Peripheral Blood DNA Methylation Research Articles

Epigenome-wide association study of lung cancer among never smokers in two prospective cohorts in Shanghai, China

BackgroundThe aetiology of lung cancer among individuals who never smoked remains elusive, despite 15% of lung cancer cases in men and 53% in women worldwide being unrelated to smoking. Epigenetic...

The Impact of Sample Storage on Blood Methylation: Towards Assessing Myelin Gene Methylation as a Biomarker for Progressive Multiple Sclerosis.

One of the major challenges in multiple sclerosis (MS) is to accurately monitor and quantify disability over time. Thus, there is a pressing need to identify new biomarkers for disease progression. Peripheral blood DNA methylation has been demonstrated to be an easily accessible and quantifiable marker in many neurodegenerative diseases. In this study, we aimed to investigate whether methylation patterns that were previously determined in chronic inactive white matter lesions of patients with progressive MS are also reflected in the blood, and whether the latter can serve as a biomarker for disease progression in MS. While our initial analysis revealed differences in the blood methylation state of important myelin-related genes between patients with progressive MS and controls, these findings could not be validated in other independent patient cohorts. Subsequent investigation suggests that sample storage can selectively influence DNA methylation patterns, potentially hindering accurate epigenetic analysis. Therefore, sample storage time should be taken into consideration during the initial sample selection stage in biomarker studies.

Multimodal Associations of FKBP5 Methylation with Emotion-Regulatory Brain Circuits

Understanding the biological processes underlying individual differences in emotion regulation and stress responsivity is a key challenge for translational neuroscience. The gene FKBP5 is a core regulator in molecular stress signaling that is implicated in the development of psychiatric disorders. Yet it remains unclear how FKBP5 DNA methylation (DNAm) in peripheral blood relates to individual differences in measures of neural structure and function, and their relevance to daily-life stress responsivity. Here, we characterize multimodal correlates of FKBP5 DNAm by combining epigenetic data with neuroimaging and Ambulatory Assessment in a sample of 395 healthy individuals. First, we show that FKBP5 demethylation as a psychiatric risk factor relates to an anxiety-associated reduction of gray matter volume in the ventromedial prefrontal cortex (vmPFC), a brain area that is involved in emotion regulation and mental health risk and resilience. This effect of epigenetic upregulation of FKBP5 on neuronal structure is more pronounced where FKBP5 is epigenetically downregulated at baseline. Leveraging 208 functional MRI scans during a well-established emotion processing task we find that FKBP5 DNAm in peripheral blood is associated with functional difference of prefrontal-limbic circuits modulating affective responsivity to daily stressors, which we measured using ecological momentary assessment in daily life. Overall, we demonstrate how FKBP5 contributes to interindividual differences in neural and real-life affect regulation via structural and functional changes in prefrontal-limbic brain circuits.

P592 Higher predictive power of epigenetic signatures for response to vedolizumab and ustekinumab in anti-TNF naïve patients with active Crohn’s disease

