ABSTRACTCharacterizing the complex relationship between genetic, epigenetic and transcriptomic variation has the potential to increase understanding about the mechanisms underpinning health and disease phenotypes. In this study, we describe the most comprehensive analysis of common genetic variation on DNA methylation (DNAm) to date, using the Illumina EPIC array to profile samples from the UK Household Longitudinal study. We identified 12,689,548 significant DNA methylation quantitative trait loci (mQTL) associations (P < 6.52x10-14) occurring between 2,907,234 genetic variants and 93,268 DNAm sites, including a large number not identified using previous DNAm-profiling methods. We demonstrate the utility of these data for interpreting the functional consequences of common genetic variation associated with > 60 human traits, using Summary data–based Mendelian Randomization (SMR) to identify 1,662 pleiotropic associations between 36 complex traits and 1,246 DNAm sites. We also use SMR to characterize the relationship between DNAm and gene expression, identifying 6,798 pleiotropic associations between 5,420 DNAm sites and the transcription of 1,702 genes. Our mQTL database and SMR results are available via a searchable online database (http://www.epigenomicslab.com/online-data-resources/) as a resource to the research community.

Abstract

Importance: Genetic factors increase risk of dementia, but the extent to which this can be offset by lifestyle factors is unknown.

Objective: to investigate whether a healthy lifestyle is associated with lower risk of dementia regardless of genetic risk.

Design, Setting, and Participants: a retrospective cohort study that included adults of European ancestry aged at least 60 years without cognitive impairment or dementia at baseline. Participants joined the UK Biobank study from 2006 to 2010 and were followed up until 2016 or 2017.

Exposures: a polygenic risk score for dementia with low (lowest quintile), intermediate (quintiles 2 to 4), and high (highest quintile) risk categories and a weighted healthy lifestyle score, including no current smoking, regular physical activity, healthy diet, and moderate alcohol consumption, categorized into favorable, intermediate, and unfavorable lifestyles.

Main Outcomes and Measures: Incident all-cause dementia, ascertained through hospital inpatient and death records.

Results: a total of 196 383 individuals (mean [SD] age, 64.1 [2.9] years; 52.7% were women) were followed up for 1 545 433 person-years (median [interquartile range] follow-up, 8.0 [7.4-8.6] years). Overall, 68.1% of participants followed a favorable lifestyle, 23.6% followed an intermediate lifestyle, and 8.2% followed an unfavorable lifestyle. Twenty percent had high polygenic risk scores, 60% had intermediate risk scores, and 20% had low risk scores. of the participants with high genetic risk, 1.23% (95% CI, 1.13%-1.35%) developed dementia compared with 0.63% (95% CI, 0.56%-0.71%) of the participants with low genetic risk (adjusted hazard ratio, 1.91 [95% CI, 1.64-2.23]). of the participants with a high genetic risk and unfavorable lifestyle, 1.78% (95% CI, 1.38%-2.28%) developed dementia compared with 0.56% (95% CI, 0.48%-0.66%) of participants with low genetic risk and favorable lifestyle (hazard ratio, 2.83 [95% CI, 2.09-3.83]). There was no significant interaction between genetic risk and lifestyle factors (P = .99). Among participants with high genetic risk, 1.13% (95% CI, 1.01%-1.26%) of those with a favorable lifestyle developed dementia compared with 1.78% (95% CI, 1.38%-2.28%) with an unfavorable lifestyle (hazard ratio, 0.68 [95% CI, 0.51-0.90]).

Conclusions and Relevance: Among older adults without cognitive impairment or dementia, both an unfavorable lifestyle and high genetic risk were significantly associated with higher dementia risk. A favorable lifestyle was associated with a lower dementia risk among participants with high genetic risk.

© 2019, the Author(s). Common genetic variation contributes a substantial proportion of risk for both schizophrenia and bipolar disorder. Furthermore, there is evidence of significant, but not complete, overlap in genetic risk between the two disorders. It has been hypothesised that genetic variants conferring risk for these disorders do so by influencing brain development, leading to the later emergence of symptoms. The comparative profile of risk gene expression for schizophrenia and bipolar disorder across development over different brain regions however remains unclear. Using genotypes derived from genome-wide associations studies of the largest available cohorts of patients and control subjects, we investigated whether genes enriched for schizophrenia and bipolar disorder association show a bias for expression across any of 13 developmental stages in prefrontal cortical and subcortical brain regions. We show that genetic association with schizophrenia is positively correlated with expression in the prefrontal cortex during early midfetal development and early infancy, and negatively correlated with expression during late childhood, which stabilises in adolescence. In contrast, risk-associated genes for bipolar disorder did not exhibit a bias towards expression at any prenatal stage, although the pattern of postnatal expression was similar to that of schizophrenia. These results highlight the dynamic expression of genes harbouring risk for schizophrenia and bipolar disorder across prefrontal cortex development and support the hypothesis that prenatal neurodevelopmental events are more strongly associated with schizophrenia than bipolar disorder.

BACKGROUND: Previous studies have reported associations between attention-deficit/hyperactivity disorder symptoms and DNA methylation in children. We report the first epigenome-wide association study meta-analysis of adult attention-deficit/hyperactivity disorder symptoms, based on peripheral blood DNA methylation (Infinium HumanMethylation450K array) in three population-based adult cohorts. METHODS: an epigenome-wide association study was performed in the Netherlands Twin Register (N = 2258, mean age 37 years), Dunedin Multidisciplinary Health and Development Study (N = 800, age 38 years), and Environmental Risk Longitudinal Twin Study (N = 1631, age 18 years), and results were combined through meta-analysis (total sample size N = 4689). Region-based analyses accounting for the correlation between nearby methylation sites were also performed. RESULTS: One epigenome-wide significant differentially methylated position was detected in the Dunedin study, but meta-analysis did not detect differentially methylated positions that were robustly associated across cohorts. In region-based analyses, six significant differentially methylation regions (DMRs) were identified in the Netherlands Twin Register, 19 in the Dunedin study, and none in the Environmental Risk Longitudinal Twin Study. of these DMRs, 92% were associated with methylation quantitative trait loci, and 68% showed moderate to large blood-brain correlations for DNA methylation levels. DMRs included six nonoverlapping DMRs (three in the Netherlands Twin Register, three in the Dunedin study) in the major histocompatibility complex, which were associated with expression of genes in the major histocompatibility complex, including C4A and C4B, previously implicated in schizophrenia. CONCLUSIONS: Our findings point at new candidate loci involved in immune and neuronal functions that await further replication. Our work also illustrates the need for further research to examine to what extent epigenetic associations with psychiatric traits depend on characteristics such as age, comorbidities, exposures, and genetic background.

