Elucidating distinct molecular signatures of Lewy body dementias.

Dementia with Lewy bodies and Parkinson's disease dementia are common neurodegenerative diseases that share similar neuropathological profiles and spectra of clinical symptoms but are primarily differentiated by the order in which symptoms manifest. The question of whether a distinct molecular pathological profile could distinguish these disorders is yet to be answered. However, in recent years, studies have begun to investigate genomic, epigenomic, transcriptomic and proteomic differences that may differentiate these disorders, providing novel insights in to disease etiology. In this review, we present an overview of the clinical and pathological hallmarks of Lewy body dementias before summarizing relevant research into genetic, epigenetic, transcriptional and protein signatures in these diseases, with a particular interest in those resolving "omic" level changes. We conclude by suggesting future research directions to address current gaps and questions present within the field.

Epigenetic insights into neuropsychiatric and cognitive symptoms in Parkinson's disease: A DNA co-methylation network analysis.

Parkinson's disease is a highly heterogeneous disorder, encompassing a complex spectrum of clinical presentation including motor, sleep, cognitive and neuropsychiatric symptoms. We aimed to investigate genome-wide DNA methylation networks in post-mortem Parkinson's disease brain samples and test for region-specific association with common neuropsychiatric and cognitive symptoms. Of traits tested, we identify a co-methylation module in the substantia nigra with significant correlation to depressive symptoms and with ontological enrichment for terms relevant to neuronal and synaptic processes. Notably, expression of the genes annotated to the methylation loci present within this module are found to be significantly enriched in neuronal subtypes within the substantia nigra. These findings highlight the potential involvement of neuronal-specific changes within the substantia nigra with regard to depressive symptoms in Parkinson's disease.

From methylation to myelination: epigenomic and transcriptomic profiling of chronic inactive demyelinated multiple sclerosis lesions.

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-captured OPCs using pyrosequencing. The CRISPR-dCas9-DNMT3a/TET1 system was used to epigenetically edit human-iPSC-derived oligodendrocytes to assess the effect on cellular differentiation. Our data show hypermethylation of CpGs within genes that cluster in gene ontologies related to myelination and axon ensheathment. Cell type-specific validation indicates a region-dependent hypermethylation of MBP, encoding for myelin basic protein, in OPCs obtained from white matter lesions compared to NAWM-derived OPCs. By altering the DNA methylation state of specific CpGs within the promotor region of MBP, using epigenetic editing, we show that cellular differentiation and myelination can be bidirectionally manipulated using the CRISPR-dCas9-DNMT3a/TET1 system in vitro. Our data indicate that OPCs within chronically demyelinated MS lesions acquire an inhibitory phenotype, which translates into hypermethylation of crucial myelination-related genes. Altering the epigenetic status of MBP can restore the differentiation capacity of OPCs and possibly boost (re)myelination.

Genome-wide DNA methylation analysis of aggressive behaviour: a longitudinal population-based study.

BACKGROUND: Human aggression is influenced by an interplay between genetic predisposition and experience across the life span. This interaction is thought to occur through epigenetic mechanisms, inducing differential gene expression, thereby moderating neuronal cell and circuit function, and thus shaping aggressive behaviour. METHODS: Genome-wide DNA methylation (DNAm) levels were measured in peripheral blood obtained from 95 individuals participating in the Estonian Children Personality Behaviours and Health Study (ECPBHS) at 15 and 25 years of age. We examined the association between aggressive behaviour, as measured by Life History of Aggression (LHA) total score and DNAm levels both assessed at age 25. We further examined the pleiotropic effect of genetic variants regulating LHA-associated differentially methylated positions (DMPs) and multiple traits related to aggressive behaviours. Lastly, we tested whether the DNA methylomic loci identified in association with LHA at age 25 were also present at age 15. RESULTS: We found one differentially methylated position (DMP) (cg17815886; p = 1.12 × 10-8 ) and five differentially methylated regions (DMRs) associated with LHA after multiple testing adjustments. The DMP annotated to the PDLIM5 gene, and DMRs resided in the vicinity of four protein-encoding genes (TRIM10, GTF2H4, SLC45A4, B3GALT4) and a long intergenic non-coding RNA (LINC02068). We observed evidence for the colocalization of genetic variants associated with top DMPs and general cognitive function, educational attainment and cholesterol levels. Notably, a subset of the DMPs associated with LHA at age 25 also displayed altered DNAm patterns at age 15 with high accuracy in predicting aggression. CONCLUSIONS: Our findings highlight the potential role of DNAm in the development of aggressive behaviours. We observed pleiotropic genetic variants associated with identified DMPs, and various traits previously established to be relevant in shaping aggression in humans. The concordance of DNAm signatures in adolescents and young adults may have predictive value for inappropriate and maladaptive aggression later in life.

