The Impact of Childhood Mental Health and Substance Use on Methylation Aging Into Adulthood.

OBJECTIVE: To test whether childhood mental health symptoms, substance use, and early adversity accelerate the rate of DNA methylation (DNAm) aging from adolescence to adulthood. METHOD: DNAm was assayed from blood samples in 381 participants in both adolescence (mean [SD] age = 13.9 [1.6] years) and adulthood (mean [SD] age = 25.9 [2.7] years). Structured diagnostic interviews were completed with participants and their parents at multiple childhood observations (1,950 total) to assess symptoms of common mental health disorders (attention-deficit/hyperactivity disorder, oppositional defiant disorder, conduct disorder, anxiety, and depression) and common types of substance use (alcohol, cannabis, nicotine) and early adversities. RESULTS: Neither childhood mental health symptoms nor substance use variables were associated with DNAm aging cross-sectionally. In contrast, the following mental health symptoms and substance variables were associated with accelerated DNAm aging from adolescence to adulthood: depressive symptoms (b = 0.314, SE = 0.127, p = .014), internalizing symptoms (b = 0.108, SE = 0.049, p = .029), weekly cannabis use (b =1.665, SE = 0.591, p = .005), and years of weekly cannabis use (b = 0.718, SE = 0.283, p = .012). In models testing all individual variables simultaneously, the combined effect of the variables was equivalent to a potential difference of 3.17 to 3.76 years in DNAm aging. A final model tested a variable assessing cumulative exposure to mental health symptoms, substance use, and early adversities. This cumulative variable was strongly associated with accelerated aging (b = 0.126, SE = 0.044, p = .005). CONCLUSION: Mental health symptoms and substance use accelerated DNAm aging into adulthood in a manner consistent with a shared risk mechanism.

Meta-Analyses of Genome-Wide Association Studies for Postpartum Depression.

OBJECTIVE: Postpartum depression (PPD) is a common subtype of major depressive disorder (MDD) that is more heritable, yet is understudied in psychiatric genetics. The authors conducted meta-analyses of genome-wide association studies (GWASs) to investigate the genetic architecture of PPD. METHOD: Meta-analyses were conducted on 18 cohorts of European ancestry (17,339 PPD cases and 53,426 controls), one cohort of East Asian ancestry (975 cases and 3,780 controls), and one cohort of African ancestry (456 cases and 1,255 controls), totaling 18,770 PPD cases and 58,461 controls. Post-GWAS analyses included 1) single-nucleotide polymorphism (SNP)-based heritability ([Formula: see text]), 2) genetic correlations between PPD and other phenotypes, and 3) enrichment of the PPD GWAS findings in 27 human tissues and 265 cell types from the mouse central and peripheral nervous system. RESULTS: No SNP achieved genome-wide significance in the European or the trans-ancestry meta-analyses. The [Formula: see text] of PPD was 0.14 (SE=0.02). Significant genetic correlations were estimated for PPD with MDD, bipolar disorder, anxiety disorders, posttraumatic stress disorder, insomnia, age at menarche, and polycystic ovary syndrome. Cell-type enrichment analyses implicate inhibitory neurons in the thalamus and cholinergic neurons within septal nuclei of the hypothalamus, a pattern that differs from MDD. CONCLUSIONS: While more samples are needed to reach genome-wide levels of significance, the results presented confirm PPD as a polygenic and heritable phenotype. There is also evidence that despite a high correlation with MDD, PPD may have unique genetic components. Cell enrichment results suggest GABAergic neurons, which converge on a common mechanism with the only medication approved by the U.S. Food and Drug Administration for PPD (brexanolone).

Fine-grained cell-type specific association studies with human bulk brain data using a large single-nucleus RNA sequencing based reference panel.

Brain disorders are leading causes of disability worldwide. Gene expression studies provide promising opportunities to better understand their etiology but it is critical that expression is studied on a cell-type level. Cell-type specific association studies can be performed with bulk expression data using statistical methods that capitalize on cell-type proportions estimated with the help of a reference panel. To create a fine-grained reference panel for the human prefrontal cortex, we performed an integrated analysis of the seven largest single nucleus RNA-seq studies. Our panel included 17 cell-types that were robustly detected across all studies, subregions of the prefrontal cortex, and sex and age groups. To estimate the cell-type proportions, we used an empirical Bayes estimator that substantially outperformed three estimators recommended previously after a comprehensive evaluation of methods to estimate cell-type proportions from brain transcriptome data. This is important as being able to precisely estimate the cell-type proportions may avoid unreliable results in downstream analyses particularly for the multiple cell-types that had low abundances. Transcriptome-wide association studies performed with permuted bulk expression data showed that it is possible to perform transcriptome-wide association studies for even the rarest cell-types without an increased risk of false positives.