Large-scale epigenome-wide association meta-analyses have identified multiple 'signatures'' of smoking. Drawing on these findings, we describe the construction of a polyepigenetic DNA methylation score that indexes smoking behavior and that can be utilized for multiple purposes in population health research. To validate the score, we use data from two birth cohort studies: the Dunedin Longitudinal Study, followed to age-38 years, and the Environmental Risk Study, followed to age-18 years. Longitudinal data show that changes in DNA methylation accumulate with increased exposure to tobacco smoking and attenuate with quitting. Data from twins discordant for smoking behavior show that smoking influences DNA methylation independently of genetic and environmental risk factors. Physiological data show that changes in DNA methylation track smoking-related changes in lung function and gum health over time. Moreover, DNA methylation changes predict corresponding changes in gene expression in pathways related to inflammation, immune response, and cellular trafficking. Finally, we present prospective data about the link between adverse childhood experiences (ACEs) and epigenetic modifications; these findings document the importance of controlling for smoking-related DNA methylation changes when studying biological embedding of stress in life-course research. We introduce the polyepigenetic DNA methylation score as a tool both for discovery and theory-guided research in epigenetic epidemiology.

Autism spectrum disorder (ASD) encompasses a collection of complex neuropsychiatric disorders characterized by deficits in social functioning, communication and repetitive behaviour. Building on recent studies supporting a role for developmentally moderated regulatory genomic variation in the molecular aetiology of ASD, we quantified genome-wide patterns of DNA methylation in 223 post-mortem tissues samples isolated from three brain regions [prefrontal cortex, temporal cortex and cerebellum (CB)] dissected from 43 ASD patients and 38 non-psychiatric control donors. We identified widespread differences in DNA methylation associated with idiopathic ASD (iASD), with consistent signals in both cortical regions that were distinct to those observed in the CB. Individuals carrying a duplication on chromosome 15q (dup15q), representing a genetically defined subtype of ASD, were characterized by striking differences in DNA methylationacross a discrete domain spanning an imprinted gene cluster within the duplicated region. In addition to the dramatic cis-effects on DNA methylation observed in dup15q carriers, we identified convergent methylomic signatures associated with both iASD and dup15q, reflecting the findings from previous studies of gene expression and H3K27ac. Cortical co-methylation network analysis identified a number of co-methylated modules significantly associated with ASD that are enriched for genomic regions annotated to genes involved in the immune system, synaptic signalling and neuronal regulation. Our study represents the first systematic analysis of DNA methylation associated with ASD across multiple brain regions, providing novel evidence for convergent molecular signatures associated with both idiopathic and syndromic autism.

BACKGROUND: There has been a steady increase in the number of studies aiming to identify DNA methylation differences associated with complex phenotypes. Many of the challenges of epigenetic epidemiology regarding study design and interpretation have been discussed in detail, however there are analytical concerns that are outstanding and require further exploration. In this study we seek to address three analytical issues. First, we quantify the multiple testing burden and propose a standard statistical significance threshold for identifying DNA methylation sites that are associated with an outcome. Second, we establish whether linear regression, the chosen statistical tool for the majority of studies, is appropriate and whether it is biased by the underlying distribution of DNA methylation data. Finally, we assess the sample size required for adequately powered DNA methylation association studies. RESULTS: We quantified DNA methylation in the Understanding Society cohort (n = 1175), a large population based study, using the Illumina EPIC array to assess the statistical properties of DNA methylation association analyses. By simulating null DNA methylation studies, we generated the distribution of p-values expected by chance and calculated the 5% family-wise error for EPIC array studies to be 9 × 10- 8. Next, we tested whether the assumptions of linear regression are violated by DNA methylation data and found that the majority of sites do not satisfy the assumption of normal residuals. Nevertheless, we found no evidence that this bias influences analyses by increasing the likelihood of affected sites to be false positives. Finally, we performed power calculations for EPIC based DNA methylation studies, demonstrating that existing studies with data on ~ 1000 samples are adequately powered to detect small differences at the majority of sites. CONCLUSION: We propose that a significance threshold of P 

Methylome-wide association studies (MWASs) are promising complements to sequence variation studies. We used existing sequencing-based methylation data, which assayed the majority of all 28 million CpGs in the human genome, to perform an MWAS for schizophrenia in blood, while controlling for cell-type heterogeneity with a recently generated platform-specific reference panel. Next, we compared the MWAS results with findings from 3 existing large-scale array-based schizophrenia methylation studies in blood that assayed up to ~450 000 CpGs. Our MWAS identified 22 highly significant loci (P < 5 × 10-8) and 852 suggestively significant loci (P < 1 × 10-5). The top finding (P = 5.62 × 10-11, q = 0.001) was located in MFN2, which encodes mitofusin-2 that regulates Ca2+ transfer from the endoplasmic reticulum to mitochondria in cooperation with DISC1. The second-most significant site (P = 1.38 × 10-9, q = 0.013) was located in ALDH1A2, which encodes an enzyme for astrocyte-derived retinoic acid-a key neuronal morphogen with relevance for schizophrenia. Although the most significant MWAS findings were not assayed on the arrays, we observed significant enrichment of overlapping findings with 2 of the 3 array datasets (P = 0.0315, 0.0045, 0.1946). Overrepresentation analysis of Gene Ontology terms for the genes in the significant overlaps suggested high similarity in the biological functions detected by the different datasets. Top terms were related to immune and/or stress responses, cell adhesion and motility, and a broad range of processes essential for neurodevelopment.

