Tissue-specific patterns of allelically-skewed DNA methylation

While DNA methylation is usually thought to be symmetrical across both alleles, there are some notable exceptions. Genomic imprinting and X chromosome inactivation are two well-studied sources of allele-specific methylation (ASM), but recent research has indicated a more complex pattern in which genotypic variation can be associated with allelically-skewed DNA methylation in cis. Given the known heterogeneity of DNA methylation across tissues and cell types we explored inter- and intra-individual variation in ASM across several regions of the human brain and whole blood from multiple individuals. Consistent with previous studies, we find widespread ASM with >4% of the ~220,000 loci interrogated showing evidence of allelically-skewed DNA methylation. We identify ASM flanking known imprinted regions, and show that ASM sites are enriched in DNase I hypersensitivity sites and often located in an extended genomic context of intermediate DNA methylation. We also detect examples of genotype-driven ASM, some of which are also tissue-specific. These findings contribute to our understanding about the nature of differential DNA methylation across tissues and have important implications for genetic studies of complex disease. As a resource to the community, ASM patterns across each of the tissues studied are available in a searchable online database: http://epigenetics.essex.ac.uk/ASMBrainBlood

Genome-wide DNA Methylation Meta-Analysis in the Brains of Suicide Completers

Suicide is the second leading cause of death globally among young people representing a significant global health burden. Although the molecular correlates of suicide remains poorly understood, it has been hypothesised that epigenomic processes may play a role. The objective of this study was to identify suicide-associated DNA methylation changes in the human brain by utilising previously published and unpublished methylomic datasets. We analysed prefrontal cortex (PFC, n = 211) and cerebellum (CER, n = 114) DNA methylation profiles from suicide completers and non-psychiatric, sudden-death controls, meta-analysing data from independent cohorts for each brain region separately. We report evidence for altered DNA methylation at several genetic loci in suicide cases compared to controls in both brain regions with suicide-associated differentially methylated positions enriched among functional pathways relevant to psychiatric phenotypes and suicidality, including nervous system development (PFC) and regulation of long-term synaptic depression (CER). In addition, we examined the functional consequences of variable DNA methylation within a PFC suicide-associated differentially methylated region (PSORS1C3 DMR) using a dual luciferase assay and examined expression of nearby genes. DNA methylation within this region was associated with decreased expression of firefly luciferase but was not associated with expression of nearby genes, PSORS1C3 and POU5F1. Our data suggest that suicide is associated with DNA methylation, offering novel insights into the molecular pathology associated with suicidality

Genome-wide DNA Methylation Meta-analysis in the Brains of Suicide Completers

Suicide is the second leading cause of death globally among young people representing a significant global health burden. Although the molecular correlates of suicide remains poorly understood, it has been hypothesised that epigenomic processes may play a role. The objective of this study was to identify suicide-associated DNA methylation changes in the human brain by utilising previously published and unpublished methylomic datasets. We analysed prefrontal cortex (PFC, n = 211) and cerebellum (CER, n = 114) DNA methylation profiles from suicide completers and non-psychiatric, sudden-death controls, meta-analysing data from independent cohorts for each brain region separately. We report evidence for altered DNA methylation at several genetic loci in suicide cases compared to controls in both brain regions with suicide-associated differentially methylated positions enriched among functional pathways relevant to psychiatric phenotypes and suicidality, including nervous system development (PFC) and regulation of long-term synaptic depression (CER). In addition, we examined the functional consequences of variable DNA methylation within a PFC suicide-associated differentially methylated region (PSORS1C3 DMR) using a dual luciferase assay and examined expression of nearby genes. DNA methylation within this region was associated with decreased expression of firefly luciferase but was not associated with expression of nearby genes, PSORS1C3 and POU5F1. Our data suggest that suicide is associated with DNA methylation, offering novel insights into the molecular pathology associated with suicidality

Sites of active gene regulation in the prenatal frontal cortex and their role in neuropsychiatric disorders