Methylome-wide association study of anxiety disorders.

Anxiety Disorders (ANX) such as panic disorder, generalized anxiety disorder, and phobias, are highly prevalent conditions that are moderately heritable. Evidence suggests that DNA methylation may play a role, as it is involved in critical adaptations to changing environments. Applying an enrichment-based sequencing approach covering nearly 28 million autosomal CpG sites, we conducted a methylome-wide association study (MWAS) of lifetime ANX in 1132 participants (618 cases/514 controls) from the Netherlands Study of Depression and Anxiety. Using epigenomic deconvolution, we performed MWAS for the main cell types in blood: granulocytes, T-cells, B-cells and monocytes. Cell-type specific analyses identified 280 and 82 methylome-wide significant associations (q-value < 0.1) in monocytes and granulocytes, respectively. Our top finding in monocytes was located in ZNF823 on chromosome 19 (p = 1.38 × 10-10) previously associated with schizophrenia. We observed significant overlap (p < 1 × 10-06) with the same direction of effect in monocytes (210 sites), T-cells (135 sites), and B-cells (727 sites) between this Discovery MWAS signal and a comparable replication dataset from the Great Smoky Mountains Study (N = 433). Overlapping Discovery-Replication MWAS signal was enriched for findings from published GWAS of ANX, major depression, and post-traumatic stress disorder. In monocytes, two specific sites in the FZR1 gene showed significant replication after Bonferroni correction with an additional 15 nominally replicated sites in monocytes and 4 in T-cells. FZR1 regulates neurogenesis in the hippocampus, and its knockout leads to impairments in associative fear memory and long-term potentiation in mice. In the largest and most extensive methylome-wide study of ANX, we identified replicable methylation sites located in genes of potential relevance for brain mechanisms of psychiatric conditions.

A method to improve the reproducibility of findings from epigenome- and transcriptome-wide association studies.

Reproducibility is a cornerstone of scientific progress. In epigenome- and transcriptome-wide association studies (E/TWAS) failure to reproduce may be the result of false discoveries. Whereas multiple methods exist to control false discoveries due to sampling error, minimizing false discoveries due to outliers and other data artefacts remains challenging. We propose a robust E/TWAS approach that outperforms alternative methods to improve reproducibility such as split-half replication. Furthermore, robust E/TWAS results in only a minor loss of power if there are no outliers and can in the presence of outliers, likely a more realistic scenario, even be more powerful than regular E/TWAS.

Genes implicated by a methylome-wide schizophrenia study in neonatal blood show differential expression in adult brain samples.

Schizophrenia is a disabling disorder involving genetic predisposition in combination with environmental influences that likely act via dynamic alterations of the epigenome and the transcriptome but its detailed pathophysiology is largely unknown. We performed cell-type specific methylome-wide association study of neonatal blood (N = 333) from individuals who later in life developed schizophrenia and controls. Suggestively significant associations (P < 1.0 × 10-6) were detected in all cell-types and in whole blood with methylome-wide significant associations in monocytes (P = 2.85 × 10-9-4.87 × 10-9), natural killer cells (P = 1.72 × 10-9-7.82 × 10-9) and B cells (P = 3.8 × 10-9). Validation of methylation findings in post-mortem brains (N = 596) from independent schizophrenia cases and controls showed significant enrichment of transcriptional differences (enrichment ratio = 1.98-3.23, P = 2.3 × 10-3-1.0 × 10-5), with specific highly significant differential expression for, for example, BDNF (t = -6.11, P = 1.90 × 10-9). In addition, expression difference in brain significantly predicted schizophrenia (multiple correlation = 0.15-0.22, P = 3.6 × 10-4-4.5 × 10-8). In summary, using a unique design combining pre-disease onset (neonatal) blood methylomic data and post-disease onset (post-mortem) brain transcriptional data, we have identified genes of likely functional relevance that are associated with schizophrenia susceptibility, rather than confounding disease associated artifacts. The identified loci may be of clinical value as a methylation-based biomarker for early detection of increased schizophrenia susceptibility.

A single-nucleus transcriptomics study of alcohol use disorder in the nucleus accumbens.

