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Background: Despite compelling evidence that cigarette smoking impacts the risk of developing multiple sclerosis (MS), little is known about smoking-associated changes in the primary exposed lung cells of patients. Objectives: We aimed to examine molecular changes occurring in bronchoalveolar lavage (BAL) cells from MS patients in relation to smoking and in comparison to healthy controls (HCs). Methods: We profiled DNA methylation in BAL cells from female MS ( n = 17) and HC ( n = 22) individuals, using Illumina Infinium EPIC and performed RNA-sequencing in non-smokers. Results: The most prominent changes were found in relation to smoking, with 1376 CpG sites (adjusted P Conclusions: Our study provides insights into the impact of smoking on lung inflammation and immunopathogenesis of MS.

Background While smoking is known to associate with development of multiple diseases, the underlying mechanisms are still poorly understood. Tobacco smoking can modify the chemical integrity of DNA leading to changes in transcriptional activity, partly through an altered epigenetic state. We aimed to investigate the impact of smoking on lung cells collected from bronchoalveolar lavage (BAL). Methods We profiled changes in DNA methylation (5mC) and its oxidised form hydroxymethylation (5hmC) using conventional bisulphite (BS) treatment and oxidative bisulphite treatment with Illumina Infinium MethylationEPIC BeadChip, and examined gene expression by RNA-seq in healthy smokers. Findings We identified 1667 total 5mC + 5hmC, 1756 5mC and 67 5hmC differentially methylated positions (DMPs) between smokers and non-smokers (FDR-adjusted P 0.15). Both 5mC DMPs and to a lesser extent 5mC + 5hmC were predominantly hypomethylated. In contrast, almost all 5hmC DMPs were hypermethylated, supporting the hypothesis that smoking-associated oxidative stress can lead to DNA demethylation, via the established sequential oxidation of which 5hmC is the first step. While we confirmed differential methylation of previously reported smoking-associated 5mC + 5hmC CpGs using former generations of BeadChips in alveolar macrophages, the large majority of identified DMPs, 5mC + 5hmC (1639/1667), 5mC (1738/1756), and 5hmC (67/67), have not been previously reported. Most of these novel smoking-associating sites are specific to the EPIC BeadChip and, interestingly, many of them are associated to FANTOM5 enhancers. Transcriptional changes affecting 633 transcripts were consistent with DNA methylation profiles and converged to alteration of genes involved in migration, signalling and inflammatory response of immune cells. Interpretation Collectively, these findings suggest that tobacco smoke exposure epigenetically modifies BAL cells, possibly involving a continuous active demethylation and subsequent increased activity of inflammatory processes in the lungs. Fund The study was supported by the Swedish Research Council, the Swedish Heart-Lung Foundation, the Stockholm County Council (ALF), the King Gustav's and Queen Victoria's Freemasons' Foundation, Knut and Alice Wallenberg Foundation, Neuro Sweden, and the Swedish MS foundation.

Background Multiple Sclerosis (MS) is a chronic inflammatory disease and a leading cause of progressive neurological disability among young adults. DNA methylation, which intersects genes and environment to control cellular functions on a molecular level, may provide insights into MS pathogenesis. Methods We measured DNA methylation in CD4+ T cells (n = 31), CD8+ T cells (n = 28), CD14+ monocytes (n = 35) and CD19+ B cells (n = 27) from relapsing-remitting (RRMS), secondary progressive (SPMS) patients and healthy controls (HC) using Infinium HumanMethylation450 arrays. Monocyte (n = 25) and whole blood (n = 275) cohorts were used for validations. Findings B cells from MS patients displayed most significant differentially methylated positions (DMPs), followed by monocytes, while only few DMPs were detected in T cells. We implemented a non-parametric combination framework (omicsNPC) to increase discovery power by combining evidence from all four cell types. Identified shared DMPs co-localized at MS risk loci and clustered into distinct groups. Functional exploration of changes discriminating RRMS and SPMS from HC implicated lymphocyte signaling, T cell activation and migration. SPMS-specific changes, on the other hand, implicated myeloid cell functions and metabolism. Interestingly, neuronal and neurodegenerative genes and pathways were also specifically enriched in the SPMS cluster. Interpretation We utilized a statistical framework (omicsNPC) that combines multiple layers of evidence to identify DNA methylation changes that provide new insights into MS pathogenesis in general, and disease progression, in particular. Fund This work was supported by the Swedish Research Council, Stockholm County Council, AstraZeneca, European Research Council, Karolinska Institutet and Margaretha af Ugglas Foundation.