Childhood psychotic experiences (PEs), such as seeing or hearing things that others do not, or extreme paranoia, are relatively common with around 1 in 20 children reporting them at age 12. Childhood PEs are often distressing and can be predictive of schizophrenia, other psychiatric disorders, and suicide attempts in adulthood, particularly if they persist during adolescence. Previous research has demonstrated that methylomic signatures in blood could be potential biomarkers of psychotic phenomena. This study explores the association between DNA methylation (DNAm) and the emergence, persistence, and remission of PEs in childhood and adolescence. DNAm profiles were obtained from the ALSPAC cohort at birth, age 7, and age 15/17 (n = 901). PEs were assessed through interviews with participants at ages 12 and 18. We identified PE-associated probes (p 

Schizophrenia (SCZ) is associated with high mortality. DNA methylation levels vary over the life course, and pre-selected combinations of methylation array probes can be used to estimate "methylation age" (mAge). mAge correlates highly with chronological age but when it differs, termed mAge acceleration, it has been previously associated with all-cause mortality. We tested the association between mAge acceleration and mortality in SCZ and controls. We selected 190 SCZ cases and 190 controls from the Sweden Schizophrenia Study. Cases were identified from the Swedish Hospital Discharge Register with ≥5 specialist treatment contacts and ≥5 antipsychotic prescriptions. Controls had no psychotic disorder or antipsychotics. Subjects were selected if they had died or survived during follow-up (2:1 oversampling). Extracted DNA was assayed on the Illumina MethylationEPIC array. mAge was regressed on age at sampling to obtain mAge acceleration. Using Cox proportional hazards regression, the association between mAge acceleration and mortality was tested. After quality control, the following were available: n = 126 SCZ died, 63 SCZ alive, 127 controls died, 62 controls alive. In the primary analyses, we did not find a significant association between mAge acceleration and SCZ mortality (adjusted p > 0.005). Sensitivity analyses excluding SCZ cases with pre-existing cancer demonstrated a significant association between the Hannum mAge acceleration and mortality (hazard ratio = 1.13, 95% confidence interval = 1.04-1.22, p = 0.005). Per our pre-specified criteria, we did not confirm our primary hypothesis that mAge acceleration would predict subsequent mortality in people with SCZ, but we cannot rule out smaller effects or effects in patient subsets.

BACKGROUND: Alzheimer's disease is a progressive neurodegenerative disorder that is hypothesized to involve epigenetic dysfunction. Previous studies of DNA modifications in Alzheimer's disease have been unable to distinguish between DNA methylation and DNA hydroxymethylation. DNA hydroxymethylation has been shown to be enriched in the human brain, although its role in Alzheimer's disease has not yet been fully explored. Here, we utilize oxidative bisulfite conversion, in conjunction with the Illumina Infinium Human Methylation 450K microarray, to identify neuropathology-associated differential DNA methylation and DNA hydroxymethylation in the entorhinal cortex. RESULTS: We identified one experiment-wide significant differentially methylated position residing in the WNT5B gene. Next, we investigated pathology-associated regions consisting of multiple adjacent loci. We identified one significant differentially hydroxymethylated region consisting of four probes spanning 104 bases in the FBXL16 gene. We also identified two significant differentially methylated regions: one consisting of two probes in a 93 base-pair region in the ANK1 gene and the other consisting of six probes in a 99-base pair region in the ARID5B gene. We also highlighted three regions that show alterations in unmodified cytosine: two probes in a 39-base pair region of ALLC, two probes in a 69-base pair region in JAG2, and the same six probes in ARID5B that were differentially methylated. Finally, we replicated significant ANK1 disease-associated hypermethylation and hypohydroxymethylation patterns across eight CpG sites in an extended 118-base pair region in an independent cohort using oxidative-bisulfite pyrosequencing. CONCLUSIONS: Our study represents the first epigenome-wide association study of both DNA methylation and hydroxymethylation in Alzheimer's disease entorhinal cortex. We demonstrate that previous estimates of DNA hypermethylation in ANK1 in Alzheimer's disease were underestimates as it is confounded by hypohydroxymethylation.

X-chromosome inactivation (XCI), i.e. the inactivation of one of the female X chromosomes, restores equal expression of X-chromosomal genes between females and males. However, ~10% of genes show variable degrees of escape from XCI between females, although little is known about the causes of variable XCI. Using a discovery data-set of 1867 females and 1398 males and a replication sample of 3351 females, we show that genetic variation at three autosomal loci is associated with female-specific changes in X-chromosome methylation. Through cis-eQTL expression analysis, we map these loci to the genes SMCHD1/METTL4, TRIM6/HBG2, and ZSCAN9. Low-expression alleles of the loci are predominantly associated with mild hypomethylation of CpG islands near genes known to variably escape XCI, implicating the autosomal genes in variable XCI. Together, these results suggest a genetic basis for variable escape from XCI and highlight the potential of a population genomics approach to identify genes involved in XCI.

MotivationThe datasets generated by DNA methylation analyses are getting bigger. With the release of the HumanMethylationEPIC micro-array and datasets containing thousands of samples, analyses of these large datasets using R are becoming impractical due to large memory requirements. As a result there is an increasing need for computationally efficient methodologies to perform meaningful analysis on high dimensional data.ResultsHere we introduce the bigmelon R package, which provides a memory efficient workflow that enables users to perform the complex, large scale analyses required in epigenome wide association studies (EWAS) without the need for large RAM. Building on top of the CoreArray Genomic Data Structure file format and libraries packaged in the gdsfmt package, we provide a practical workflow that facilitates the reading-in, preprocessing, quality control and statistical analysis of DNA methylation data.We demonstrate the capabilities of the bigmelon package using a large dataset consisting of 1193 human blood samples from the Understanding Society: UK Household Longitudinal Study, assayed on the EPIC micro-array platform.Availability and implementationThe bigmelon package is available on Bioconductor (http://bioconductor.org/packages/bigmelon/). The Understanding Society dataset is available at https://www.understandingsociety.ac.uk/about/health/data upon request.Supplementary informationSupplementary data are available at Bioinformatics online.

INTRODUCTION: Alzheimer's disease is a neurodegenerative disorder that is hypothesized to involve epigenetic dysregulation of gene expression in the brain. METHODS: We performed an epigenome-wide association study to identify differential DNA methylation associated with neuropathology in prefrontal cortex and superior temporal gyrus samples from 147 individuals, replicating our findings in two independent data sets (N = 117 and 740). RESULTS: We identify elevated DNA methylation associated with neuropathology across a 48-kb region spanning 208 CpG sites within the HOXA gene cluster. A meta-analysis of the top-ranked probe within the HOXA3 gene (cg22962123) highlighted significant hypermethylation across all three cohorts (P = 3.11 × 10-18). DISCUSSION: We present robust evidence for elevated DNA methylation associated with Alzheimer's disease neuropathology spanning the HOXA gene cluster on chromosome 7. These data add to the growing evidence highlighting a role for epigenetic variation in Alzheimer's disease, implicating the HOX gene family as a target for future investigation.