Common genetic variation appears to largely influence risk for neuropsychiatric disorders through effects on gene regulation. It is therefore possible to shed light on the biology of these conditions by testing for enrichment of associated genetic variation within regulatory genomic regions operating in specific tissues or cell types. Here, we have used the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) to map open chromatin (an index of active regulatory genomic regions) in bulk tissue, NeuN+ and NeuN− nuclei from the prenatal human frontal cortex, and tested enrichment of single-nucleotide polymorphism (SNP) heritability for five neuropsychiatric disorders (autism spectrum disorder, attention deficit hyperactivity disorder [ADHD], bipolar disorder, major depressive disorder, and schizophrenia) within these regions. We observed significant enrichment of SNP heritability for ADHD, major depressive disorder, and schizophrenia within open chromatin regions (OCRs) mapped in bulk fetal frontal cortex, and for all five tested neuropsychiatric conditions when we restricted these sites to those overlapping histone modifications indicative of enhancers (H3K4me1) or promoters (H3K4me3) in fetal brain. SNP heritability for neuropsychiatric disorders was significantly enriched in OCRs identified in fetal frontal cortex NeuN− as well as NeuN+ nuclei overlapping fetal brain H3K4me1 or H3K4me3 sites. We additionally demonstrate the utility of our mapped OCRs for prioritizing potentially functional SNPs at genome-wide significant risk loci for neuropsychiatric disorders. Our data provide evidence for an early neurodevelopmental component to a range of neuropsychiatric conditions and highlight an important role for regulatory genomic regions active within both NeuN+ and NeuN− cells of the prenatal brain

Genome-wide methylomic analysis of monozygotic twins discordant for adolescent depression

BACKGROUND Adolescent depression is a common neuropsychiatric disorder that often continues into adulthood and is associated with a wide range of poor outcomes including suicide. Although numerous studies have looked at genetic markers associated with depression, the role of epigenetic variation remains relatively unexplored. METHODS Monozygotic (MZ) twins were selected from an adolescent twin study designed to investigate the interplay of genetic and environmental factors in the development of emotional and behavioral difficulties. There were 18 pairs of MZ twins identified in which one member scored consistently higher (group mean within the clinically significant range) on self-rated depression than the other. We assessed genome-wide patterns of DNA methylation in twin buccal cell DNA using the Infinium HumanMethylation450 BeadChip from Illumina. Quality control and data preprocessing was undertaken using the wateRmelon package. Differentially methylated probes (DMPs) were identified using an analysis strategy taking into account both the significance and the magnitude of DNA methylation differences. The top differentially methylated DMP was successfully validated by bisulfite-pyrosequencing, and identified DMPs were tested in postmortem brain samples obtained from patients with major depressive disorder (n = 14) and matched control subjects (n = 15). RESULTS Two reproducible depression-associated DMPs were identified, including the top-ranked DMP that was located within STK32C, which encodes a serine/threonine kinase, of unknown function. CONCLUSIONS Our data indicate that DNA methylation differences are apparent in MZ twins discordant for adolescent depression and that some of the disease-associated variation observed in buccal cell DNA is mirrored in adult brain tissue obtained from individuals with clinical depression

Genome-wide characterization of mitochondrial DNA methylation in human brain

BackgroundThere is growing interest in the role of DNA methylation in regulating the transcription of mitochondrial genes, particularly in brain disorders characterized by mitochondrial dysfunction. Here, we present a novel approach to interrogate the mitochondrial DNA methylome at single base resolution using targeted bisulfite sequencing. We applied this method to investigate mitochondrial DNA methylation patterns in post-mortem superior temporal gyrus and cerebellum brain tissue from seven human donors.ResultsWe show that mitochondrial DNA methylation patterns are relatively low but conserved, with peaks in DNA methylation at several sites, such as within the D-LOOP and the genes MT-ND2, MT-ATP6, MT-ND4, MT-ND5 and MT-ND6, predominantly in a non-CpG context. The elevated DNA methylation we observe in the D-LOOP we validate using pyrosequencing. We identify loci that show differential DNA methylation patterns associated with age, sex and brain region. Finally, we replicate previously reported differentially methylated regions between brain regions from a methylated DNA immunoprecipitation sequencing study.ConclusionsWe have annotated patterns of DNA methylation at single base resolution across the mitochondrial genome in human brain samples. Looking to the future this approach could be utilized to investigate the role of mitochondrial epigenetic mechanisms in disorders that display mitochondrial dysfunction

