Infinium HumanMethylation450 BeadChip Array Research Articles

DNA methylation patterns of circadian and ultradian genes are altered in the peripheral blood of patients with hidradenitis suppurativa.

Hidradenitis suppurativa (HS) is a chronic inflammatory skin condition that affects hair follicles in areas with apocrine sweat glands, such as the underarms, groin, and buttocks. The pathogenesis of HS is not fully understood, but considering the key role played by the biological clock in the control of immune/inflammatory processes the derangement of circadian and ultradian pathways could be hypothesized. We analyzed genome-wide DNA methylation patterns in peripheral blood from 24 HS cases and 24 controls using the Infinium HumanMethylation450 BeadChip array (Illumina), followed by bioinformatics and statistical analyses. We found that several circadian and ultradian genes were differentially methylated in HS patients, predominantly exhibiting hypomethylation. These genes were enriched in pathways such as MAPK and WNT cascades, acute phase response, cytokine release, inflammation, innate immune response, xenobiotic detoxification, and oxidative stress response. Altered DNA methylation patterns of genes related to circadian and ultradian pathways could contribute to immune system derangement and inflammatory processes chronicization in addition to other comorbidities hallmarking HS onset and progression, at the same time representing possible druggable targets.

Placental DNA methylation signatures of prenatal air pollution exposure and potential effects on birth outcomes: an analysis of three prospective cohorts.

Pregnancy air pollution exposure (PAPE) has been linked to a wide range of adverse birth and childhood outcomes, but there is a paucity of data on its influence on the placental epigenome, which can regulate the programming of physiological functions and affect child development. This study aimed to investigate the association between prenatal air pollutant exposure concentrations and changes in placental DNA methylation patterns, and to explore the potential windows of susceptibility and sex-specific alterations. This multi-site study used three prospective population-based mother-child cohorts: EDEN, PELAGIE, and SEPAGES, originating from four French geographical regions (Nancy, Poitiers, Brittany, and Grenoble). Pregnant women were included between 2003 and 2006 for EDEN and PELAGIE, and between 2014 and 2017 for SEPAGES. The main eligibility criteria were: being older than 18 years, having a singleton pregnancy, and living and planning to deliver in one of the maternity clinics in one of the study areas. A total of 1539 mother-child pairs were analysed, measuring placental DNA methylation using Illumina BeadChips. We used validated spatiotemporally resolved models to estimate PM2·5, PM10, and NO2 exposure over each trimester of pregnancy at the maternal residential address. We conducted a pooled adjusted epigenome-wide association study to identify differentially methylated 5'-C-phosphate-G-3' (CpG) sites and regions (assessed using the Infinium HumanMethylationEPIC BeadChip array, n=871), including sex-specific and sex-linked alterations, and independently validated our results (assessed using the Infinium HumanMethylation450 BeadChip array, n=668). We identified four CpGs and 28 regions associated with PAPE in the total population, 469 CpGs and 87 regions in male infants, and 150 CpGs and 66 regions in female infants. We validated 35% of the CpGs available. More than 30% of the identified CpGs were related to one (or more) birth outcome and most significant alterations were enriched for neural development, immunity, and metabolism related genes. The 28 regions identified for both sexes overlapped with imprinted genes (four genes), and were associated with neurodevelopment (nine genes), immune system (seven genes), and metabolism (five genes). Most associations were observed for the third trimester for female infants (134 of 150 CpGs), and throughout pregnancy (281 of 469 CpGs) and the first trimester (237 of 469 CpGs) for male infants. These findings highlight the molecular pathways through which PAPE might affect child health in a widespread and sex-specific manner, identifying the genes involved in the major physiological functions of a developing child. Further studies are needed to elucidate whether these epigenetic changes persist and affect health later in life. French Agency for National Research, Fondation pour la Recherche Médicale, Fondation de France, and the Plan Cancer.

Novel insights into the whole-blood DNA methylome of asthma in ethnically diverse children and youth.

