Genome-wide Methylation Differences Research Articles

Hypomethylation of Wnt signaling regulator genes in developmental language disorder.

Background: Developmental language disorder (DLD) is a neurodevelopmental disorder. Considering the pivotal role of epigenetics in neurodevelopment, we examined any altered DNA methylation between DLD and control subjects. Materials & methods: We looked into genome-wide methylation differences between DLD and control groups. The findings were validated by quantitative PCR (qPCR). Results: In the DLD group, differential methylation of CpG sites was observed in the Wnt signaling regulator genes APCDD1, AMOTL1, LRP5, MARK2, TMEM64, TRABD2B, VEPH1 and WNT2B. Hypomethylation of APCDD1, LRP5 and WNT2B was confirmed by qPCR. Conclusion: This is the first report associating Wnt signaling with DLD. The findings are relevant in the light of the essential role of Wnt in myelination, and of the altered myelination in DLD.

The Effect of Maternal Diet and Physical Activity on the Epigenome of the Offspring.

The aim of this review was to examine the current literature regarding the effect of maternal lifestyle interventions (i.e., diet and physical activity) on the epigenome of the offspring. PubMed, Scopus and Cochrane-CENTRAL were screened until 8 July 2023. Only randomized controlled trials (RCTs) where a lifestyle intervention was compared to no intervention (standard care) were included. Outcome variables included DNA methylation, miRNA expression, and histone modifications. A qualitative approach was used for the consideration of the studies' results. Seven studies and 1765 mother-child pairs were assessed. The most common types of intervention were dietary advice, physical activity, and following a specific diet (olive oil). The included studies correlated the lifestyle and physical activity intervention in pregnancy to genome-wide or gene-specific differential methylation and miRNA expression in the cord blood or the placenta. An intervention of diet and physical activity in pregnancy was found to be associated with slight changes in the epigenome (DNA methylation and miRNA expression) in fetal tissues. The regions involved were related to adiposity, metabolic processes, type 2 diabetes, birth weight, or growth. However, not all studies showed significant differences in DNA methylation. Further studies with similar parameters are needed to have robust and comparable results and determine the biological role of such modifications.

Whole genome methylation sequencing reveals epigenetic landscape and abnormal expression of FABP5 in extramammary Paget's disease.

Extramammary Paget's disease (EMPD) is a rare cutaneous malignant tumor with a high recurrence rate after surgery. However, the genetic and epigenetic alterations underlying its pathogenesis remain unknown. DNA methylation is an important epigenetic modification involved in many biological processes. In this study, enzymatic methyl-sequencing (EM-seq) technique was used to investigate the landscape of genome-wide DNA methylation from three pairs of tumor tissues and adjacent tissues of patients with EMPD. Additionally, we conducted histopathological examinations to assess the expression of fatty acid-binding protein 5 (FABP5) in another three paired samples from EMPD patients. The cluster analysis showed the good quality of the samples. A differential methylation region (DMR) heat map was used to quantitatively characterize genome-wide methylation differences between tumors and controls. Global DNA methylation level is lower in EMPD tissue compared to matched controls, indicating that DNA methylation discriminates between tumor and normal skin. And the top hypomethylation gene on the promoter region in tumor tissues was FABP5 on chromosome 8 with 38.44% decreased median methylation. We next identified the expression of FABP5 in paired tumors and adjacent tissues in three additional patients with EMPD. Immunofluorescence results showed FABP5 highly expressed in tumor tissues and co-located with CK7, CK20 and EMA. GO and KEGG enrichment analysis showed DMR genes on promoter are mainly enriched in the calcium ion transport, GTPase mediated signal transduction, Rap1 signaling pathway and GnRH signaling pathway. Taken together, our findings provide the first description of the whole genome methylation map of EMPD and identify FABP5 as a pathogenic target of EMPD.

