Illumina Infinium MethylationEPIC BeadChip Research Articles

Hypermethylation of tumor suppressor lncRNA MEF2C-AS1 frequently happened in patients at all stages of colorectal carcinogenesis

BackgroundThe novel long noncoding RNA MEF2C-AS1 has been identified to play suppressor roles during tumorigenesis. DNA methylation has a regulatory effect on gene expression in cancer initiation and progression. However, the methylation status of MEF2C-AS1 and its role in colorectal cancer (CRC) development remain unclear.MethodsThe expression and methylation levels of MEF2C-AS1 were systematically analyzed among 31 cancers with available qualified data in GEPIA and UCSC Xena databases. Then, the MEF2C-AS1 methylation status was firstly examined among 12 CRCs by Illumina Infinium MethylationEPIC BeadChip in in-house step 1 and further quantified among 48 CRCs by the MassARRAY method in in-house step 2. Subsequently, its methylation and expression levels were quantified among 81 non-advanced adenomas (NAAs), 81 advanced adenomas (AAs), and 286 CRCs using the MassARRAY method, and among 34 NAAs, 45 AAs, and 75 CRCs by qRT-PCR, in in-house step 3, respectively. The effect of MEF2C-AS1 methylation on CRC survival was analyzed by the Kaplan–Meier method. Additionally, in vitro cell proliferation, migration and invasion assays, and bioinformatics analysis were performed to explore the role of MEF2C-AS1 in colorectal carcinogenesis.ResultsLower expression and higher methylation of MEF2C-AS1 were found in CRC by online databases. In the comparisons of lesion tissues with adjacent normal tissues, MEF2C-AS1 hypermethylation of each individual site and mean level was found among CRC patients in in-house step 1 and step 2, more meaningfully, among NAA patients, AA patients, and CRC patients at all stages during colorectal carcinogenesis in in-house step 3 (all p < 0.05). Further comparisons demonstrated significant differences between CRC and NAA (p = 0.025), AA and NAA (p = 0.020). Moreover, MEF2C-AS1 hypermethylation was associated with poorer disease-specific survival of CRC patients (p = 0.044). In addition, hypermethylation and lower expression of MEF2C-AS1 were verified in RKO cells, and the MEF2C-AS1 overexpression significantly suppressed RKO cell proliferation, migration, and invasion.ConclusionsThe findings reveal that MEF2C-AS1 hypermethylation might be an early driven event during colorectal carcinogenesis. It might serve as a promising prognostic biomarker for CRC survival. Our study also indicates the potential tumor-suppressing role of MEF2C-AS1 in CRC.

Methylation associated with long- or short-term survival in glioblastoma patients from the Nordic phase 3 trial.

Patients with glioblastoma (GBM) have a poor outcome, but even among patients receiving the same therapies and with good prognostic factors, one can find those with exceptionally short and long survival. From the Nordic trial, which randomized GBM patients of 60 years or older between two radiotherapy arms (60 Gy or 34 Gy) or temozolomide (TMZ), we selected 59 with good prognostic factors. These selected GBM patients were equally distributed according to treatment and MGMT promoter methylation status but had long or short survival. Methylation profiling with the Illumina Infinium Methylation EPIC BeadChip arrays was performed and utilized for methylation-based CNS tumor classification, and pathway enrichment analysis of differentially methylated CpG sites (DMCs), as well as calculation of epigenetic age acceleration with three different algorithms, to compare the long and short survival groups. Samples identified by the classifier as non-GBM IDH wildtype were excluded. DMCs between long- and short-term survivors were found in patients with methylated MGMT promoter treated with TMZ (123,510), those with unmethylated MGMT treated with 60Gy radiotherapy (4,086), and with methylated MGMT promoter treated with 34Gy radiotherapy (39,649). Long-term survivors with methylated MGMT promoter treated with TMZ exhibited hypermethylation of the Wnt signaling and the platelet activation, signaling, and aggregation pathways. The joint analysis of radiotherapy arms revealed 319 DMCs between long- and short-term survivors with unmethylated MGMT and none for samples with methylated MGMT promoter. An analysis comparing epigenetic age acceleration between patients with long- and short-term survival across all treatment arms showed a decreased epigenetic age acceleration for the latter. We identified DMCs for both TMZ and RT-treated patients and epigenetic age acceleration as a potential prognostic marker, but further systematic analysis of larger patient cohorts is necessary for confirmation of their prognostic and/or predictive properties.