The epigenetic mechanisms of asthma remain largely understudied in African Americans and Hispanics/Latinos, two populations disproportionately affected by asthma. We aimed to identify markers, regions and processes with differential patterns of DNA methylation (DNAm) in whole blood by asthma status in ethnically diverse children and youth, and to assess their functional consequences. DNAm levels were profiled with the Infinium MethylationEPIC or HumanMethylation450 BeadChip arrays among 1226 African Americans or Hispanics/Latinos and assessed for differential methylation per asthma status at the CpG and region (differentially methylated region (DMR)) level. Novel associations were validated in blood and/or nasal epithelium from ethnically diverse children and youth. The functional and biological implications of the markers identified were investigated by combining epigenomics with transcriptomics from study participants. 128 CpGs and 196 DMRs were differentially methylated after multiple testing corrections, including 92.3% and 92.8% novel associations, respectively. 41 CpGs were replicated in other Hispanics/Latinos, prioritising cg17647904 (NCOR2) and cg16412914 (AXIN1) as asthma DNAm markers. Significant DNAm markers were enriched in previous associations for asthma, fractional exhaled nitric oxide, bacterial infections, immune regulation or eosinophilia. Functional annotation highlighted epigenetically regulated gene networks involved in corticosteroid response, host defence and immune regulation. Several implicated genes are targets for approved or experimental drugs, including TNNC1 and NDUFA12. Many differentially methylated loci previously associated with asthma were validated in our study. We report novel whole-blood DNAm markers for asthma underlying key processes of the disease pathophysiology and confirm the transferability of previous asthma DNAm associations to ethnically diverse populations.

526 Clinical Characteristics and Biomarkers of Aggressive Nonfunctional Pituitary Adenomas and Pituitary Carcinomas

INTRODUCTION: Nonfunctioning pituitary adenomas (NFPAs) are benign tumors of the pituitary gland. While surgery is the treatment of choice, a subset of aggressive NFPAs is associated with multiple recurrences and treatments. Pituitary carcinomas are a rare group of pituitary tumors, comprised of both functional and non-functional, which metastasize to other locations. METHODS: A retrospective review of 601 consecutive patients who underwent endoscopic transsphenoidal surgery between 2010 and 2018 at one institution was conducted. Among 209 NFPA patients, 29 patients had aggressive NFPAs, with ≥2 recurrences and ≥3 treatments. We used Infinium HumanMethylation450 BeadChip arrays along with R (R Core 2014) and Bioconductor for methylation analysis of patients with indolent NFPAs (n = 6), aggressive NFPAs (n = 8), pituitary carcinomas (n = 9), and normal pituitary glands (n=3). RESULTS: In multivariate analysis of clinical features, tumor volume and proliferative index were higher in aggressive NFPAs compared to indolent NFPAs. Although significantly different, these features were not specific to aggressive NFPAs. Methylation analysis showed no significant global methylation differences among all samples. However, we identified significant probe-wise differentiation in methylation of over 100 genes between indolent and aggressive NFPA groups. Heat map representation of the most significant differentially methylated genes demonstrated similarity in methylation patterns between the aggressive NFPA and pituitary carcinoma group, which were distinct from the indolent group. CONCLUSIONS: Tumor volume and proliferative index were significantly increased in the aggressive compared to the indolent group. In DNA methylation analyses, we identified numerous hypermethylated genes in the aggressive and pituitary carcinoma groups, which were distinct from the indolent group. DNA methylation may provide a useful predictive biomarker to identify aggressive NFPA types and potentially reveal common mechanistic links between aggressive NFPAs and pituitary carcinomas.

DNA methylation patterns associated with breast cancer prognosis that are specific to tumor subtype and menopausal status.

Tumor subtype and menopausal status are strong predictors of breast cancer (BC) prognosis. We aimed to find and validate subtype- or menopausal-status-specific changes in tumor DNA methylation (DNAm) associated with all-cause mortality or BC progression. Associations between site-specific tumor DNAm and BC prognosis were estimated among The Cancer Genome Atlas participants (n = 692) with Illumina Infinium HumanMethylation450 BeadChip array data. All-cause mortality and BC progression were modeled using Cox proportional hazards models stratified by tumor subtypes, adjusting for age, race, stage, menopausal status, tumor purity, and cell type proportion. Effect measure modification by subtype and menopausal status were evaluated by incorporating a product term with DNAm. Site-specific inference was used to identify subtype- or menopausal-status-specific differentially methylated regions (DMRs) and functional pathways. The validation of the results was carried out on an independent dataset (GSE72308; n = 180). We identified a total of fifteen unique CpG probes that were significantly associated ( with survival outcomes in subtype- or menopausal-status-specific manner. Seven probes were associated with overall survival (OS) or progression-free interval (PFI) for women with luminal A subtype, and four probes were associated with PFI for women with luminal B subtype. Five probes were associated with PFI for post-menopausal women. A majority of significant probes showed a lower risk of OS or BC progression with higher DNAm. We identified subtype- or menopausal-status-specific DMRs and functional pathways of which top associated pathways differed across subtypes or menopausal status. None of significant probes from site-specific analyses met genome-wide significant level in validation analyses while directions and magnitudes of coefficients showed consistent pattern. We have identified subtype- or menopausal-status-specific DNAm biomarkers, DMRs and functional pathways associated with all-cause mortality or BC progression, albeit with limited validation. Future studies with larger independent cohort of non-post-menopausal women with non-luminal A subtypes are warranted for identifying subtype- and menopausal-status-specific DNAm biomarkers for BC prognosis.

