Immuno-epigenetic signature derived in saliva associates with the encephalopathy of prematurity and perinatal inflammatory disorders.

BACKGROUND: Preterm birth is closely associated with a phenotype that includes brain dysmaturation and neurocognitive impairment, commonly termed Encephalopathy of Prematurity (EoP), of which systemic inflammation is considered a key driver. DNA methylation (DNAm) signatures of inflammation from peripheral blood associate with poor brain imaging outcomes in adult cohorts. However, the robustness of DNAm inflammatory scores in infancy, their relation to comorbidities of preterm birth characterised by inflammation, neonatal neuroimaging metrics of EoP, and saliva cross-tissue applicability are unknown. METHODS: Using salivary DNAm from 258 neonates (n = 155 preterm, gestational age at birth 23.28 - 34.84 weeks, n = 103 term, gestational age at birth 37.00 - 42.14 weeks), we investigated the impact of a DNAm surrogate for C-reactive protein (DNAm CRP) on brain structure and other clinically defined inflammatory exposures. We assessed i) if DNAm CRP estimates varied between preterm infants at term equivalent age and term infants, ii) how DNAm CRP related to different types of inflammatory exposure (maternal, fetal and postnatal) and iii) whether elevated DNAm CRP associated with poorer measures of neonatal brain volume and white matter connectivity. RESULTS: Higher DNAm CRP was linked to preterm status (-0.0107 ± 0.0008, compared with -0.0118 ± 0.0006 among term infants; p < 0.001), as well as perinatal inflammatory diseases, including histologic chorioamnionitis, sepsis, bronchopulmonary dysplasia, and necrotising enterocolitis (OR range |2.00 | to |4.71|, p < 0.01). Preterm infants with higher DNAm CRP scores had lower brain volume in deep grey matter, white matter, and hippocampi and amygdalae (ß range |0.185| to |0.218|). No such associations were observed for term infants. Association magnitudes were largest for measures of white matter microstructure among preterms, where elevated epigenetic inflammation associated with poorer global measures of white matter integrity (ß range |0.206| to |0.371|), independent of other confounding exposures. CONCLUSIONS: Inflammatory-related DNAm captures the allostatic load of inflammatory burden in preterm infants. Such DNAm measures complement biological and clinical metrics when investigating the determinants of neurodevelopmental differences.

Complex trait methylation scores in the prediction of major depressive disorder.

BACKGROUND: DNA methylation (DNAm) is associated with time-varying environmental factors that contribute to major depressive disorder (MDD) risk. We sought to test whether DNAm signatures of lifestyle and biochemical factors were associated with MDD to reveal dynamic biomarkers of MDD risk that may be amenable to lifestyle interventions. METHODS: Here, we calculated methylation scores (MS) at multiple p-value thresholds for lifestyle (BMI, smoking, alcohol consumption, and educational attainment) and biochemical (high-density lipoprotein (HDL) and total cholesterol) factors in Generation Scotland (GS) (N=9,502) and in a replication cohort (ALSPACadults, N=565), using CpG sites reported in previous well-powered methylome-wide association studies. We also compared their predictive accuracy for MDD to a MDD MS in an independent GS sub-sample (N=4,432). FINDINGS: Each trait MS was significantly associated with its corresponding phenotype in GS (ßrange=0.089-1.457) and in ALSPAC (ßrange=0.078-2.533). Each MS was also significantly associated with MDD before and after adjustment for its corresponding phenotype in GS (ßrange=0.053-0.145). After accounting for relevant lifestyle factors, MS for educational attainment (ß=0.094) and alcohol consumption (MSp-value<0.01-0.5; ßrange=-0.069-0.083) remained significantly associated with MDD in GS. Smoking (AUC=0.569) and educational attainment (AUC=0.585) MSs could discriminate MDD from controls better than the MDD MS (AUC=0.553) in the independent GS sub-sample. Analyses implicating MDD did not replicate across ALSPAC, although the direction of effect was consistent for all traits when adjusting for the MS corresponding phenotypes. INTERPRETATION: We showed that lifestyle and biochemical MS were associated with MDD before and after adjustment for their corresponding phenotypes (pnominal<0.05), but not when smoking, alcohol consumption, and BMI were also included as covariates. MDD results did not replicate in the smaller, female-only independent ALSPAC cohort (NALSPAC=565; NGS=9,502), potentially due to demographic differences or low statistical power, but effect sizes were consistent with the direction reported in GS. DNAm scores for modifiable MDD risk factors may contribute to disease vulnerability and, in some cases, explain additional variance to their observed phenotypes. FUNDING: Wellcome Trust.