Abstract Background Despite the significant progress in IBD care with biological therapies, the current 'trial-and-error' method remains suboptimal. Previous results from our EPIC-CD study demonstrated that two distinct epigenetic panels of 25 and 68 DNA methylation markers, respectively, were associated with (non-)response to vedolizumab (VDZ) and ustekinumab (USTE) in active Crohn’s disease (CD)1. Data from clinical trials have demonstrated that response rates in patients naïve to biological treatment are higher. We therefore aimed to evaluate the performance of our models in bionaive and anti-TNF exposed subpopulations. Methods We prospectively measured peripheral blood DNA methylation profiles from 184 adult CD patients prior to treatment with VDZ or USTE in a discovery (n=126) and independent validation (n=58) cohort using the Illumina EPIC BeadChip array. Between 26 and 52 weeks, patients were categorized as responders or non-responders based on a combination of endoscopic response (≥50% reduction in SES-CD score), steroid-free clinical response (≥3-point drop in Harvey-Bradshaw Index (HBI) or HBI ≤4 AND no systemic steroids), and/or biochemical response (≥50% reduction in C-reactive protein (CRP) and fecal calprotectin or a CRP ≤5 g/mL and fecal calprotectin ≤250 µg/g). Epigenetic biomarker identification and classification analyses were conducted through stability selection gradient boosting on the discovery cohort. Subsequently, the performance of the identified epigenetic biomarkers was assessed independently on the validation cohort, with patients stratified by prior anti-TNF exposure. Results Out of the 58 patients in the validation cohort, 25 received treatment with VDZ, and 33 with USTE, with 52% (n=13) and 36% (n=12) respectively naïve to anti-TNF treatment. Baseline characteristics between anti-TNF naive and anti-TNF exposed patients showed no significant differences, despite a slight significant age difference in the vedolizumab group, where anti-TNF exposed patients were significantly younger (table 1). In the overall independent validation cohort, the prediction models demonstrated an AUC of 0.75 for both VDZ and USTE. However, after stratification by prior anti-TNF exposure, we observed increased performance of our models among anti-TNF naive patients for both VDZ (AUCnon-exposed=0.85 versus AUCexposed=0.66) and USTE (AUCnon-exposed=0.97 versus AUCexposed=0.63 (figure 1). Conclusion Our findings demonstrate that prior anti-TNF exposure leads to somewhat reduced predictive power of our epigenetic models for Vedolizumab and Ustekinumab response. The predictive models may therefore find its optimal application in bio-naive patients, although they remain significant in anti-TNF exposed patients.

Novel DNA methylome biomarkers associated with adalimumab response in rheumatoid arthritis patients.

Rheumatoid arthritis (RA) patients are currently treated with biological agents mostly aimed at cytokine blockade, such as tumor necrosis factor-alpha (TNFα). Currently, there are no biomarkers to predict therapy response to these agents. Here, we aimed to predict response to adalimumab (ADA) treatment in RA patients using DNA methylation in peripheral blood (PBL). DNA methylation profiling on whole peripheral blood from 92 RA patients before the start of ADA treatment was determined using Illumina HumanMethylationEPIC BeadChip array. After 6 months, treatment response was assessed according to the European Alliance of Associations for Rheumatology (EULAR) criteria for disease activity. Patients were classified as responders (Disease Activity Score in 28 Joints (DAS28) < 3.2 or decrease of 1.2 points) or as non-responders (DAS28 > 5.1 or decrease of less than 0.6 points). Machine learning models were built through stability-selected gradient boosting to predict response prior to ADA treatment with predictor DNA methylation markers. Of the 94 RA patients, we classified 49 and 43 patients as responders and non-responders, respectively. We were capable of differentiating responders from non-responders with a high performance (area under the curve (AUC) 0.76) using a panel of 27 CpGs. These classifier CpGs are annotated to genes involved in immunological and pathophysiological pathways related to RA such as T-cell signaling, B-cell pathology, and angiogenesis. Our findings indicate that the DNA methylome of PBL provides discriminative capabilities in discerning responders and non-responders to ADA treatment and may therefore serve as a tool for therapy prediction.

EPIGENETIC AGING AND RHEUMATOID ARTHRITIS

Abstract Indicators of biological aging with rheumatoid arthritis (RA), a chronic autoimmune disease, are not yet fully known. In this cross-sectional study, we tested associations between plasma indicators of epigenetic aging using online data (GSE42861) regarding RA and DNA methylation from the Swedish Epidemiological Investigation of Rheumatoid Arthritis (EIRA). Peripheral blood DNA methylation of participants 18-70 years old was assessed using Illumina HumanMethylation450 arrays. Horvath’s online DNA Methylation Age Calculator determined DNAm estimate of Telomere length (DNAmTL), Hannum’s, Horvath’s 2013 and 2018 epigenetic ages, PhenoAge and GrimAge and the respective age accelerations. Association of RA prevalence with epigenetic age were determined using linear regression, correcting for false discovery rate. We identified significant associations of RA with age acceleration measures of Horvath 2013 (Estimate:-1.34 ; FDR p-value: 1.0 × 10-2) and Horvath 2018 (Estimate:-1.32; FDR p-value: 4.0 × 10-5), extrinsic age acceleration (Estimate: 1.34 ; FDR p-value: 1.0 × 10-2), PhenoAge acceleration (Estimate: 2.31; FDR p-value: 1.1 × 10-5), GrimAge (Estimate: 2.54; FDR p-value: 1.0 × 10-2) and GrimAge acceleration (Estimate: 3.15 ; FDR p-value: 1.7 × 10-17), adjusting for sex and smoking (FDR p-value&amp;lt;=0.05). In sex stratified analyses, first-generation clocks were associated only in women, but no sex specific effects for PhenoAge or GrimAge accelerations were identified. The second generation ‘biological’ clocks PhenoAge, GrimAge and its components showed higher epigenetic age acceleration in RA cases compared to healthy controls. We conclude that second generation indicators of epigenetic aging provide promise as markers of biological aging in people with RA.