The diagnostic potential of plaque-specific methylation patterns in cell-free dna

Background and Aims : There is an urgent and unmet need for early, non-invasive diagnosis of atherosclerosis that can identify high-risk patients with potentially symptomatic plaques. Recent breakthroughs in the use of circulating fragments of cell-free DNA (cfDNA), have provided major advances in early cancer detection. Furthermore, it has been shown that, using tissue-specific methylation patterns, the contribution of different tissue origins to cfDNA can be quantified. Here we aim to identify plaque-specific methylation patterns and unravel the biological processes behind this epigenetic signature. We hypothesize that an increased contribution of plaque-derived cfDNA, as a proxy for the accumulation of atherosclerotic plaque, can be measured in cardiovascular disease patients.Methods: DNA methylation patterns were obtained for plaque samples from 50 female and 50 male patients that underwent carotid endarterectomy (CEA) from the Athero-Express Biobank using the Infinium HumanMethylation450 Beadchip Array. cfDNA methylation from 18 patients, spanning three cardiovascular clinical cohorts, was measured using the NEBNext Enzymatic Methyl-seq kit.Results: We were able to identify a plaque-specific methylation signature encompassing 200 CpG sides, of which a portion was found to be involved in atherosclerotic processes. Furthermore, we built a comprehensive human methylation atlas using methylation data on 18 different tissue and cell types, including plaque, which is used for the deconvolution of cfDNA.Conclusions: Our plaque-centric methylation atlas together with our well-established biobanks, offer the unique opportunity to investigate cfDNA composition in patients spanning various types of cardiovascular disease. We believe this comprehensive methylation framework would help unravel the potential of cfDNA as an innovative non-invasive biomarker for cardiovascular disease. Background and Aims : There is an urgent and unmet need for early, non-invasive diagnosis of atherosclerosis that can identify high-risk patients with potentially symptomatic plaques. Recent breakthroughs in the use of circulating fragments of cell-free DNA (cfDNA), have provided major advances in early cancer detection. Furthermore, it has been shown that, using tissue-specific methylation patterns, the contribution of different tissue origins to cfDNA can be quantified. Here we aim to identify plaque-specific methylation patterns and unravel the biological processes behind this epigenetic signature. We hypothesize that an increased contribution of plaque-derived cfDNA, as a proxy for the accumulation of atherosclerotic plaque, can be measured in cardiovascular disease patients. Methods: DNA methylation patterns were obtained for plaque samples from 50 female and 50 male patients that underwent carotid endarterectomy (CEA) from the Athero-Express Biobank using the Infinium HumanMethylation450 Beadchip Array. cfDNA methylation from 18 patients, spanning three cardiovascular clinical cohorts, was measured using the NEBNext Enzymatic Methyl-seq kit. Results: We were able to identify a plaque-specific methylation signature encompassing 200 CpG sides, of which a portion was found to be involved in atherosclerotic processes. Furthermore, we built a comprehensive human methylation atlas using methylation data on 18 different tissue and cell types, including plaque, which is used for the deconvolution of cfDNA. Conclusions: Our plaque-centric methylation atlas together with our well-established biobanks, offer the unique opportunity to investigate cfDNA composition in patients spanning various types of cardiovascular disease. We believe this comprehensive methylation framework would help unravel the potential of cfDNA as an innovative non-invasive biomarker for cardiovascular disease.

Further Introduction of DNA Methylation (DNAm) Arrays in Regular Diagnostics

Methylation tests have been used for decades in regular DNA diagnostics focusing primarily on Imprinting disorders or specific loci annotated to specific disease associated gene promotors. With the introduction of DNA methylation (DNAm) arrays such as the Illumina Infinium HumanMethylation450 Beadchip array or the Illumina Infinium Methylation EPIC Beadchip array (850 k), it has become feasible to study the epigenome in a timely and cost-effective way. This has led to new insights regarding the complexity of well-studied imprinting disorders such as the Beckwith Wiedemann syndrome, but it has also led to the introduction of tests such as EpiSign, implemented as a diagnostic test in which a single array experiment can be compared to databases with known episignatures of multiple genetic disorders, especially neurodevelopmental disorders. The successful use of such DNAm tests is rapidly expanding. More and more disorders are found to be associated with discrete episignatures which enables fast and definite diagnoses, as we have shown. The first examples of environmentally induced clinical disorders characterized by discrete aberrant DNAm are discussed underlining the broad application of DNAm testing in regular diagnostics. Here we discuss exemplary findings in our laboratory covering this broad range of applications and we discuss further use of DNAm tests in the near future.