Targeted Methylation Profiling of Single Laser-Capture Microdissected Post-Mortem Brain Cells by Adapted Limiting Dilution Bisulfite Pyrosequencing (LDBSP).

A reoccurring issue in neuroepigenomic studies, especially in the context of neurodegenerative disease, is the use of (heterogeneous) bulk tissue, which generates noise during epigenetic profiling. A workable solution to this issue is to quantify epigenetic patterns in individually isolated neuronal cells using laser capture microdissection (LCM). For this purpose, we established a novel approach for targeted DNA methylation profiling of individual genes that relies on a combination of LCM and limiting dilution bisulfite pyrosequencing (LDBSP). Using this approach, we determined cytosine-phosphate-guanine (CpG) methylation rates of single alleles derived from 50 neurons that were isolated from unfixed post-mortem brain tissue. In the present manuscript, we describe the general workflow and, as a showcase, demonstrate how targeted methylation analysis of various genes, in this case, RHBDF2, OXT, TNXB, DNAJB13, PGLYRP1, C3, and LMX1B, can be performed simultaneously. By doing so, we describe an adapted data analysis pipeline for LDBSP, allowing one to include and correct CpG methylation rates derived from multi-allele reactions. In addition, we show that the efficiency of LDBSP on DNA derived from LCM neurons is similar to the efficiency obtained in previously published studies using this technique on other cell types. Overall, the method described here provides the user with a more accurate estimation of the DNA methylation status of each target gene in the analyzed cell pools, thereby adding further validity to this approach.

Machine learning-based prediction of cognitive outcomes in de novo Parkinson's disease.

Cognitive impairment is a debilitating symptom in Parkinson's disease (PD). We aimed to establish an accurate multivariate machine learning (ML) model to predict cognitive outcome in newly diagnosed PD cases from the Parkinson's Progression Markers Initiative (PPMI). Annual cognitive assessments over an 8-year time span were used to define two cognitive outcomes of (i) cognitive impairment, and (ii) dementia conversion. Selected baseline variables were organized into three subsets of clinical, biofluid and genetic/epigenetic measures and tested using four different ML algorithms. Irrespective of the ML algorithm used, the models consisting of the clinical variables performed best and showed better prediction of cognitive impairment outcome over dementia conversion. We observed a marginal improvement in the prediction performance when clinical, biofluid, and epigenetic/genetic variables were all included in one model. Several cerebrospinal fluid measures and an epigenetic marker showed high predictive weighting in multiple models when included alongside clinical variables.

Longitudinal alterations in mRNA expression of the BDNF neurotrophin signaling cascade in blood correlate with changes in depression scores in patients undergoing electroconvulsive therapy.

Electroconvulsive therapy (ECT) appears to be the most effective treatment for severe depression. However, its mechanisms of action are incompletely understood. Evidence suggests ECT enhances neuroplasticity and neurogenesis. While studies on ECT-induced neuroplasticity focused on brain-derived neurotrophic factor (BDNF), other factors of the BDNF/TrkB signaling cascade remain underinvestigated. We assessed longitudinal changes in depression scores, serum BDNF protein levels, and mRNA expression of BDNF/TrkB related genes (BDNF, AKT1, ERK1, CREB), NR3C1 and IGF1 in peripheral blood in 19 treatment-resistant depressed patients undergoing ECT. We also analysed DNA methylation patterns at various timepoints to explore possible epigenetic regulation of mRNA expression. Using multilevel regression, we found a negative association between depression scores and blood-based mRNA expression of BDNF/TrkB related genes and NR3C1. Expression of BDNF, ERK1 and NR3C1 increased significantly over time (BDNF: ß = 0.0295, p = 0.003; ERK1: ß = 0.0170, p = 0.034; NR3C1: ß = 0.0035, p = 0.050). For these three genes changes in mRNA expression were highly correlated (R = 0.59 - 0.88) with changes in DNA methylation for multiple CpG sites in the respective genes. Also, serum BDNF protein levels increased across the study period (ß = 0.11, p = 0.001). Our findings show that the antidepressant effects of ECT are associated with changes in expression of BDNF and its signaling molecules and that these molecular markers can be detected in peripheral blood. Alterations in DNA methylation could be a key mechanism whereby ECT influences gene expression.

