Quantification of the pace of biological aging in humans through a blood test, the DunedinPoAm DNA methylation algorithm

Biological aging is the gradual, progressive decline in system integrity that occurs with advancing chronological age, causing morbidity and disability. Measurements of the pace of aging are needed as surrogate endpoints in trials of therapies designed to prevent disease by slowing biological aging. We report a blood-DNA-methylation measure that is sensitive to variation in pace of biological aging among individuals born the same year. We first modeled change-over-time in 18 biomarkers tracking organ-system integrity across 12 years of follow-up in n = 954 members of the Dunedin Study born in 1972-1973. Rates of change in each biomarker over ages 26-38 years were composited to form a measure of aging-related decline, termed Pace-of-Aging. Elastic-net regression was used to develop a DNA-methylation predictor of Pace-of-Aging, called DunedinPoAm for Dunedin(P)ace(o)f(A)ging(m)ethylation. Validation analysis in cohort studies and the CALERIE trial provide proof-of-principle for DunedinPoAm as a single-time-point measure of a person's pace of biological aging.This article is freely available via Open Access. Click on the Publisher URL to access it via the publisher's site.U24 AG047121/AG/NIA NIH HHS/United States R21 AG054846/AG/NIA NIH HHS/United States R21AG054846/AG/NIA NIH HHS/United States R01AG061378/AG/NIA NIH HHS/United States R01 HD077482/HD/NICHD NIH HHS/United States R01HD077482/National Institute of Child Health and Development G1002190/MRC_/Medical Research Council/United Kingdom CBD Network Fellowship/CIFAR R01 AG032282/AG/NIA NIH HHS/United States P30 ES009089/ES/NIEHS NIH HHS/United States HD077482/National Institute of Child Health and Development R01 ES025225/ES/NIEHS NIH HHS/United States R01 ES021733/ES/NIEHS NIH HHS/United States R01 AG061378/AG/NIA NIH HHS/United States U24AG047121/AG/NIA NIH HHS/United States R01 ES027747/ES/NIEHS NIH HHS/United States MR/P005918/1/MRC_/Medical Research Council/United Kingdom Wellcome Trust/United Kingdom AG032282/AG/NIA NIH HHS/United States R01AG032282/AG/NIA NIH HHS/United Statespublished version, accepted version, submitted versio

Genome-Wide DNA Methylation Patterns in Persistent Attention-Deficit/Hyperactivity Disorder and in Association With Impulsive and Callous Traits

Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder that often persists into adulthood. ADHD and related personality traits, such as impulsivity and callousness, are caused by genetic and environmental factors and their interplay. Epigenetic modifications of DNA, including methylation, are thought to mediate between such factors and behavior and may behave as biomarkers for disorders. Here, we set out to study DNA methylation in persistent ADHD and related traits. We performed epigenome-wide association studies (EWASs) on peripheral whole blood from participants in the NeuroIMAGE study (age range 12-23 years). We compared participants with persistent ADHD (n = 35) with healthy controls (n = 19) and with participants with remittent ADHD (n = 19). Additionally, we performed EWASs of impulsive and callous traits derived from the Conners Parent Rating Scale and the Callous-Unemotional Inventory, respectively, across all participants. For every EWAS, the linear regression model analyzed included covariates for age, sex, smoking scores, and surrogate variables reflecting blood cell type composition and genetic background. We observed no epigenome-wide significant differences in single CpG site methylation between participants with persistent ADHD and healthy controls or participants with remittent ADHD. However, epigenome-wide analysis of differentially methylated regions provided significant findings showing that hypermethylated regions in the APOB and LPAR5 genes were associated with ADHD persistence compared to ADHD remittance (p = 1.68 * 10-24 and p = 9.06 * 10-7, respectively); both genes are involved in cholesterol signaling. Both findings appeared to be linked to genetic variation in cis. We found neither significant epigenome-wide single CpG sites nor regions associated with impulsive and callous traits; the top-hits from these analyses were annotated to genes involved in neurotransmitter release and the regulation of the biological clock. No link to genetic variation was observed for these findings, which thus might reflect environmental influences. In conclusion, in this pilot study with a small sample size, we observed several DNA-methylation-disorder/trait associations of potential significance for ADHD and the related behavioral traits. Although we do not wish to draw conclusions before replication in larger, independent samples, cholesterol signaling and metabolism may be of relevance for the onset and/or persistence of ADHD.This article is freely available via Open Access. Click on the Publisher URL to access it via the publisher's site.This research was supported by an internal grant from the Donders Centre for Medical Neuroscience of Radboudumc. Support was also received from the Dutch National Science Agenda for the NWA NeurolabNL project (grant 400 17 602), and from the European Community's Horizon 2020 Programme (H2020/2014–2020) under grant agreement n° 728018 (Eat2beNICE). BF was also supported by a personal grant from the Netherlands Organization for Scientific Research (NWO) Vici Innovation Program (grant 016-130-669). The NeuroIMAGE project was supported by NIH Grant R01MH62873, NWO Large Investment Grant 1750102007010, and grants from Radboud University Medical Center, University Medical Center Groningen and Accare, and VU University Amsterdam. This work was also supported by grants from NWO Brain & Cognition (433-09-242 and 056-13-015) and from ZonMW (60-60600-97-193).published version, accepted version, submitted versio