Blood DNA methylation and liver cancer in American Indians: evidence from the Strong Heart Study

PurposeLiver cancer incidence among American Indians/Alaska Natives has risen over the past 20 years. Peripheral blood DNA methylation may be associated with liver cancer and could be used as a biomarker for cancer risk. We evaluated the association of blood DNA methylation with risk of liver cancer.MethodsWe conducted a prospective cohort study in 2324 American Indians, between age 45 and 75 years, from Arizona, Oklahoma, North Dakota and South Dakota who participated in the Strong Heart Study between 1989 and 1991. Liver cancer deaths (n = 21) were ascertained using death certificates obtained through 2017. The mean follow-up duration (SD) for non-cases was 25.1 (5.6) years and for cases, 11.0 (8.8) years. DNA methylation was assessed from blood samples collected at baseline using MethylationEPIC BeadChip 850 K arrays. We used Cox regression models adjusted for age, sex, center, body mass index, low-density lipoprotein cholesterol, smoking, alcohol consumption, and immune cell proportions to examine the associations.ResultsWe identified 9 CpG sites associated with liver cancer. cg16057201 annotated to MRFAP1) was hypermethylated among cases vs. non-cases (hazard ratio (HR) for one standard deviation increase in methylation was 1.25 (95% CI 1.14, 1.37). The other eight CpGs were hypomethylated and the corresponding HRs (95% CI) ranged from 0.58 (0.44, 0.75) for cg04967787 (annotated to PPRC1) to 0.77 (0.67, 0.88) for cg08550308. We also assessed 7 differentially methylated CpG sites associated with liver cancer in previous studies. The adjusted HR for cg15079934 (annotated to LPS1) was 1.93 (95% CI 1.10, 3.39).ConclusionsBlood DNA methylation may be associated with liver cancer mortality and may be altered during the development of liver cancer.

DNA methylation sites in early adulthood characterised by pubertal timing and development: a twin study

BackgroundPuberty is a highly heritable and variable trait, with environmental factors having a role in its eventual timing and development. Early and late pubertal onset are both associated with various diseases developing later in life, and epigenetic characterisation of pubertal timing and development could lead to important insights. Blood DNA methylation, reacting to both genotype and environment, has been associated with puberty; however, such studies are relatively scarce. We investigated peripheral blood DNA methylation profiles (using Illumina 450 K and EPIC platforms) of 1539 young adult Finnish twins associated with pubertal development scale (PDS) at ages 12 and 14 as well as pubertal age (PA).ResultsFixed effect meta-analysis of the two platforms on 347,521 CpGs in common identified 58 CpG sites associated (p < 1 × 10−5) with either PDS or PA. All four CpGs associated with PA and 45 CpGs associated with PDS were sex-specific. Thirteen CpGs had a high heritability (h2: 0.51–0.98), while one CpG site (mapped to GET4) had a high shared environmental component accounting for 68% of the overall variance in methylation at the site. Utilising twin discordance analysis, we found 6 CpG sites (5 associated with PDS and 1 with PA) that had an environmentally driven association with puberty. Furthermore, genes with PDS- or PA-associated CpGs were consistently linked to various developmental processes and diseases such as breast, prostate and ovarian cancer, while methylation quantitative trait loci of associated CpG sites were enriched in immune pathways developing during puberty.ConclusionsBy identifying puberty-associated DNA methylation sites and examining the effects of sex, environment and genetics, we shed light on the intricate interplay between environment and genetics in the context of puberty. Through our comprehensive analysis, we not only deepen the understanding of the significance of both genetic and environmental factors in the complex processes of puberty and its timing, but also gain insights into potential links with disease risks.