DNA Methylation and Protein Markers of Chronic Inflammation and Their Associations With Brain and Cognitive Aging.

BACKGROUND AND OBJECTIVES: To investigate chronic inflammation in relation to cognitive aging by comparison of an epigenetic and serum biomarker of C-reactive protein and their associations with neuroimaging and cognitive outcomes. METHODS: At baseline, participants (n = 521) were cognitively normal, around 73 years of age (mean 72.4, SD 0.716), and had inflammation, vascular risk (cardiovascular disease history, hypertension, diabetes, smoking, alcohol consumption, body mass index), and neuroimaging (structural and diffusion MRI) data available. Baseline inflammatory status was quantified by a traditional measure of peripheral inflammation-serum C-reactive protein (CRP)-and an epigenetic measure (DNA methylation [DNAm] signature of CRP). Linear models were used to examine the inflammation-brain health associations; mediation analyses were performed to interrogate the relationship between chronic inflammation, brain structure, and cognitive functioning. RESULTS: We demonstrate that DNAm CRP shows significantly (on average 6.4-fold) stronger associations with brain health outcomes than serum CRP. DNAm CRP is associated with total brain volume (ß = -0.197, 95% confidence interval [CI] -0.28 to -0.12, p FDR = 8.42 × 10-6), gray matter volume (ß = -0.200, 95% CI -0.28 to -0.12, p FDR = 1.66 × 10-5), and white matter volume (ß = -0.150, 95% CI -0.23 to -0.07, p FDR = 0.001) and regional brain atrophy. We also find that DNAm CRP has an inverse association with global and domain-specific (speed, visuospatial, and memory) cognitive functioning and that brain structure partially mediates this CRP-cognitive association (up to 29.7%), dependent on lifestyle and health factors. DISCUSSION: These results support the hypothesis that chronic inflammation may contribute to neurodegenerative brain changes that underlie differences in cognitive ability in later life and highlight the potential of DNAm proxies for indexing chronic inflammatory status. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that a DNAm signature of CRP levels is more strongly associated with brain health outcomes than serum CRP levels.

Associations between major psychiatric disorder polygenic risk scores and blood-based markers in UK biobank.

Major depressive disorder (MDD), schizophrenia (SCZ), and bipolar disorder (BD) have both shared and discrete genetic risk factors, and are associated with peripheral abnormalities. The relationships between such genetic architectures and blood-based markers are, however, unclear. We investigated relationships between polygenic risk scores (PRS) for these disorders and peripheral markers in the UK Biobank cohort. We calculated polygenic risk scores for n = 367,329 (MDD PRS), n = 366,465 (SCZ PRS), and n = 366,383 (BD PRS) UK Biobank cohort subjects. We then examined associations between disorder PRS and 58 inflammatory/immune, hematological, bone, cardiovascular, hormone, liver, renal and diabetes-associated blood markers using two generalized linear regression models: 'minimally adjusted' controlling for variables such as age and sex, and 'fully adjusted' including additional lifestyle covariates: BMI, alcohol and smoking status, and medication intake. There were 38/58 MDD PRS, 32/58 SCZ PRS, and 20/58 BD PRS-blood marker associations detected for our minimally adjusted model. Of these, 13/38 (MDD PRS), 14/32 (SCZ PRS), and 10/20 (BD PRS) associations remained significant after controlling for lifestyle factors. Many were disorder-specific, with 8/13 unique MDD PRS associations identified. Several disorder-specific associations for MDD and SCZ were immune-related, with mostly positive and negative associations identified for MDD and SCZ PRS respectively. This study suggests that MDD, SCZ and BD have both shared and distinct peripheral markers associated with disorder-specific genetic risk. The results also implicate inflammatory dysfunction in MDD and SCZ, albeit with differences in patterns between the two conditions, and enrich our understanding of potential underlying pathophysiological mechanisms in major psychiatric disorders.