Autism spectrum disorder (ASD) is a severe neurodevelopmental disorder characterized by deficits in social communication and restricted, repetitive behaviors, interests, or activities. The etiology of ASD involves both inherited and environmental risk factors, with epigenetic processes hypothesized as one mechanism by which both genetic and non-genetic variation influence gene regulation and pathogenesis. The aim of this study was to identify DNA methylation biomarkers of ASD detectable at birth.

BACKGROUND: Genetic influences on gene expression in the human fetal brain plausibly impact upon a variety of postnatal brain-related traits, including susceptibility to neuropsychiatric disorders. However, to date, there have been no studies that have mapped genome-wide expression quantitative trait loci (eQTL) specifically in the human prenatal brain. RESULTS: We performed deep RNA sequencing and genome-wide genotyping on a unique collection of 120 human brains from the second trimester of gestation to provide the first eQTL dataset derived exclusively from the human fetal brain. We identify high confidence cis-acting eQTL at the individual transcript as well as whole gene level, including many mapping to a common inversion polymorphism on chromosome 17q21. Fetal brain eQTL are enriched among risk variants for postnatal conditions including attention deficit hyperactivity disorder, schizophrenia, and bipolar disorder. We further identify changes in gene expression within the prenatal brain that potentially mediate risk for neuropsychiatric traits, including increased expression of C4A in association with genetic risk for schizophrenia, increased expression of LRRC57 in association with genetic risk for bipolar disorder, and altered expression of multiple genes within the chromosome 17q21 inversion in association with variants influencing the personality trait of neuroticism. CONCLUSIONS: We have mapped eQTL operating in the human fetal brain, providing evidence that these confer risk to certain neuropsychiatric disorders, and identifying gene expression changes that potentially mediate susceptibility to these conditions.

Major depressive disorder (MDD) is a common illness accompanied by considerable morbidity, mortality, costs, and heightened risk of suicide. We conducted a genome-wide association meta-analysis based in 135,458 cases and 344,901 controls and identified 44 independent and significant loci. The genetic findings were associated with clinical features of major depression and implicated brain regions exhibiting anatomical differences in cases. Targets of antidepressant medications and genes involved in gene splicing were enriched for smaller association signal. We found important relationships of genetic risk for major depression with educational attainment, body mass, and schizophrenia: lower educational attainment and higher body mass were putatively causal, whereas major depression and schizophrenia reflected a partly shared biological etiology. All humans carry lesser or greater numbers of genetic risk factors for major depression. These findings help refine the basis of major depression and imply that a continuous measure of risk underlies the clinical phenotype.

Heterozygous mutation of the transcription factor HNF1B is the most common cause of monogenetic developmental renal disease. Disease-associated mutations fall into two categories: HNF1B intragenic mutations and a 1.3 Mb deletion at chromosome 17q12. An increase in neurodevelopmental disorders has been observed in individuals harbouring the 17q12 deletion but not in patients with HNF1B coding mutations.Previous investigations have concentrated on identifying a genetic cause for the increase in behavioural problems seen in 17q12 deletion carriers. We have taken the alternative approach of investigating the DNA methylation profile of these two HNF1B genotype groups along with controls matched for age, gender and diabetes status using the Illumina 450K DNA methylation array (total sample n = 60).We identified a number of differentially methylated probes (DMPs) that were associated with HNF1B-associated disease and passed our stringent experiment-wide significance threshold. These associations were largely driven by the deletion patients and the majority of the significant probes mapped to the 17q12 deletion locus. The observed changes in DNA methylation at this locus were not randomly dispersed and occurred in clusters, suggesting a regulatory mechanism reacting to haploinsufficiency across the entire deleted region.Along with these deletion-specific changes in DNA methylation, we also identified a shared DNA methylation signature in both mutation and deletion patient groups indicating that haploinsufficiency of HNF1B impacts on the methylome of a number of genes, giving further insight to the role of HNF1B.

© 2018 the Authors Characterizing the complex relationship between genetic, epigenetic, and transcriptomic variation has the potential to increase understanding about the mechanisms underpinning health and disease phenotypes. We undertook a comprehensive analysis of common genetic variation on DNA methylation (DNAm) by using the Illumina EPIC array to profile samples from the UK Household Longitudinal study. We identified 12,689,548 significant DNA methylation quantitative trait loci (mQTL) associations (p < 6.52 × 10−14) occurring between 2,907,234 genetic variants and 93,268 DNAm sites, including a large number not identified by previous DNAm-profiling methods. We demonstrate the utility of these data for interpreting the functional consequences of common genetic variation associated with > 60 human traits by using summary-data-based Mendelian randomization (SMR) to identify 1,662 pleiotropic associations between 36 complex traits and 1,246 DNAm sites. We also use SMR to characterize the relationship between DNAm and gene expression and thereby identify 6,798 pleiotropic associations between 5,420 DNAm sites and the transcription of 1,702 genes. Our mQTL database and SMR results are available via a searchable online database as a resource to the research community.

Cognitive functions are important correlates of health outcomes across the life-course. Individual differences in cognitive functions are partly heritable. Epigenetic modifications, such as DNA methylation, are susceptible to both genetic and environmental factors and may provide insights into individual differences in cognitive functions. Epigenome-wide meta-analyses for blood-based DNA methylation levels at ~420,000 CpG sites were performed for seven measures of cognitive functioning using data from 11 cohorts. CpGs that passed a Bonferroni correction, adjusting for the number of CpGs and cognitive tests, were assessed for: longitudinal change; being under genetic control (methylation QTLs); and associations with brain health (structural MRI), brain methylation and Alzheimer's disease pathology. Across the seven measures of cognitive functioning (meta-analysis n range: 2557-6809), there were epigenome-wide significant (P 

Accelerated DNA methylation age is linked to all-cause mortality and environmental factors, but studies of associations with socioeconomic position are limited. Researchers generally use small selected samples, and it is unclear how findings obtained with 2 commonly used methods for calculating methylation age (the Horvath method and the Hannum method) translate to general population samples including younger and older adults. Among 1,099 United Kingdom adults aged 28-98 years in 2011-2012, we assessed the relationship of Horvath and Hannum DNA methylation age acceleration with a range of social position measures: current income and employment, education, income and unemployment across a 12-year period, and childhood social class. Accounting for confounders, participants who had been less advantaged in childhood were epigenetically "older" as adults: in comparison with participants who had professional/managerial parents, Hannum age was 1.07 years higher (95% confidence interval: 0.20, 1.94) for participants with parents in semiskilled/unskilled occupations and 1.85 years higher (95% confidence interval: 0.67, 3.02) for those without a working parent at age 14 years. No other robust associations were seen. Results accord with research implicating early life circumstances as critical for DNA methylation age in adulthood. Since methylation age acceleration as measured by the Horvath and Hannum estimators appears strongly linked to chronological age, researchers examining associations with the social environment must take steps to avoid age-related confounding.