DNA methylation signatures of Alzheimer's disease neuropathology in the cortex are primarily driven by variation in non-neuronal cell-types.

Alzheimer's disease (AD) is a chronic neurodegenerative disease characterized by the progressive accumulation of amyloid-beta and neurofibrillary tangles of tau in the neocortex. We profiled DNA methylation in two regions of the cortex from 631 donors, performing an epigenome-wide association study of multiple measures of AD neuropathology. We meta-analyzed our results with those from previous studies of DNA methylation in AD cortex (total n = 2013 donors), identifying 334 cortical differentially methylated positions (DMPs) associated with AD pathology including methylomic variation at loci not previously implicated in dementia. We subsequently profiled DNA methylation in NeuN+ (neuronal-enriched), SOX10+ (oligodendrocyte-enriched) and NeuN-/SOX10- (microglia- and astrocyte-enriched) nuclei, finding that the majority of DMPs identified in 'bulk' cortex tissue reflect DNA methylation differences occurring in non-neuronal cells. Our study highlights the power of utilizing multiple measures of neuropathology to identify epigenetic signatures of AD and the importance of characterizing disease-associated variation in purified cell-types.

Epigenomic features related to microglia are associated with attenuated effect of APOE ε4 on Alzheimer's disease risk in humans.

Not all apolipoprotein E (APOE) ε4 carriers who survive to advanced age develop Alzheimer's disease (AD); factors attenuating the risk of ε4 on AD may exist. Guided by the top ε4-attenuating signals from methylome-wide association analyses (N = 572, ε4+ and ε4-) of neurofibrillary tangles and neuritic plaques, we conducted a meta-analysis for pathological AD within the ε4+ subgroups (N = 235) across four independent collections of brains. Cortical RNA-seq and microglial morphology measurements were used in functional analyses. Three out of the four significant CpG dinucleotides were captured by one principal component (PC1), which interacts with ε4 on AD, and is associated with expression of innate immune genes and activated microglia. In ε4 carriers, reduction in each unit of PC1 attenuated the odds of AD by 58% (odds ratio = 2.39, 95% confidence interval = [1.64,3.46], P = 7.08 × 10-6 ). An epigenomic factor associated with a reduced proportion of activated microglia (epigenomic factor of activated microglia, EFAM) appears to attenuate the risk of ε4 on AD.

Characterization of DNA Methylomic Signatures in Induced Pluripotent Stem Cells During Neuronal Differentiation.

In development, differentiation from a pluripotent state results in global epigenetic changes, although the extent to which this occurs in induced pluripotent stem cell-based neuronal models has not been extensively characterized. In the present study, induced pluripotent stem cell colonies (33Qn1 line) were differentiated and collected at four time-points, with DNA methylation assessed using the Illumina Infinium Human Methylation EPIC BeadChip array. Dynamic changes in DNA methylation occurring during differentiation were investigated using a data-driven trajectory inference method. We identified a large number of Bonferroni-significant loci that showed progressive alterations in DNA methylation during neuronal differentiation. A gene-gene interaction network analysis identified 60 densely connected genes that were influential in the differentiation of neurons, with STAT3 being the gene with the highest connectivity.

A meta-analysis of epigenome-wide association studies in Alzheimer's disease highlights novel differentially methylated loci across cortex.

Epigenome-wide association studies of Alzheimer's disease have highlighted neuropathology-associated DNA methylation differences, although existing studies have been limited in sample size and utilized different brain regions. Here, we combine data from six DNA methylomic studies of Alzheimer's disease (N = 1453 unique individuals) to identify differential methylation associated with Braak stage in different brain regions and across cortex. We identify 236 CpGs in the prefrontal cortex, 95 CpGs in the temporal gyrus and ten CpGs in the entorhinal cortex at Bonferroni significance, with none in the cerebellum. Our cross-cortex meta-analysis (N = 1408 donors) identifies 220 CpGs associated with neuropathology, annotated to 121 genes, of which 84 genes have not been previously reported at this significance threshold. We have replicated our findings using two further DNA methylomic datasets consisting of a further >600 unique donors. The meta-analysis summary statistics are available in our online data resource ( www.epigenomicslab.com/ad-meta-analysis/ ).