Gene expression studies offer promising opportunities to better understand the processes underlying alcohol use disorder (AUD). As cell types differ in their function, gene expression profiles will typically vary across cell types. When studying bulk tissue, failure to account for this cellular diversity has a detrimental impact on the ability to detect disease associations. We therefore assayed the transcriptomes of 32,531 individual nuclei extracted from the nucleus accumbens (NAc) of nine donors with AUD and nine controls (72% male). Our study identified 17 clearly delineated cell types. We detected 26 transcriptome-wide significant differentially expressed genes (DEGs) that mainly involved medium spiny neurons with both D1-type and D2-type dopamine receptors, microglia (MGL) and oligodendrocytes. A higher than expected number of DEGs replicated in an existing single nucleus gene expression study of alcohol dependence in the prefrontal cortex (enrichment ratio 1.91, p value 0.019) with two genes remaining significant after a Bonferroni correction. Our most compelling result involved CD53 in MGL that replicated in the same cell type in the prefrontal cortex and was previously implicated in studies of DNA methylation, bulk gene expression and genetic variants. Several DEGs were previously reported to be associated with AUD (e.g., PER1 and MGAT5). The DEGs for MSN.3 seemed involved in neurodegeneration, disruption of circadian rhythms, alterations in glucose metabolism and changes in synaptic plasticity. For MGL, the DEGs implicated neuroinflammation and immune-related processes and for OLI, disruptions in myelination. This identification of the specific cell-types from which the association signals originate will be key for designing proper follow-up experiments and, eventually, novel clinical interventions.

DNA methylation signatures of childhood trauma predict psychiatric disorders and other adverse outcomes 17 years after exposure.

Childhood trauma is robustly linked to a broad range of adverse outcomes with consequences persisting far into adulthood. We conducted a prospective longitudinal study to predict psychiatric disorders and other adverse outcomes from trauma-related methylation changes 16.9 years after trauma exposure in childhood. Methylation was assayed using a sequencing-based approach that provides near-complete coverage of all 28 million sites in the blood methylome. Methylation data involved 673 assays from 489 participants aged 13.6 years (SD = 1.9) with outcomes measures collected at age 30.4 (SD = 2.26). For a subset of 303 participants we also generated methylation data in adulthood. Trauma-related methylation risk scores (MRSs) significantly predicted adult depression, externalizing problems, nicotine dependence, alcohol use disorder, serious medical problems, social problems and poverty. The predictive power of the MRSs was higher than that of reported trauma and could not be explained by the reported trauma, correlations with demographic variables, or a continuity of the predicted health problems from childhood to adulthood. Rather than measuring the occurrence of traumatic events, the MRSs seemed to capture the subject-specific impact of trauma. The majority of predictive sites did not remain associated with the outcomes suggesting the signatures of trauma do not become biologically embedded in the blood methylome. Instead, the long-term effects of trauma therefore seemed more consistent with a developmental mechanism where the initial subject-specific impacts of trauma are magnified over time. The MRSs have the potential to be a novel clinical biomarker for the assessment of trauma-related health risks.

Transcriptome-wide association study for postpartum depression implicates altered B-cell activation and insulin resistance.

Postpartum depression (PPD) affects 1 in 7 women and has negative mental health consequences for both mother and child. However, the precise biological mechanisms behind the disorder are unknown. Therefore, we performed the largest transcriptome-wide association study (TWAS) for PPD (482 cases, 859 controls) to date using RNA-sequencing in whole blood and deconvoluted cell types. No transcriptional changes were observed in whole blood. B-cells showed a majority of transcriptome-wide significant results (891 transcripts representing 789 genes) with pathway analyses implicating altered B-cell activation and insulin resistance. Integration of other data types revealed cell type-specific DNA methylation loci and disease-associated eQTLs (deQTLs), but not hormones/neuropeptides (estradiol, progesterone, oxytocin, BDNF), serve as regulators for part of the transcriptional differences between cases and controls. Further, deQTLs were enriched for several brain region-specific eQTLs, but no overlap with MDD risk loci was observed. Altogether, our results constitute a convergence of evidence for pathways most affected in PPD with data across different biological mechanisms.

Early adversities accelerate epigenetic aging into adulthood: a 10-year, within-subject analysis.

