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1.  Genome-wide Methylomic Analysis of Monozygotic Twins Discordant for Adolescent Depression 
Biological Psychiatry  2014;76(12):977-983.
Background
Adolescent depression is a common neuropsychiatric disorder that often continues into adulthood and is associated with a wide range of poor outcomes including suicide. Although numerous studies have looked at genetic markers associated with depression, the role of epigenetic variation remains relatively unexplored.
Methods
Monozygotic (MZ) twins were selected from an adolescent twin study designed to investigate the interplay of genetic and environmental factors in the development of emotional and behavioral difficulties. There were 18 pairs of MZ twins identified in which one member scored consistently higher (group mean within the clinically significant range) on self-rated depression than the other. We assessed genome-wide patterns of DNA methylation in twin buccal cell DNA using the Infinium HumanMethylation450 BeadChip from Illumina. Quality control and data preprocessing was undertaken using the wateRmelon package. Differentially methylated probes (DMPs) were identified using an analysis strategy taking into account both the significance and the magnitude of DNA methylation differences. The top differentially methylated DMP was successfully validated by bisulfite-pyrosequencing, and identified DMPs were tested in postmortem brain samples obtained from patients with major depressive disorder (n = 14) and matched control subjects (n = 15).
Results
Two reproducible depression-associated DMPs were identified, including the top-ranked DMP that was located within STK32C, which encodes a serine/threonine kinase, of unknown function.
Conclusions
Our data indicate that DNA methylation differences are apparent in MZ twins discordant for adolescent depression and that some of the disease-associated variation observed in buccal cell DNA is mirrored in adult brain tissue obtained from individuals with clinical depression.
doi:10.1016/j.biopsych.2014.04.013
PMCID: PMC4252163  PMID: 24929637
Adolescent depression; depression; DNA methylation; epigenetic; genomics; monozygotic twins
2.  Protection from genetic diathesis in ADHD: Possible complementary roles of exercise 
Objective
The degree of functional impairment and adverse developmental outcomes in individuals with attention-deficit/hyperactivity disorder (ADHD) likely reflect interplay between genes and environment. To establish whether physical exercise might reduce the level of ADHD symptoms or ADHD-related impairments, we conducted a comprehensive review of the effect of exercise in children with ADHD. Findings on the impact of exercise in animals and typically developing humans, and an overview of putative mechanisms involved are also presented to provide the context in which to understand this review.
Method
The electronic databases PubMed, OVID and Web of Knowledge were searched for all studies investigating the effect of exercise in children and adolescents with ADHD, as well as animal models of ADHD behaviours (available in January 2013). Of 2,150 initially identified records, 16 were included.
Results
Animal studies indicate that exercise, especially early in development, may be beneficial for ADHD symptom reduction. The limited research investigating the effect of exercise in children and adolescents with ADHD suggests that exercise may improve executive functioning and behavioural symptoms associated with ADHD. While animal research suggests brain-derived neurotrophic factor (BDNF) and catecholamines (CAs) play a role in mediating these effects, the association between BDNF and ADHD remains unclear in humans.
Conclusions
The potential protective qualities of exercise with regard to reducing symptoms and impairments commonly associated may hold promise for the future. Further research is needed to firmly establish whether there are clinically significant effects of exercise on the severity of ADHD symptoms, impairments and associated developmental outcomes.
doi:10.1016/j.jaac.2013.05.018
PMCID: PMC4257065  PMID: 23972692
3.  Epigenetic studies in Alzheimer’s disease: current findings, caveats and considerations for future studies 
Alzheimer’s disease (AD) is a sporadic, chronic neurodegenerative disease, usually occurring late in life. The last decade has witnessed tremendous advances in our understanding about the genetic basis of AD, but a large amount of the variance in disease risk remains to be explained. Epigenetic mechanisms, which developmentally regulate gene expression via modifications to DNA, histone proteins and chromatin, have been hypothesised to play a role in other complex neurobiological diseases, and studies to identify genome-wide epigenetic changes in AD are currently under way. However, the simple brute-force approach that has been successfully employed in genome-wide association studies is unlikely to be successful in epigenome-wide association studies of neurodegeneration. A more academic approach to understanding the role of epigenetic variation in AD is required, with careful consideration of study design, methodological approaches, tissue-specificity, and causal inference. In this article we review the empirical literature supporting a role for epigenetic processes in AD, and discuss important considerations and future directions for this new and emerging field of research.
doi:10.1002/ajmg.b.32201
PMCID: PMC3947441  PMID: 24038819
Dementia; DNA methylation; brain; neurodegeneration; genetics
4.  Methylomic profiling of human brain tissue supports a neurodevelopmental origin for schizophrenia 
Genome Biology  2014;15(10):483.