Identification of novel hypermethylated or hypomethylated CpG sites and genes associated with anthracycline-induced cardiomyopathy

Anthracycline-induced cardiomyopathy is a leading cause of late morbidity in childhood cancer survivors. Aberrant DNA methylation plays a role in de novo cardiovascular disease. Epigenetic processes could play a role in anthracycline-induced cardiomyopathy but remain unstudied. We sought to examine if genome-wide differential methylation at ‘CpG’ sites in peripheral blood DNA is associated with anthracycline-induced cardiomyopathy. This report used participants from a matched case–control study; 52 non-Hispanic White, anthracycline-exposed childhood cancer survivors with cardiomyopathy were matched 1:1 with 52 survivors with no cardiomyopathy. Paired ChAMP (Chip Analysis Methylation Pipeline) with integrated reference-based deconvolution of adult peripheral blood DNA methylation was used to analyze data from Illumina HumanMethylation EPIC BeadChip arrays. An epigenome-wide association study (EWAS) was performed, and the model was adjusted for GrimAge, sex, interaction terms of age at enrollment, chest radiation, age at diagnosis squared, and cardiovascular risk factors (CVRFs: diabetes, hypertension, dyslipidemia). Prioritized genes were functionally validated by gene knockout in human induced pluripotent stem cell cardiomyocytes (hiPSC-CMs) using CRISPR/Cas9 technology. DNA-methylation EPIC array analyses identified 32 differentially methylated probes (DMP: 15 hyper-methylated and 17 hypo-methylated probes) that overlap with 23 genes and 9 intergenic regions. Three hundred and fifty-four differential methylated regions (DMRs) were also identified. Several of these genes are associated with cardiac dysfunction. Knockout of genes EXO6CB, FCHSD2, NIPAL2, and SYNPO2 in hiPSC-CMs increased sensitivity to doxorubicin. In addition, EWAS analysis identified hypo-methylation of probe ‘cg15939386’ in gene RORA to be significantly associated with anthracycline-induced cardiomyopathy. In this genome-wide DNA methylation profile study, we observed significant differences in DNA methylation at the CpG level between anthracycline-exposed childhood cancer survivors with and without cardiomyopathy, implicating differential DNA methylation of certain genes could play a role in pathogenesis of anthracycline-induced cardiomyopathy.

Genome-wide DNA methylation and transcriptome analyses reveal the key gene for wool type variation in sheep

BackgroundWool fibers are valuable materials for textile industry. Typical wool fibers are divided into medullated and non-medullated types, with the former generated from primary wool follicles and the latter by either primary or secondary wool follicles. The medullated wool is a common wool type in the ancestors of fine wool sheep before breeding. The fine wool sheep have a non-medullated coat. However, the critical period determining the type of wool follicles is the embryonic stage, which limits the phenotypic observation and variant contrast, making both selection and studies of wool type variation fairly difficult.ResultsDuring the breeding of a modern fine (MF) wool sheep population with multiple-ovulation and embryo transfer technique, we serendipitously discovered lambs with ancestral-like coarse (ALC) wool. Whole-genome resequencing confirmed ALC wool lambs as a variant type from the MF wool population. We mapped the significantly associated methylation locus on chromosome 4 by using whole genome bisulfite sequencing signals, and in turn identified the SOSTDC1 gene as exons hypermethylated in ALC wool lambs compare to their half/full sibling MF wool lambs. Transcriptome sequencing found that SOSTDC1 was expressed dozens of times more in ALC wool lamb skin than that of MF and was at the top of all differentially expressed genes. An analogy with the transcriptome of coarse/fine wool breeds revealed that differentially expressed genes and enriched pathways at postnatal lamb stage in ALC/MF were highly similar to those at the embryonic stage in the former. Further experiments validated that the SOSTDC1 gene was specifically highly expressed in the nucleus of the dermal papilla of primary wool follicles.ConclusionIn this study, we conducted genome-wide differential methylation site association analysis on differential wool type trait, and located the only CpG locus that strongly associated with primary wool follicle development. Combined with transcriptome analysis, SOSTDC1 was identified as the only gene at this locus that was specifically overexpressed in the primary wool follicle stem cells of ALC wool lamb skin. The discovery of this key gene and its epigenetic regulation contributes to understanding the domestication and breeding of fine wool sheep.

Genome-wide DNA methylation analysis reveals different methylation patterns in Chinese indigenous sheep with different type of tail.