BACKGROUND: Numerous risk factors for dementia are well established, though the causal nature of these associations remains unclear. OBJECTIVE: to systematically review Mendelian randomization (MR) studies investigating causal relationships between risk factors and global cognitive function or dementia. METHODS: We searched five databases from inception to February 2017 and conducted citation searches including MR studies investigating the association between any risk factor and global cognitive function, all-cause dementia or dementia subtypes. Two reviewers independently assessed titles and abstracts, full-texts, and study quality. RESULTS: We included 18 MR studies investigating education, lifestyle factors, cardiovascular factors and related biomarkers, diabetes related and other endocrine factors, and telomere length. Studies were of predominantly good quality, however eight received low ratings for sample size and statistical power. The most convincing causal evidence was found for an association of shorter telomeres with increased risk of Alzheimer's disease (AD). Causal evidence was weaker for smoking quantity, vitamin D, homocysteine, systolic blood pressure, fasting glucose, insulin sensitivity, and high-density lipoprotein cholesterol. Well-replicated associations were not present for most exposures and we cannot fully discount survival and diagnostic bias, or the potential for pleiotropic effects. CONCLUSIONS: Genetic evidence supported a causal association between telomere length and AD, whereas limited evidence for other risk factors was largely inconclusive with tentative evidence for smoking quantity, vitamin D, homocysteine, and selected metabolic markers. The lack of stronger evidence for other risk factors may reflect insufficient statistical power. Larger well-designed MR studies would therefore help establish the causal status of these dementia risk factors.

Identifying genes regulating the pace of epigenetic ageing represents a new frontier in genome-wide association studies (GWASs). Here using 1,796 brain samples from 1,163 individuals, we carry out a GWAS of two DNA methylation-based biomarkers of brain age: the epigenetic ageing rate and estimated proportion of neurons. Locus 17q11.2 is significantly associated (P=4.5 × 10-9) with the ageing rate across five brain regions and harbours a cis-expression quantitative trait locus for EFCAB5 (P=3.4 × 10-20). Locus 1p36.12 is significantly associated (P=2.2 × 10-8) with epigenetic ageing of the prefrontal cortex, independent of the proportion of neurons. Our GWAS of the proportion of neurons identified two genome-wide significant loci (10q26 and 12p13.31) and resulted in a gene set that overlaps significantly with sets found by GWAS of age-related macular degeneration (P=1.4 × 10-12), ulcerative colitis (P

OBJECTIVE: Genome-wide association studies (GWAS) have been successful at identifying associations with stroke and stroke subtypes, but have not yet identified any associations solely with small vessel stroke (SVS). SVS comprises one quarter of all ischemic stroke and is a major manifestation of cerebral small vessel disease, the primary cause of vascular cognitive impairment. Studies across neurological traits have shown that younger-onset cases have an increased genetic burden. We leveraged this increased genetic burden by performing an age-at-onset informed GWAS meta-analysis, including a large younger-onset SVS population, to identify novel associations with stroke. METHODS: We used a three-stage age-at-onset informed GWAS to identify novel genetic variants associated with stroke. On identifying a novel locus associated with SVS, we assessed its influence on other small vessel disease phenotypes, as well as on messenger RNA (mRNA) expression of nearby genes, and on DNA methylation of nearby CpG sites in whole blood and in the fetal brain. RESULTS: We identified an association with SVS in 4,203 cases and 50,728 controls on chromosome 16q24.2 (odds ratio [OR; 95% confidence interval {CI}] = 1.16 [1.10-1.22]; p = 3.2 × 10-9 ). The lead single-nucleotide polymorphism (rs12445022) was also associated with cerebral white matter hyperintensities (OR [95% CI] = 1.10 [1.05-1.16]; p = 5.3 × 10-5 ; N = 3,670), but not intracerebral hemorrhage (OR [95% CI] = 0.97 [0.84-1.12]; p = 0.71; 1,545 cases, 1,481 controls). rs12445022 is associated with mRNA expression of ZCCHC14 in arterial tissues (p = 9.4 × 10-7 ) and DNA methylation at probe cg16596957 in whole blood (p = 5.3 × 10-6 ). INTERPRETATION: 16q24.2 is associated with SVS. Associations of the locus with expression of ZCCHC14 and DNA methylation suggest the locus acts through changes to regulatory elements. Ann Neurol 2017;81:383-394.

Major depressive disorder (MDD) represents a major social and economic health issue and constitutes a major risk factor for suicide. The molecular pathology of suicidal depression remains poorly understood, although it has been hypothesised that regulatory genomic processes are involved in the pathology of both MDD and suicidality. In this study, genome-wide patterns of DNA methylation were assessed in depressed suicide completers (n=20) and compared with non-psychiatric, sudden-death controls (n=20) using tissue from two cortical brain regions (Brodmann Area 11 (BA11) and Brodmann Area 25 (BA25)). Analyses focused on identifying differentially methylated regions (DMRs) associated with suicidal depression and epigenetic variation were explored in the context of polygenic risk scores for major depression and suicide. Weighted gene co-methylation network analysis was used to identify modules of co-methylated loci associated with depressed suicide completers and polygenic burden for MDD and suicide attempt. We identified a DMR upstream of the PSORS1C3 gene, subsequently validated using bisulfite pyrosequencing and replicated in a second set of suicide samples, which is characterised by significant hypomethylation in both cortical brain regions in MDD suicide cases. We also identified discrete modules of co-methylated loci associated with polygenic risk burden for suicide attempt, but not major depression. Suicide-associated co-methylation modules were enriched among gene networks implicating biological processes relevant to depression and suicidality, including nervous system development and mitochondria function. Our data suggest that there are coordinated changes in DNA methylation associated with suicide that may offer novel insights into the molecular pathology associated with depressed suicide completers.

