-based investigations into how interindividual epigenetic variation modulates risk of disease.

Maternal One-Carbon Metabolism and Infant DNA Methylation between Contrasting Seasonal Environments: A Case Study from The Gambia

BackgroundThe periconceptional period is a time in which environmentally induced changes to the epigenome could have significant consequences for offspring health. Metastable epialleles (MEs) are genomic loci demonstrating interindividual variation in DNA methylation with intraindividual crosstissue correlation, suggesting that methylation states are established in the very early embryo before gastrulation. In our previous Gambian studies, we have shown that ME methylation states in the offspring are predicted by maternal concentrations of certain nutritional biomarkers around the time of conception. ObjectiveWe aimed to assess whether the profile of maternal biomarker predictors of offspring methylation differs between rainy and dry seasons in a population of rural Gambians, using a larger set of 50 recently identified MEs. MethodsWe measured 1-carbon biomarkers in maternal plasma back-extrapolated to conception, and cytosine-phosphate-guanine (CpG) methylation at 50 ME loci in their infants’ blood at a mean age of 3.3 mo (n = 120 mother-child pairs). We tested for interactions between seasonality and effects of biomarker concentrations on mean ME methylation z score. We used backward stepwise linear regression to select the profile of nutritional predictors of methylation in each season and repeated this analysis with biomarker principal components (PCs) to capture biomarker covariation. ResultsWe found preliminary evidence of seasonal differences in biomarker-methylation associations for folate, choline, and homocysteine (interaction P values ≤0.03). Furthermore, in stratified analyses, biomarker predictors of methylation changed between seasons. In the dry season, vitamin B-2 and methionine were positive predictors. In the rainy season, however, choline and vitamin B-6 were positive predictors, and folate and vitamin B-12 were negative predictors. PC1 captured covariation in the folate metabolism cycle and predicted methylation in dry season conceptions. PC2 represented the betaine remethylation pathway and predicted rainy season methylation. ConclusionsUnderlying nutritional status may modify the association between nutritional biomarkers and methylation, and should be considered in future studies.

A survey of inter-individual variation in DNA methylation identifies environmentally responsive co-regulated networks of epigenetic variation in the human genome.

While population studies have resulted in detailed maps of genetic variation in humans, to date there are few robust maps of epigenetic variation. We identified sites containing clusters of CpGs with high inter-individual epigenetic variation, termed Variably Methylated Regions (VMRs) in five purified cell types. We observed that VMRs occur preferentially at enhancers and 3’ UTRs. While the majority of VMRs have high heritability, a subset of VMRs within the genome show highly correlated variation in trans, forming co-regulated networks that have low heritability, differ between cell types and are enriched for specific transcription factor binding sites and biological pathways of functional relevance to each tissue. For example, in T cells we defined a network of 95 co-regulated VMRs enriched for genes with roles in T-cell activation; in fibroblasts a network of 34 co-regulated VMRs comprising all four HOX gene clusters enriched for control of tissue growth; and in neurons a network of 18 VMRs enriched for roles in synaptic signaling. By culturing genetically-identical fibroblasts under varying environmental conditions, we experimentally demonstrated that some VMR networks are responsive to the environment, with methylation levels at these loci changing in a coordinated fashion in trans dependent on cellular growth. Intriguingly these environmentally-responsive VMRs showed a strong enrichment for imprinted loci (p<10−80), suggesting that these are particularly sensitive to environmental conditions. Our study provides a detailed map of common epigenetic variation in the human genome, showing that both genetic and environmental causes underlie this variation.

