Emma Dempster

BackgroundThe study of biological age acceleration may help identify at-risk individuals and reduce the rising global burden of age-related diseases. Using DNA methylation (DNAm) clocks, we investigated biological aging in schizophrenia (SCZ), a mental illness that is associated with an increased prevalence of age-related disabilities and morbidities. In a whole blood DNAm sample of 1090 SCZ cases and 1206 controls across four European cohorts, we performed a meta-analysis of differential aging using three DNAm clocks (i.e., Hannum, Horvath, and Levine). To dissect how DNAm aging contributes to SCZ, we integrated information on duration of illness and SCZ polygenic risk, as well as stratified our analyses by chronological age and biological sex.ResultsWe found that blood-based DNAm aging is significantly altered in SCZ independent from duration of the illness since onset. We observed sex-specific …

BackgroundDue to interindividual variation in the cellular composition of the human cortex, it is essential that covariates that capture these differences are included in epigenome-wide association studies using bulk tissue. As experimentally derived cell counts are often unavailable, computational solutions have been adopted to estimate the proportion of different cell types using DNA methylation data. Here, we validate and profile the use of an expanded reference DNA methylation dataset incorporating two neuronal and three glial cell subtypes for quantifying the cellular composition of the human cortex.ResultsWe tested eight reference panels containing different combinations of neuronal- and glial cell types and characterised their performance in deconvoluting cell proportions from computationally reconstructed or empirically derived human cortex DNA methylation data. Our analyses demonstrate that while …

Increased understanding of the functional complexity of the genome has led to growing recognition about the role of epigenetic/transcriptional variation in health and disease. Current analyses of the human brain, however, are limited by the use of “bulk” tissue, comprising a heterogeneous mix of different neural cell types. Because epigenetic processes play a critical role in determining cell type-specific patterns of gene regulation it is important to consider cellular composition in regulatory genomic studies of human post-mortem tissue, and there is a need for methods to purify populations of specific cell-types. This protocol builds on a previous protocol that uses fluorescence-activated nuclei sorting (FANS) to isolate and profile nuclei from multiple different human brain cell-types from frozen post-mortem tissue. Because NeuN is not an optimal marker for neuronal nuclei from fetal cortex, we have optimized a method using a SATB2 antibody to purify nuclei from excitatory neurons in both fetal and postnatal cortex. Purified populations of nuclei are amenable to simultaneous profiling of i) DNA modifications (via bisulfite sequencing/array), ii) histone modifications (via CUT&Tag), iii) open chromatin analysis (via ATAC-seq), and iv) gene expression (via RNA-seq).

Background Dementia with Lewy bodies (DLB), Parkinson’s disease (PD) and Parkinson’s disease dementia (PDD) are termed Lewy body diseases (LBDs). These neurodegenerative diseases are classified by the accumulation of alpha‐synuclein in neurons, forming Lewy bodies (LB). Whilst pathologically similar, symptomatic staging is different. Previous genome wide association studies (GWAS) have identified classic Alzheimer’s disease (AD) and PD loci as risk loci in DLB. Therefore, we have used polygenic risk score (PRS) analysis to investigate the contribution of AD and PD variants in the LBDs. Method DNA was extracted from bulk brain tissue from 481 individuals (162 controls, 95 DLB, 156 PDD and 68 PD) and genotyped on the Illumina global screening array. After a strict quality control and imputation pipeline, PRSs for multiple relevant traits using reference GWAS summary statistics were calculated …

The second-generation antipsychotic clozapine is used as a medication for treatment-resistant schizophrenia. It has previously been associated with epigenetic changes in pre-clinical rodent models and cross-sectional studies of treatment-resistant schizophrenia. Cross-sectional studies are susceptible to confounding, however, and cannot disentangle the effects of diagnosis and medication. We therefore profiled DNA methylation in sequential blood samples (n=126) from two independent cohorts of patients (n=38) with treatment-resistant schizophrenia spectrum disorders who commenced clozapine after study enrolment and were followed up for up to six months. We identified significant non-linear changes in cell-type proportion estimates derived from DNA methylation data - specifically B-cells - associated with time on clozapine. Mixed effects regression models were used to identify changes in DNA methylation at specific sites associated with time on clozapine, identifying 37 differentially methylated positions (DMPs) (p < 5x10-5 ) in a linear model and 90 DMPs in a non-linear quadratic model. We compared these results to data from our previous epigenome-wide association study (EWAS) meta-analysis of psychosis, finding evidence that many previously identified DMPs associated with schizophrenia and treatment-resistant schizophrenia might reflect exposure to clozapine. In conclusion, our results indicate that clozapine exposure is associated with changes in DNA methylation and cellular composition. Our study shows that medication effects might confound many case-control studies of neuropsychiatric disorders performed in blood.