Most genetic variants identified in genome-wide association studies (GWASs) of complex traits are thought to act by affecting gene regulation rather than directly altering the protein product. As a consequence, the actual genes involved in disease are not necessarily the most proximal to the associated variants. By integrating data from GWAS analyses with those from genetic studies of regulatory variation, it is possible to identify variants pleiotropically associated with both a complex trait and measures of gene regulation. In this study, we used summary-data-based Mendelian randomization (SMR), a method developed to identify variants pleiotropically associated with both complex traits and gene expression, to identify variants associated with complex traits and DNA methylation. We used large DNA methylation quantitative trait locus (mQTL) datasets generated from two different tissues (blood and fetal brain) to prioritize genes for >40 complex traits with robust GWAS data and found considerable overlap with the results of SMR analyses performed with expression QTL (eQTL) data. We identified multiple examples of variable DNA methylation associated with GWAS variants for a range of complex traits, demonstrating the utility of this approach for refining genetic association signals.

BACKGROUND: DNA methylation is an important epigenetic mechanism involved in gene regulation, with alterations in DNA methylation in the nuclear genome being linked to numerous complex diseases. Mitochondrial DNA methylation is a phenomenon that is receiving ever-increasing interest, particularly in diseases characterized by mitochondrial dysfunction; however, most studies have been limited to the investigation of specific target regions. Analyses spanning the entire mitochondrial genome have been limited, potentially due to the amount of input DNA required. Further, mitochondrial genetic studies have been previously confounded by nuclear-mitochondrial pseudogenes. Methylated DNA Immunoprecipitation Sequencing is a technique widely used to profile DNA methylation across the nuclear genome; however, reads mapped to mitochondrial DNA are often discarded. Here, we have developed an approach to control for nuclear-mitochondrial pseudogenes within Methylated DNA Immunoprecipitation Sequencing data. We highlight the utility of this approach in identifying differences in mitochondrial DNA methylation across regions of the human brain and pre-mortem blood. RESULTS: We were able to correlate mitochondrial DNA methylation patterns between the cortex, cerebellum and blood. We identified 74 nominally significant differentially methylated regions (p 

Genetic association studies provide evidence for a substantial polygenic component to schizophrenia, although the neurobiological mechanisms underlying the disorder remain largely undefined. Building on recent studies supporting a role for developmentally regulated epigenetic variation in the molecular aetiology of schizophrenia, this study aimed to identify epigenetic variation associated with both a diagnosis of schizophrenia and elevated polygenic risk burden for the disease across multiple brain regions. Genome-wide DNA methylation was quantified in 262 post-mortem brain samples, representing tissue from four brain regions (prefrontal cortex, striatum, hippocampus and cerebellum) from 41 schizophrenia patients and 47 controls. We identified multiple disease-associated and polygenic risk score-associated differentially methylated positions and regions, which are not enriched in genomic regions identified in genetic studies of schizophrenia and do not reflect direct genetic effects on DNA methylation. Our study represents the first analysis of epigenetic variation associated with schizophrenia across multiple brain regions and highlights the utility of polygenic risk scores for identifying molecular pathways associated with aetiological variation in complex disease.

Epigenetic disruption has been implicated in many diseases of aging, and age-associated DNA methylation changes at specific genomic loci in humans are strongly correlated with chronological age. The aim of this study was to explore the specificity of selected age-associated differentially methylated positions (aDMPs) identified in human epidemiological studies by quantifying DNA methylation across multiple tissues in homologous regions of the murine genome. We selected four high-confidence aDMPs (located in the vicinity of the ELOVL2, GLRA1, MYOD1 and PDE4C genes) and quantified DNA methylation across these regions in four tissues (blood, lung, cerebellum and hippocampus) from male and female C57BL/6J mice, ranging in age from fetal (embryonic day 17) to 630 days. We observed tissue-specific age-associated changes in DNA methylation that was directionally consistent with those observed in humans. These findings lend further support to the notion that changes in DNA methylation are associated with chronological age and suggest that these processes are often conserved across tissues and between mammalian species. Our data highlight the relevance of utilizing model systems, in which environmental and genetic influences can be carefully controlled, for the further study of these phenomena.

BACKGROUND: Schizophrenia is a highly heritable, neuropsychiatric disorder characterized by episodic psychosis and altered cognitive function. Despite success in identifying genetic variants associated with schizophrenia, there remains uncertainty about the causal genes involved in disease pathogenesis and how their function is regulated. RESULTS: We performed a multi-stage epigenome-wide association study, quantifying genome-wide patterns of DNA methylation in a total of 1714 individuals from three independent sample cohorts. We have identified multiple differentially methylated positions and regions consistently associated with schizophrenia across the three cohorts; these effects are independent of important confounders such as smoking. We also show that epigenetic variation at multiple loci across the genome contributes to the polygenic nature of schizophrenia. Finally, we show how DNA methylation quantitative trait loci in combination with Bayesian co-localization analyses can be used to annotate extended genomic regions nominated by studies of schizophrenia, and to identify potential regulatory variation causally involved in disease. CONCLUSIONS: This study represents the first systematic integrated analysis of genetic and epigenetic variation in schizophrenia, introducing a methodological approach that can be used to inform epigenome-wide association study analyses of other complex traits and diseases. We demonstrate the utility of using a polygenic risk score to identify molecular variation associated with etiological variation, and of using DNA methylation quantitative trait loci to refine the functional and regulatory variation associated with schizophrenia risk variants. Finally, we present strong evidence for the co-localization of genetic associations for schizophrenia and differential DNA methylation.