Identification of an association between coronary heart disease and ITGB2 methylation in peripheral blood by a case-control study

BackgroundBlood DNA methylation was associated with coronary heart disease (CHD) risk in Caucasians. We investigated the association between DNA methylation in peripheral blood at the reported loci and CHD in the Chinese population. MethodsThe integrin subunit beta 2 (ITGB2) gene was identified in 196 CHD cases and 184 controls, and its methylation level was determined by mass spectrometry. Logistic regression was used to assess the association. ResultsHypomethylation of ITGB2 was significantly associated with heart failure CHD and NYHA Ⅰ&Ⅱ CHD patients with minor to medium cardiac function impairment (ITGB2_CpG_11/cg08422803, OR per −10 % methylation = 1.15 and 1.16; p = 0.012 and 0.018 by Bonferroni correction, respectively). Hypomethylation of ITGB2_CpG_11/cg08422803 was a risk factor for CHD in people < 65 years and males (p < 0.05 after Bonferroni correction). The combination of ITGB2 methylation and conventional CHD risk factors could efficiently discriminate CHD, heart failure CHD, NYHA I&II CHD, and myocardial infarction CHD patients from controls (AUC = 0.78, 0.81, 0.80, and 0.81, respectively). ConclusionBlood-based ITGB2 methylation has the potential as a biomarker for CHD. The combination of ITGB2 methylation and conventional CHD risk factors may improve the risk assessment and detection of CHD.

Peripheral blood DNA methylation and neuroanatomical responses to HDACi treatment that rescues neurological deficits in a Kabuki syndrome mouse model

BackgroundRecent findings from studies of mouse models of Mendelian disorders of epigenetic machinery strongly support the potential for postnatal therapies to improve neurobehavioral and cognitive deficits. As several of these therapies move into human clinical trials, the search for biomarkers of treatment efficacy is a priority. A potential postnatal treatment of Kabuki syndrome type 1 (KS1), caused by pathogenic variants in KMT2D encoding a histone-lysine methyltransferase, has emerged using a mouse model of KS1 (Kmt2d+/βGeo). In this mouse model, hippocampal memory deficits are ameliorated following treatment with the histone deacetylase inhibitor (HDACi), AR-42. Here, we investigate the effect of both Kmt2d+/βGeo genotype and AR-42 treatment on neuroanatomy and on DNA methylation (DNAm) in peripheral blood. While peripheral blood may not be considered a “primary tissue” with respect to understanding the pathophysiology of neurodevelopmental disorders, it has the potential to serve as an accessible biomarker of disease- and treatment-related changes in the brain.MethodsHalf of the KS1 and wildtype mice were treated with 14 days of AR-42. Following treatment, fixed brain samples were imaged using MRI to calculate regional volumes. Blood was assayed for genome-wide DNAm at over 285,000 CpG sites using the Illumina Infinium Mouse Methylation array. DNAm patterns and brain volumes were analyzed in the four groups of animals: wildtype untreated, wildtype AR-42 treated, KS1 untreated and KS1 AR-42 treated.ResultsWe defined a DNAm signature in the blood of KS1 mice, that overlapped with the human KS1 DNAm signature. We also found a striking 10% decrease in total brain volume in untreated KS1 mice compared to untreated wildtype, which correlated with DNAm levels in a subset KS1 signature sites, suggesting that disease severity may be reflected in blood DNAm. Treatment with AR-42 ameliorated DNAm aberrations in KS1 mice at a small number of signature sites.ConclusionsAs this treatment impacts both neurological deficits and blood DNAm in mice, future KS clinical trials in humans could be used to assess blood DNAm as an early biomarker of therapeutic efficacy.