Schizophrenia-associated differential DNA methylation in brain is distributed across the genome and annotated to MAD1L1, a locus at which DNA methylation and transcription phenotypes share genetic variation with schizophrenia risk

DNA methylation (DNAm), the addition of a methyl group to a cytosine in DNA, plays an important role in the regulation of gene expression. Single-nucleotide polymorphisms (SNPs) associated with schizophrenia (SZ) by genome-wide association studies (GWAS) often influence local DNAm levels. Thus, DNAm alterations, acting through effects on gene expression, represent one potential mechanism by which SZ-associated SNPs confer risk. In this study, we investigated genome-wide DNAm in postmortem superior temporal gyrus from 44 subjects with SZ and 44 non-psychiatric comparison subjects using Illumina Infinium MethylationEPIC BeadChip microarrays, and extracted cell-type-specific methylation signals by applying tensor composition analysis. We identified SZ-associated differential methylation at 242 sites, and 44 regions containing two or more sites (FDR cutoff of q = 0.1) and determined a subset of these were cell-type specific. We found mitotic arrest deficient 1-like 1 (MAD1L1), a gene within an established GWAS risk locus, harbored robust SZ-associated differential methylation. We investigated the potential role of MAD1L1 DNAm in conferring SZ risk by assessing for colocalization among quantitative trait loci for methylation and gene transcripts (mQTLs and tQTLs) in brain tissue and GWAS signal at the locus using multiple-trait-colocalization analysis. We found that mQTLs and tQTLs colocalized with the GWAS signal (posterior probability >0.8). Our findings suggest that alterations in MAD1L1 methylation and transcription may mediate risk for SZ at the MAD1L1-containing locus. Future studies to identify how SZ-associated differential methylation affects MAD1L1 biological function are indicated.

Identification of an Epigenetic Signature for Coronary Heart Disease in Postmenopausal Women's PBMC DNA.

Menopause is accompanied with an increased risk of cardiovascular disease. DNA methylation may have a significant impact on postmenopausal women's development of coronary heart disease. DNA methylation alterations in peripheral blood mononuclear cells (PBMCs) from women with coronary heart disease and healthy controls were detected using the Illumina Infinium MethylationEPIC BeadChip platform in this work. We employed Sangerbox technology and the GO and KEGG databases to further study the pathogenesis of coronary heart disease in postmenopausal women. After that, we used functional epigenetic module analysis and Cytoscape to remove the hub genes from the protein–protein interaction networks. Five genes (FOXA2, PTRD, CREB1, CTNAP2, and FBN2) were the hub genes. Lipid accumulation, endothelial cell failure, inflammatory responses, monocyte recruitment and aggregation, and other critical biological processes were all influenced by these genes. Finally, we employed methylation-specific PCR to demonstrate that FOXA2 was methylated at a high level in postmenopausal women with coronary heart disease. To better understand coronary heart disease in postmenopausal women's molecular mechanisms, our study examine the major factors contributing to the state of DNA methylation modification, which will help discover novel diagnostic tools and treatment options.

Cell-free DNA in maternal blood and artificial intelligence: accurate prenatal detection of fetal congenital heart defects

DNA cytosine nucleotide methylation (epigenomics and epigenetics) is an important mechanism for controlling gene expression in cardiac development. Combined artificial intelligence and whole-genome epigenomic analysis of circulating cell-free DNA in maternal blood has the potential for the detection of fetal congenital heart defects. This study aimed to use genome-wide DNA cytosine methylation and artificial intelligence analyses of circulating cell-free DNA for the minimally invasive detection of fetal congenital heart defects. In this prospective study, whole-genome cytosine nucleotide methylation analysis was performed on circulating cell-free DNA using the Illumina Infinium MethylationEPIC BeadChip array. Multiple artificial intelligence approaches were evaluated for the detection of congenital hearts. The Ingenuity Pathway Analysis program was used to identify gene pathways that were epigenetically altered and important in congenital heart defect pathogenesis to further elucidate the pathogenesis of isolated congenital heart defects. There were 12 cases of isolated nonsyndromic congenital heart defects and 26 matched controls. A total of 5918 cytosine nucleotides involving 4976 genes had significantly altered methylation, that is, a P value of <.05 along with ≥5% whole-genome cytosine nucleotide methylation difference, in congenital heart defect cases vs controls. Artificial intelligence analysis of the methylation data achieved excellent congenital heart defect predictive accuracy (areas under the receiver operating characteristic curve, ≥0.92). For example, an artificial intelligence model using a combination of 5 whole-genome cytosine nucleotide markers achieved an area under the receiver operating characteristic curve of 0.97 (95% confidence interval, 0.87-1.0) with 98% sensitivity and 94% specificity. We found epigenetic changes in genes and gene pathways involved in the following important cardiac developmental processes: "cardiovascular system development and function," "cardiac hypertrophy," "congenital heart anomaly," and "cardiovascular disease." This lends biologic plausibility to our findings. This study reported the feasibility of minimally invasive detection of fetal congenital heart defect using artificial intelligence and DNA methylation analysis of circulating cell-free DNA for the prediction of fetal congenital heart defect. Furthermore, the findings supported an important role of epigenetic changes in congenital heart defect development.