Background
Schizophrenia is a severe neuropsychiatric disorder that is hypothesized to result from disturbances in early brain development. There is mounting evidence to support a role for developmentally regulated epigenetic variation in the molecular etiology of the disorder. Here, we describe a systematic study of schizophrenia-associated methylomic variation in the adult brain and its relationship to changes in DNA methylation across human fetal brain development.
Results
We profile methylomic variation in matched prefrontal cortex and cerebellum brain tissue from schizophrenia patients and controls, identifying disease-associated differential DNA methylation at multiple loci, particularly in the prefrontal cortex, and confirming these differences in an independent set of adult brain samples. Our data reveal discrete modules of co-methylated loci associated with schizophrenia that are enriched for genes involved in neurodevelopmental processes and include loci implicated by genetic studies of the disorder. Methylomic data from human fetal cortex samples, spanning 23 to 184 days post-conception, indicates that schizophrenia-associated differentially methylated positions are significantly enriched for loci at which DNA methylation is dynamically altered during human fetal brain development.
Conclusions
Our data support the hypothesis that schizophrenia has an important early neurodevelopmental component, and suggest that epigenetic mechanisms may mediate these effects.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-014-0483-2) contains supplementary material, which is available to authorized users.
doi:10.1186/s13059-014-0483-2
PMCID: PMC4262979  PMID: 25347937
5.  Epigenetic regulation of adult neural stem cells: implications for Alzheimer’s disease 
Experimental evidence has demonstrated that several aspects of adult neural stem cells (NSCs), including their quiescence, proliferation, fate specification and differentiation, are regulated by epigenetic mechanisms. These control the expression of specific sets of genes, often including those encoding for small non-coding RNAs, indicating a complex interplay between various epigenetic factors and cellular functions.
Previous studies had indicated that in addition to the neuropathology in Alzheimer’s disease (AD), plasticity-related changes are observed in brain areas with ongoing neurogenesis, like the hippocampus and subventricular zone. Given the role of stem cells e.g. in hippocampal functions like cognition, and given their potential for brain repair, we here review the epigenetic mechanisms relevant for NSCs and AD etiology. Understanding the molecular mechanisms involved in the epigenetic regulation of adult NSCs will advance our knowledge on the role of adult neurogenesis in degeneration and possibly regeneration in the AD brain.
doi:10.1186/1750-1326-9-25
PMCID: PMC4080757  PMID: 24964731
Adult neurogenesis; Epigenetics; Alzheimer’s disease; DNA methylation; Histone modifications; MicroRNAs; Stem cell; Induced pluripotent stem cell
6.  Hypermethylation in the ZBTB20 gene is associated with major depressive disorder 
Genome Biology  2014;15(4):R56.
Background
Although genetic variation is believed to contribute to an individual’s susceptibility to major depressive disorder, genome-wide association studies have not yet identified associations that could explain the full etiology of the disease. Epigenetics is increasingly believed to play a major role in the development of common clinical phenotypes, including major depressive disorder.
Results
Genome-wide MeDIP-Sequencing was carried out on a total of 50 monozygotic twin pairs from the UK and Australia that are discordant for depression. We show that major depressive disorder is associated with significant hypermethylation within the coding region of ZBTB20, and is replicated in an independent cohort of 356 unrelated case-control individuals. The twins with major depressive disorder also show increased global variation in methylation in comparison with their unaffected co-twins. ZBTB20 plays an essential role in the specification of the Cornu Ammonis-1 field identity in the developing hippocampus, a region previously implicated in the development of major depressive disorder.
Conclusions
Our results suggest that aberrant methylation profiles affecting the hippocampus are associated with major depressive disorder and show the potential of the epigenetic twin model in neuro-psychiatric disease.
doi:10.1186/gb-2014-15-4-r56
PMCID: PMC4072999  PMID: 24694013
7.  Transcriptomic changes in the frontal cortex associated with paternal age 
Molecular Autism  2014;5:24.
Background
Advanced paternal age is robustly associated with several human neuropsychiatric disorders, particularly autism. The precise mechanism(s) mediating the paternal age effect are not known, but they are thought to involve the accumulation of de novo (epi)genomic alterations. In this study we investigate differences in the frontal cortex transcriptome in a mouse model of advanced paternal age.
Findings
Transcriptomic profiling was undertaken for medial prefrontal cortex tissue dissected from the male offspring of young fathers (2 month old, 4 sires, n = 16 offspring) and old fathers (10 month old, 6 sires, n = 16 offspring) in a mouse model of advancing paternal age. We found a number of differentially expressed genes in the offspring of older fathers, many previously implicated in the aetiology of autism. Pathway analysis highlighted significant enrichment for changes in functional networks involved in inflammation and inflammatory disease, which are also implicated in autism.