BackgroundMultiple Sclerosis (MS), the leading cause of non-traumatic neurological disability in young adults, is a chronic inflammatory and neurodegenerative disease of the central nervous system (CNS). Due to the poor accessibility to the target organ, CNS-confined processes underpinning the later progressive form of MS remain elusive thereby limiting treatment options. We aim to examine DNA methylation, a stable epigenetic mark of genome activity, in glial cells to capture relevant molecular changes underlying MS neuropathology.MethodsWe profiled DNA methylation in nuclei of glial cells, isolated from 38 post-mortem normal-appearing white matter (NAWM) specimens of MS patients (n=8) in comparison to white matter of control individuals (n=14), using Infinium MethylationEPIC BeadChip.FindingsWe identified 1,226 significant (genome-wide adjusted P-value < 0.05) differentially methylated positions (DMPs) between MS patients and controls. Functional annotation of the altered DMP-genes uncovered alterations of processes related to cellular motility, cytoskeleton dynamics, metabolic processes, synaptic support, neuroinflammation and signaling, such as Wnt and TGF-β pathways. A fraction of the affected genes displayed transcriptional differences in the brain of MS patients, as reported by publically available transcriptomic data. Cell type-restricted annotation of DMP-genes attributed alteration of cytoskeleton rearrangement and extracellular matrix remodelling to all glial cell types, while some processes, including ion transport, Wnt/TGF-β signaling and immune processes were more specifically linked to oligodendrocytes, astrocytes and microglial cells, respectively.ConclusionOur findings strongly suggest that NAWM glial cells are highly altered, even in the absence of lesional insult, collectively exhibiting a multicellular reaction in response to diffuse inflammation.

Multiple sclerosis (MS), a chronic inflammatory demyelinating and neurodegenerative disease of the central nervous system, is today a leading cause of unpredictable lifelong disability in young adults. The treatment of patients in progressive stages remains highly challenging, alluding to our limited understanding of the underlying pathological processes. In this review, we provide insights into the mechanisms underpinning MS progression from a perspective of epigenetics, that refers to stable and mitotically heritable, yet reversible, changes in the genome activity and gene expression. We first recapitulate findings from epigenetic studies examining the brain tissue of progressive MS patients, which support a contribution of DNA and histone modifications in impaired oligodendrocyte differentiation, defective myelination/remyelination and sustained neuro-axonal vulnerability. We next explore possibilities for identifying factors affecting progression using easily accessible tissues such as blood by comparing epigenetic signatures in peripheral immune cells and brain tissue. Despite minor overlap at individual methylation sites, nearly 30% of altered genes reported in peripheral immune cells of progressive MS patients were found in brain tissue, jointly converging on alterations of neuronal functions. We further speculate about the mechanisms underlying shared epigenetic patterns between blood and brain, which likely imply the influence of internal (genetic control) and/or external (e.g. smoking and ageing) factors imprinting a common signature in both compartments. Overall, we propose that epigenetics might shed light on clinically relevant mechanisms involved in disease progression and open new avenues for the treatment of progressive MS patients in the future.

Psychiatric disorders are clinically heterogeneous and debilitating chronic diseases resulting from a complex interplay between gene variants and environmental factors. Epigenetic processes, such as DNA methylation and histone posttranslational modifications, instruct the cell/tissue to correctly interpret external signals and adjust its functions accordingly. Given that epigenetic modifications are sensitive to environment, stable, and reversible, epigenetic studies in psychiatry could represent a promising approach to better understanding and treating disease. In the present review, we aim to discuss the clinical opportunities and challenges arising from the epigenetic research in psychiatry. Using selected examples, we first recapitulate key findings supporting the role of adverse life events, alone or in combination with genetic risk, in epigenetic programming of neuropsychiatric systems. Epigenetic studies further report encouraging findings about the use of methylation changes as diagnostic markers of disease phenotype and predictive tools of progression and response to treatment. Then we discuss the potential of using targeted epigenetic pharmacotherapy, combined with psychosocial interventions, for future personalized medicine for patients. Finally, we review the methodological limitations that could hinder interpretation of epigenetic data in psychiatry. They mainly arise from heterogeneity at the individual and tissue level and require future strategies in order to reinforce the biological relevance of epigenetic data and its translational use in psychiatry. Overall, we suggest that epigenetics could provide new insights into a more comprehensive interpretation of mental illness and might eventually improve the nosology, treatment, and prevention of psychiatric disorders.