Elucidating functional pathways associated with schizophrenia risk through analysis of brain gene expression

Schizophrenia is a highly heritable, common psychiatric disorder. Although onset generally occurs during adolescence, multiple lines of evidence point to a neurodevelopmental insult that occurs many years prior to the presentation of symptoms. Many different approaches have been used to elucidate the genetic risk factors and their impact; however, few unequivocal facts have been established. With a considerable amount of data publically available, integrative approaches look to leverage multiple data sources to identify coherent themes. This thesis investigates the neurodevelopmental hypothesis of schizophrenia by incorporating results from genome-wide association studies and copy number variation studies with gene expression datasets, with the overall aim of identifying functional pathways that may be disrupted in the aetiology of the disorder. This study used foetal and developmental expression datasets of the human brain and statistical approaches to characterise the expression profiles of schizophrenia risk genes. Both spatial profiles in the mid-foetal brain and temporal profiles across development were considered. Data from genome-wide association studies and copy number variation studies were used to test for an enrichment of risk genes; in addition the genetic overlap with bipolar disorder identified through genome-wide association studies was used for validation. Gene sets with a common expression profile enriched for schizophrenia variants were used to identify biological pathways and assessed for their polygenic contribution to schizophrenia risk. The results of this thesis converged on a common developmental expression profile for schizophrenia risk genes. Genes identified with this profile were shown to harbour multiple, common risk variants for schizophrenia and were implicated in epigenetic processes relating to the regulation of gene transcription. Together this suggests that schizophrenia associated genes are involved in brain development, particularly during foetal stages, and may play a role in the regulation of this process

Epigenome-wide association study of attention-deficit/hyperactivity disorder in adults