BACKGROUND: Single variant approaches have been successful in identifying DNA methylation quantitative trait loci (mQTL), although as with complex traits they lack the statistical power to identify the effects from rare genetic variants. We have undertaken extensive analyses to identify regions of low frequency and rare variants that are associated with DNA methylation levels. METHODS: We used repeated measurements of DNA methylation from five different life stages in human blood, taken from the Avon Longitudinal Study of Parents and Children (ALSPAC) cohort. Variants were collapsed across CpG islands and their flanking regions to identify variants collectively associated with methylation, where no single variant was individually responsible for the observed signal. All analyses were undertaken using the sequence kernel association test. RESULTS: for loci where no individual variant mQTL was observed based on a single variant analysis, we identified 95 unique regions where the combined effect of low frequency variants (MAF ≤ 5%) provided strong evidence of association with methylation. For loci where there was previous evidence of an individual variant mQTL, a further 3 regions provided evidence of association between multiple low frequency variants and methylation levels. Effects were observed consistently across 5 different time points in the lifecourse and evidence of replication in the TwinsUK and Exeter cohorts was also identified. CONCLUSION: We have demonstrated the potential of this novel approach to mQTL analysis by analysing the combined effect of multiple low frequency or rare variants. Future studies should benefit from applying this approach as a complementary follow up to single variant analyses.

DNA methylation (DNAm) levels lend themselves for defining an epigenetic biomarker of aging known as the 'epigenetic clock'. Our genome-wide association study (GWAS) of cerebellar epigenetic age acceleration identifies five significant (P

Although mutations within the TREM2 gene have been robustly associated with Alzheimer's disease, it is not known whether alterations in the regulation of this gene are also involved in pathogenesis. Here, we present data demonstrating increased DNA methylation in the superior temporal gyrus in Alzheimer's disease brain at a CpG site located 289 bp upstream of the transcription start site of the TREM2 gene in 3 independent study cohorts using 2 different technologies (Illumina Infinium 450K methylation beadchip and pyrosequencing). A meta-analysis across all 3 cohorts reveals consistent AD-associated hypermethylation (p = 3.47E-08). This study highlights that extending genetic studies of TREM2 in AD to investigate epigenetic changes may nominate additional mechanisms by which disruption to this gene increases risk.

We characterized DNA methylation quantitative trait loci (mQTLs) in a large collection (n = 166) of human fetal brain samples spanning 56-166 d post-conception, identifying >16,000 fetal brain mQTLs. Fetal brain mQTLs were primarily cis-acting, enriched in regulatory chromatin domains and transcription factor binding sites, and showed substantial overlap with genetic variants that were also associated with gene expression in the brain. Using tissue from three distinct regions of the adult brain (prefrontal cortex, striatum and cerebellum), we found that most fetal brain mQTLs were developmentally stable, although a subset was characterized by fetal-specific effects. Fetal brain mQTLs were enriched amongst risk loci identified in a recent large-scale genome-wide association study (GWAS) of schizophrenia, a severe psychiatric disorder with a hypothesized neurodevelopmental component. Finally, we found that mQTLs can be used to refine GWAS loci through the identification of discrete sites of variable fetal brain methylation associated with schizophrenia risk variants.

BACKGROUND: the most widely utilized approaches for quantifying DNA methylation involve the treatment of genomic DNA with sodium bisulfite; however, this method cannot distinguish between 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Previous studies have shown that 5hmC is enriched in the brain, although little is known about its genomic distribution and how it differs between anatomical regions and individuals. In this study, we combine oxidative bisulfite (oxBS) treatment with the Illumina Infinium 450K BeadArray to quantify genome-wide patterns of 5hmC in two distinct anatomical regions of the brain from multiple individuals. RESULTS: We identify 37,145 and 65,563 sites passing our threshold for detectable 5hmC in the prefrontal cortex and cerebellum respectively, with 23,445 loci common across both brain regions. Distinct patterns of 5hmC are identified in each brain region, with notable differences in the genomic location of the most hydroxymethylated loci between these brain regions. Tissue-specific patterns of 5hmC are subsequently confirmed in an independent set of prefrontal cortex and cerebellum samples. CONCLUSIONS: This study represents the first systematic analysis of 5hmC in the human brain, identifying tissue-specific hydroxymethylated positions and genomic regions characterized by inter-individual variation in DNA hydroxymethylation. This study demonstrates the utility of combining oxBS-treatment with the Illumina 450k methylation array to systematically quantify 5hmC across the genome and the potential utility of this approach for epigenomic studies of brain disorders.

Given the tissue-specific nature of epigenetic processes, the assessment of disease-relevant tissue is an important consideration for epigenome-wide association studies (EWAS). Little is known about whether easily accessible tissues, such as whole blood, can be used to address questions about interindividual epigenomic variation in inaccessible tissues, such as the brain. We quantified DNA methylation in matched DNA samples isolated from whole blood and 4 brain regions (prefrontal cortex, entorhinal cortex, superior temporal gyrus, and cerebellum) from 122 individuals. We explored co-variation between tissues and the extent to which methylomic variation in blood is predictive of interindividual variation identified in the brain. For the majority of DNA methylation sites, interindividual variation in whole blood is not a strong predictor of interindividual variation in the brain, although the relationship with cortical regions is stronger than with the cerebellum. Variation at a subset of probes is strongly correlated across tissues, even in instances when the actual level of DNA methylation is significantly different between them. A substantial proportion of this co-variation, however, is likely to result from genetic influences. Our data suggest that for the majority of the genome, a blood-based EWAS for disorders where brain is presumed to be the primary tissue of interest will give limited information relating to underlying pathological processes. These results do not, however, discount the utility of using a blood-based EWAS to identify biomarkers of disease phenotypes manifest in the brain. We have generated a searchable database for the interpretation of data from blood-based EWAS analyses ( http://epigenetics.essex.ac.uk/bloodbrain/).

Childhood psychotic symptoms are associated with increased rates of schizophrenia, other psychiatric disorders, and suicide attempts in adulthood; thus, elucidating early risk indicators is crucial to target prevention efforts. There is considerable discordance for psychotic symptoms between monozygotic twins, indicating that child-specific non-genetic factors must be involved. Epigenetic processes may constitute one of these factors and have not yet been investigated in relation to childhood psychotic symptoms. Therefore, this study explored whether differences in DNA methylation at age 10 were associated with monozygotic twin discordance for psychotic symptoms at age 12. The Environmental Risk (E-Risk) Longitudinal Twin Study cohort of 2,232 children (1,116 twin pairs) was assessed for age-12 psychotic symptoms and 24 monozygotic twin pairs discordant for symptoms were identified for methylomic comparison. Children provided buccal samples at ages 5 and 10. DNA was bisulfite modified and DNA methylation was quantified using the Infinium HumanMethylation450 array. Differentially methylated positions (DMPs) associated with psychotic symptoms were subsequently tested in post-mortem prefrontal cortex tissue from adult schizophrenia patients and age-matched controls. Site-specific DNA methylation differences were observed at age 10 between monozygotic twins discordant for age-12 psychotic symptoms. Similar DMPs were not found at age 5. The top-ranked psychosis-associated DMP (cg23933044), located in the promoter of the C5ORF42 gene, was also hypomethylated in post-mortem prefrontal cortex brain tissue from schizophrenia patients compared to unaffected controls. These data tentatively suggest that epigenetic variation in peripheral tissue is associated with childhood psychotic symptoms and may indicate susceptibility to schizophrenia and other mental health problems.