BACKGROUND: Longitudinal studies are needed to clarify whether early adversities are associated with advanced methylation age or if they actually accelerate methylation aging. This study test whether different dimensions of childhood adversity accelerate biological aging from childhood to adulthood, and, if so, via which mechanisms. METHODS: 381 participants provided one blood sample in childhood (average age 15.0; SD = 2.3) and another in young adulthood (average age 23.1; SD = 2.8). Participants and their parents provided a median of 6 childhood assessments (total = 1,950 childhood observations), reporting exposures to different types of adversity dimensions (i.e. threat, material deprivation, loss, unpredictability). The blood samples were assayed to estimate DNA methylation age in both childhood and adulthood and also change in methylation age across this period. RESULTS: Cross-sectional associations between the childhood adversity dimensions and childhood measures of methylation age were non-significant. In contrast, multiple adversity dimensions were associated with accelerated within-person change in methylation age from adolescence to young adulthood. These associations attenuated in model testing all dimensions at the same time. Accelerated aging increased with increasing number of childhood adversities: Individuals with highest number of adversities experienced 2+ additional years of methylation aging compared to those with no exposure to childhood adversities. The association between total childhood adversity exposure and accelerated aging was partially explained by childhood depressive symptoms, but not anxiety or behavioral symptoms. CONCLUSIONS: Early adversities accelerate epigenetic aging long after they occur, in proportion to the total number of such experiences, and in a manner consistent with a shared effect that crosses multiple early dimensions of risk.

A methylation study implicates the rewiring of brain neural circuits during puberty in the emergence of sex differences in depression symptoms.

BACKGROUND: Women are 1.5-3 times more likely to suffer from depression than men. This sex bias first emerges during puberty and then persists across the reproductive years. As the cause remains largely elusive, we performed a methylation-wide association study (MWAS) to generate novel hypotheses. METHODS: We assayed nearly all 28 million possible methylation sites in blood in 595 blood samples from 487 participants aged 9-17. MWASs were performed to identify methylation sites associated with increasing sex differences in depression symptoms as a function of pubertal stage. Epigenetic deconvolution was applied to perform analyses on a cell-type specific level. RESULTS: In monocytes, a substantial number of significant associations were detected after controlling the FDR at 0.05. These results could not be explained by plasma testosterone/estradiol or current/lifetime trauma. Our top results in monocytes were significantly enriched (ratio of 2.48) for genes in the top of a large genome-wide association study (GWAS) meta-analysis of depression and neurodevelopment-related Gene Ontology (GO) terms that remained significant after correcting for multiple testing. Focusing on our most robust findings (70 genes overlapping with the GWAS meta-analysis and the significant GO terms), we find genes coding for members of each of the major classes of axon guidance molecules (netrins, slits, semaphorins, ephrins, and cell adhesion molecules). Many of these genes were previously implicated in rodent studies of brain development and depression-like phenotypes, as well as human methylation, gene expression and GWAS studies. CONCLUSIONS: Our study suggests that the emergence of sex differences in depression may be related to the differential rewiring of brain circuits between boys and girls during puberty.

Dual methylation and hydroxymethylation study of alcohol use disorder.

Using an integrative, multi-tissue design, we sought to characterize methylation and hydroxymethylation changes in blood and brain associated with alcohol use disorder (AUD). First, we used epigenomic deconvolution to perform cell-type-specific methylome-wide association studies within subpopulations of granulocytes/T-cells/B-cells/monocytes in 1132 blood samples. Blood findings were then examined for overlap with AUD-related associations with methylation and hydroxymethylation in 50 human post-mortem brain samples. Follow-up analyses investigated if overlapping findings mediated AUD-associated transcription changes in the same brain samples. Lastly, we replicated our blood findings in an independent sample of 412 individuals and aimed to replicate published alcohol methylation findings using our results. Cell-type-specific analyses in blood identified methylome-wide significant associations in monocytes and T-cells. The monocyte findings were significantly enriched for AUD-related methylation and hydroxymethylation in brain. Hydroxymethylation in specific sites mediated AUD-associated transcription in the same brain samples. As part of the most comprehensive methylation study of AUD to date, this work involved the first cell-type-specific methylation study of AUD conducted in blood, identifying and replicating a finding in DLGAP1 that may be a blood-based biomarker of AUD. In this first study to consider the role of hydroxymethylation in AUD, we found evidence for a novel mechanism for cognitive deficits associated with AUD. Our results suggest promising new avenues for AUD research.

A targeted solution for estimating the cell-type composition of bulk samples.