Disease-associated epigenetic changes in monozygotic twins discordant for schizophrenia and bipolar disorder

Studies of the major psychoses, schizophrenia (SZ) and bipolar disorder (BD), have traditionally focused on genetic and environmental risk factors, although more recent work has highlighted an additional role for epigenetic processes in mediating susceptibility. Since monozygotic (MZ) twins share a common DNA sequence, their study represents an ideal design for investigating the contribution of epigenetic factors to disease etiology. We performed a genome-wide analysis of DNA methylation on peripheral blood DNA samples obtained from a unique sample of MZ twin pairs discordant for major psychosis. Numerous loci demonstrated disease-associated DNA methylation differences between twins discordant for SZ and BD individually, and together as a combined major psychosis group. Pathway analysis of our top loci highlighted a significant enrichment of epigenetic changes in biological networks and pathways directly relevant to psychiatric disorder and neurodevelopment. The top psychosis-associated, differentially methylated region, significantly hypomethylated in affected twins, was located in the promoter of ST6GALNAC1 overlapping a previously reported rare genomic duplication observed in SZ. The mean DNA methylation difference at this locus was 6%, but there was considerable heterogeneity between families, with some twin pairs showing a 20% difference in methylation. We subsequently assessed this region in an independent sample of postmortem brain tissue from affected individuals and controls, finding marked hypomethylation (>25%) in a subset of psychosis patients. Overall, our data provide further evidence to support a role for DNA methylation differences in mediating phenotypic differences between MZ twins and in the etiology of both SZ and BD

Variation in 5-hydroxymethylcytosine across human cortex and cerebellum

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

The quantification of COMT mRNA in post mortem cerebellum tissue: diagnosis, genotype, methylation and expression

BACKGROUND: The COMT gene is located on chromosome 22q11, a region strongly implicated in the aetiology of several psychiatric disorders, in particular schizophrenia. Previous research has suggested that activity and expression of COMT is altered in schizophrenia, and is mediated by one or more polymorphisms within the gene, including the functional Val(158)Met polymorphism. METHOD: In this study we examined the expression levels of COMT mRNA using quantitative RT-PCR in 60 post mortem cerebellum samples derived from individuals with schizophrenia, bipolar disorder, depression, and no history of psychopathology. Furthermore, we have examined the methylation status of two CpG sites in the promoter region of the gene. RESULTS: We found no evidence of altered COMT expression or methylation in any of the psychiatric diagnoses examined. We did, however, find evidence to suggest that genotype is related to COMT gene expression, replicating the findings of two previous studies. Specifically, val(158)met (rs165688; Val allele) rs737865 (G allele) and rs165599 (G allele) all showed reduced expression (P < 0.05). Finally, we observe a strong sexual dimorphism in COMT expression, with females exhibiting significantly greater levels of COMT mRNA. CONCLUSION: The expression of COMT does not appear to be altered in the cerebellum of individuals suffering from schizophrenia, bipolar disorder or depression, but does appear to be influenced by single nucleotide polymorphisms within the gene

Full-length transcript sequencing of human and mouse cerebral cortex identifies widespread isoform diversity and alternative splicing.

Alternative splicing is a post-transcriptional regulatory mechanism producing distinct mRNA molecules from a single pre-mRNA with a prominent role in the development and function of the central nervous system. We used long-read isoform sequencing to generate full-length transcript sequences in the human and mouse cortex. We identify novel transcripts not present in existing genome annotations, including transcripts mapping to putative novel (unannotated) genes and fusion transcripts incorporating exons from multiple genes. Global patterns of transcript diversity are similar between human and mouse cortex, although certain genes are characterized by striking differences between species. We also identify developmental changes in alternative splicing, with differential transcript usage between human fetal and adult cortex. Our data confirm the importance of alternative splicing in the cortex, dramatically increasing transcriptional diversity and representing an important mechanism underpinning gene regulation in the brain. We provide transcript-level data for human and mouse cortex as a resource to the scientific community