Genome-wide methylation analysis of early-onset schizophrenia.

Schizophrenia (SCZ) is a debilitating disease with a complex genetic cause in which age at onset may reflect genetic vulnerability. Though there has been some association between genetic polymorphisms and age of onset, there has been little exploration of the role of epigenetic processes. We sought to explore the influence of DNA methylation, a key epigenetic mechanism, and its association with the age of onset of illness. One hundred thirty-eight participants aged 18-75 years and previously diagnosed with SCZ spectrum disorders by the Structured Clinical Interview for the Diagnostic and Statistical Manual of Mental Disorders (SCID DSM-5) were recruited. Venous blood was collected and genome-wide DNA methylation was quantified using the Illumina Infinium HumanMethylation450 BeadChip array. Individual CpG sites and regions of differential methylation were explored by the age of onset; covariates included age, sex, as well as white blood cell composition. Binary grouping (early vs. late onset) revealed four intergenic CpG sites on chromosome 2 that were above the expected P-value threshold, with hypermethylation of the CpG site cg10392614 most strongly associated with early-onset SCZ. The four most strongly associated CpG sites, including cg 10392614, were intergenic. Continuous analysis revealed the top CpG site to be cg11723066 , which is linked to the JAM3 gene, with hypomethylation associated with earlier onset; however, results were below the expected P-value threshold. Studies on DNA methylation in the first-episode psychosis population may help further our understanding of the role of epigenetics in the age of onset of SCZ.

Abstract 5706: Epigenetic signatures in association with breast cancer mortality and prognosis that are specific to tumor subtype and menopausal status

Abstract Background: Despite advancements in screening and treatment options for breast cancer (BC), mortality from BC has increased, accounting for 15% of new cancer deaths in the U.S. in 2020. BC is heterogeneous cancer with different molecular subtypes defined by hormone-receptor status, and the prognoses of BC patients differ by their menopausal status in conjunction with their molecular subtypes. Epigenetic dysregulation patterns can serve as principal predictors of BC progression and mortality by capturing these differences in biology that are linked to BC outcomes, leading to personalized treatment plans. In this study, we aimed to detect subtype- or menopausal-specific DNA methylation (DNAm) in BC tumor tissues across the genome that are associated with BC progression or all-cause mortality. Methods: Associations between site-specific DNAm in tumor and BC survival were evaluated using the Illumina Infinium HumanMethylation450 BeadChip array data from the Cancer Genome Atlas participants. Cox proportional hazards models were used in the strata by tumor subtypes and menopausal status, adjusting for age, race, cancer stage, tumor purity, and cell type proportion. Detected site-specific DNAm was further analyzed to identify subtype- or menopausal-specific differentially methylated regions (DMRs) and functional pathways. The validation of the results was carried out on an independent dataset. Results: We identified eight CpG probes that were associated with survival outcomes in subtype- or menopausal-specific manner. Seven probes among women with luminal A subtype were associated with all-cause mortality or progression-free interval (PFI); four probes among women with luminal B were associated with PFI; and five probes among post-menopausal women were associated with PFI. Of them, six probes presented a lower risk of all-cause mortality or BC progression with higher DNAm. The identified DMRs differed by tumor subtype and menopausal status, and a majority of DMRs were driven by several weaker associations in the same direction among proximal loci. Also, we identified subtype- or menopausal-specific functional genomic pathways. Particularly, a pathway of insulin regulation was associated with all-cause mortality and PFI among pre-menopausal women. The detected site-specific probes were validated with consistent directions and magnitudes of effects on prognosis across two independent datasets. Conclusion: We identified subtype- or menopausal-specific DNAm in BC tumor tissues, DMRs, and functional pathways in association with all-cause mortality or BC progression. Our findings warrant future studies with larger independent datasets for replication and functional implications of detected DNAm. Our detected genome-wide-associated-CpG loci could improve prognosis prediction for BC patients, thus contributing to tailored therapeutic regimens. Citation Format: Do Hyun Kim, Alexandra M. Binder, Hua Zhou, Su Yon Jung. Epigenetic signatures in association with breast cancer mortality and prognosis that are specific to tumor subtype and menopausal status [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5706.

Prediction of tissue origin of adenocarcinomas in the esophagogastric junction by DNA methylation.