Clinico-biological refinement of BCL11B-related disorder and identification of an episignature: A series of 20 unreported individuals

PurposeBCL11B-related disorder (BCL11B-RD) arises from rare genetic variants within the BCL11B gene, resulting in a distinctive clinical spectrum encompassing syndromic neurodevelopmental disorder, with or without intellectual disability, associated with facial features and impaired immune function. This study presents an in-depth clinico-biological analysis of 20 newly reported individuals with BCL11B-RD, coupled with a characterization of genome-wide DNA methylation patterns of this genetic condition. MethodsThrough an international collaboration, clinical and molecular data from 20 individuals were systematically gathered, and a comparative analysis was conducted between this series and existing literature. We further scrutinized peripheral blood DNA methylation profile of individuals with BCL11B-RD, contrasting them with healthy controls and other neurodevelopmental disorders marked by established episignature. ResultsOur findings unveil rarely documented clinical manifestations, notably including Rubinstein-Taybi-like facial features, craniosynostosis, and autoimmune disorders, all manifesting within the realm of BCL11B-RD. We refine the intricacies of T cell compartment alterations of BCL11B-RD, revealing decreased levels naive CD4+ T cells and recent thymic emigrants while concurrently observing an elevated proportion of effector-memory expressing CD45RA CD8+ T cells (TEMRA). Finally, a distinct DNA methylation episignature exclusive to BCL11B-RD is unveiled. ConclusionThis study serves to enrich our comprehension of the clinico-biological landscape of BCL11B-RD, potentially furnishing a more precise framework for diagnosis and follow-up of individuals carrying pathogenic BCL11B variant. Moreover, the identification of a unique DNA methylation episignature offers a valuable diagnosis tool for BCL11B-RD, thereby facilitating routine clinical practice by empowering physicians to reevaluate variants of uncertain significance within the BCL11B gene.

DNA Methylation Patterns in Relation to Acute Severity and Duration of Anxiety and Depression.

Depression and anxiety are common mental disorders that often occur together. Stress is an important risk factor for both disorders, affecting pathophysiological processes through epigenetic changes that mediate gene-environment interactions. In this study, we explored two proposed models about the dynamic nature of DNA methylation in anxiety and depression: a stable change, in which DNA methylation accumulates over time as a function of the duration of clinical symptoms of anxiety and depression, or a flexible change, in which DNA methylation correlates with the acute severity of clinical symptoms. Symptom severity was assessed using clinical questionnaires for anxiety and depression (BDI-II, IDS-C, and HAM-A), and the current episode and the total lifetime symptom duration was obtained from patients' medical records. Peripheral blood DNA methylation levels were determined for the BDNF, COMT, and SLC6A4 genes. We found a significant negative correlation between COMT_1 amplicon methylation and acute symptom scores, with BDI-II (R(22) = 0.190, p = 0.033), IDS-C (R(22) = 0.199, p = 0.029), and HAM-A (R(22) = 0.231, p = 0.018) all showing a similar degree of correlation. Our results suggest that DNA methylation follows flexible dynamics, with methylation levels closely associated with acute clinical presentation rather than with the duration of anxiety and depression. These results provide important insights into the dynamic nature of DNA methylation in anxiety and affective disorders and contribute to our understanding of the complex interplay between stress, epigenetics, and individual phenotype.

Hippocampal and peripheral blood DNA methylation signatures correlate at the gene and pathway level in a mouse model of autism.