BACKGROUND: To avoid false-positive findings and detect cell-type specific associations in methylation and transcription investigations with bulk samples, it is critical to know the proportions of the major cell-types. RESULTS: We present a novel approach that allows for precise estimation of cell-type proportions using only a few highly informative methylation markers. The most reliable estimates were obtained with 17 amplicons (34 CpGs) using the MuSiC estimator, for which the average correlations between the estimated and the true cell-type proportions were 0.889. Furthermore, the estimates were not significantly different from the true values (P = 0.95) indicating that the estimator is unbiased and the standard deviation of the estimates further indicate high precision. Moreover, the overall variability of the estimates as measured by the Root Mean Squared Error (RMSE), which is a function of both bias and precision, was low (mean RMSE = 0.038). Taken together, these results indicate that the approach produced reliable estimates that are both unbiased and highly precise. CONCLUSION: This cost-effective approach for estimating cell-type proportions in bulk samples allows for enhanced targeted analysis, which in turn will minimize the risk of reporting false-positive findings and allowing for detection of cell-type specific associations. The approach is applicable across platforms and can be extended to assess cell-type proportions for various tissues.

Methylomic Investigation of Problematic Adolescent Cannabis Use and Its Negative Mental Health Consequences.

OBJECTIVE: The impact of adolescent cannabis use is a pressing public health question owing to the high rates of use and links to negative outcomes. This study considered the association between problematic adolescent cannabis use and methylation. METHOD: Using an enrichment-based sequencing approach, a methylome-wide association study (MWAS) was performed of problematic adolescent cannabis use in 703 adolescent samples from the Great Smoky Mountain Study. Using epigenomic deconvolution, MWASs were performed for the main cell types in blood: granulocytes, T cells, B cells, and monocytes. Enrichment testing was conducted to establish overlap between cannabis-associated methylation differences and variants associated with negative mental health effects of adolescent cannabis use. RESULTS: Whole-blood analyses identified 45 significant CpGs, and cell type-specific analyses yielded 32 additional CpGs not identified in the whole-blood MWAS. Significant overlap was observed between the B-cell MWAS and genetic studies of education attainment and intelligence. Furthermore, the results from both T cells and monocytes overlapped with findings from an MWAS of psychosis conducted in brain tissue. CONCLUSION: In one of the first methylome-wide association studies of adolescent cannabis use, several methylation sites located in genes of importance for potentially relevant brain functions were identified. These findings resulted in several testable hypotheses by which cannabis-associated methylation can impact neurological development and inflammation response as well as potential mechanisms linking cannabis-associated methylation to potential downstream mental health effects.

An integrative study of five biological clocks in somatic and mental health.

Biological clocks have been developed at different molecular levels and were found to be more advanced in the presence of somatic illness and mental disorders. However, it is unclear whether different biological clocks reflect similar aging processes and determinants. In ~3000 subjects, we examined whether five biological clocks (telomere length, epigenetic, transcriptomic, proteomic, and metabolomic clocks) were interrelated and associated to somatic and mental health determinants. Correlations between biological aging indicators were small (all r < 0.2), indicating little overlap. The most consistent associations of advanced biological aging were found for male sex, higher body mass index (BMI), metabolic syndrome, smoking, and depression. As compared to the individual clocks, a composite index of all five clocks showed most pronounced associations with health determinants. The large effect sizes of the composite index and the low correlation between biological aging indicators suggest that one's biological age is best reflected by combining aging measures from multiple cellular levels.

Test-statistic inflation in methylome-wide association studies.

Recent years have seen a surge of methylome-wide association studies (MWAS). We observed that many of these studies suffer from test statistic inflation that is most likely caused by commonly used quality control (QC) pipelines not going far enough to remove technical artefacts. To support this claim, we reanalysed GEO datasets with an improved QC pipeline that reduced test-statistic inflation parameter lambda from the original mean/median of 20.16/15.17 to 3.07/1.14. Furthermore, the mean/median number of methylome-wide significant findings was reduced by 65,688/57,805 loci after more thorough QC. To avoid such false positives we argue for more extensive QC and that reporting the test-statistic inflation parameter lambda become standard for all MWAS allowing readers to better assess the risk of false discoveries.

DNA methylation and histone acetylation changes to cytochrome P450 2E1 regulation in normal aging and impact on rates of drug metabolism in the liver.