Evolutionary conservation of DNA methylation in CpG sites within ultraconserved noncoding elements

ABSTRACTUltraconserved noncoding elements (UCNEs) constitute less than 1 Mb of vertebrate genomes and are impervious to accumulating mutations. About 4000 UCNEs exist in vertebrate genomes, each at least 200 nucleotides in length, sharing greater than 95% sequence identity between human and chicken. Despite extreme sequence conservation over 400 million years of vertebrate evolution, we show both ordered interspecies and within-species interindividual variation in DNA methylation in these regions. Here, we surveyed UCNEs with high CpG density in 56 species finding half to be intermediately methylated and the remaining near 0% or 100%. Intermediately methylated UCNEs displayed a greater range of methylation between mouse tissues. In a human population, most UCNEs showed greater variation than the LINE1 transposon, a frequently used epigenetic biomarker. Global methylation was found to be inversely correlated to hydroxymethylation across 60 vertebrates. Within UCNEs, DNA methylation is flexible, conserved between related species, and relaxed from the underlying sequence selection pressure, while remaining heritable through speciation.

Inter-individual variation in DNA methylation is largely restricted to tissue-specific differentially methylated regions in maize

BackgroundVariation in DNA methylation across distinct genetic populations, or in response to specific biotic or abiotic stimuli, has typically been studied in leaf DNA from pooled individuals using either reduced representation bisulfite sequencing, whole genome bisulfite sequencing (WGBS) or methylation sensitive amplified polymorphism (MSAP). The latter represents a useful alterative when sample size is large, or when analysing methylation changes in genomes that have yet to be sequenced. In this study we compared variation in methylation across ten individual leaf and endosperm samples from maize hybrid and inbred lines using MSAP. We also addressed the methodological implications of analysing methylation variation using pooled versus individual DNA samples, in addition to the validity of MSAP compared to WGBS. Finally, we analysed a subset of variable and non-variable fragments with respect to genomic location, vicinity to repetitive elements and expression patterns across leaf and endosperm tissues.ResultsOn average, 30% of individuals showed inter-individual methylation variation, mostly of leaf and endosperm-specific differentially methylated DNA regions. With the exception of low frequency demethylation events, the bulk of inter-individual methylation variation (84 and 80% in leaf and endosperm, respectively) was effectively captured in DNA from pooled individuals. Furthermore, available genome-wide methylation data largely confirmed MSAP leaf methylation profiles. Most variable methylation that mapped within genes was associated with CG methylation, and many of such genes showed tissue-specific expression profiles. Finally, we found that the hAT DNA transposon was the most common class II transposable element found in close proximity to variable DNA regions.ConclusionsThe relevance of our results with respect to future studies of methylation variation is the following: firstly, the finding that inter-individual methylation variation is largely restricted to tissue-specific differentially methylated DNA regions, underlines the importance of tissue-type when analysing the methylation response to a defined stimulus. Secondly, we show that pooled sample-based MSAP studies are methodologically appropriate to study methylation variation. Thirdly, we confirm that MSAP is a powerful tool when WGBS is not required or feasible, for example in plant species that have yet to be sequenced.

Interindividual Variation in DNA Methylation at a Putative POMC Metastable Epiallele Is Associated with Obesity

The estimated heritability of human BMI is close to 75%, but identified genetic variants explain only a small fraction of interindividual body-weight variation. Inherited epigenetic variants identified in mouse models named "metastable epialleles" could in principle explain this "missing heritability." We provide evidence that methylation in a variably methylated region (VMR) in the pro-opiomelanocortin gene (POMC), particularly in postmortem human laser-microdissected melanocyte-stimulating hormone (MSH)-positive neurons, is strongly associated with individual BMI. Using cohorts from different ethnic backgrounds, including a Gambian cohort, we found evidence suggesting that methylation of the POMC VMR is established in the early embryo and that offspring methylation correlates with the paternal somatic methylation pattern. Furthermore, it is associated with levels of maternal one-carbon metabolites at conception and stable during postnatal life. Together, these data suggest that the POMC VMR may be a human metastable epiallele that influences body-weight regulation.