Autism spectrum disorders (ASD) are polygenic multifactorial disorders influenced by environmental factors. ASD-related differential DNA methylation has been found in human peripheral tissues, such as placenta, paternal sperm, buccal epithelium, and blood. However, these data lack direct comparison of DNA methylation levels with brain tissue from the same individual to determine the extent that peripheral tissues are surrogates for behavior-related disorders. Here, whole genome methylation profiling at all the possible sites throughout the mouse genome (>25 million) from both brain and blood tissues revealed novel insights into the systemic contributions of DNA methylation to ASD. Sixty-six differentially methylated regions (DMRs) share the same genomic coordinates in these two tissues, many of which are linked to risk genes for neurodevelopmental disorders and intellectual disabilities (e.g. Prkch, Ptn, Hcfc1, Mid1, and Nfia). Gene ontological pathways revealed a significant number of common terms between brain and blood (N = 65 terms), and nearly half (30/65) were associated with brain/neuronal development. Furthermore, seven DMR-associated genes among these terms contain methyl-sensitive transcription factor sequence motifs within the DMRs of both tissues; four of them (Cux2, Kcnip2, Fgf13, and Mrtfa) contain the same methyl-sensitive transcription factor binding sequence motifs (HES1/2/5, TBX2 and TFAP2C), suggesting DNA methylation influences the binding of common transcription factors required for gene expression. Together, these findings suggest that peripheral blood is a good surrogate tissue for brain and support that DNA methylation contributes to altered gene regulation in the pathogenesis of ASD.

The role and mechanism of myeloperoxidase in dermatomyositis

ObjectiveDermatomyositis (DM) is the best known subtype of idiopathic inflammatory myopathies. The hallmarks of DM muscle pathology including microangiopathy, inflammatory infiltration, and perifascicular atrophy. Recent findings have revealed pathogenetic effects of myeloperoxidase (MPO) by causing oxidative damage and regulating abnormal immunity in multiple disease conditions. In this study, we aimed to explore the role of MPO in the pathogenesis of DM. MethodsThe peripheral blood mononuclear cell (PBMC) mRNA expression and DNA methylation of MPO were verified using real-time qPCR and bisulfite pyrosequencing, respectively. Plasma MPO levels were measured with enzyme-linked immunosorbent assay, and their relationships with clinical characteristics were analyzed. The expression and distribution of MPO in muscle were tested by immunofluorescence. Purified human native MPO protein was used to stimulate human dermal microvascular endothelial cells (HDMECs) and skeletal muscle myotubes. The cell viability, tube forming capacity, permeability, adhesion molecule expressions in HDMECs, and atrophy and programmed cell death pathways in myotubes were then observed. ResultsMPO gene methylation was decreased, while mRNA expression and plasma levels were increased in DM. Plasma MPO of DM patients was positively correlated with serum creatine kinase (CK). MPO mainly distributed around endomysia capillaries and perifascicular atrophy in DM muscle biopsies, and was co-localized with CD4+, CD8+ T cells and CD19+ B cells. MPO not only could influence the cell viability, tube forming capacity, permeability and expression of adhesion molecules (including ICAM 1, VCAM 1 and E-selectin) of HDMECs, but also could cause atrophy of myotubes. ConclusionsOur study disclosed, for the first time, that MPO plays an important role in promoting inflammatory infiltration and inducing muscle damage in DM patients. MPO may be a potential biomarker for DM muscle involvement and MPO targeted drugs may be promising in DM treatment.

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.

Hypomethylation of DYRK4 in peripheral blood is associated with increased lung cancer risk.

Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. It is urgent to identify new biomarkers for the early detection of LC. DNA methylation in peripheral blood has been reported to be associated with cancers. We conducted two independent case-control studies and a nested case-control study (168 LC cases and 167 controls in study Ⅰ, 677 LC cases and 833 controls in study Ⅱ, 147 precancers and 21 controls in the nested case-control study). The methylation levels of DYRK4 CpG sites were measured using mass spectrometry and their correlations with LC were analyzed by logistic regression and nonparametric tests. Bonferroni correction was used for the multiple comparisons. LC-related decreased DYRK4 methylation was discovered in Study I and validated in Study II (the odds ratios [ORs] for the lowest vs. highest quartile of all three DYRK4 CpG sites ranged from 1.64 to 2.09, all p < 0.001). Combining the two studies, hypomethylation of DYRK4 was observed in stage I cases (ORs per -10% methylation ranged from 1.16 to 1.38, all p < 5.9E-04), and could be enhanced by male gender (ORs ranged from 1.77 to 4.17 via interquartile analyses, all p < 0.017). Hypomethylation of DYRK4_A_CpG_2 was significantly correlated with tumor size, length, and stage (p = 0.034, 0.002, and 0.002, respectively) in LC cases. Our study disclosed the association between DYRK4 hypomethylation in peripheral blood and LC, suggesting the feasibility of blood-based DNA methylation as new biomarker for LC detection.