Novel epigenetic clock for fetal brain development predicts prenatal age for cellular stem cell models and derived neurons.

Induced pluripotent stem cells (iPSCs) and their differentiated neurons (iPSC-neurons) are a widely used cellular model in the research of the central nervous system. However, it is unknown how well they capture age-associated processes, particularly given that pluripotent cells are only present during the earliest stages of mammalian development. Epigenetic clocks utilize coordinated age-associated changes in DNA methylation to make predictions that correlate strongly with chronological age. It has been shown that the induction of pluripotency rejuvenates predicted epigenetic age. As existing clocks are not optimized for the study of brain development, we developed the fetal brain clock (FBC), a bespoke epigenetic clock trained in human prenatal brain samples in order to investigate more precisely the epigenetic age of iPSCs and iPSC-neurons. The FBC was tested in two independent validation cohorts across a total of 194 samples, confirming that the FBC outperforms other established epigenetic clocks in fetal brain cohorts. We applied the FBC to DNA methylation data from iPSCs and embryonic stem cells and their derived neuronal precursor cells and neurons, finding that these cell types are epigenetically characterized as having an early fetal age. Furthermore, while differentiation from iPSCs to neurons significantly increases epigenetic age, iPSC-neurons are still predicted as being fetal. Together our findings reiterate the need to better understand the limitations of existing epigenetic clocks for answering biological research questions and highlight a limitation of iPSC-neurons as a cellular model of age-related diseases.

Altered sphingolipid function in Alzheimer's disease; a gene regulatory network approach.

Sphingolipids (SLs) are bioactive lipids involved in various important physiological functions. The SL pathway has been shown to be affected in several brain-related disorders, including Alzheimer's disease (AD). Recent evidence suggests that epigenetic dysregulation plays an important role in the pathogenesis of AD as well. Here, we use an integrative approach to better understand the relationship between epigenetic and transcriptomic processes in regulating SL function in the middle temporal gyrus of AD patients. Transcriptomic analysis of 252 SL-related genes, selected based on GO term annotations, from 46 AD patients and 32 healthy age-matched controls, revealed 103 differentially expressed SL-related genes in AD patients. Additionally, methylomic analysis of the same subjects revealed parallel hydroxymethylation changes in PTGIS, GBA, and ITGB2 in AD. Subsequent gene regulatory network-based analysis identified 3 candidate genes, that is, SELPLG, SPHK1 and CAV1 whose alteration holds the potential to revert the gene expression program from a diseased towards a healthy state. Together, this epigenomic and transcriptomic approach highlights the importance of SL-related genes in AD, and may provide novel biomarkers and therapeutic alternatives to traditionally investigated biological pathways in AD.

Meta-analysis of genome-wide DNA methylation identifies shared associations across neurodegenerative disorders.

BACKGROUND: People with neurodegenerative disorders show diverse clinical syndromes, genetic heterogeneity, and distinct brain pathological changes, but studies report overlap between these features. DNA methylation (DNAm) provides a way to explore this overlap and heterogeneity as it is determined by the combined effects of genetic variation and the environment. In this study, we aim to identify shared blood DNAm differences between controls and people with Alzheimer's disease, amyotrophic lateral sclerosis, and Parkinson's disease. RESULTS: We use a mixed-linear model method (MOMENT) that accounts for the effect of (un)known confounders, to test for the association of each DNAm site with each disorder. While only three probes are found to be genome-wide significant in each MOMENT association analysis of amyotrophic lateral sclerosis and Parkinson's disease (and none with Alzheimer's disease), a fixed-effects meta-analysis of the three disorders results in 12 genome-wide significant differentially methylated positions. Predicted immune cell-type proportions are disrupted across all neurodegenerative disorders. Protein inflammatory markers are correlated with profile sum-scores derived from disease-associated immune cell-type proportions in a healthy aging cohort. In contrast, they are not correlated with MOMENT DNAm-derived profile sum-scores, calculated using effect sizes of the 12 differentially methylated positions as weights. CONCLUSIONS: We identify shared differentially methylated positions in whole blood between neurodegenerative disorders that point to shared pathogenic mechanisms. These shared differentially methylated positions may reflect causes or consequences of disease, but they are unlikely to reflect cell-type proportion differences.