Prediction of tissue origin of esophagogastric junction (EGJ) adenocarcinomas can be important for therapeutic decision, but no molecular marker is available. Here, we aimed to develop such a marker taking advantage of tissue-specific profiles of DNA methylation. DNA methylation profiles of gastric adenocarcinomas (GACs) were obtained by an Infinium HumanMethylation450 BeadChip array, and those of esophageal adenocarcinoma (EACs) were obtained from the TCGA database. DNA from formalin-fixed paraffin-embedded (FFPE) samples was analyzed by bisulfite pyrosequencing. In the screening set, 51 of 145,841 CpG sites in CpG islands were methylated at significantly higher levels in 30 GACs compared to those in 30 EACs. Among them, SLC46A3 and cg09177106 were unmethylated in all the 30 EACs. Predictive powers of these two markers were successfully confirmed in an independent validation set (18 GACs and 18 EACs) (SLC46A3, sensitivity = 77.8%, specificity = 100%; cg09177106, sensitivity = 83.3%, specificity = 94.4%), and could be applied to FFPE samples (37 GACs and 18 EACs) (SLC46A3, P = 0.0001; cg09177106, P = 0.0028). On the other hand, EAC-specific markers informative in the FFPE samples could not be isolated. Using these GAC-specific markers, nine of 46 (19.6%) TCGA EGJ adenocarcinomas were predicted to be GACs. Two GAC-specific markers, SLC46A3 and cg09177106, had a high specificity for identifying the tissue origin of EGJ adenocarcinoma.

Genome-wide DNA methylation changes in oral submucous fibrosis.

Oral submucous fibrosis (OSF) is a debilitating potentially malignant condition of the buccal cavity characterized by extensive extracellular matrix deposition resulting in stiffness and trismus. As OSF is a progressive disease, we hypothesized that there would be extensive epigenetic changes in OSF tissues. Using the Infinium HumanMethylation450 BeadChip Array, we analyzed gross DNA methylation changes in seven OSF tissues compared to five controls. Comparison with transcriptomic data and pathway analyses was conducted to find commonly regulated genes. A total of 3,294 differentially methylated regions mapping to 857 genes were identified. Comparison with transcriptome data revealed 38 downregulated-hypermethylated genes and 55 hypomethylated-upregulated genes. Using methylation-specific and qRT-PCR, aberrant hypomethylation and increased expression of FGF13, RPS6KA3, and ACSL4 genes were confirmed. Pathways involved in insulin signaling, ubiquitin-mediated proteolysis, nicotine addiction, and RAS/MAPK pathways were dysregulated, among others. Intriguingly, numerous genes located on the X chromosome were dysregulated in OSF tissues as the transcript for XIST gene was downregulated due to hypermethylation of the XIST promoter. This study highlights global epigenetic dysregulation of tissues of the oral cavity in OSF patients and hints at possible X chromosomal dysregulation, previously not implicated in the pathogenesis of OSF.

Methylation and molecular profiles of ependymoma: Influence of patient age and tumor anatomic location

Ependymomas are tumors of the central nervous system that can occur in patients of all ages. Guidelines from the World Health Organization (WHO) for the grading of ependymomas consider patient age, tumor resection range, tumor location and histopathological grade. However, recent studies have suggested that a greater focus on both tumor location and patient age in terms of transcriptomic, genetic, and epigenetic analyses may provide a more accurate assessment of clinical prognosis than the grading system proposed by WHO guidelines. The current study identified the differences and similarities in ependymoma characteristics using three different molecular analyses and methylation arrays. Primary intracranial ependymoma tissues were obtained from 13 Korean patients (9 adults and 4 children), after which whole-exome sequencing (WES), ion-proton comprehensive cancer panel (CCP) analysis, RNA sequencing, and Infinium HumanMethylation450 BeadChip array analysis was performed. Somatic mutations, copy number variations, and fusion genes were identified. It was observed that the methylation status and differentially expressed genes were significantly different according to tumor location and patient age. Several novel gene fusions and somatic mutations were identified, including a yes-associated protein 1 fusion mutation in a child with a good prognosis. Moreover, the methylation microarray revealed that genes associated with neurogenesis and neuron differentiation were hypermethylated in the adult group, whereas genes in the homeobox gene family were hypermethylated in the supratentorial (ST) group. The results confirmed the existence of significantly differentially expressed tumor-specific genes based on tumor location and patient age. These results provided valuable insight into the epigenetic and genetic profiles of intracranial ependymomas and uncovered potential strategies for the identification of location- and age-based ependymoma-related prognostic factors.