Conclusions
We observed widespread alterations to the transcriptome associated with advanced paternal age with an enrichment of genes associated with inflammation, an interesting observation given previous evidence linking the immune system to several neuropsychiatric disorders including autism.
doi:10.1186/2040-2392-5-24
PMCID: PMC3998024  PMID: 24655730
Autism; Advanced paternal age; Gene expression; Transcriptome; Inflammation; Immune response; Brain
8.  Long-Term Effects of Gestational Nicotine Exposure and Food-Restriction on Gene Expression in the Striatum of Adolescent Rats 
PLoS ONE  2014;9(2):e88896.
Gestational exposure to environmental toxins such as nicotine may result in detectable gene expression changes in later life. To investigate the direct toxic effects of prenatal nicotine exposure on later brain development, we have used transcriptomic analysis of striatal samples to identify gene expression differences between adolescent Lister Hooded rats exposed to nicotine in utero and controls. Using an additional group of animals matched for the reduced food intake experienced in the nicotine group, we were also able to assess the impact of imposed food-restriction on gene expression profiles. We found little evidence for a role of gestational nicotine exposure on altered gene expression in the striatum of adolescent offspring at a significance level of p<0.01 and |log2 fold change >0.5|, although we cannot exclude the possibility of nicotine-induced changes in other brain regions, or at other time points. We did, however, find marked gene expression differences in response to imposed food-restriction. Food-restriction resulted in significant group differences for a number of immediate early genes (IEGs) including Fos, Fosb, Fosl2, Arc, Junb, Nr4a1 and Nr4a3. These genes are associated with stress response pathways and therefore may reflect long-term effects of nutritional deprivation on the development of the stress system.
doi:10.1371/journal.pone.0088896
PMCID: PMC3929494  PMID: 24586432
9.  Epigenetic Studies of Schizophrenia: Progress, Predicaments, and Promises for the Future 
Schizophrenia Bulletin  2012;39(1):11-16.
Increased understanding about the functional complexity of the genome has led to growing recognition about the role of epigenetic variation in the etiology of schizophrenia. Epigenetic processes act to dynamically control gene expression independently of DNA sequence variation and are known to regulate key neurobiological and cognitive processes in the brain. To date, our knowledge about the role of epigenetic processes in schizophrenia is limited and based on analyses of small numbers of samples obtained from a range of different cell and tissue types. Moving forward, it will be important to establish cause and effect in epigenetic studies of schizophrenia and broaden our horizons beyond DNA methylation. Rather than investigating genetic and epigenetic factors independently, an integrative etiological research paradigm based on the combination of genomic, transcriptomic, and epigenomic analyses is required.
doi:10.1093/schbul/sbs139
PMCID: PMC3523917  PMID: 23211398
schizophrenia; epigenetics; DNA methylation; genetics; epidemiology
10.  A data-driven approach to preprocessing Illumina 450K methylation array data 
BMC Genomics  2013;14:293.
Background
As the most stable and experimentally accessible epigenetic mark, DNA methylation is of great interest to the research community. The landscape of DNA methylation across tissues, through development and in disease pathogenesis is not yet well characterized. Thus there is a need for rapid and cost effective methods for assessing genome-wide levels of DNA methylation. The Illumina Infinium HumanMethylation450 (450K) BeadChip is a very useful addition to the available methods for DNA methylation analysis but its complex design, incorporating two different assay methods, requires careful consideration. Accordingly, several normalization schemes have been published. We have taken advantage of known DNA methylation patterns associated with genomic imprinting and X-chromosome inactivation (XCI), in addition to the performance of SNP genotyping assays present on the array, to derive three independent metrics which we use to test alternative schemes of correction and normalization. These metrics also have potential utility as quality scores for datasets.
Results
The standard index of DNA methylation at any specific CpG site is β = M/(M + U + 100) where M and U are methylated and unmethylated signal intensities, respectively. Betas (βs) calculated from raw signal intensities (the default GenomeStudio behavior) perform well, but using 11 methylomic datasets we demonstrate that quantile normalization methods produce marked improvement, even in highly consistent data, by all three metrics. The commonly used procedure of normalizing betas is inferior to the separate normalization of M and U, and it is also advantageous to normalize Type I and Type II assays separately. More elaborate manipulation of quantiles proves to be counterproductive.
Conclusions
Careful selection of preprocessing steps can minimize variance and thus improve statistical power, especially for the detection of the small absolute DNA methylation changes likely associated with complex disease phenotypes. For the convenience of the research community we have created a user-friendly R software package called wateRmelon, downloadable from bioConductor, compatible with the existing methylumi, minfi and IMA packages, that allows others to utilize the same normalization methods and data quality tests on 450K data.
doi:10.1186/1471-2164-14-293
PMCID: PMC3769145  PMID: 23631413
11.  Transcriptomic Analysis of Autistic Brain Reveals Convergent Molecular Pathology 
Nature  2011;474(7351):380-384.