DNA methylation signatures of adolescent victimization: analysis of a longitudinal monozygotic twin sample.

Accumulating evidence suggests that individuals exposed to victimization at key developmental stages may have different epigenetic fingerprints compared to those exposed to no/minimal stressful events, however results are inconclusive. This study aimed to strengthen causal inference regarding the impact of adolescent victimization on the epigenome by controlling for genetic variation, age, gender, and shared environmental exposures. We conducted longitudinal epigenome-wide association analyses (EWAS) on DNA methylation (DNAm) profiles of 118 monozygotic (MZ) twin pairs from the Environmental Risk study with and without severe adolescent victimization generated using buccal DNA collected at ages 5, 10 and 18, and the Illumina EPIC array. Additionally, we performed cross-sectional EWAS on age-18 blood and buccal DNA from the same individuals to elucidate tissue-specific signatures of severe adolescent victimization. Our analyses identified 20 suggestive differentially methylated positions (DMPs) (P < 5e-05), with altered DNAm trajectories between ages 10-18 associated with severe adolescent victimization (∆Beta range = -5.5%-5.3%). Age-18 cross-sectional analyses revealed 72 blood (∆Beta range = -2.2%-3.4%) and 42 buccal (∆Beta range = -3.6%-4.6%) suggestive severe adolescent victimization-associated DMPs, with some evidence of convergent signals between these two tissue types. Downstream regional analysis identified significant differentially methylated regions (DMRs) in LGR6 and ANK3 (Sidák P = 5e-09 and 4.07e-06), and one upstream of CCL27 (Sidák P = 2.80e-06) in age-18 blood and buccal EWAS, respectively. Our study represents the first longitudinal MZ twin analysis of DNAm and severe adolescent victimization, providing initial evidence for altered DNA methylomic signatures in individuals exposed to adolescent victimization.

Increased isoform-specific phosphodiesterase 4D expression is associated with pathology and cognitive impairment in Alzheimer's disease.

Pharmacological phosphodiesterase 4D (PDE4D) inhibition shows therapeutic potential to restore memory function in Alzheimer's disease (AD), but will likely evoke adverse side effects. As PDE4D encodes multiple isoforms, targeting specific isoforms may improve treatment efficacy and safety. Here, we investigated whether PDE4D isoform expression and PDE4D DNA methylation is affected in AD and whether expression changes are associated with severity of pathology and cognitive impairment. In post-mortem temporal lobe brain material from AD patients (n = 42) and age-matched controls (n = 40), we measured PDE4D isoform expression and PDE4D DNA (hydroxy)methylation using quantitative polymerase chain reaction and Illumina 450k Beadarrays, respectively. Linear regression revealed increased PDE4D1, -D3, -D5, and -D8 expression in AD with concurrent (hydroxy)methylation changes in associated promoter regions. Moreover, increased PDE4D1 and -D3 expression was associated with higherplaque and tau pathology levels, higher Braak stages, and progressed cognitive impairment. Future studies should indicate functional roles of specific PDE4D isoforms and the efficacy and safety of their selective inhibition to restore memory function in AD.

An epigenome-wide association study of Alzheimer's disease blood highlights robust DNA hypermethylation in the HOXB6 gene.

A growing number of epigenome-wide association studies have demonstrated a role for DNA methylation in the brain in Alzheimer's disease. With the aim of exploring peripheral biomarker potential, we have examined DNA methylation patterns in whole blood collected from 284 individuals in the AddNeuroMed study, which included 89 nondemented controls, 86 patients with Alzheimer's disease, and 109 individuals with mild cognitive impairment, including 38 individuals who progressed to Alzheimer's disease within 1 year. We identified significant differentially methylated regions, including 12 adjacent hypermethylated probes in the HOXB6 gene in Alzheimer's disease, which we validated using pyrosequencing. Using weighted gene correlation network analysis, we identified comethylated modules of genes that were associated with key variables such as APOE genotype and diagnosis. In summary, this study represents the first large-scale epigenome-wide association study of Alzheimer's disease and mild cognitive impairment using blood. We highlight the differences in various loci and pathways in early disease, suggesting that these patterns relate to cognitive decline at an early stage.