Neural Tube Defects and ZIC4 Hypomethylation in Relation to Polycyclic Aromatic Hydrocarbon Exposure.

BackgroundEpigenetic dysregulation is one of the postulated underlying mechanisms of neural tube defects (NTDs). Polycyclic aromatic hydrocarbons (PAHs), a group of environmental pollutants that are reported as a risk factor of NTDs, may cause decreased genome-wide DNA methylation. With DNA extracted from neural tissues, this study identified gene(s) whose hypomethylation was related to elevated risk for NTDs and examined whether its hypomethylation is related to PAH exposure.ResultsUsing data profiled by Infinium HumanMethylation450 BeadChip array from 10 NTD cases and eight controls, ZIC4, CASP8, RAB32, RARA, and TRAF6 were identified to be the top five genes in NTD-related hypomethylated gene families. Among all identified genes, ZIC4 had the largest number of differently methylated CpG sites (n = 13) in the promoter region and 5′ UTR. Significantly decreased methylation in the ZIC4 promoter region and 5′ UTR was verified in an independent cohort of 80 cases and 32 controls (p < 0.001) utilizing the Sequenom EpiTYPER platform. Hypomethylation of ZIC4 was associated with a higher risk of NTDs [adjusted OR = 1.08; 95% confidence interval (CI): 1.03, 1.13] in a logistic regression model. Mean methylation levels in the promoter region and 5′ UTR of ZIC4 tended to be inversely associated with levels of high-molecular-weight PAHs in fetal liver among NTD fetuses (β [95% CI]: −0.045 [−0.091, 0.001], p = 0.054). Six and three CpG sites in the ZIC4 promoter region and 5′ UTR were inversely correlated with antioxidant indicators and protein oxidation markers (ρ: −0.45 to −0.75, p < 0.05) in fetal neural tissues, respectively. In a whole-embryo cultured mouse model, hypomethylation of the Zic4 promoter region and 5′ UTR and upregulation of Zic4 were observed, coupled with increased NTD rates after BaP exposure. The antioxidant N-acetyl-L-cysteine normalized the changes observed in the BaP exposure group.ConclusionHypomethylation of the ZIC4 promoter region and 5′ UTR may increase the risk for NTDs; oxidative stress is likely to play a role in the methylation change of Zic4 in response to PAH exposure in NTD formation.

Genome-wide investigation of DNA methylation in congenital adrenal hyperplasia

Patients with congenital adrenal hyperplasia (CAH) are at risk of long-term cognitive and metabolic sequelae with some of the effects being attributed to the chronic glucocorticoid treatment that they receive. Our pilot study investigates genome-wide DNA methylation in patients with CAH to determine whether there is preliminary evidence for epigenomic reprogramming as well as any relationship to patient outcome. Here, we analysed CD4 + T cell DNA from 28 patients with CAH (mean age = 18.5 ± 6.5 years [y]) and 37 population controls (mean age = 17.0 ± 6.1 y) with the Infinium-HumanMethylation450 BeadChip array to measure genome-wide locus-specific DNA methylation levels. Effects of CAH, phenotype and CYP21A2 genotype on methylation were investigated as well as the association between differentially methylated CpGs and glucose homeostasis, blood lipid profile, and cognitive functions. In addition, we report data on a small cohort of 11 patients (mean age = 19.1, ±6.0 y) with CAH who were treated prenatally with dexamethasone (DEX) in addition to postnatal glucocorticoid treatment.We identified two CpGs to be associated with patient phenotype: cg18486102 (located in the FAIM2 gene; rho = 0.58, adjusted p = 0.027) and cg02404636 (located in the SFI1 gene; rho = 0.58, adjusted p = 0.038). cg02404636 was also associated with genotype (rho = 0.59, adjusted p = 0.024). Higher levels of serum C-peptide was also observed in patients with CAH (p = 0.044). Additionally, levels of C-peptide and HbA1c were positively correlated with patient phenotype (p = 0.044 and p = 0.034) and genotype (p = 0.044 and p = 0.033), respectively. No significant association was found between FAIM2 methylation and cognitive or metabolic outcome. However, SFI1 TSS methylation was associated with fasting plasma HDL cholesterol levels (p = 0.035).In conclusion, in this pilot study, higher methylation levels in CpG sites covering FAIM2 and SFI1 were associated with disease severity. Hypermethylation in these genes may have implications for long-term cognitive and metabolic outcome in patients with CAH, although the data must be interpreted with caution due to the small sample size. Additional studies in larger cohorts are therefore warranted.