Attention-deficit/hyperactivity disorder (ADHD) is a highly heritable neurodevelopmental disorder that often persists into adulthood. There is growing evidence that epigenetic dysregulation participates in ADHD. Given that only a limited number of epigenome-wide association studies (EWASs) of ADHD have been conducted so far and they have mainly focused on pediatric and population-based samples, we performed an EWAS in a clinical sample of adults with ADHD. We report one CpG site and four regions differentially methylated between patients and controls, which are located in or near genes previously involved in autoimmune diseases, cancer or neuroticism. Our sensitivity analyses indicate that smoking status is not responsible for these results and that polygenic risk burden for ADHD does not greatly impact the signatures identified. Additionally, we show an overlap of our EWAS findings with genetic signatures previously described for ADHD and with epigenetic signatures for smoking behavior and maternal smoking. These findings support a role of DNA methylation in ADHD and emphasize the need for additional efforts in larger samples to clarify the role of epigenetic mechanisms on ADHD across the lifespan.This article is freely available via Open Access. Click on the Publisher URL to access it via the publisher's site.Paula Rovira, Dr. Sánchez-Mora, Dr. Pagerols, Laura Vilar-Ribó, Lorena Arribas, Gemma Shireby, Dr. Hannon, Prof. Mill, Dr. Soler Artigas and Dr. Ribasés reported no biomedical financial interests or potential conflicts of interest. Vanesa Richarte has served on the speakers for Eli Lilly, Rubio and Shire in the last 5 years. She has received travel awards from Eli Lilly and Co. and Shire for participating in psychiatric meetings. The ADHD Program has received unrestricted educational and research support from Eli Lilly and Co., JanssenCilag, Shire, Rovi, Psious and Laboratorios Rubió in the past 2 years. Montserrat Corrales received travel awards for taking part in psychiatric meetings from Shire. Christian Fadeuilhe received fees to give talks for Shire, Ferrer, Italfarmaco and Otsuka in the last 5 years. He also received travel awards (air tickets +hotel) for taking part in psychiatric meetings from Janssen-Cilag, Rubió, Shire, Lundbeck, Otsuka and Ferrer. Prof. Casas has received travel grants and research support from Eli Lilly and Co., Janssen-Cilag, Shire and Lundbeck and served as consultant for Eli Lilly and Co., Janssen-Cilag, Shire and Lundbeck. Prof. Ramos-Quiroga was on the speakers’ bureau and/or acted as consultant for Eli-Lilly, Janssen-Cilag, Novartis, Shire, Lundbeck, Almirall, Braingaze, Sincrolab, Medice, and Rubió in the last 5 years. He also received travel awards (air tickets + hotel) for taking part in psychiatric meetings from Janssen-Cilag, Rubió, Shire, Medice and Eli- Lilly. The Department of Psychiatry chaired by him received unrestricted educational and research support from the following companies in the last 5 years: Eli-Lilly, Lundbeck, Janssen- Cilag,Actelion, Shire, Ferrer, Oryzon, Roche, Psious, and Rubiópublished version, accepted version, submitted versio

Patterns of Reliability: Assessing the Reproducibility and Integrity of DNA Methylation Measurement

DNA methylation plays an important role in both normal human development and risk of disease. The most utilized method of assessing DNA methylation uses BeadChips, generating an epigenome-wide "snapshot" of >450,000 observations (probe measurements) per assay. However, the reliability of each of these measurements is not equal, and little consideration is paid to consequences for research. We correlated repeat measurements of the same DNA samples using the Illumina HumanMethylation450K and the Infinium MethylationEPIC BeadChips in 350 blood DNA samples. Probes that were reliably measured were more heritable and showed consistent associations with environmental exposures, gene expression, and greater cross-tissue concordance. Unreliable probes were less replicable and generated an unknown volume of false negatives. This serves as a lesson for working with DNA methylation data, but the lessons are equally applicable to working with other data: as we advance toward generating increasingly greater volumes of data, failure to document reliability risks harming reproducibility.This article is freely available via Open Access. Click on the Publisher URL to access it via the publisher's site.R01 AG049789/AG/NIA NIH HHS/United States R01 HD077482/HD/NICHD NIH HHS/United States MR/R005176/1/MRC_/Medical Research Council/United Kingdom MR/M008924/1/MRC_/Medical Research Council/United Kingdom R01 AG032282/AG/NIA NIH HHS/United State

Epigenome-wide Association Study of Attention-Deficit/Hyperactivity Disorder Symptoms in Adults

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.This article is freely available via Open Access. Click on the Publisher URL to access it via the publisher's site.R01 HD077482/HD/NICHD NIH HHS/United States G1002190/MRC_/Medical Research Council/United Kingdom MR/P014100/1/MRC_/Medical Research Council/United Kingdom MR/P005918/1/MRC_/Medical Research Council/United Kingdom R01 AG032282/AG/NIA NIH HHS/United Statespublished version, accepted version (12 month embargo

Methylation age acceleration does not predict mortality in schizophrenia

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.This article is freely available via Open Access. Click on the Publisher URL to access it via the publisher's site.This work was supported by the Swedish Research Council (Vetenskapsrådet, award D0886501 to PFS) and the UK Medical Research Council (award MR/K013807/1 to JM). The Sweden Schizophrenia Study was supported by NIMH R01 MH077139.published version, accepted version, submitted versio

Interindividual methylomic variation across blood, cortex, and cerebellum: implications for epigenetic studies of neurological and neuropsychiatric phenotypes

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/)

Methylation age acceleration does not predict mortality in schizophrenia.

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