Epigenetic prediction of major depressive disorder.

Variation in DNA methylation (DNAm) is associated with lifestyle factors such as smoking and body mass index (BMI) but there has been little research exploring its ability to identify individuals with major depressive disorder (MDD). Using penalised regression on genome-wide CpG methylation, we tested whether DNAm risk scores (MRS), trained on 1223 MDD cases and 1824 controls, could discriminate between cases (n = 363) and controls (n = 1417) in an independent sample, comparing their predictive accuracy to polygenic risk scores (PRS). The MRS explained 1.75% of the variance in MDD (ß = 0.338, p = 1.17 × 10-7) and remained associated after adjustment for lifestyle factors (ß = 0.219, p = 0.001, R2 = 0.68%). When modelled alongside PRS (ß = 0.384, p = 4.69 × 10-9) the MRS remained associated with MDD (ß = 0.327, p = 5.66 × 10-7). The MRS was also associated with incident cases of MDD who were well at recruitment but went on to develop MDD at a later assessment (ß = 0.193, p = 0.016, R2 = 0.52%). Heritability analyses found additive genetic effects explained 22% of variance in the MRS, with a further 19% explained by pedigree-associated genetic effects and 16% by the shared couple environment. Smoking status was also strongly associated with MRS (ß = 0.440, p ≤ 2 × 10-16). After removing smokers from the training set, the MRS strongly associated with BMI (ß = 0.053, p = 0.021). We tested the association of MRS with 61 behavioural phenotypes and found that whilst PRS were associated with psychosocial and mental health phenotypes, MRS were more strongly associated with lifestyle and sociodemographic factors. DNAm-based risk scores of MDD significantly discriminated MDD cases from controls in an independent dataset and may represent an archive of exposures to lifestyle factors that are relevant to the prediction of MDD.

A meta-analysis of genome-wide association studies of epigenetic age acceleration.

'Epigenetic age acceleration' is a valuable biomarker of ageing, predictive of morbidity and mortality, but for which the underlying biological mechanisms are not well established. Two commonly used measures, derived from DNA methylation, are Horvath-based (Horvath-EAA) and Hannum-based (Hannum-EAA) epigenetic age acceleration. We conducted genome-wide association studies of Horvath-EAA and Hannum-EAA in 13,493 unrelated individuals of European ancestry, to elucidate genetic determinants of differential epigenetic ageing. We identified ten independent SNPs associated with Horvath-EAA, five of which are novel. We also report 21 Horvath-EAA-associated genes including several involved in metabolism (NHLRC, TPMT) and immune system pathways (TRIM59, EDARADD). GWAS of Hannum-EAA identified one associated variant (rs1005277), and implicated 12 genes including several involved in innate immune system pathways (UBE2D3, MANBA, TRIM46), with metabolic functions (UBE2D3, MANBA), or linked to lifespan regulation (CISD2). Both measures had nominal inverse genetic correlations with father's age at death, a rough proxy for lifespan. Nominally significant genetic correlations between Hannum-EAA and lifestyle factors including smoking behaviours and education support the hypothesis that Hannum-based epigenetic ageing is sensitive to variations in environment, whereas Horvath-EAA is a more stable cellular ageing process. We identified novel SNPs and genes associated with epigenetic age acceleration, and highlighted differences in the genetic architecture of Horvath-based and Hannum-based epigenetic ageing measures. Understanding the biological mechanisms underlying individual differences in the rate of epigenetic ageing could help explain different trajectories of age-related decline.