The rising prevalence and legalization of cannabis worldwide have underscored the need for a comprehensive understanding of its biological impact, particularly on mental health. Epigenetic mechanisms, specifically DNA methylation, have gained increasing recognition as vital factors in the interplay between risk factors and mental health. This study aimed to explore the effects of current cannabis use and potency on DNA methylation in two independent cohorts of individuals experiencing first-episode psychosis (FEP) compared to control subjects. The combined sample consisted of 682 participants (cannabis users (n= 188) and never users (n= 494)). DNA methylation profiles were generated on blood-derived DNA samples using the Illumina DNA methylation array platform. Each cohort was analysed individually and then meta-analysed. We identified one CpG site (cg11669285) in the CAVIN1 gene that showed differential methylation with current cannabis use, surpassing the array-wide significance threshold, and independent of the tobacco-related epigenetic signature. Furthermore, a CpG site localized in the MCU gene (cg11669285) achieved array-wide significance in the analysis of high-potency cannabis use. Pathway and regional analyses identified cannabis-related epigenetic deviations in genes linked to immune and mitochondrial function, both of which are known to be influenced by cannabinoids. Notably, the analyses focused on first-onset psychoses identified differential methylation at nominal significance in a CpG site situated in the TRPV2 gene, which is known to be activated by cannabinoids. Overall, these findings contribute …

Development of the human pancreas requires the precise temporal control of gene expression via epigenetic mechanisms and the binding of key transcription factors. We quantified genome-wide patterns of DNA methylation in human fetal pancreatic samples from donors aged 6 to 21 post-conception weeks. We found dramatic changes in DNA methylation across pancreas development, with >21% of sites characterized as developmental differentially methylated positions (dDMPs) including many annotated to genes associated with monogenic diabetes. An analysis of DNA methylation in postnatal pancreas tissue showed that the dramatic temporal changes in DNA methylation occurring in the developing pancreas are largely limited to the prenatal period. Significant differences in DNA methylation were observed between males and females at a number of autosomal sites, with a small proportion of sites showing sex-specific DNA methylation trajectories across pancreas development. Pancreas dDMPs were not distributed equally across the genome, with a depletion of developmentally-dynamic sites in regulatory domains characterized by open chromatin and the binding of known pancreatic development transcription factors. Finally, we compared our pancreas dDMPs to previous findings from the human brain, identifying some similarities but also tissue-specific developmental changes in DNA methylation. To our knowledge, this represents the most extensive exploration of DNA methylation patterns during human fetal pancreas development, confirming the prenatal period as a time of major epigenomic plasticity.

INTRODUCTION Given the established association between DNA methylation and the pathophysiology of dementia and its plausible role as a molecular mediator of lifestyle and environment, blood-derived DNA methylation data could enable early detection of dementia risk. METHODS In conjunction with an extensive array of machine learning techniques, we employed whole blood genome-wide DNA methylation data as a surrogate for 14 modifiable and non-modifiable factors in the assessment of dementia risk in two independent cohorts of Alzheimer's disease (AD) and Parkinson's disease (PD). RESULTS We established a multivariate methylation risk score (MMRS) to identify the status of mild cognitive impairment (MCI) cross-sectionally, independent of age and sex. We further demonstrated significant predictive capability of this score for the prospective onset of cognitive decline in AD and PD. DISCUSSION Our work shows the potential of employing blood-derived DNA methylation data in the assessment of dementia risk.