Telomere length correlates with subtelomeric DNA methylation in long-term mindfulness practitioners

Mindfulness and meditation techniques have proven successful for the reduction of stress and improvement in general health. In addition, meditation is linked to longevity and longer telomere length, a proposed biomarker of human aging. Interestingly, DNA methylation changes have been described at specific subtelomeric regions in long-term meditators compared to controls. However, the molecular basis underlying these beneficial effects of meditation on human health still remains unclear. Here we show that DNA methylation levels, measured by the Infinium HumanMethylation450 BeadChip (Illumina) array, at specific subtelomeric regions containing GPR31 and SERPINB9 genes were associated with telomere length in long-term meditators with a strong statistical trend when correcting for multiple testing. Notably, age showed no association with telomere length in the group of long-term meditators. These results may suggest that long-term meditation could be related to epigenetic mechanisms, in particular gene-specific DNA methylation changes at distinct subtelomeric regions.

Maternal Gestational Diabetes Mellitus and Newborn DNA Methylation: Findings From the Pregnancy and Childhood Epigenetics Consortium.

Maternal gestational diabetes mellitus (GDM) has been associated with adverse outcomes in the offspring. Growing evidence suggests that the epigenome may play a role, but most previous studies have been small and adjusted for few covariates. The current study meta-analyzed the association between maternal GDM and cord blood DNA methylation in the Pregnancy and Childhood Epigenetics (PACE) consortium. Seven pregnancy cohorts (3,677 mother-newborn pairs [317 with GDM]) contributed results from epigenome-wide association studies, using DNA methylation data acquired by the Infinium HumanMethylation450 BeadChip array. Associations between GDM and DNA methylation were examined using robust linear regression, with adjustment for potential confounders. Fixed-effects meta-analyses were performed using METAL. Differentially methylated regions (DMRs) were identified by taking the intersection of results obtained using two regional approaches: comb-p and DMRcate. Two DMRs were identified by both comb-p and DMRcate. Both regions were hypomethylated in newborns exposed to GDM in utero compared with control subjects. One DMR (chr 1: 248100345-248100614) was located in the OR2L13 promoter, and the other (chr 10: 135341870-135342620) was located in the gene body of CYP2E1. Individual CpG analyses did not reveal any differentially methylated loci based on a false discovery rate-adjusted P value threshold of 0.05. Maternal GDM was associated with lower cord blood methylation levels within two regions, including the promoter of OR2L13, a gene associated with autism spectrum disorder, and the gene body of CYP2E1, which is upregulated in type 1 and type 2 diabetes. Future studies are needed to understand whether these associations are causal and possible health consequences.

Airway epithelial cell isolation techniques affect DNA methylation profiles with consequences for analysis of asthma related perturbations to DNA methylation

The airway epithelium forms the interface between the inhaled environment and the lung. The airway epithelium is dysfunctional in asthma and epigenetic mechanisms are considered a contributory factor. We hypothesised that the DNA methylation profiles of cultured primary airway epithelial cells (AECs) would differ between cells isolated from individuals with asthma (n = 17) versus those without asthma (n = 16). AECs were isolated from patients by two different isolation techniques; pronase digestion (9 non-asthmatic, 8 asthmatic) and bronchial brushings (7 non-asthmatic and 9 asthmatic). DNA methylation was assessed using an Illumina Infinium HumanMethylation450 BeadChip array. DNA methylation of AECs clustered by isolation technique and linear regression identified 111 CpG sites differentially methylated between isolation techniques in healthy individuals. As a consequence, the effect of asthmatic status on DNA methylation was assessed within AEC samples isolated using the same technique. In pronase isolated AECs, 15 DNA regions were differentially methylated between asthmatics and non-asthmatics. In bronchial brush isolated AECs, 849 differentially methylated DNA regions were identified with no overlap to pronase regions. In conclusion, regardless of cell isolation technique, differential DNA methylation was associated with asthmatic status in AECs, providing further evidence for aberrant DNA methylation as a signature of epithelial dysfunction in asthma.