Identification of novel differentially methylated sites with potential as clinical predictors of impaired respiratory function and COPD.

BACKGROUND: The causes of poor respiratory function and COPD are incompletely understood, but it is clear that genes and the environment play a role. As DNA methylation is under both genetic and environmental control, we hypothesised that investigation of differential methylation associated with these phenotypes would permit mechanistic insights, and improve prediction of COPD. We investigated genome-wide differential DNA methylation patterns using the recently released 850 K Illumina EPIC array. This is the largest single population, whole-genome epigenetic study to date. METHODS: Epigenome-wide association studies (EWASs) of respiratory function and COPD were performed in peripheral blood samples from the Generation Scotland: Scottish Family Health Study (GS:SFHS) cohort (n = 3781; 274 COPD cases and 2919 controls). In independent COPD incidence data (n = 149), significantly differentially methylated sites (DMSs; p < 3.6 × 10-8) were evaluated for their added predictive power when added to a model including clinical variables, age, sex, height and smoking history using receiver operating characteristic analysis. The Lothian Birth Cohort 1936 (LBC1936) was used to replicate association (n = 895) and prediction (n = 178) results. FINDINGS: We identified 28 respiratory function and/or COPD associated DMSs, which mapped to genes involved in alternative splicing, JAK-STAT signalling, and axon guidance. In prediction analyses, we observed significant improvement in discrimination between COPD cases and controls (p < .05) in independent GS:SFHS (p = .016) and LBC1936 (p = .010) datasets by adding DMSs to a clinical model. INTERPRETATION: Identification of novel DMSs has provided insight into the molecular mechanisms regulating respiratory function and aided prediction of COPD risk. Further studies are needed to assess the causality and clinical utility of identified associations. FUND: Wellcome Trust Strategic Award 10436/Z/14/Z.

Associations between vascular risk factors and brain MRI indices in UK Biobank.

AIMS: Several factors are known to increase risk for cerebrovascular disease and dementia, but there is limited evidence on associations between multiple vascular risk factors (VRFs) and detailed aspects of brain macrostructure and microstructure in large community-dwelling populations across middle and older age. METHODS AND RESULTS: Associations between VRFs (smoking, hypertension, pulse pressure, diabetes, hypercholesterolaemia, body mass index, and waist-hip ratio) and brain structural and diffusion MRI markers were examined in UK Biobank (N = 9722, age range 44-79 years). A larger number of VRFs was associated with greater brain atrophy, lower grey matter volume, and poorer white matter health. Effect sizes were small (brain structural R2 ≤1.8%). Higher aggregate vascular risk was related to multiple regional MRI hallmarks associated with dementia risk: lower frontal and temporal cortical volumes, lower subcortical volumes, higher white matter hyperintensity volumes, and poorer white matter microstructure in association and thalamic pathways. Smoking pack years, hypertension and diabetes showed the most consistent associations across all brain measures. Hypercholesterolaemia was not uniquely associated with any MRI marker. CONCLUSION: Higher levels of VRFs were associated with poorer brain health across grey and white matter macrostructure and microstructure. Effects are mainly additive, converging upon frontal and temporal cortex, subcortical structures, and specific classes of white matter fibres. Though effect sizes were small, these results emphasize the vulnerability of brain health to vascular factors even in relatively healthy middle and older age, and the potential to partly ameliorate cognitive decline by addressing these malleable risk factors.

An epigenome-wide association study of sex-specific chronological ageing.