The study was aimed to analyze the difference of genome-wide DNA differential methylation in Lanzhou Large-tailed sheep, Altay sheep and Tibetan sheep, which the typical breeds with different type tails, as to screen the differentially methylated genes (DMGs) that affect the type of tails. In this study, three Lanzhou Large-tailed sheep, three Altay sheep and three Tibetan sheep were detected by whole genome bisulfite sequencing (WGBS). The degree of genome-wide DNA methylation, differentially methylated regions (DMRs) and DMGs were analyzed. The candidate genes affecting the tail type of sheep were identified by GO and KEGG pathway enrichment analysis of DMGs. we identified 68,603 different methylated regions (DMCs) and 75 differentially methylated genes (DMGs) associated with these DMCs. Functional analysis showed that these DMGs were mainly enriched in biological process, cellular component and molecular function, Some of the genes in these pathways are involved in fat metabolism: NFATC4, LPIN2, MGAT2 and MAT2B. Our results may help to further understand the epigenetic regulation mechanisms of deposition of fat in the tail of sheep and provide new basic data for the study of local sheep.

DNA Methylation and Prospects for Predicting the Therapeutic Effect of Neoadjuvant Chemotherapy for Triple-Negative and Luminal B Breast Cancer.

Despite advances in the diagnosis and treatment of breast cancer (BC), the main cause of deaths is resistance to existing therapies. An approach to improve the effectiveness of therapy in patients with aggressive BC subtypes is neoadjuvant chemotherapy (NACT). Yet, the response to NACT for aggressive subtypes is less than 65% according to large clinical trials. An obvious fact is the lack of biomarkers predicting the therapeutic effect of NACT. In a search for epigenetic markers, we performed genome-wide differential methylation screening by XmaI-RRBS in cohorts of NACT responders and nonresponders, for triple-negative (TN) and luminal B tumors. The predictive potential of the most discriminative loci was further assessed in independent cohorts by methylation-sensitive restriction enzyme quantitative PCR (MSRE-qPCR), a promising method for the implementation of DNA methylation markers in diagnostic laboratories. The selected most informative individual markers were combined into panels demonstrating cvAUC = 0.83 (TMEM132D and MYO15B markers panel) for TN tumors and cvAUC = 0.76 (TTC34, LTBR and CLEC14A) for luminal B tumors. The combination of methylation markers with clinical features that correlate with NACT effect (clinical stage for TN and lymph node status for luminal B tumors) produces better classifiers, with cvAUC = 0.87 for TN tumors and cvAUC = 0.83 for luminal B tumors. Thus, clinical characteristics predictive of NACT response are independently additive to the epigenetic classifier and in combination improve prediction.

Actinobacillus pleuropneumoniae encodes multiple phase-variable DNA methyltransferases that control distinct phasevarions.

Actinobacillus pleuropneumoniae is the cause of porcine pleuropneumonia, a severe respiratory tract infection that is responsible for major economic losses to the swine industry. Many host-adapted bacterial pathogens encode systems known as phasevarions (phase-variable regulons). Phasevarions result from variable expression of cytoplasmic DNA methyltransferases. Variable expression results in genome-wide methylation differences within a bacterial population, leading to altered expression of multiple genes via epigenetic mechanisms. Our examination of a diverse population of A. pleuropneumoniae strains determined that Type I and Type III DNA methyltransferases with the hallmarks of phase variation were present in this species. We demonstrate that phase variation is occurring in these methyltransferases, and show associations between particular Type III methyltransferase alleles and serovar. Using Pacific BioSciences Single-Molecule, Real-Time (SMRT) sequencing and Oxford Nanopore sequencing, we demonstrate the presence of the first ever characterised phase-variable, cytosine-specific Type III DNA methyltransferase. Phase variation of distinct Type III DNA methyltransferase in A. pleuropneumoniae results in the regulation of distinct phasevarions, and in multiple phenotypic differences relevant to pathobiology. Our characterisation of these newly described phasevarions in A. pleuropneumoniae will aid in the selection of stably expressed antigens, and direct and inform development of a rationally designed subunit vaccine against this major veterinary pathogen.