FGF5 methylation is a sensitivity marker of esophageal squamous cell carcinoma to definitive chemoradiotherapy

Definitive chemoradiotherapy (dCRT) is the major treatment for esophageal squamous cell carcinoma (ESCC), and prediction of the response to dCRT is important so as not to miss an opportunity to cure an ESCC. Nevertheless, few validated markers are available. Here, we aimed to identify a highly reproducible marker using multi-layer omics analysis. 117 ESCC samples from 67 responders and 50 non-responders were divided into screening, validation, and re-validation sets. In the screening cohort (n = 41), somatic mutations in 114 genes showed no association with dCRT response. Genome-wide DNA methylation analysis using Infinium HumanMethylation450 BeadChip array identified four genic regions significantly associated with dCRT response. Among them, FGF5 methylation was validated to be associated with dCRT response (n = 34; P = 0.001), and further re-validated (n = 42; P = 0.020) by bisulfite-pyrosequencing. The sensitivity and specificity in the combined validation and re-validation sets (n = 76) were 45% and 90%, respectively, by using the cut-off value established in the screening set, and FGF5 methylation had predictive power independent from clinicopathological parameters. In ESCC cell lines, FGF5 promoter methylation repressed its expression. FGF5 expression was induced by cisplatin (CDDP) treatment in three unmethylated cell lines, but not in two methylated cell lines. Exogenous FGF5 overexpression in a cell line with its methylation conferred resistance to CDDP. In non-cancerous esophageal tissues, FGF5 was not expressed, and its methylation was present in a small fraction of cells. These results showed that FGF5 methylation is a validated marker for ESCC sensitivity to dCRT.

Experiencing community and domestic violence is associated with epigenetic changes in DNA methylation of BDNF and CLPX in adolescents.

Experiencing violence changes behavior, shapes personalities, and poses a risk factor for mental disorders. This association might be mediated through epigenetic modifications that affect gene expression, such as DNA methylation. The present study investigated the impact of community and domestic violence on DNA methylation measured in saliva collected from 375 individuals including three generations: grandmothers (n = 126), mothers (n = 125), and adolescents (n = 124, 53% female). Using the Infinium HumanMethylation450 BeadChip array, in adolescents, we detected two CpG sites that showed an association of DNA methylation and lifetime exposure to community and domestic violence even after FDR correction: BDNF_cg06260077 (logFC −0.454, p = 3.71E‐07), and CLPX_cg01908660 (logFC = −0.372, p = 1.38E‐07). Differential DNA methylation of the CpG BDNF_cg06260077 associated with exposure to violence was also observed in the maternal but not the grandmaternal generation. BDNF (brain‐derived neurotrophic factor) and CLPX (caseinolytic mitochondrial matrix peptidase chaperone subunit) genes are involved in neural development. Our results thus reveal altered molecular mechanisms of developmental and intergenerational trajectories in survivors of repeated violent experiences.

The DNA methylation profile of non-coding RNAs improves prognosis prediction for pancreatic adenocarcinoma

BackgroundCompelling lines of evidence indicate that DNA methylation of non-coding RNAs (ncRNAs) plays critical roles in various tumour progression. In addition, the differential methylation of ncRNAs can predict prognosis of patients. However, little is known about the clear relationship between DNA methylation profile of ncRNAs and the prognosis of pancreatic adenocarcinoma (PAC) patients.MethodsThe data of DNA methylation, RNA-seq, miRNA-seq and clinical features of PAC patients were collected from TCGA database. The DNA methylation profile was obtained using the Infinium HumanMethylation450 BeadChip array. LASSO regression was performed to construct two methylation-based classifiers. The risk score of methylation-based classifiers was calculated for each patient, and the accuracy of the classifiers in predicting overall survival (OS) was examined by ROC curve analysis. In addition, Cox regression models were utilized to assess whether clinical variables and the classifiers were independent prognostic factors for OS. The targets of miRNA and the genes co-expressed with lncRNA were identified with DIANA microT-CDS and the Multi-Experiment Matrix (MEM), respectively. Moreover, DAVID Bioinformatics Resources were applied to analyse the functional enrichment of these targets and co-expressed genes.ResultsA total of 4004 CpG sites of miRNA and 11,259 CpG sites of lncRNA were screened. Among these CpG sites, 8 CpG sites of miRNA and 7 CpG sites of lncRNA were found with regression coefficients. By multiplying the sum of methylation degrees of the selected CpGs with these coefficients, two methylation-based classifiers were constructed. The classifiers have shown good performance in predicting the survival rate of PAC patients at varying follow-up times. Interestingly, both of these two classifiers were predominant and independent factors for OS. Furthermore, functional enrichment analysis demonstrated that aberrantly methylated miRNAs and lncRNAs are related to calcium ion transmembrane transport and MAPK, Ras and calcium signalling pathways.ConclusionIn the present study, we identified two methylation-based classifiers of ncRNA associated with OS in PAC patients through a comprehensive analysis of miRNA and lncRNA profiles. We are the first group to demonstrate a relationship between the aberrant DNA methylation of ncRNAs and the prognosis of PAC, and this relationship would contribute to individualized PAC therapy.