Autism spectrum disorder (ASD) is a common, highly heritable neuro-developmental condition characterized by marked genetic heterogeneity1–3. Thus, a fundamental question is whether autism represents an etiologically heterogeneous disorder in which the myriad genetic or environmental risk factors perturb common underlying molecular pathways in the brain4. Here, we demonstrate consistent differences in transcriptome organization between autistic and normal brain by gene co-expression network analysis. Remarkably, regional patterns of gene expression that typically distinguish frontal and temporal cortex are significantly attenuated in the ASD brain, suggesting abnormalities in cortical patterning. We further identify discrete modules of co-expressed genes associated with autism: a neuronal module enriched for known autism susceptibility genes, including the neuronal specific splicing factor A2BP1/FOX1, and a module enriched for immune genes and glial markers. Using high-throughput RNA-sequencing we demonstrate dysregulated splicing of A2BP1-dependent alternative exons in ASD brain. Moreover, using a published autism GWAS dataset, we show that the neuronal module is enriched for genetically associated variants, providing independent support for the causal involvement of these genes in autism. In contrast, the immune-glial module showed no enrichment for autism GWAS signals, indicating a non-genetic etiology for this process. Collectively, our results provide strong evidence for convergent molecular abnormalities in ASD, and implicate transcriptional and splicing dysregulation as underlying mechanisms of neuronal dysfunction in this disorder.
doi:10.1038/nature10110
PMCID: PMC3607626  PMID: 21614001
12.  Epigenetic and genetic variation at the IGF2/H19 imprinting control region on 11p15.5 is associated with cerebellum weight 
Epigenetics  2012;7(2):155-163.
IGF2 is a paternally expressed imprinted gene with an important role in development and brain function. Allele-specific expression of IGF2 is regulated by DNA methylation at three differentially methylated regions (DMRs) spanning the IGF2/H19 domain on human 11p15.5. We have comprehensively assessed DNA methylation and genotype across the three DMRs and the H19 promoter using tissue from a unique collection of well-characterized and neuropathologically-dissected post-mortem human cerebellum samples (n = 106) and frontal cortex samples (n = 51). We show that DNA methylation, particularly in the vicinity of a key CTCF-binding site (CTCF3) in the imprinting control region (ICR) upstream of H19, is strongly correlated with cerebellum weight. DNA methylation at CTCF3 uniquely explains ∼25% of the variance in cerebellum weight. In addition, we report that genetic variation in this ICR is strongly associated with cerebellum weight in a parental-origin specific manner, with maternally-inherited alleles associated with a 16% increase in cerebellum weight compared with paternally-inherited alleles. Given the link between structural brain abnormalities and neuropsychiatric disease, an understanding of the epigenetic and parent-of-origin specific genetic factors associated with brain morphology provides important clues about the etiology of disorders such as schizophrenia and autism.
doi:10.4161/epi.7.2.18910
PMCID: PMC3335909  PMID: 22395465
epigenetic; DNA methylation; genomic imprinting; cerebellum; IGF2; H19; brain; expression; frontal cortex; genetic; single nucleotide polymorphism
13.  Current status and future prospects for epigenetic psychopharmacology 
Epigenetics  2012;7(1):20-28.
Mounting evidence suggest that epigenetic regulation of brain functions is important in the etiology of psychiatric disorders. These epigenetic regulatory mechanisms, such as DNA methylation and histone acetylation, are influenced by many pharmaceutical compounds including psychiatric drugs. It is therefore of interest to investigate how psychiatric drugs are of influence and what the potential is of new epigenetic drugs for psychiatric disorders. With this targeted review we summarize the current state of knowledge in order to provide insight in this developing field. Several traditional psychiatric drugs have been found to alter the epigenome and in a variety of animal studies, experimental compounds with epigenetic targets have been investigated as potential psychiatric drugs. After discussion of the most relevant epigenetic mechanisms we present the evidence for epigenetic effects for the most relevant classes of drugs.
doi:10.4161/epi.7.1.18688
PMCID: PMC3329498  PMID: 22207355
BDNF; DNA Methylation; DNMT inhibitors; epigenetics; HDAC inhibitors; Histone Modifications; neuropharmacology; psychiatry; psychotropics; treatment
14.  DNA methylation at the Igf2/H19 imprinting control region is associated with cerebellum mass in outbred mice 
Molecular Brain  2012;5:42.