Comparative and integrative analysis of transcriptomic and epigenomic-wide DNA methylation changes in African American prostate cancer

ABSTRACT African American (AA) men have the highest incidence and mortality rate from Prostate cancer (PCa) than any other racial/ethnic group. To date, PCa genomic studies have largely under-represented tumour samples from AA men. We measured genome-wide DNA methylation in benign and tumor prostate tissues from AA men using the Illumina Infunium 850 K EPIC array. mRNA expression database from a subset of the AA biospecimen were used to assess correlation of transcriptome and methylation datasets. Genome-wide methylation analysis identified 11,460 probes that were significant (p < 0.01) and differentially methylated in AA PCa compared to normal prostate tissues and showed significant (p < 0.01) inverse-correlation with mRNA expression. Ingenuity pathway analysis and Gene Ontology analysis in our AA dataset compared with TCGA dataset showed similarities in methylation patterns: top candidate genes with significant hypermethylation and corresponding down-regulated gene expression were associated with biological pathways in hemidesmosome assembly, mammary gland development, epidermis development, hormone biosynthesis, and cell communication. In addition, top candidate genes with significant hypomethylation and corresponding up-regulated gene expression were associated with biological pathways in macrophage differentiation, cAMP-dependent protein kinase activity, protein destabilization, transcription co-repression, and fatty acid biosynthesis. In contrast, differences in genome-wide methylation in our AA dataset compared with TCGA dataset were enriched for genes in steroid signalling, immune signalling, chromatin structure remodelling and RNA processing. Overall, differential methylation of AMIGO3, IER3, UPB1, GRM7, TFAP2C, TOX2, PLSCR2, ZNF292, ESR2, MIXL1, BOLL, and FGF6 were significant and uniquely associated with PCa progression in our AA cohort.

Epigenetic Regulation of Phenotypic Sexual Plasticity Inducing Skewed Sex Ratio in Zebrafish.

The plasticity of sexual phenotype in response to environmental conditions results in biased sex ratios, and their variation has an effect on population dynamics. Epigenetic modifications can modulate sex ratio variation in species, where sex is determined by genetic and environmental factors. However, the role of epigenetic mechanisms underlying skewed sex ratios is far from being clear and is still an object of debate in evolutionary developmental biology. In this study, we used zebrafish as a model animal to investigate the effect of DNA methylation on sex ratio variation in sex-biased families in response to environmental temperature. Two sex-biased families with a significant difference in sex ratio were selected for genome-wide DNA methylation analysis using reduced representation bisulfite sequencing (RRBS). The results showed significant genome-wide methylation differences between male-biased and female-biased families, with a greater number of methylated CpG sites in testes than ovaries. Likewise, pronounced differences between testes and ovaries were identified within both families, where the male-biased family exhibited a higher number of methylated sites than the female-biased family. The effect of temperature showed more methylated positions in the high incubation temperature than the control temperature. We found differential methylation of many reproduction-related genes (e.g., sox9a, nr5a2, lhx8a, gata4) and genes involved in epigenetic mechanisms (e.g., dnmt3bb.1, dimt1l, hdac11, h1m) in both families. We conclude that epigenetic modifications can influence the sex ratio variation in zebrafish families and may generate skewed sex ratios, which could have a negative consequence for population fitness in species with genotype-environment interaction sex-determining system under rapid environmental changes.

DNA methylation signatures in human neonatal blood following maternal antenatal corticosteroid treatment

Antenatal corticosteroids (ACS) are used to treat women at risk of preterm birth to improve neonatal survival. Though affected children may be at long-term risk of neurobehavioural disorders, the driving mechanisms remain unknown. Animal studies have shown that ACS exposure can lead to overlapping changes in DNA methylation between the blood and the brain, identifying gene pathways for neurodevelopment, which highlights the potential to examine peripheral blood as a surrogate for inaccessible human brain tissue. We hypothesized that differential methylation will be identified in blood of term-born neonates following ACS. Mother-infant dyads that received ACS were retrospectively identified through the Ontario Birth Study at Sinai Health Complex and matched to untreated controls for maternal age, BMI, parity and foetal sex (n = 14/group). Genome-wide methylation differences were examined at single-nucleotide resolution in DNA extracted from dried bloodspot cards using reduced representative bisulfite sequencing approaches. 505 differentially methylated CpG sites (DMCs) were identified, wherein 231 were hypermethylated and 274 were hypomethylated. These sites were annotated to 219 genes, of which USP48, SH3PXD2A, NTM, CAMK2N2, MAP6D1 were five of the top ten genes with known neurological function. Collectively, the set of hypermethylated genes were enriched for pathways of transcription regulation, while pathways of proteasome activity were enriched among the set of hypomethylated genes. This study is the first to identify DNA methylation changes in human neonatal blood following ACS. Understanding the epigenetic changes that occur in response to ACS will support future investigations to delineate the effects of prenatal glucocorticoid exposure on human development.