DNA methylation variation of human-specific Alu repeats

ABSTRACTDNA methylation is the major repression mechanism for human retrotransposons, such as the Alu family. Here, we have determined the methylation levels associated with 5238 loci belonging to 2 Alu subfamilies, AluYa5 and AluYb8, using high-throughput targeted repeat element bisulfite sequencing (HT-TREBS). The results indicate that ∼90% of loci are repressed by high methylation levels. Of the remaining loci, many of the hypomethylated elements are found near gene promoters and show high levels of DNA methylation variation. We have characterized this variation in the context of tumorigenesis and interindividual differences. Comparison of a primary breast tumor and its matched normal tissue revealed early DNA methylation changes in ∼1% of AluYb8 elements in response to tumorigenesis. Simultaneously, AluYa5/Yb8 elements proximal to promoters also showed differences in methylation of up to one order of magnitude, even between normal individuals. Overall, the current study demonstrates that early loss of methylation occurs during tumorigenesis in a subset of young Alu elements, suggesting their potential clinical relevance. However, approaches such as deep-bisulfite-sequencing of individual loci using HT-TREBS are required to distinguish clinically relevant loci from the background observed for AluYa5/Yb8 elements in general with regard to high levels of interindividual variation in DNA methylation.

Abstract PL02-01: Nutrition, epigenetics, and cancer

Abstract Transient nutritional exposures during critical ontogenic periods can cause persistent changes in gene expression, metabolism, and risk of various diseases including cancer. We have been investigating whether such ‘developmental programming’ occurs via nutritional influences on developmental epigenetics. Our studies in agouti viable yellow and axin fused mice showed that developmental establishment of DNA methylation at ‘metastable epialleles’ is especially sensitive to maternal nutritional status around the time of conception. Lately, using a multiple-tissue screen for interindividual variation in DNA methylation, we have identified human genomic regions that appear to be metastable epialleles. Stochastic establishment of DNA methylation at these loci is affected by maternal nutrition around the time of conception, consistent across multiple tissues, and stable for many years. Most recently, our studies using genome-wide bisulfite sequencing have identified candidate metastable epialleles that are associated with genes implicated in human cancer, potentially providing opportunities for cancer risk assessment and prevention. Citation Format: Robert A. Waterland. Nutrition, epigenetics, and cancer. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr PL02-01.

Independent genomewide screens identify the tumor suppressor VTRNA2-1 as a human epiallele responsive to periconceptional environment.

BackgroundInterindividual epigenetic variation that occurs systemically must be established prior to gastrulation in the very early embryo and, because it is systemic, can be assessed in easily biopsiable tissues. We employ two independent genome-wide approaches to search for such variants.ResultsFirst, we screen for metastable epialleles by performing genomewide bisulfite sequencing in peripheral blood lymphocyte (PBL) and hair follicle DNA from two Caucasian adults. Second, we conduct a genomewide screen for genomic regions at which PBL DNA methylation is affected by season of conception in rural Gambia. Remarkably, both approaches identify the genomically imprinted VTRNA2-1 as a top environmentally responsive epiallele. We demonstrate systemic and stochastic interindividual variation in DNA methylation at the VTRNA2-1 differentially methylated region in healthy Caucasian and Asian adults and show, in rural Gambians, that periconceptional environment affects offspring VTRNA2-1 epigenotype, which is stable over at least 10 years. This unbiased screen also identifies over 100 additional candidate metastable epialleles, and shows that these are associated with cis genomic features including transposable elements.ConclusionsThe non-coding VTRNA2-1 transcript (also called nc886) is a putative tumor suppressor and modulator of innate immunity. Thus, these data indicating environmentally induced loss of imprinting at VTRNA2-1 constitute a plausible causal pathway linking early embryonic environment, epigenetic alteration, and human disease. More broadly, the list of candidate metastable epialleles provides a resource for future studies of epigenetic variation and human disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0660-y) contains supplementary material, which is available to authorized users.