Peripheral blood DNA methylation signatures and response to tofacitinib in moderate-to-severe ulcerative colitis.

Predictive biomarkers for treatment efficacy of ulcerative colitis (UC) treatments are lacking. Here, we performed a longitudinal study investigating the association and potential predictive power of genome-wide peripheral blood (PB) DNA methylation signatures and response to tofacitinib treatment in UC. We recruited moderate-to-severe UC patients starting tofacitinib treatment and measured PB DNA methylation profiles at baseline (T1), after 8 weeks (T2), and in a subset (n=8), after a median of 20 weeks (T3) using the Illumina Infinium HumanMethylation EPIC BeadChip. After 8 weeks, we categorized responders (R) from non-responders (NR) based on a centrally read endoscopic response (decrease in endoscopic mayo score ≥1 or UCEIS ≥2) combined with corticosteroid-free clinical- and/or biochemical response. T1 PB samples were used for biomarker identification, while T2 and publicly available intra-class correlation (ICC) data were used for stability analyses. RNA-sequencing was performed to understand the downstream effects of the predictor CpG loci. In total, 16 R and 15 NR patients with a median disease duration of 7 (4-12) years and overall comparable patient characteristics at baseline were analyzed. We identified a panel of 53 differentially methylated positions (DMPs) associated with response to tofacitinib (AUROC 0.74). Most DMPs (77%) demonstrated both short- and long-term hyper stability (ICC ≥0.90), irrespective of inflammatory status. Gene expression analysis showed lower FGFR2 (pBH=0.011) and LRPAP1 (pBH=0.020), and higher OR2L13 (pBH=0.016) expression at T1 in R compared to NR. Our observations demonstrate the utility of genome-wide PB DNA methylation signatures to predict response to tofacitinib.

Genome-Scale Methylation Analysis Identifies Immune Profiles and Age Acceleration Associations with Bladder Cancer Outcomes.

Immune profiles have been associated with bladder cancer outcomes and may have clinical applications for prognosis. However, associations of detailed immune cell subtypes with patient outcomes remain underexplored and may contribute crucial prognostic information for better managing bladder cancer recurrence and survival. Bladder cancer case peripheral blood DNA methylation was measured using the Illumina HumanMethylationEPIC array. Extended cell-type deconvolution quantified 12 immune cell-type proportions, including memory, naïve T and B cells, and granulocyte subtypes. DNA methylation clocks determined biological age. Cox proportional hazards models tested associations of immune cell profiles and age acceleration with bladder cancer outcomes. The partDSA algorithm discriminated 10-year overall survival groups from clinical variables and immune cell profiles, and a semi-supervised recursively partitioned mixture model (SS-RPMM) with DNA methylation data was applied to identify a classifier for 10-year overall survival. Higher CD8T memory cell proportions were associated with better overall survival [HR = 0.95, 95% confidence interval (CI) = 0.93-0.98], while higher neutrophil-to-lymphocyte ratio (HR = 1.36, 95% CI = 1.23-1.50), CD8T naïve (HR = 1.21, 95% CI = 1.04-1.41), neutrophil (HR = 1.04, 95% CI = 1.03-1.06) proportions, and age acceleration (HR = 1.06, 95% CI = 1.03-1.08) were associated with worse overall survival in patient with bladder cancer. partDSA and SS-RPMM classified five groups of subjects with significant differences in overall survival. We identified associations between immune cell subtypes and age acceleration with bladder cancer outcomes. The findings of this study suggest that bladder cancer outcomes are associated with specific methylation-derived immune cell-type proportions and age acceleration, and these factors could be potential prognostic biomarkers.

Brain serotonin 1A receptor binding: relationship to peripheral blood DNA methylation, recent life stress and childhood adversity in unmedicated major depression.