Background
Insulin-like growth factor 2 (Igf2) is a paternally expressed imprinted gene regulating fetal growth, playing an integral role in the development of many tissues including the brain. The parent-of-origin specific expression of Igf2 is largely controlled by allele-specific DNA methylation at CTCF-binding sites in the imprinting control region (ICR), located immediately upstream of the neighboring H19 gene. Previously we reported evidence of a negative correlation between DNA methylation in this region and cerebellum weight in humans.
Results
We quantified cerebellar DNA methylation across all four CTCF binding sites spanning the murine Igf2/H19 ICR in an outbred population of Heterogeneous Stock (HS) mice (n = 48). We observe that DNA methylation at the second and third CTCF binding sites in the Igf2/H19 ICR shows a negative relationship with cerebellar mass, reflecting the association observed in human post-mortem cerebellum tissue.
Conclusions
Given the important role of the cerebellum in motor control and cognition, and the link between structural cerebellar abnormalities and neuropsychiatric phenotypes, the identification of epigenetic factors associated with cerebellum growth and development may provide important insights about the etiology of psychiatric disorders.
doi:10.1186/1756-6606-5-42
PMCID: PMC3541153  PMID: 23216893
Igf2; H19; Epigenetics; DNA methylation; Cerebellum; Brain; Mouse; Genotype; Genomic imprinting
15.  Schizophrenia is associated with dysregulation of a Cdk5 activator that regulates synaptic protein expression and cognition 
Brain  2011;134(8):2408-2421.
Cyclin-dependent kinase 5 is activated by small subunits, of which p35 is the most abundant. The functions of cyclin-dependent kinase 5 signalling in cognition and cognitive disorders remains unclear. Here, we show that in schizophrenia, a disorder associated with impaired cognition, p35 expression is reduced in relevant brain regions. Additionally, the expression of septin 7 and OPA1, proteins downstream of truncated p35, is decreased in schizophrenia. Mimicking a reduction of p35 in heterozygous knockout mice is associated with cognitive endophenotypes. Furthermore, a reduction of p35 in mice results in protein changes similar to schizophrenia post-mortem brain. Hence, heterozygous p35 knockout mice model both cognitive endophenotypes and molecular changes reminiscent of schizophrenia. These changes correlate with reduced acetylation of the histone deacetylase 1 target site H3K18 in mice. This site has previously been shown to be affected by truncated p35. By restoring H3K18 acetylation with the clinically used specific histone deacetylase 1 inhibitor MS-275 both cognitive and molecular endophenotypes of schizophrenia can be rescued in p35 heterozygous knockout mice. In summary, we suggest that reduced p35 expression in schizophrenia has an impact on synaptic protein expression and cognition and that these deficits can be rescued, at least in part, by the inhibition of histone deacetylase 1.
doi:10.1093/brain/awr155
PMCID: PMC3155706  PMID: 21772061
animal models; brain; cognition; schizophrenia; signalling
16.  Functional annotation of the human brain methylome identifies tissue-specific epigenetic variation across brain and blood 
Genome Biology  2012;13(6):R43.
Background
Dynamic changes to the epigenome play a critical role in establishing and maintaining cellular phenotype during differentiation, but little is known about the normal methylomic differences that occur between functionally distinct areas of the brain. We characterized intra- and inter-individual methylomic variation across whole blood and multiple regions of the brain from multiple donors.
Results
Distinct tissue-specific patterns of DNA methylation were identified, with a highly significant over-representation of tissue-specific differentially methylated regions (TS-DMRs) observed at intragenic CpG islands and low CG density promoters. A large proportion of TS-DMRs were located near genes that are differentially expressed across brain regions. TS-DMRs were significantly enriched near genes involved in functional pathways related to neurodevelopment and neuronal differentiation, including BDNF, BMP4, CACNA1A, CACA1AF, EOMES, NGFR, NUMBL, PCDH9, SLIT1, SLITRK1 and SHANK3. Although between-tissue variation in DNA methylation was found to greatly exceed between-individual differences within any one tissue, we found that some inter-individual variation was reflected across brain and blood, indicating that peripheral tissues may have some utility in epidemiological studies of complex neurobiological phenotypes.
Conclusions
This study reinforces the importance of DNA methylation in regulating cellular phenotype across tissues, and highlights genomic patterns of epigenetic variation across functionally distinct regions of the brain, providing a resource for the epigenetics and neuroscience research communities.
doi:10.1186/gb-2012-13-6-r43
PMCID: PMC3446315  PMID: 22703893
17.  A genetic association study of DNA methylation levels in the DRD4 gene region finds associations with nearby SNPs 
Background
Dopamine receptor D4(DRD4) polymorphisms have been associated with a number of psychiatric disorders, but little is known about the mechanism of these associations. DNA methylation is linked to the regulation of gene expression and plays a vital role in normal cellular function, with abnormal DNA methylation patterns implicated in a range of disorders. Recent evidence suggests DNA methylation can be influenced by cis-acting DNA sequence variation, that is, DNA sequence variation located nearby on the same chromosome.