Novel region discovery method for Infinium 450K DNA methylation data reveals changes associated with aging in muscle and neuronal pathways

We describe a methodology for detecting differentially methylated regions (DMRs) and variably methylated regions (VMRs), in data from Infinium 450K arrays that are very widely used in epigenetic studies. Region detection is more specific than single CpG analysis as it increases the extent of common findings between studies, and is more powerful as it reduces the multiple testing problem inherent in epigenetic whole-genome association studies (EWAS). In addition, results driven by single erroneous probes are removed. We have used multiple publicly available Infinium 450K data sets to generate a consensus list of DMRs for age, supporting the hypothesis that aging is associated with specific epigenetic modifications. The consensus aging DMRs are significantly enriched for muscle biogenesis pathways. We find a massive increase in VMRs with age and in regions of the genome associated with open chromatin and neurotransmission. Old age VMRs are significantly enriched for neurotransmission pathways. EWAS studies should investigate the role of this interindividual variation in DNA methylation, in the age-associated diseases of sarcopenia and dementia.

Influence of DNMT Genotype on Global and Site Specific DNA Methylation Patterns in Neonates and Pregnant Women

This study examines the relationship between common genetic variation within DNA methyltransferase genes and inter-individual variation in DNA methylation. Eleven polymorphisms spanning DNMT1 and DNMT3B were genotyped. Global and gene specific (IGF2, IGFBP3, ZNT5) DNA methylation was quantified by LUMA and bisulfite Pyrosequencing assays, respectively, in neonatal cord blood and in maternal peripheral blood. Associations between maternal genotype and maternal methylation (n ≈ 333), neonatal genotype and neonatal methylation (n ≈ 454), and maternal genotype and neonatal methylation (n ≈ 137) were assessed. The findings of this study provide some support to the hypothesis that genetic variation in DNA methylating enzymes influence DNA methylation at global and gene-specific levels; however observations were not robust to correction for multiple testing. More comprehensive analysis of the influence of genetic variation on global and site specific DNA methylation is warranted.

Characterization of the DNA Methylome and its Interindividual Variation in Human Peripheral Blood Monocytes

Peripheral blood monocytes (PBMs) play multiple and critical roles in the immune response, and abnormalities in PBMs have been linked to a variety of human disorders. However, the DNA methylation landscape in PBMs is largely unknown. In this study, we characterized epigenome-wide DNA methylation profiles in purified PBMs. PBMs were isolated from freshly collected peripheral blood from 18 unrelated healthy postmenopausal Caucasian females. Epigenome-wide DNA methylation profiles (the methylome) were characterized by using methylated DNA immunoprecipitation combined with high-throughput sequencing. 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. Although CpG-rich and -poor promoters showed distinct methylation patterns, interestingly, a negative correlation between promoter methylation levels and gene transcription levels was consistently observed across promoters with high to low CpG densities. Importantly, we observed substantial interindividual variation in DNA methylation across the individual PBM methylomes and the pattern of this interindividual variation varied between different genomic features, with highly variable regions enriched for repetitive DNA elements. Furthermore, we observed a modest but significant excess (p < 2.2 × 10(-16)) of genes showing a negative correlation between interindividual promoter methylation and transcription levels. These significant genes were enriched in biological processes that are closely related to PBM functions, suggesting that alteration in DNA methylation is likely to be an important mechanism contributing to the interindividual variation in PBM function, and PBM-related phenotypic and disease-susceptibility variation in humans. This study represents a comprehensive analysis of the human PBM methylome and its interindividual variation. Our data provide a valuable resource for future epigenomic and multiomic studies, exploring biological and disease-related regulatory mechanisms in PBMs.

Phylogenetic and DNA methylation analysis reveal novel regions of variable methylation in the mouse IAP class of transposons