Aging is associated with reduced liver function that may increase the risk for adverse drug reactions in older adults. We hypothesized that age-related changes to epigenetic regulation of genes involved in drug metabolism may contribute to this effect. We reviewed published epigenome-wide studies of human blood and identified the cytochrome P450 2E1 (CYP2E1) gene as a top locus exhibiting epigenetic changes with age. To investigate potential functional changes with age in the liver, the primary organ of drug metabolism, we obtained liver tissue from mice aged 4-32 months from the National Institute on Aging. We assayed global DNA methylation (5-methylcytosine, 5mC), hydroxymethylation (5-hydroxymethylcytosine, 5hmC), and locus-specific 5mC and histone acetylation changes around mouse Cyp2e1. The mouse livers exhibit significant global decreases in 5mC and 5hmC with age. Furthermore, 5mC significantly increased with age at two regulatory regions of Cyp2e1 in tandem with decreases in its gene and protein expressions. H3K9ac levels also changed with age at both regulatory regions of Cyp2e1 investigated, while H3K27ac did not. To test if these epigenetic changes are associated with varying rates of drug metabolism, we assayed clearance of the CYP2E1-specific probe drug chlorzoxazone in microsome extracts from the same livers. CYP2E1 intrinsic clearance is associated with DNA methylation and H3K9ac levels at the Cyp2e1 locus but not with chronological age. This suggests that age-related epigenetic changes may influence rates of hepatic drug metabolism. In the future, epigenetic biomarkers could prove useful to guide dosing regimens in older adults.

Cell Type-Specific Methylome-wide Association Studies Implicate Neurotrophin and Innate Immune Signaling in Major Depressive Disorder.

BACKGROUND: We sought to characterize methylation changes in brain and blood associated with major depressive disorder (MDD). As analyses of bulk tissue may obscure association signals and hamper the biological interpretation of findings, these changes were studied on a cell type-specific level. METHODS: In 3 collections of human postmortem brain (n = 206) and 1 collection of blood samples (N = 1132) of MDD cases and controls, we used epigenomic deconvolution to perform cell type-specific methylome-wide association studies within subpopulations of neurons/glia for the brain data and granulocytes/T cells/B cells/monocytes for the blood data. Sorted neurons/glia from a fourth postmortem brain collection (n = 58) were used for validation purposes. RESULTS: Cell type-specific methylome-wide association studies identified multiple findings in neurons/glia that were detected across brain collections and were reproducible in physically sorted nuclei. Cell type-specific analyses in blood samples identified methylome-wide significant associations in T cells, monocytes, and whole blood that replicated findings from a past methylation study of MDD. Pathway analyses implicated p75 neurotrophin receptor/nerve growth factor signaling and innate immune toll-like receptor signaling in MDD. Top results in neurons, glia, bulk brain, T cells, monocytes, and whole blood were enriched for genes supported by genome-wide association studies for MDD and other psychiatric disorders. CONCLUSIONS: We both replicated and identified novel MDD-methylation associations in human brain and blood samples at a cell type-specific level. Our results provide mechanistic insights into how the immune system may interact with the brain to affect MDD susceptibility. Importantly, our findings involved associations with MDD in human samples that implicated many closely related biological pathways. These disease-linked sites and pathways represent promising new therapeutic targets for MDD.

Methylome-wide association findings for major depressive disorder overlap in blood and brain and replicate in independent brain samples.

We present the first large-scale methylome-wide association studies (MWAS) for major depressive disorder (MDD) to identify sites of potential importance for MDD etiology. Using a sequencing-based approach that provides near-complete coverage of all 28 million common CpGs in the human genome, we assay methylation in MDD cases and controls from both blood (N = 1132) and postmortem brain tissues (N = 61 samples from Brodmann Area 10, BA10). The MWAS for blood identified several loci with P ranging from 1.91 × 10-8 to 4.39 × 10-8 and a resampling approach showed that the cumulative association was significant (P = 4.03 × 10-10) with the signal coming from the top 25,000 MWAS markers. Furthermore, a permutation-based analysis showed significant overlap (P = 5.4 × 10-3) between the MWAS findings in blood and brain (BA10). This overlap was significantly enriched for a number of features including being in eQTLs in blood and the frontal cortex, CpG islands and shores, and exons. The overlapping sites were also enriched for active chromatin states in brain including genic enhancers and active transcription start sites. Furthermore, three loci located in GABBR2, RUFY3, and in an intergenic region on chromosome 2 replicated with the same direction of effect in the second brain tissue (BA25, N = 60) from the same individuals and in two independent brain collections (BA10, N = 81 and 64). GABBR2 inhibits neuronal activity through G protein-coupled second-messenger systems and RUFY3 is implicated in the establishment of neuronal polarity and axon elongation. In conclusion, we identified and replicated methylated loci associated with MDD that are involved in biological functions of likely importance to MDD etiology.

Independent Methylome-Wide Association Studies of Schizophrenia Detect Consistent Case-Control Differences.

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.