In the progressive phase of multiple sclerosis (MS), the hampered differentiation capacity of oligodendrocyte precursor cells (OPCs) eventually results in remyelination failure. We have previously shown that DNA methylation of Id2/Id4 is highly involved in OPC differentiation and remyelination. In this study, we took an unbiased approach by determining genome-wide DNA methylation patterns within chronically demyelinated MS lesions and investigated how certain epigenetic signatures relate to OPC differentiation capacity. We compared genome-wide DNA methylation and transcriptional profiles between chronically demyelinated MS lesions and matched normal-appearing white matter (NAWM), making use of post-mortem brain tissue (n = 9/group). DNA methylation differences that inversely correlated with mRNA expression of their corresponding genes were validated for their cell-type specificity in laser …

SR, AI, and CM: conceptualization. SR, NN, WT, and CM: methodology. SR and NN: data collection. WT and MB: data analysis. SR, NN, and WT: writing—original draft preparation. SR, NN, WT, MB, LT, AY, and CM: writing—review and editing. MD and ED: epigenetic analysis. JR, LT, ED, AY, and CM: supervision. All authors contributed to the article and approved the submitted version.

Increasing evidence suggests that alternative splicing plays an important role in Alzheimer's disease (AD), a devastating neurodegenerative disorder involving the intracellular aggregation of hyperphosphorylated tau. We used long-read cDNA sequencing to profile transcript diversity in the entorhinal cortex of wild-type (WT) and transgenic (TG) mice harboring a mutant form of human tau. Whole transcriptome profiling showed that previously reported gene-level expression differences between WT and TG mice reflect changes in the abundance of specific transcripts. Ultradeep targeted long-read cDNA sequencing of genes implicated in AD revealed hundreds of novel isoforms and identified specific transcripts associated with the development of tau pathology. Our results highlight the importance of differential transcript usage, even in the absence of gene-level expression alterations, as a mechanism underpinning gene regulation in the development of neuropathology. Our transcript annotations and a novel informatics pipeline for the analysis of long-read transcript sequencing data are provided as a resource to the community.

Studies conducted in psychotic disorders have shown that DNA-methylation (DNAm) is sensitive to the impact of Childhood Adversity (CA). However, whether it mediates the association between CA and psychosis is yet to be explored. Epigenome wide association studies (EWAS) using the Illumina Infinium-Methylation EPIC array in peripheral blood tissue from 366 First-episode of psychosis and 517 healthy controls was performed. Adversity scores were created for abuse, neglect and composite adversity with the Childhood Trauma Questionnaire (CTQ). Regressions examining (I) CTQ scores with psychosis; (II) with DNAm EWAS level and (III) between DNAm and caseness, adjusted for a variety of confounders were conducted. Divide-Aggregate Composite-null Test for the composite null-hypothesis of no mediation effect was conducted. Enrichment analyses were conducted with missMethyl package and the …

BackgroundThere is mounting evidence to support a role for developmentally regulated epigenetic variation in the molecular etiology of schizophrenia. Previous analyses of epigenetic variation in the human brain have been limited to the study of bulk tissue, which is a heterogeneous mix of different cell types. We sought to characterize cell-type-specific epigenetic signatures across human cortex development and relate this to differences identified in schizophrenia.MethodsTo obtain purified populations of neural cell types from post-mortem prefrontal cortex tissue, we used fluorescence-activated nuclei sorting (FANS) to gate and select NeuN+/SATB2+ (neuronal), SOX10+ (oligodendrocytes) and IRF8+ (microglial) immunolabeled nuclei populations derived from prefrontal cortex tissue from 150 schizophrenia cases and 150 controls, profiling DNA methylation using the Illumina EPIC BeadArray. In parallel we …

The majority of epigenetic epidemiology studies to date have generated genome-wide profiles from bulk tissues (e.g., whole blood) however these are vulnerable to confounding from variation in cellular composition. Proxies for cellular composition can be mathematically derived from the bulk tissue profiles using a deconvolution algorithm; however, there is no method to assess the validity of these estimates for a dataset where the true cellular proportions are unknown. In this study, we describe, validate and characterize a sample level accuracy metric for derived cellular heterogeneity variables. The CETYGO score captures the deviation between a sample’s DNA methylation profile and its expected profile given the estimated cellular proportions and cell type reference profiles. We demonstrate that the CETYGO score consistently distinguishes inaccurate and incomplete deconvolutions when applied to …