BackgroundSelect retrotransposons in the long terminal repeat (LTR) class exhibit interindividual variation in DNA methylation that is altered by developmental environmental exposures. Yet, neither the full extent of variability at these “metastable epialleles,” nor the phylogenetic relationship underlying variable elements is well understood. The murine metastable epialleles, Avy and CabpIAP, result from independent insertions of an intracisternal A particle (IAP) mobile element, and exhibit remarkably similar sequence identity (98.5%).ResultsUtilizing the C57BL/6 genome we identified 10802 IAP LTRs overall and a subset of 1388 in a family that includes Avy and CabpIAP. Phylogenetic analysis revealed two duplication and divergence events subdividing this family into three clades. To characterize interindividual variation across clades, liver DNA from 17 isogenic mice was subjected to combined bisulfite and restriction analysis (CoBRA) for 21 separate LTR transposons (7 per clade). The lowest and highest mean methylation values were 59% and 88% respectively, while methylation levels at individual LTRs varied widely, ranging from 9% to 34%. The clade with the most conserved elements had significantly higher mean methylation across LTRs than either of the two diverged clades (p = 0.040 and p = 0.017). Within each mouse, average methylation across all LTRs was not significantly different (71%-74%, p > 0.99).ConclusionsCombined phylogenetic and DNA methylation analysis allows for the identification of novel regions of variable methylation. This approach increases the number of known metastable epialleles in the mouse, which can serve as biomarkers for environmental modifications to the epigenome.

Global DNA methylation and risk of subclinical atherosclerosis in young adults: The Pathobiological Determinants of Atherosclerosis in Youth (PDAY) study

ObjectiveThe association between hepatic global DNA methylation measured using pyrosequencing technology and the risk of subclinical atherosclerosis was examined in the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) study. PDAY is a bi-racial investigation of the natural history of atherosclerosis and its risk factors involving 3013 individuals aged 15–34 years who underwent autopsy after dying of unrelated causes in 1987–1994. MethodsRaised atherosclerotic lesions were defined as the sum of the percentages of intimal surface area detected in the right coronary artery and left half of the abdominal and thoracic aorta harboring fibrous plaques, complicated lesions, and calcified lesions during a postmortem pathological examination. To conduct the case–control study, 300 cases selected with the highest raised lesion scores were paired with 300 controls without raised lesions after matching for age, race, and gender. ResultsGlobal DNA methylation was not associated with disease risk in the study population considered as a whole using conditional logistic regression models to analyze matched pairs. Since the estimation of the risk of atherosclerosis associated with inter-individual variation in DNA methylation was similar if unconditional logistic regression was used, subgroup analyses were carried out after adjusting for matching variables. A modest association with methylation levels below the median value was found in white but not in African-American study participants (odds ratio=1.59, 95% confidence interval=1.02–2.49, p=0.04). ConclusionsHepatic global DNA methylation does not appear to be a definitive determinant of atherosclerosis burden in a postmortem sample of young adults.

Inter- and Intra-Individual Variation in Allele-Specific DNA Methylation and Gene Expression in Children Conceived using Assisted Reproductive Technology

Epidemiological studies have reported a higher incidence of rare disorders involving imprinted genes among children conceived using assisted reproductive technology (ART), suggesting that ART procedures may be disruptive to imprinted gene methylation patterns. We examined intra- and inter-individual variation in DNA methylation at the differentially methylated regions (DMRs) of the IGF2/H19 and IGF2R loci in a population of children conceived in vitro or in vivo. We found substantial variation in allele-specific methylation at both loci in both groups. Aberrant methylation of the maternal IGF2/H19 DMR was more common in the in vitro group, and the overall variance was also significantly greater in the in vitro group. We estimated the number of trophoblast stem cells in each group based on approximation of the variance of the binomial distribution of IGF2/H19 methylation ratios, as well as the distribution of X chromosome inactivation scores in placenta. Both of these independent measures indicated that placentas of the in vitro group were derived from fewer stem cells than the in vivo conceived group. Both IGF2 and H19 mRNAs were significantly lower in placenta from the in vitro group. Although average birth weight was lower in the in vitro group, we found no correlation between birth weight and IGF2 or IGF2R transcript levels or the ratio of IGF2/IGF2R transcript levels. Our results show that in vitro conception is associated with aberrant methylation patterns at the IGF2/H19 locus. However, very little of the inter- or intra-individual variation in H19 or IGF2 mRNA levels can be explained by differences in maternal DMR DNA methylation, in contrast to the expectations of current transcriptional imprinting models. Extraembryonic tissues of embryos cultured in vitro appear to be derived from fewer trophoblast stem cells. It is possible that this developmental difference has an effect on placental and fetal growth.

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.