CDK13-related disorder: Report of a series of 18 previously unpublished individuals and description of an epigenetic signature

PurposeRare genetic variants in CDK13 are responsible for CDK13-related disorder (CDK13-RD), with main clinical features being developmental delay or intellectual disability, facial features, behavioral problems, congenital heart defect, and seizures. In this paper, we report 18 novel individuals with CDK13-RD and provide characterization of genome-wide DNA methylation. MethodsWe obtained clinical phenotype and neuropsychological data for 18 and 10 individuals, respectively, and compared this series with the literature. We also compared peripheral blood DNA methylation profiles in individuals with CDK13-RD, controls, and other neurodevelopmental disorders episignatures. Finally, we developed a support vector machine–based classifier distinguishing CDK13-RD and non–CDK13-RD samples. ResultsWe reported health and developmental parameters, clinical data, and neuropsychological profile of individuals with CDK13-RD. Genome-wide differential methylation analysis revealed a global hypomethylated profile in individuals with CDK13-RD in a highly sensitive and specific model that could aid in reclassifying variants of uncertain significance. ConclusionWe describe the novel features such as anxiety disorder, cryptorchidism, and disrupted sleep in CDK13-RD. We define a CDK13-RD DNA methylation episignature as a diagnostic tool and a defining functional feature of the evolving clinical presentation of this disorder. We also show overlap of the CDK13 DNA methylation profile in an individual with a functionally and clinically related CCNK-related disorder.

Genome-wide locus-specific DNA methylation repatterning may facilitate rapid evolution of mercury resistance in rice.

Albeit a relatively stable epigenetic modification, DNA methylation in plants can be repatterned and play important roles in response to biotic and abiotic stresses. However, whether DNA methylation dynamics may contribute to cope with mercury (Hg) stress in plants remains to be fully investigated. To probe the potential roles of DNA methylation dynamics in coping with Hg stress in rice. Whole-genome bisulfite sequencing was used to profile the DNA methylation patterns of a rice Hg-resistant line (RHg) selected from a heterozygous mutant of the DNA methyltransferase 1 gene (OsMET1+/-), together with its Hg-sensitive wild-type plants of cv. Nipponbare (Nip) under both normal and Hg stress conditions. Genome-wide locus-specific differential methylation regions (DMRs) were detected between RHg and Nip under normal condition, the predominant DMR patterns were CG hypo-DMRs, CHG hypo-DMRs and CHH hyper-DMRs. In both lines, more hyper- than hypo-DMRs were detected at all three sequence contexts (CG, CHG and CHH) under Hg stress relative to normal condition. Comparison of DNA methylation changes in the two lines under Hg stress indicates that RHg had a more dynamic methylome than the control (Nip). Original DMRs in RHg trended to transform to opposite status (from hyper- to hypo- or vice versa) under Hg stress condition. Gene ontology analysis revealed that Hg-resistance-related DMGs were enriched in diverse biological processes. Our results suggest genome-wide locus-specific DNA methylation repatterning can facilitate rapid acquisition of Hg resistance in rice.

Invertebrate methylomes provide insight into mechanisms of environmental tolerance and reveal methodological biases.