Epigenome-wide association studies in Alzheimer's disease; achievements and challenges.

Alzheimer's disease (AD) represents a devastating progressive neurodegenerative disease with a complex pathophysiology, affecting millions of people worldwide. Recent epigenome-wide association studies suggest a key role for epigenetic mechanisms in its development and course. Despite the fact that current evidence on the role of epigenetic dysregulation in aging and AD is convincing, the pioneering field of neuroepigenetics is still facing many challenges that need to be addressed to fundamentally increase our understanding about the underlying mechanisms of this neurodegenerative disorder. This perspective paper describes the current state of play for epigenetic research into AD and discusses how new methodological advances in the field of epigenetics and related data science disciplines could further spur the development of novel therapeutic agents and biomarker assays.

Psychosis-associated DNA methylomic variation in Alzheimer's disease cortex.

Psychotic symptoms are a common and debilitating feature of Alzheimer's disease (AD) and are associated with a more rapid course of decline. Current evidence from postmortem and neuroimaging studies implicates frontal, temporal, and parietal lobes, with reported disruptions in monoaminergic pathways. However, the molecular mechanisms underlying this remain unclear. In the present study, we investigated methylomic variation associated with AD psychosis in 3 key brain regions implicated in the etiology of psychosis (prefrontal cortex, entorhinal cortex, and superior temporal gyrus) in postmortem brain samples from 29 AD donors with psychosis and 18 matched AD donors without psychosis. We identified psychosis-associated methylomic changes in a number of loci, with these genes being enriched in known schizophrenia-associated genetic and epigenetic variants. One of these known loci resided in the AS3MT gene-previously implicated in schizophrenia in a large GWAS meta-analysis. We used bisulfite-pyrosequencing to confirm hypomethylation across 4 neighboring CpG sites in the ASM3T gene. Finally, our regional analysis nominated multiple CpG sites in TBX15 and WT1, which are genes that have been previously implicated in AD. Thus one potential implication from our study is whether psychosis-associated variation drives reported associations in AD case-control studies.

Longitudinal epigenome-wide association studies of three male military cohorts reveal multiple CpG sites associated with post-traumatic stress disorder.

BACKGROUND: Epigenetic mechanisms have been suggested to play a role in the development of post-traumatic stress disorder (PTSD). Here, blood-derived DNA methylation data (HumanMethylation450 BeadChip) collected prior to and following combat exposure in three cohorts of male military members were analyzed to assess whether DNA methylation profiles are associated with the development of PTSD. A total of 123 PTSD cases and 143 trauma-exposed controls were included in the analyses. The Psychiatric Genomics Consortium (PGC) PTSD EWAS QC pipeline was used on all cohorts, and results were combined using a sample size weighted meta-analysis in a two-stage design. In stage one, we jointly analyzed data of two new cohorts (N = 126 and 78) for gene discovery, and sought to replicate significant findings in a third, previously published cohort (N = 62) to assess the robustness of our results. In stage 2, we aimed at maximizing power for gene discovery by combining all three cohorts in a meta-analysis. RESULTS: Stage 1 analyses identified four CpG sites in which, conditional on pre-deployment DNA methylation, post-deployment DNA methylation was significantly associated with PTSD status after epigenome-wide adjustment for multiple comparisons. The most significant (intergenic) CpG cg05656210 (p = 1.0 × 10-08) was located on 5q31 and significantly replicated in the third cohort. In addition, 19 differentially methylated regions (DMRs) were identified, but failed replication. Stage 2 analyses identified three epigenome-wide significant CpGs, the intergenic CpG cg05656210 and two additional CpGs located in MAD1L1 (cg12169700) and HEXDC (cg20756026). Interestingly, cg12169700 had an underlying single nucleotide polymorphism (SNP) which was located within the same LD block as a recently identified PTSD-associated SNP in MAD1L1. Stage 2 analyses further identified 12 significant differential methylated regions (DMRs), 1 of which was located in MAD1L1 and 4 were situated in the human leukocyte antigen (HLA) region. CONCLUSIONS: This study suggests that the development of combat-related PTSD is associated with distinct methylation patterns in several genomic positions and regions. Our most prominent findings suggest the involvement of the immune system through the HLA region and HEXDC, and MAD1L1 which was previously associated with PTSD.