BACKGROUND: Advanced age is associated with cognitive and physical decline and is a major risk factor for a multitude of disorders. There is also a gap in life expectancy between males and females. DNA methylation differences have been shown to be associated with both age and sex. Here, we investigate age-by-sex differences in blood-based DNA methylation in an unrelated cohort of 2586 individuals between the ages of 18 and 87 years, with replication in a further 4450 individuals between the ages of 18 and 93 years. METHODS: Linear regression models were applied, with stringent genome-wide significance thresholds (p < 3.6 × 10-8) used in both the discovery and replication data. A second, highly conservative mixed linear model method that better controls the false-positive rate was also applied, using the same genome-wide significance thresholds. RESULTS: Using the linear regression method, 52 autosomal and 597 X-linked CpG sites, mapping to 251 unique genes, replicated with concordant effect size directions in the age-by-sex interaction analysis. The site with the greatest difference mapped to GAGE10, an X-linked gene. Here, DNA methylation levels remained stable across the male adult age range (DNA methylation by age r = 0.02) but decreased across female adult age range (DNA methylation by age r = - 0.61). One site (cg23722529) with a significant age-by-sex interaction also had a quantitative trait locus (rs17321482) that is a genome-wide significant variant for prostate cancer. The mixed linear model method identified 11 CpG sites associated with the age-by-sex interaction. CONCLUSION: The majority of differences in age-associated DNA methylation trajectories between sexes are present on the X chromosome. Several of these differences occur within genes that have been implicated in sexually dimorphic traits.

Epigenetic signatures of starting and stopping smoking.

BACKGROUND: Multiple studies have made robust associations between differential DNA methylation and exposure to cigarette smoke. But whether a DNA methylation phenotype is established immediately upon exposure, or only after prolonged exposure is less well-established. Here, we assess DNA methylation patterns from peripheral blood samples in current smokers in response to dose and duration of exposure, along with the effects of smoking cessation on DNA methylation in former smokers. METHODS: Dimensionality reduction was applied to DNA methylation data at 90 previously identified smoking-associated CpG sites for over 4900 individuals in the Generation Scotland cohort. K-means clustering was performed to identify clusters associated with current and never smoker status based on these methylation patterns. Cluster assignments were assessed with respect to duration of exposure in current smokers (years as a smoker), time since smoking cessation in former smokers (years), and dose (cigarettes per day). FINDINGS: Two clusters were specified, corresponding to never smokers (97·5% of whom were assigned to Cluster 1) and current smokers (81·1% of whom were assigned to Cluster 2). The exposure time point from which >50% of current smokers were assigned to the smoker-enriched cluster varied between 5 and 9 years in heavier smokers and between 15 and 19 years in lighter smokers. Low-dose former smokers were more likely to be assigned to the never smoker-enriched cluster in the first year following cessation. In contrast, a period of at least two years was required before the majority of former high-dose smokers were assigned to the never smoker-enriched cluster. INTERPRETATION: Our findings suggest that smoking-associated DNA methylation changes are a result of prolonged exposure to cigarette smoke, and can be reversed following cessation. The length of time in which these signatures are established and recovered is dose dependent. Should DNA methylation-based signatures of smoking status be predictive of smoking-related health outcomes, our findings may provide an additional criterion on which to stratify risk.

Investigating the relationship between DNA methylation age acceleration and risk factors for Alzheimer's disease.

INTRODUCTION: The "epigenetic clock" is a DNA methylation-based estimate of biological age and is correlated with chronological age-the greatest risk factor for Alzheimer's disease (AD). Genetic and environmental risk factors exist for AD, several of which are potentially modifiable. In this study, we assess the relationship between the epigenetic clock and AD risk factors. METHODS: Multilevel models were used to assess the relationship between age acceleration (the residual of biological age regressed onto chronological age) and AD risk factors relating to cognitive reserve, lifestyle, disease, and genetics in the Generation Scotland study (n = 5100). RESULTS: We report significant associations between age acceleration and body mass index, total cholesterol to high-density lipoprotein cholesterol ratios, socioeconomic status, high blood pressure, and smoking behavior (Bonferroni-adjusted P < .05). DISCUSSION: Associations are present between environmental risk factors for AD and age acceleration. Measures to modify such risk factors might improve the risk profile for AD and the rate of biological ageing. Future longitudinal analyses are therefore warranted.

Epigenetic prediction of complex traits and death.