There is a growing focus on the role of DNA methylation in the ability of marine invertebrates to rapidly respond to changing environmental factors and anthropogenic impacts. However, genome-wide DNA methylation studies in nonmodel organisms are currently hampered by a limited understanding of methodological biases. Here, we compare three methods for quantifying DNA methylation at single base-pair resolution-whole genome bisulfite sequencing (WGBS), reduced representation bisulfite sequencing (RRBS), and methyl-CpG binding domain bisulfite sequencing (MBDBS)-using multiple individuals from two reef-building coral species with contrasting environmental sensitivity. All methods reveal substantially greater methylation in Montipora capitata (11.4%) than the more sensitive Pocillopora acuta (2.9%). The majority of CpG methylation in both species occurs in gene bodies and flanking regions. In both species, MBDBS has the greatest capacity for detecting CpGs in coding regions at our sequencing depth, but MBDBS may be influenced by intrasample methylation heterogeneity. RRBS yields robust information for specific loci albeit without enrichment of any particular genome feature and with significantly reduced genome coverage. Relative genome size strongly influences the number and location of CpGs detected by each method when sequencing depth is limited, illuminating nuances in cross-species comparisons. As genome-wide methylation differences, supported by data across bisulfite sequencing methods, may contribute to environmental sensitivity phenotypes in critical marine invertebrate taxa, these data provide a genomic resource for investigating the functional role of DNA methylation in environmental tolerance.

Upper Aerodigestive Tract Squamous Cell Carcinomas Show Distinct Overall DNA Methylation Profiles and Different Molecular Mechanisms behind WNT Signaling Disruption.

Simple SummarySquamous cell carcinomas of the upper aerodigestive tract are highly incident, lethal, and share the same epithelial lining of origin, risk factors and genetic alterations. However, their biological and clinical behaviors differ, having an impact on patient survival. This study aimed at identifying the main DNA methylation differences between these tumors, giving an overview of the main genomic regions affected, whether DNA methylation gains or losses are more common, the impact on gene expression and the signaling pathways affected. This knowledge will help identifying potential site-specific biomarkers as well as shedding light on whether epigenetic mechanisms explain, at least in part, the diverse behavior of upper aerodigestive tract tumors.Upper aerodigestive tract (UADT) tumors present different biological behavior and prognosis, suggesting specific molecular mechanisms underlying their development. However, they are rarely considered as single entities (particularly head and neck subsites) and share the most common genetic alterations. Therefore, there is a need for a better understanding of the global DNA methylation differences among UADT tumors. We performed a genome-wide DNA methylation analysis of esophageal (ESCC), laryngeal (LSCC), oral (OSCC) and oropharyngeal (OPSCC) squamous cell carcinomas, and their non-tumor counterparts. The unsupervised analysis showed that non-tumor tissues present markedly distinct DNA methylation profiles, while tumors are highly heterogeneous. Hypomethylation was more frequent in LSCC and OPSCC, while ESCC and OSCC presented mostly hypermethylation, with the latter showing a CpG island overrepresentation. Differentially methylated regions affected genes in 127 signaling pathways, with only 3.1% of these being common among different tumor subsites, but with different genes affected. The WNT signaling pathway, known to be dysregulated in different epithelial tumors, is a frequent hit for DNA methylation and gene expression alterations in ESCC and OPSCC, but mostly for genetic alterations in LSCC and OSCC. UADT tumor subsites present differences in genome-wide methylation regarding their profile, intensity, genomic regions and signaling pathways affected.

Genome-Wide Differential Methylation Profiles from Two Terpene-Rich Medicinal Plant Extracts Administered in Osteoarthritis Rats

Extracts from the plants Phlomis umbrosa and Dipsacus asperoides—which are widely used in Korean and Chinese traditional medicine to treat osteoarthritis and other bone diseases—were used to treat experimental osteoarthritis (OA) rats. Genome-wide differential methylation regions (DMRs) of these medicinal-plant-treated rats were profiled as therapeutic evidence associated with traditional medicine, and they need to be investigated further using detailed molecular research to extrapolate traditional practices to modern medicine. In total, 49 protein-encoding genes whose expression is differentially regulated during disease progression and recovery have been discovered via systematic bioinformatic analysis and have been approved/proposed as druggable targets for various bone diseases by the US food and drug administration. Genes encoding proteins involved in the PI3K/AKT pathway were found to be enriched, likely as this pathway plays a crucial role during OA progression as well as during the recovery process after treatment with the aforementioned plant extracts. The four sub-networks of PI3K/AKT were highly regulated by these plant extracts. Overall, 29 genes were seen in level 2 (51–75%) DMRs and were correlated highly with OA pathogenesis. Here, we propose that these genes could serve as targets to study OA; moreover, the iridoid and triterpenoid phytochemicals obtained from these two plants may serve as potential therapeutic agents.