Methods
To investigate the potential influence of cis-acting genetic elements within DRD4, we analysed DRD4 promoter DNA methylation levels in the transformed lymphoblastoid cell-line DNA of 89 individuals (from 30 family-trios). Five SNPs located +/− 10kb of the promoter region were interrogated for associations with DNA methylation levels.
Results
Four significant SNP associations were found with DNA methylation (rs3758653, rs752306, rs11246228 and rs936465). The associations of rs3758653 and rs936465 with DNA methylation were tested and nominally replicated (p-value < 0.05) in post-mortem brain tissue from an independent sample (N = 18). Interestingly, the DNA methylation patterns observed in post-mortem brain tissue were similar to those observed in transformed lymphoblastoid cell line DNA.
Conclusions
The link reported between DNA sequence and DNA methylation offers a possible functional role to seemingly non-functional SNP associations. DRD4 has been implicated in several psychiatric disease phenotypes and our results shed light upon the possible mode of action of SNP associations in this region.
doi:10.1186/1744-9081-8-31
PMCID: PMC3538530  PMID: 22691691
18.  Epigenome-Wide Scans Identify Differentially Methylated Regions for Age and Age-Related Phenotypes in a Healthy Ageing Population 
PLoS Genetics  2012;8(4):e1002629.
Age-related changes in DNA methylation have been implicated in cellular senescence and longevity, yet the causes and functional consequences of these variants remain unclear. To elucidate the role of age-related epigenetic changes in healthy ageing and potential longevity, we tested for association between whole-blood DNA methylation patterns in 172 female twins aged 32 to 80 with age and age-related phenotypes. Twin-based DNA methylation levels at 26,690 CpG-sites showed evidence for mean genome-wide heritability of 18%, which was supported by the identification of 1,537 CpG-sites with methylation QTLs in cis at FDR 5%. We performed genome-wide analyses to discover differentially methylated regions (DMRs) for sixteen age-related phenotypes (ap-DMRs) and chronological age (a-DMRs). Epigenome-wide association scans (EWAS) identified age-related phenotype DMRs (ap-DMRs) associated with LDL (STAT5A), lung function (WT1), and maternal longevity (ARL4A, TBX20). In contrast, EWAS for chronological age identified hundreds of predominantly hyper-methylated age DMRs (490 a-DMRs at FDR 5%), of which only one (TBX20) was also associated with an age-related phenotype. Therefore, the majority of age-related changes in DNA methylation are not associated with phenotypic measures of healthy ageing in later life. We replicated a large proportion of a-DMRs in a sample of 44 younger adult MZ twins aged 20 to 61, suggesting that a-DMRs may initiate at an earlier age. We next explored potential genetic and environmental mechanisms underlying a-DMRs and ap-DMRs. Genome-wide overlap across cis-meQTLs, genotype-phenotype associations, and EWAS ap-DMRs identified CpG-sites that had cis-meQTLs with evidence for genotype–phenotype association, where the CpG-site was also an ap-DMR for the same phenotype. Monozygotic twin methylation difference analyses identified one potential environmentally-mediated ap-DMR associated with total cholesterol and LDL (CSMD1). Our results suggest that in a small set of genes DNA methylation may be a candidate mechanism of mediating not only environmental, but also genetic effects on age-related phenotypes.
Author Summary
Epigenetic patterns vary during healthy ageing and development. Age-related DNA methylation changes have been implicated in cellular senescence and longevity, yet the causes and functional consequences of these variants remain unclear. To understand the biological mechanisms involved in potential longevity and rate of healthy ageing, we performed genome-wide association of epigenetic and genetic variation with both chronological age and age-related phenotypes. We identified hundreds of DNA methylation variants significantly associated with age and replicated these in an independent sample of young adult twins. Only a small proportion of these variants were also associated with age-related phenotypes. Therefore, the majority of age-related epigenetic changes do not contribute to rate of healthy ageing at later stages in life. Our results suggest that age-related changes in methylation occur throughout an individual's lifespan and that a proportion of these may be initiated from an early age. Intriguingly, a fraction of the age differentially methylated regions also associated with genetic variants in our sample, suggesting that DNA methylation may be a candidate mechanism of mediating not only environmental but also genetic effects on age-related phenotypes.
doi:10.1371/journal.pgen.1002629
PMCID: PMC3330116  PMID: 22532803
19.  Commentary: The seven plagues of epigenetic epidemiology 
doi:10.1093/ije/dyr225
PMCID: PMC3304528  PMID: 22269254
20.  Disease-associated epigenetic changes in monozygotic twins discordant for schizophrenia and bipolar disorder 
Human Molecular Genetics  2011;20(24):4786-4796.