Multiple sclerosis (MS) is a chronic inflammatory and demyelinating disease of the central nervous system. MS likely results from a complex interplay between predisposing causal gene variants (the strongest influence coming from HLA class II locus) and environmental risk factors such as smoking, infectious mononucleosis, and lack of sun exposure/vitamin D. However, little is known about the mechanisms underlying MS development and progression. Moreover, the clinical heterogeneity and variable response to treatment represent additional challenges to a comprehensive understanding and efficient treatment of disease. Epigenetic processes, such as DNA methylation and histone posttranslational modifications, integrate influences from the genes and the environment to regulate gene expression accordingly. Studying epigenetic modifications, which are stable and reversible, may provide an alternative approach to better understand and manage disease. We here aim to review findings from epigenetic studies in MS and further discuss the challenges and clinical opportunities arising from epigenetic research, many of which apply to other diseases with similar complex etiology. A growing body of evidence supports a role of epigenetic processes in the mechanisms underlying immune pathogenesis and nervous system dysfunction in MS. However, disparities between studies shed light on the need to consider possible confounders and methodological limitations for a better interpretation of the data. Nevertheless, translational use of epigenetics might offer new opportunities in epigenetic-based diagnostics and therapeutic tools for a personalized care of MS patients.

Background Little is known about how genetics and epigenetics interplay in depression. Evidence suggests that genetic variants may change vulnerability to depression by modulating DNA methylation (DNAm) and non-coding RNA (ncRNA) levels. Therefore, the aim of the study was to investigate the effect of the genetic variation, previously identified in the largest genome-wide association study for depression, on proximal DNAm and ncRNA levels. Results We performed DNAm quantitative trait locus (meQTL) analysis in two independent cohorts (total n = 435 healthy individuals), testing associations between 102 single-nucleotide polymorphisms (SNPs) and DNAm levels in whole blood. We identified and replicated 64 SNP-CpG pairs (padj. < 0.05) with meQTL effect. Lower DNAm at cg02098413 located in the HACE1 promoter conferred by the risk allele (C allele) at rs1933802 was associated with higher risk for depression (praw = 0.014, DNAm = 2.3%). In 1202 CD14+ cells sorted from blood, DNAm at cg02088412 positively correlated with HACE1 mRNA expression. Investigation in postmortem brain tissue of adults diagnosed with major depressive disorder (MDD) indicated 1% higher DNAm at cg02098413 in neurons and lower HACE1 mRNA expression in CA1 hippocampus of MDD patients compared with healthy controls (p = 0.008 and 0.012, respectively). Expression QTL analysis in blood of 74 adolescent revealed that hsa-miR-3664-5p was associated with rs7117514 (SHANK2) (padj. = 0.015, mRNA difference = 5.2%). Gene ontology analysis of the miRNA target genes highlighted implication in neuronal processes. Conclusions Collectively, our findings from a multi-tissue (blood and brain) and multi-layered (genetic, epigenetic, transcriptomic) approach suggest that genetic factors may influence depression by modulating DNAm and miRNA levels. Alterations at HACE1 and SHANK2 loci imply potential mechanisms, such as oxidative stress in the brain, underlying depression. Our results deepened the knowledge of molecular mechanisms in depression and suggest new epigenetic targets that should be further evaluated.

Background Due to limited access to brain tissue, the precise mechanisms underlying neuro-axonal dysfunction in neurological disorders such as multiple sclerosis (MS) are largely unknown. In that context, profiling DNA methylation, which is a stable and cell type-specific regulatory epigenetic mark of genome activity, offers a unique opportunity to characterize the molecular mechanisms underpinning brain pathology in situ. We examined DNA methylation patterns of neuronal nuclei isolated from post-mortem brain tissue to infer processes that occur in neurons of MS patients. Results We isolated subcortical neuronal nuclei from post-mortem white matter tissue of MS patients and non-neurological controls using flow cytometry. We examined bulk DNA methylation changes (total n = 29) and further disentangled true DNA methylation (5mC) from neuron-specific DNA hydroxymethylation (5hmC) (n = 17), using Illumina Infinium 450K arrays. We performed neuronal sub-type deconvolution using glutamate and GABA methylation profiles to further reduce neuronal sample heterogeneity. In total, we identified 2811 and 1534 significant (genome-wide adjusted P value