Human brain region-specific variably methylated regions are enriched for heritability of distinct neuropsychiatric traits

BackgroundDNA methylation dynamics in the brain are associated with normal development and neuropsychiatric disease and differ across functionally distinct brain regions. Previous studies of genome-wide methylation differences among human brain regions focus on limited numbers of individuals and one to two brain regions.ResultsUsing GTEx samples, we generate a resource of DNA methylation in purified neuronal nuclei from 8 brain regions as well as lung and thyroid tissues from 12 to 23 donors. We identify differentially methylated regions between brain regions among neuronal nuclei in both CpG (181,146) and non-CpG (264,868) contexts, few of which were unique to a single pairwise comparison. This significantly expands the knowledge of differential methylation across the brain by 10-fold. In addition, we present the first differential methylation analysis among neuronal nuclei from basal ganglia tissues and identify unique CpG differentially methylated regions, many associated with ion transport. We also identify 81,130 regions of variably CpG methylated regions, i.e., variable methylation among individuals in the same brain region, which are enriched in regulatory regions and in CpG differentially methylated regions. Many variably methylated regions are unique to a specific brain region, with only 202 common across all brain regions, as well as lung and thyroid. Variably methylated regions identified in the amygdala, anterior cingulate cortex, and hippocampus are enriched for heritability of schizophrenia.ConclusionsThese data suggest that epigenetic variation in these particular human brain regions could be associated with the risk for this neuropsychiatric disorder.

Relationship of DNA methylation and cognitive and neuropsychiatric symptoms among older adults

<h3>Introduction</h3> Accelerated biological aging is related to worse brain aging. However, the interface of Alzheimer's Disease (AD) and the biology of aging is little understood. The study of epigenetics and epigenetic aging may yield advances in prevention and treatment of AD by identifying new therapeutic targets or approaches. Further, signals from epigenetic measures may provide novel mechanistic links between neuropsychiatric symptoms (NPS) and cognitive outcomes in aging; NPS are highly prevalent in Alzheimer's Disease and related dementia (ADRD) and even appear in pre-clinical and early stages. <h3>Methods</h3> We included 45 adults aged 60+ years who are participants in a depression prevention ancillary study (VITAL-DEP) to the VITamin D and OmegA-3 TriaL, a randomized trial of fish oil and vitamin D supplements. We identified the presence of NPS using established behavioral measures. Neuropsychiatric Inventory (NPI) severity scores were mapped from dozens of item-level NPS variables extracted from mood and anxiety questionnaires and structured psychiatric diagnostic interviews in the VITAL-DEP protocol. We defined 3 groups based on cognitive status: normal cognition with or without NPS (n=20), and mild cognitive impairment (MCI) with or without NPS (n=25). Genomic DNA was extracted from leukocytes, and DNA methylation was assayed using Illumina Infinium MethylationEPIC BeadArray technology (Methyl850K chip) at baseline and year-2 follow-up. Epigenetic aging metrics [DNA methylation age (DNAmAge), AgeAccel, DNAmGrimAge, DNAmPhenoAge] were computed using the DNA epigenetic clock - Horvath method. <h3>Results</h3> The mean age of participants was 69.8 (range=60.3-83.4) years; percentages of who were females=48.9% and with post-graduate education=62.2%. In preliminary analysis, epigenetic measures were significantly correlated with chronological age (Spearman correlation (rho): for DNAmAge=0.51, p<0.001; for DNAmGrimAge=0.84, p<0.01; for DNAmPhenoAge=0.65, p<0.01). Furthermore, DNAmAge was significantly inversely correlated with NPI score (rho=-0.36, p=0.02). Participants with MCI had higher DNAmPhenoAge and DNAmGrimAge compared to those with normal cognition [median (interquartile range): for DNAmPhenoAge=56.1 vs. 54.4 years; for DNAmGrimAge=69.1 vs. 68.5 years). Additional results of longitudinal 2-year change in DNAmAge and genome-wide differential methylation analyses among participants with MCI, with or without NPS, and normal cognition will be presented. <h3>Conclusions</h3> Our preliminary results suggest that accelerated biological aging may be related to cognitive and neuropsychiatric phenotypes.