Studies of the major psychoses, schizophrenia (SZ) and bipolar disorder (BD), have traditionally focused on genetic and environmental risk factors, although more recent work has highlighted an additional role for epigenetic processes in mediating susceptibility. Since monozygotic (MZ) twins share a common DNA sequence, their study represents an ideal design for investigating the contribution of epigenetic factors to disease etiology. We performed a genome-wide analysis of DNA methylation on peripheral blood DNA samples obtained from a unique sample of MZ twin pairs discordant for major psychosis. Numerous loci demonstrated disease-associated DNA methylation differences between twins discordant for SZ and BD individually, and together as a combined major psychosis group. Pathway analysis of our top loci highlighted a significant enrichment of epigenetic changes in biological networks and pathways directly relevant to psychiatric disorder and neurodevelopment. The top psychosis-associated, differentially methylated region, significantly hypomethylated in affected twins, was located in the promoter of ST6GALNAC1 overlapping a previously reported rare genomic duplication observed in SZ. The mean DNA methylation difference at this locus was 6%, but there was considerable heterogeneity between families, with some twin pairs showing a 20% difference in methylation. We subsequently assessed this region in an independent sample of postmortem brain tissue from affected individuals and controls, finding marked hypomethylation (>25%) in a subset of psychosis patients. Overall, our data provide further evidence to support a role for DNA methylation differences in mediating phenotypic differences between MZ twins and in the etiology of both SZ and BD.
doi:10.1093/hmg/ddr416
PMCID: PMC3221539  PMID: 21908516
21.  A longitudinal study of epigenetic variation in twins 
Epigenetics  2010;5(6):516-526.
DNA methylation is a key epigenetic mechanism involved in the developmental regulation of gene expression. Alterations in DNA methylation are established contributors to inter-individual phenotypic variation and have been associated with disease susceptibility. The degree to which changes in loci-specific DNA methylation are under the influence of heritable and environmental factors is largely unknown. In this study, we quantitatively measured DNA methylation across the promoter regions of the dopamine receptor 4 gene (DRD4), the serotonin transporter gene (SLC6A4/SERT) and the X-linked monoamine oxidase A gene (MAOA) using DNA sampled at both ages 5 and 10 years in 46 MZ twinpairs and 45 DZ twin-pairs (total n = 182). Our data suggest that DNA methylation differences are apparent already in early childhood, even between genetically identical individuals, and that individual differences in methylation are not stable over time. Our longitudinal-developmental study suggests that environmental influences are important factors accounting for interindividual DNA methylation differences, and that these influences differ across the genome. The observation of dynamic changes in DNA methylation over time highlights the importance of longitudinal research designs for epigenetic research.
doi:10.4161/epi.5.6.12226
PMCID: PMC3322496  PMID: 20505345
epigenetics; DNA methylation; twin; heritability; dynamic; environment
22.  A Longitudinal Twin Study of Skewed X Chromosome-Inactivation 
PLoS ONE  2011;6(3):e17873.
X-chromosome inactivation (XCI) is a pivotal epigenetic mechanism involved in the dosage compensation of X-linked genes between males and females. In any given cell, the process of XCI in early female development is thought to be random across alleles and clonally maintained once established. Recent studies, however, suggest that XCI might not always be random and that skewed inactivation may become more prevalent with age. The factors influencing such XCI skewing and its changes over time are largely unknown. To elucidate the influence of stochastic, heritable and environmental factors in longitudinal changes in XCI, we examined X inactivation profiles in a sample of monozygotic (MZ) (n = 23) and dizygotic (DZ) (n = 22) female twin-pairs at ages 5 and 10 years. Compared to MZ twins who were highly concordant for allelic XCI ratios, DZ twins showed much lower levels of concordance. Whilst XCI patterns were moderately stable between ages 5 and 10 years, there was some drift over time with an increased prevalence of more extreme XCI skewing at age 10. To our knowledge, this study represents the earliest longitudinal assessment of skewed XCI patterns, and suggests that skewed XCI may already be established in early childhood. Our data also suggest a link between MZ twinning and the establishment of allelic XCI ratios, and demonstrate that acquired skewing in XCI after establishment is primarily mediated by stochastic mechanisms. These data have implications for our understanding about sex differences in complex disease, and the potential causes of phenotypic discordance between MZ female twins.
doi:10.1371/journal.pone.0017873
PMCID: PMC3062559  PMID: 21445353
23.  Integrated Genetic and Epigenetic Analysis Identifies Haplotype-Specific Methylation in the FTO Type 2 Diabetes and Obesity Susceptibility Locus 
PLoS ONE  2010;5(11):e14040.