BACKGROUND: Genome-wide DNA methylation (DNAm) profiling has allowed for the development of molecular predictors for a multitude of traits and diseases. Such predictors may be more accurate than the self-reported phenotypes and could have clinical applications. RESULTS: Here, penalized regression models are used to develop DNAm predictors for ten modifiable health and lifestyle factors in a cohort of 5087 individuals. Using an independent test cohort comprising 895 individuals, the proportion of phenotypic variance explained in each trait is examined for DNAm-based and genetic predictors. Receiver operator characteristic curves are generated to investigate the predictive performance of DNAm-based predictors, using dichotomized phenotypes. The relationship between DNAm scores and all-cause mortality (n = 212 events) is assessed via Cox proportional hazards models. DNAm predictors for smoking, alcohol, education, and waist-to-hip ratio are shown to predict mortality in multivariate models. The predictors show moderate discrimination of obesity, alcohol consumption, and HDL cholesterol. There is excellent discrimination of current smoking status, poorer discrimination of college-educated individuals and those with high total cholesterol, LDL with remnant cholesterol, and total:HDL cholesterol ratios. CONCLUSIONS: DNAm predictors correlate with lifestyle factors that are associated with health and mortality. They may supplement DNAm-based predictors of age to identify the lifestyle profiles of individuals and predict disease risk.

Impact of cannabis use on thalamic volume in people at familial high risk of schizophrenia.

BACKGROUND: No longitudinal study has yet examined the association between substance use and brain volume changes in a population at high risk of schizophrenia. AIMS: To examine the effects of cannabis on longitudinal thalamus and amygdala-hippocampal complex volumes within a population at high risk of schizophrenia. METHOD: Magnetic resonance imaging scans were obtained from individuals at high genetic risk of schizophrenia at the point of entry to the Edinburgh High-Risk Study (EHRS) and approximately 2 years later. Differential thalamic and amygdala-hippocampal complex volume change in high-risk individuals exposed (n = 25) and not exposed (n = 32) to cannabis in the intervening period was investigated using repeated-measures analysis of variance. RESULTS: Cannabis exposure was associated with bilateral thalamic volume loss. This effect was significant on the left (F = 4.47, P = 0.04) and highly significant on the right (F= 7.66, P= 0.008). These results remained significant when individuals using other illicit drugs were removed from the analysis. CONCLUSIONS: These are the first longitudinal data to demonstrate an association between thalamic volume loss and exposure to cannabis in currently unaffected people at familial high risk of developing schizophrenia. This observation may be important in understanding the link between cannabis exposure and the subsequent development of schizophrenia.

The impact of substance use on brain structure in people at high risk of developing schizophrenia.

Ventricular enlargement and reduced prefrontal volume are consistent findings in schizophrenia. Both are present in first episode subjects and may be detectable before the onset of clinical disorder. Substance misuse is more common in people with schizophrenia and is associated with similar brain abnormalities. We employ a prospective cohort study with nested case control comparison design to investigate the association between substance misuse, brain abnormality, and subsequent schizophrenia. Substance misuse history, imaging data, and clinical information were collected on 147 subjects at high risk of schizophrenia and 36 controls. Regions exhibiting a significant relationship between level of use of alcohol, cannabis or tobacco, and structure volume were identified. Multivariate regression then elucidated the relationship between level of substance use and structure volumes while accounting for correlations between these variables and correcting for potential confounders. Finally, we established whether substance misuse was associated with later risk of schizophrenia. Increased ventricular volume was associated with alcohol and cannabis use in a dose-dependent manner. Alcohol consumption was associated with reduced frontal lobe volume. Multiple regression analyses found both alcohol and cannabis were significant predictors of these abnormalities when simultaneously entered into the statistical model. Alcohol and cannabis misuse were associated with an increased subsequent risk of schizophrenia. We provide prospective evidence that use of cannabis or alcohol by people at high genetic risk of schizophrenia is associated with brain abnormalities and later risk of psychosis. A family history of schizophrenia may render the brain particularly sensitive to the risk-modifying effects of these substances.