Multiple Sclerosis (MS) is a leading cause of lifelong disability in young adults. The disease strikes individuals in their most productive years with incurable and progressive course that results in development of fatigue and accumulation of physical and cognitive disability. MS is characterized by autoimmune destruction of the myelin and subsequent neurodegeneration. This chronic disease of the central nervous system is likely triggered by environmental factors such as smoking, lack of sun exposure/vitamin D deficiency and infection, in genetically predisposed individuals, the strongest influence coming from HLA-DRB1 variants within the HLA class II locus. However, the mechanisms underlying susceptibility to MS are still puzzling and specific clinical translations are lacking. Emerging evidence suggests the implication of epigenetic mechanisms such as DNA methylation in the pathogenesis of MS. In this chapter, we aimed to review findings from DNA methylation studies in MS and discuss their clinical relevance. We first present a critical overview of the outcomes of DNA methylation studies in immune cells and brain tissue from MS patients. We then discuss emerging evidence supporting a role of DNA methylation in mediating the effect from the major genetic risk variant HLA-DRB1*15:01 and environmental risk factors, smoking and vitamin D deficiency, in MS. We also describe the potential of DNA methylation-based biomarkers and therapies for precision medicine in MS. We expect that the encouraging findings from DNA methylation studies in MS might open new avenues for a better understanding and treatment MS patients.

Evidence for parent-of-origin effects in complex diseases such as Multiple Sclerosis (MS) strongly suggests a role for epigenetic mechanisms in their pathogenesis. In this review, we describe the importance of accounting for parent-of-origin when identifying new risk variants for complex diseases and discuss how genomic imprinting, one of the best-characterized epigenetic mechanisms causing parent-of-origin effects, may impact etiology of complex diseases. While the role of imprinted genes in growth and development is well established, the contribution and molecular mechanisms underlying the impact of genomic imprinting in immune functions and inflammatory diseases are still largely unknown. Here we discuss emerging roles of imprinted genes in the regulation of inflammatory responses with a particular focus on the Dlk1 cluster that has been implicated in etiology of experimental MS-like disease and Type 1 Diabetes. Moreover, we speculate on the potential wider impact of imprinting via the action of imprinted microRNAs, which are abundantly present in the Dlk1 locus and predicted to fine-tune important immune functions. Finally, we reflect on how unrelated imprinted genes or imprinted genes together with non-imprinted genes can interact in so-called imprinted gene networks (IGN) and suggest that IGNs could partly explain observed parent-of-origin effects in complex diseases. Unveiling the mechanisms of parent-of-origin effects is therefore likely to teach us not only about the etiology of complex diseases but also about the unknown roles of this fascinating phenomenon underlying uneven genetic contribution from our parents. This article is part of a Directed Issue entitled: Epigenetics dynamics in development and disease.

Cigarette smoking is an established environmental risk factor for Multiple Sclerosis (MS), a chronic inflammatory and neurodegenerative disease, although a mechanistic basis remains largely unknown. We aimed at investigating how smoking affects blood DNA methylation in MS patients, by assaying genome-wide DNA methylation and comparing smokers, former smokers and never smokers in two Swedish cohorts, differing for known MS risk factors. Smoking affects DNA methylation genome-wide significantly, an exposure-response relationship exists and the time since smoking cessation affects methylation levels. The results also show that the changes were larger in the cohort bearing the major genetic risk factors for MS (female sex and HLA risk haplotypes). Furthermore, CpG sites mapping to genes with known genetic or functional role in the disease are differentially methylated by smoking. Modeling of the methylation levels for a CpG site in the AHRR gene indicates that MS modifies the effect of smoking on methylation changes, by significantly interacting with the effect of smoking load. Alongside, we report that the gene expression of AHRR increased in MS patients after smoking. Our results suggest that epigenetic modifications may reveal the link between a modifiable risk factor and the pathogenetic mechanisms.

Background: Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system caused by genetic and environmental factors. DNA methylation, an epigenetic mechanism that controls genome activity, may provide a link between genetic and environmental risk factors. Objective: We sought to identify DNA methylation changes in CD4+ T cells in patients with relapsing-remitting (RR-MS) and secondary-progressive (SP-MS) disease and healthy controls (HC). Methods: We performed DNA methylation analysis in CD4+ T cells from RR-MS, SP-MS, and HC and associated identified changes with the nearby risk allele, smoking, age, and gene expression. Results: We observed significant methylation differences in the VMP1/MIR21 locus, with RR-MS displaying higher methylation compared to SP-MS and HC. VMP1/MIR21 methylation did not correlate with a known MS risk variant in VMP1 or smoking but displayed a significant negative correlation with age and the levels of mature miR-21 in CD4+ T cells. Accordingly, RR-MS displayed lower levels of miR-21 compared to SP-MS, which might reflect differences in age between the groups, and healthy individuals and a significant enrichment of up-regulated miR-21 target genes. Conclusion: Disease-related changes in epigenetic marking of MIR21 in RR-MS lead to differences in miR-21 expression with a consequence on miR-21 target genes.