Childhood and lifetime adversity may reduce brain serotonergic (5-HT) neurotransmission by epigenetic mechanisms. We tested the relationships of childhood adversity and recent stress to serotonin 1A (5-HT1A) receptor genotype, DNA methylation of this gene in peripheral blood monocytes and in vivo 5-HT1A receptor binding potential (BPF) determined by positron emission tomography (PET) in 13 a priori brain regions, in participants with major depressive disorder (MDD) and healthy volunteers (controls). Medication-free participants with MDD (n = 192: 110 female, 81 male, 1 other) and controls (n = 88: 48 female, 40 male) were interviewed about childhood adversity and recent stressors and genotyped for rs6295. DNA methylation was assayed at three upstream promoter sites (-1019, -1007, -681) of the 5-HT1A receptor gene. A subgroup (n = 119) had regional brain 5-HT1A receptor BPF quantified by PET. Multi-predictor models were used to test associations between diagnosis, recent stress, childhood adversity, genotype, methylation and BPF. Recent stress correlated positively with blood monocyte methylation at the -681 CpG site, adjusted for diagnosis, and had positive and region-specific correlations with 5-HT1A BPF in participants with MDD, but not in controls. In participants with MDD, but not in controls, methylation at the -1007 CpG site had positive and region-specific correlations with binding potential. Childhood adversity was not associated with methylation or BPF in participants with MDD. These findings support a model in which recent stress increases 5-HT1A receptor binding, via methylation of promoter sites, thus affecting MDD psychopathology.

Stage 2 Registered Report: Epigenetic Intergenerational Transmission: Mothers’ Adverse Childhood Experiences and DNA Methylation

Individual differences in risk for mental disorders over the lifespan are shaped by forces acting before the individual is born-in utero, but likely even earlier, during the mother's own childhood. The environmental epigenetics hypothesis proposes that sustained effects of environmental conditions on gene expression are mediated by epigenetic mechanisms. Recent human studies have shown that adversities in childhood are correlated with DNA methylation (DNAm) in adulthood. In the current study, we tested the following pre-registered hypotheses: Mothers' adverse childhood experiences (ACEs) are correlated with DNAm in peripheral blood during pregnancy (hypothesis 1) and in cord blood samples from newborn infants (hypothesis 2), and women's depression and anxiety symptoms during pregnancy mediate the association between mothers' ACE exposure and prenatal/neonatal DNA methylation (hypothesis 3). Data were from the Avon Longitudinal Study of Parents and Children Accessible Resource for Integrated Epigenomic Studies substudy. Women provided retrospective self-reports during pregnancy of ACE exposure. We conducted an epigenome-wide association study testing whether mothers' ACE exposure, cumulative score (0-10), was associated with DNAm in maternal antenatal blood and infant cord blood in more than 450,000 CpG (point on DNA sequence where cytosine and guanine base pairs are linked by a phosphate, where methylation usually occurs) sites on the Illumina 450K BeadChip. Analyses for cord blood were separated by infant sex, a pre-registered analysis. Hypothesis 1: In 896 mother-infant pairs with available methylation and ACE exposure data, there were no significant associations between mothers' ACE score and DNAm from antenatal peripheral blood, after controlling for covariates. Hypothesis 2: In infant cord blood, there were 5 CpG sites significantly differentially methylated in relation to mothers' ACEs (false discovery rate [FDR]< .05), but only in male offspring. Effect sizes were medium, with partial eta squared values ranging from 0.060 to 0.078. CpG sites were in genes related to mitochondrial function and neuronal development in the cerebellum. Hypothesis 3: There was no mediation by maternal anxiety/depression symptoms found between mothers' ACEs score and DNAm in the significant CpG sites in male cord blood. Mediation was not tested in antenatal peripheral blood, because no direct association between mothers' ACE score and antenatal peripheral blood was found. Our results show that mothers' ACE exposure is associated with DNAm in male offspring, supporting the notion that DNAm could be a marker of intergenerational biological embedding of mothers' childhood adversity. Epigenetic Intergenerational Transmission: Mothers' Adverse Childhood Experiences and DNA Methylation; https://doi.org/10.1016/j.jaac.2020.03.008.