Recent multi-dimensional approaches to the study of complex disease have revealed powerful insights into how genetic and epigenetic factors may underlie their aetiopathogenesis. We examined genotype-epigenotype interactions in the context of Type 2 Diabetes (T2D), focussing on known regions of genomic susceptibility. We assayed DNA methylation in 60 females, stratified according to disease susceptibility haplotype using previously identified association loci. CpG methylation was assessed using methylated DNA immunoprecipitation on a targeted array (MeDIP-chip) and absolute methylation values were estimated using a Bayesian algorithm (BATMAN). Absolute methylation levels were quantified across LD blocks, and we identified increased DNA methylation on the FTO obesity susceptibility haplotype, tagged by the rs8050136 risk allele A (p = 9.40×10−4, permutation p = 1.0×10−3). Further analysis across the 46 kb LD block using sliding windows localised the most significant difference to be within a 7.7 kb region (p = 1.13×10−7). Sequence level analysis, followed by pyrosequencing validation, revealed that the methylation difference was driven by the co-ordinated phase of CpG-creating SNPs across the risk haplotype. This 7.7 kb region of haplotype-specific methylation (HSM), encapsulates a Highly Conserved Non-Coding Element (HCNE) that has previously been validated as a long-range enhancer, supported by the histone H3K4me1 enhancer signature. This study demonstrates that integration of Genome-Wide Association (GWA) SNP and epigenomic DNA methylation data can identify potential novel genotype-epigenotype interactions within disease-associated loci, thus providing a novel route to aid unravelling common complex diseases.
doi:10.1371/journal.pone.0014040
PMCID: PMC2987816  PMID: 21124985
24.  Epigenetic Mediation of Environmental Influences in Major Psychotic Disorders 
Schizophrenia Bulletin  2009;35(6):1045-1056.
The major psychotic disorders schizophrenia and bipolar disorder are etiologically complex involving both heritable and nonheritable factors. The absence of consistently replicated major genetic effects, together with evidence for lasting changes in gene expression after environmental exposures, is consistent with the concept that the biologic underpinnings of these disorders are epigenetic in form rather than DNA sequence based. Psychosis-associated environmental exposures, particularly at key developmental stages, may result in long-lasting epigenetic alterations that impact on the neurobiological processes involved in pathology. Although direct evidence for epigenetic dysfunction in both schizophrenia and bipolar disorder is still limited, methodological technologies in epigenomic profiling have advanced. This means that we are at the exciting stage where it is feasible to start investigating molecular modifications to DNA and histones and examine the mechanisms by which environmental factors can act upon the genome to bring about epigenetic changes in gene expression involved in the etiology of these disorders. Given the dynamic nature of the epigenetic machinery and potential reversibility of epigenetic modifications, the understanding of such mechanisms is of key relevance for clinical psychiatry and for identifying new targets for prevention and/or intervention.
doi:10.1093/schbul/sbp104
PMCID: PMC2762629  PMID: 19783603
DNA methylation; histone modifications; development; schizophrenia; bipolar disorder; gene-environment interaction
25.  Allele-specific methylation in the human genome 
Epigenetics  2010;5(7):578-582.
Across the genome, outside of a small number of known imprinted genes and regions subject to X-inactivation in females, DNA methylation at CpG dinucleotides is often assumed to be complementary across both alleles in a diploid cell. However, recent findings suggest the reality is more complex, with the discovery that allele-specific methylation (ASM) is a common feature across the human genome. A key observation is that the majority of ASM is associated with genetic variation in cis, although a noticeable proportion is also non-cis in nature and mediated, for example, by parental origin. ASM appears to be both quantitative, characterized by subtle skewing of DNA methylation between alleles, and heterogeneous, varying across tissues and between individuals. These findings have important implications for complex disease genetics; while cis-mediated ASM provides a functional consequence for non-coding genetic variation, heterogeneous and quantitative ASM complicates the identification of disease-associated loci. We propose that non-cis ASM could contribute toward the “missing heritability” of complex diseases, rendering certain loci hemizygous and masking the direct association between genotype and phenotype. We suggest that the interpretation of results from genome-wide association studies can be improved by the incorporation of epi-allelic information and that in order to fully understand the extent and consequence of ASM in the human genome, a comprehensive sequencing-based analysis of allelic methylation patterns across tissues and individuals is required.
doi:10.4161/epi.5.7.12960
PMCID: PMC3052843  PMID: 20716955
DNA methylation; allelespecific methylation; allele-specific expression; tissue-specific methylation; epigenetics; imprinting; genome-wide association study (GWAS); genetics; complex disease; missing heritability

Results 1-25 (31)