Genome-Wide Differentially Methylated Region Analysis to Reveal Epigenetic Differences of Articular Cartilage in Kashin-Beck Disease and Osteoarthritis.

Kashin–Beck disease (KBD) is a degenerative osteoarticular disorder, and displays the significant differences with osteoarthritis (OA) regarding the etiology and molecular changes in articular cartilage. However, the underlying dysfunctions of molecular mechanisms in KBD and OA remain unclear. Here, we primarily performed the various genome-wide differential methylation analyses to reveal the distinct differentially methylated regions (DMRs) in conjunction with corresponding differentially methylated genes (DMGs), and enriched functional pathways in KBD and OA. We identified a total of 131 DMRs in KBD vs. Control, and 58 DMRs in OA vs. Controls, and the results demonstrate that many interesting DMRs are linked to DMGs, such as SMOC2 and HOXD3, which are all key genes to regulate cartilage/skeletal physiologic and pathologic process, and are further enriched in skeletal system and limb-associated pathways. Our DMR analysis indicates that KBD-associated DMRs has higher proportion than OA-associated DMRs in gene body regions. KBD-associated DMGs were enriched in wounding and coagulation-related functional pathways that may be stimulated by trace elements. The identified molecular features provide novel clues for understanding the pathogenetic and therapeutic studies of both KBD and OA.

Abstract 1205: Leveraging genome-wide methylation data to understand lung cancer risk in heavy smokers

Abstract Epigenome-wide association studies (EWAS) have been used to identify variation in methylation of cytosine-phosphate-guanine (CpG) sites associated with many phenotypes, including lung cancer. A recent meta-EWAS revealed that associations between CpG methylation and lung cancer risk differ by smoking status. Also, smoking status, intensity, and time since cessation are associated with changes in peripheral blood DNA methylation across the genome. We sought to address whether genome-wide methylation differences are associated with lung cancer risk in 316 matched lung cancer case and control pairs from the Beta Carotene and Retinol Efficacy Trial (CARET) of heavy smokers (≥20 pack years). Case-control pairs were matched on age (±5 years), sex, race/ethnicity, enrollment year (±2 years), smoking status (ever/never), occupational asbestos exposure (yes/no), and follow-up time. Genome-wide DNA methylation was assayed on the Illumina EPIC array in whole blood samples collected on average 4.1 years prior to diagnosis in cases. To improve scalability for testing, we mapped 866,326 normalized and processed EPIC array CpGs to 26,994 gene regions using the EPIC array annotation. We performed region-based variance component tests for associations between each region and lung cancer risk. We modeled associations as random effects such that changes reflecting both hyper- and hypo-methylation across different CpG sites in a gene region cumulatively contribute to the overall variance component test. The test assesses whether the variance associated with the regression coefficient for each CpG in the region is equal to zero. Since this method does not have a conditional adaptation for matched study designs, our region-based models included adjustment for age, sex, race/ethnicity, enrollment year, smoking status, and occupational asbestos exposure, as well as pack years and the first two principal components estimated from the genome-wide methylation data. Our preliminary results include 66 genes (12 in adenocarcinoma, 33 in squamous, 21 in small cell) with permutation-based P&amp;lt;0.001. The top statistically significant associations (all P&amp;lt;0.0001) were TMEM63C in adenocarcinoma, MCC in squamous cell carcinoma, and GRM5-AS1, UBE2QL1, and TNNI1 in small cell lung cancer. GWAS studies have shown associations between single nucleotide polymorphisms (SNPs) in TNNI1 and lung function, and also SNPs from the TMEM and UBE2 gene families with lung cancer risk. Our findings suggest that region-based testing may be applied for association studies of genome-wide methylation where site-specific targeting of methylation markers may be underpowered to individually associate with risk. Citation Format: Laurie Grieshober, Jincheng Shen, Stefan Graw, Matt J. Barnett, Mark D. Thornquist, Gary E. Goodman, Chu Chen, Devin C. Koestler, Carmen J. Marsit, Jennifer A. Doherty. Leveraging genome-wide methylation data to understand lung cancer risk in heavy smokers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1205.