BackgroundThe field of epigenomics holds great promise in understanding and treating disease with advances in machine learning (ML) and artificial intelligence being vitally important in this pursuit. Increasingly, research now utilises DNA methylation measures at cytosine–guanine dinucleotides (CpG) to detect disease and estimate biological traits such as aging. Given the challenge of high dimensionality of DNA methylation data, feature-selection techniques are commonly employed to reduce dimensionality and identify the most important subset of features. In this study, our aim was to test and compare a range of feature-selection methods and ML algorithms in the development of a novel DNA methylation-based telomere length (TL) estimator. We utilised both nested cross-validation and two independent test sets for the comparisons.ResultsWe found that principal component analysis in advance of elastic net …

BackgroundAlternative splicing (AS) 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. Because alternatively spliced transcripts from a single gene can produce proteins with different functions, there is increasing interest in their role in human disease. AS is particularly important and prevalent in the central nervous system, where it impacts neurodevelopment, aging and key neural functions. AS is a common feature of many neuropsychiatric diseases, with recent studies highlighting splicing differences associated with autism and schizophrenia. Novel long read sequencing approaches provide a novel opportunity to generate full-length transcript sequences and fully characterise isoform diversity.MethodsWe used Oxford Nanopore Technology (ONT) whole transcriptome …

Background An increasing number of studies implicate a role for alternative splicing in development and neuropathology of Alzheimer’s disease (AD). However, it has been historically challenging to characterise splicing events, due to the inherent limitations of traditional RNA‐sequencing (RNA‐Seq) to capture full‐length transcripts critical for transcriptome assembly. In this study, we use two complementary targeted long‐read sequencing approaches, Pacific Biosciences (PacBio) isoform sequencing (Iso‐Seq) and Oxford Nanopore Technologies (ONT) nanopore cDNA sequencing, to profile the cortex of a mouse model of tau pathology (rTg4510) with ultra‐deep sequencing of a panel of 20 genes robustly implicated in AD. Method Targeted PacBio Iso‐Seq and targeted ONT profiling were performed on RNA isolated from 24 female rTg4510 transgenic and wild‐type mice, aged 2, 4, 6 and 8 months. Following …

Background There 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. Results We 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. Conclusions We 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.

Increased understanding of the functional complexity of the genome has led to growing recognition about the role of epigenetic/transcriptional variation in health and disease. Because epigenetic processes play a critical role in determining cell type-specific patterns of gene regulation it is important to consider cellular composition in regulatory genomic studies of heterogeneous tissue like the brain. Building on a previous protocol for isolating purified populations of nuclei from different cortical cell types from human post-mortem brain tissue, this protocol uses fluorescence-activated nuclei sorting (FANS) to isolate and profile nuclei from multiple different cell types from frozen mouse cortex. This protocol can be used to robustly purify populations of neuronal (NeuN+ ve) and microglia (PU. 1+ ve) and other glial origin nuclei (NeuN-ve/PU. 1-ve) from frozen mouse cortex tissue, with each sample yielding purified populations of nuclei amenable to simultaneous analysis of i) DNA modifications (via bisulfite sequencing/microarray), ii) histone modifications (via CUT&RUN-seq), iii) chromatin accessibility (via ATAC-seq), and iv) gene expression (via RNA-seq).

Background: Despite advances in elucidating the genetic basis of schizophrenia, autism and other neurodevelopmental disorders, there remains uncertainty about the specific causal genes involved and how their function is neurodevelopmentally regulated. Many disease-associated variants are hypothesized to influence gene regulation rather than directly affecting protein structure. Because regulatory genomic signatures are cell-and developmental-stage-specific, it is important these relationships are explored in relevant cell-types and across developmental time points. Importantly, little is known about patterns of gene regulation in the later stages of fetal development and the first decade of life. In this study we profiled changes in DNA methylation (DNAm), an epigenetic modification to cytosine involved in mediating the developmental regulation of gene expression and function, across human brain development …