BACKGROUND: Asthma is the most common chronic inflammatory disorder in children. The aetiology of asthma pathology is complex and highly heterogeneous, involving the interplay between genetic and environmental risk factors that is hypothesized to involve epigenetic processes. Our aim was to explore whether methylomic variation in early childhood is associated with discordance for asthma symptoms within monozygotic (MZ) twin pairs recruited from the Environmental Risk (E-Risk) longitudinal twin study. We also aimed to identify differences in DNA methylation that are associated with asthma that develops in childhood and persists into early adulthood as these may represent useful prognostic biomarkers. RESULTS: We examined genome-wide patterns of DNA methylation in buccal cell samples collected from 37 MZ twin pairs discordant for asthma at age 10. DNA methylation at individual CpG sites demonstrated significant variability within discordant MZ twin pairs with the top-ranked nominally significant differentially methylated position (DMP) located in the HGSNAT gene. We stratified our analysis by assessing DNA methylation differences in a sub-group of MZ twin pairs who remained persistently discordant for asthma at age 18. The top-ranked nominally significant DMP associated with persisting asthma is located in the vicinity of the HLX gene, which has been previously implicated in childhood asthma. CONCLUSIONS: We identified DNA methylation differences associated with childhood asthma in peripheral DNA samples from discordant MZ twin pairs. Our data suggest that differences in DNA methylation associated with childhood asthma which persists into early adulthood are distinct from those associated with asthma which remits.

Epigenetic processes play a key role in orchestrating transcriptional regulation during development. The importance of DNA methylation in fetal brain development is highlighted by the dynamic expression of de novo DNA methyltransferases during the perinatal period and neurodevelopmental deficits associated with mutations in the methyl-CpG binding protein 2 (MECP2) gene. However, our knowledge about the temporal changes to the epigenome during fetal brain development has, to date, been limited. We quantified genome-wide patterns of DNA methylation at ∼ 400,000 sites in 179 human fetal brain samples (100 male, 79 female) spanning 23 to 184 d post-conception. We identified highly significant changes in DNA methylation across fetal brain development at >7% of sites, with an enrichment of loci becoming hypomethylated with fetal age. Sites associated with developmental changes in DNA methylation during fetal brain development were significantly underrepresented in promoter regulatory regions but significantly overrepresented in regions flanking CpG islands (shores and shelves) and gene bodies. Highly significant differences in DNA methylation were observed between males and females at a number of autosomal sites, with a small number of regions showing sex-specific DNA methylation trajectories across brain development. Weighted gene comethylation network analysis (WGCNA) revealed discrete modules of comethylated loci associated with fetal age that are significantly enriched for genes involved in neurodevelopmental processes. This is, to our knowledge, the most extensive study of DNA methylation across human fetal brain development to date, confirming the prenatal period as a time of considerable epigenomic plasticity.

Inherited alleles account for most of the genetic risk for schizophrenia. However, new (de novo) mutations, in the form of large chromosomal copy number changes, occur in a small fraction of cases and disproportionally disrupt genes encoding postsynaptic proteins. Here we show that small de novo mutations, affecting one or a few nucleotides, are overrepresented among glutamatergic postsynaptic proteins comprising activity-regulated cytoskeleton-associated protein (ARC) and N-methyl-d-aspartate receptor (NMDAR) complexes. Mutations are additionally enriched in proteins that interact with these complexes to modulate synaptic strength, namely proteins regulating actin filament dynamics and those whose messenger RNAs are targets of fragile X mental retardation protein (FMRP). Genes affected by mutations in schizophrenia overlap those mutated in autism and intellectual disability, as do mutation-enriched synaptic pathways. Aligning our findings with a parallel case-control study, we demonstrate reproducible insights into aetiological mechanisms for schizophrenia and reveal pathophysiology shared with other neurodevelopmental disorders. © 2014 Macmillan Publishers Limited. All rights reserved.

Inherited alleles account for most of the genetic risk for schizophrenia. However, new (de novo) mutations, in the form of large chromosomal copy number changes, occur in a small fraction of cases and disproportionally disrupt genes encoding postsynaptic proteins. Here we show that small de novo mutations, affecting one or a few nucleotides, are overrepresented among glutamatergic postsynaptic proteins comprising activity-regulated cytoskeleton-associated protein (ARC) and N-methyl-d-aspartate receptor (NMDAR) complexes. Mutations are additionally enriched in proteins that interact with these complexes to modulate synaptic strength, namely proteins regulating actin filament dynamics and those whose messenger RNAs are targets of fragile X mental retardation protein (FMRP). Genes affected by mutations in schizophrenia overlap those mutated in autism and intellectual disability, as do mutation-enriched synaptic pathways. Aligning our findings with a parallel case-control study, we demonstrate reproducible insights into aetiological mechanisms for schizophrenia and reveal pathophysiology shared with other neurodevelopmental disorders.

Alzheimer's disease (AD) is a chronic neurodegenerative disorder that is characterized by progressive neuropathology and cognitive decline. We performed a cross-tissue analysis of methylomic variation in AD using samples from four independent human post-mortem brain cohorts. We identified a differentially methylated region in the ankyrin 1 (ANK1) gene that was associated with neuropathology in the entorhinal cortex, a primary site of AD manifestation. This region was confirmed as being substantially hypermethylated in two other cortical regions (superior temporal gyrus and prefrontal cortex), but not in the cerebellum, a region largely protected from neurodegeneration in AD, or whole blood obtained pre-mortem from the same individuals. Neuropathology-associated ANK1 hypermethylation was subsequently confirmed in cortical samples from three independent brain cohorts. This study represents, to the best of our knowledge, the first epigenome-wide association study of AD employing a sequential replication design across multiple tissues and highlights the power of this approach for identifying methylomic variation associated with complex disease.