Genome-wide decrease in DNA methylation in adults with epilepsy treated with modified ketogenic diet: A prospective study.

The aim of this study was to investigate the impact of the modified ketogenic diet on DNA methylation in adults with epilepsy. In this prospective study, we investigated the genome-wide DNA methylation in whole blood in 58 adults with epilepsy treated with the modified ketogenic for 12 weeks. Patients were recruited from the National Center for Epilepsy, Norway, from March 1, 2011 to February 28, 2017. DNA methylation was analyzed using the Illumina Infinium MethylationEPIC BeadChip array. Analysis of variance and paired t-test were used to identify differentially methylated loci after 4 and 12 weeks of dietary treatment. A false discovery rate approach with a significance threshold of <5% was used to adjust for multiple comparisons. We observed a genome-wide decrease in DNA methylation, both globally and at specific sites, after 4 and 12 weeks of dietary treatment. A substantial share of the differentially methylated positions (CpGs) were annotated to genes associated with epilepsy (n= 7), lipid metabolism (n= 8), and transcriptional regulation (n= 10). Furthermore, five of the identified genes were related to inositol phosphate metabolism, which may represent a possible mechanism by which the ketogenic diet attenuates seizures. A better understanding of the modified ketogenic diet's influence at the molecular level may be the key to unraveling the mechanisms by which the diet can ameliorate seizures and possibly to identifying novel therapeutic targets for epilepsy.

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.

A Bayesian hierarchical model for improving measurement of 5mC and 5hmC levels: Toward revealing associations between phenotypes and methylation states.

5-hydroxymethylcytosine (5hmC) is a methylation state linked with gene regulation, commonly found in cells of the central nervous system. 5hmC is associated with demethylation of cytosines from 5-methylcytosine (5mC) to the unmethylated state. The presence of 5hmC can be inferred by a paired experiment involving bisulfite and oxidation-bisulfite treatments on the same sample, followed by a methylation assay using a platform such as the Illumina Infinium MethylationEPIC BeadChip (EPIC). Existing methods for analysis of the resulting EPIC data are not ideal. Most approaches ignore the correlation between the two experiments and any imprecision associated with DNA damage from the additional treatment. Estimates of 5mC/5hmC levels free from these limitations are desirable to reveal associations between methylation states and phenotypes. We propose a hierarchical Bayesian method called Constrained HYdroxy Methylation Estimation (CHYME) to simultaneously estimate 5mC/5hmC signals as well as any associations between these signals and covariates or phenotypes, while accounting for the potential impact of DNA damage and dependencies induced by the experimental design. Simulations show that CHYME has valid type 1 error and better power than a range of alternative methods, including the popular OxyBS method and linear models on transformed proportions. Other methods we examined suffer from hugely inflated type 1 error for inference on 5hmC proportions. We use CHYME to explore genome-wide associations between 5mC/5hmC levels and cause of death in postmortem prefrontal cortex brain tissue samples. These analyses indicate that CHYME is a useful tool to reveal phenotypic associations with 5mC/5hmC levels.

Abstract 3675: Epigenetic variations associated with therapy resistance among prostate tumors from Puerto Rican men

Abstract Prostate cancer (PCa) is the leading cause of cancer-related death in Puerto Rican (PR) men, a population at higher risk of developing the disease than other Hispanic/Latino groups. But the relationship between this phenomenon and epigenetics, and how this relationship explains PCa racial and ethnic health disparities are controversial. The purpose of this study was to expand on the knowledge regarding epigenetic differences in terms of DNA methylation to enable risk stratification and treatment selection in this genetically admixed population. In addition, we aim to assess differential DNA methylation patterns associated with drug resistance in PCa in PR Hispanic/Latino men. Since epigenetic changes and modifications represent critical components of carcinogenesis and progression, we are expecting differential methylation levels between aggressive vs. indolent, and resistant vs. non-resistant cases. Prostate tumors from PR men were stratified based on Gleason score. Methylation levels were measured by Illumina Infinium Methylation EPIC BeadChip DNA methylation platform, and bioinformatic analyses. In parallel, DNA methylation profiles were also analyzed using genomic DNA from PCa cells selected for resistance to androgen depletion, enzalutamide resistance, or docetaxel resistance. We identified 1,225 differentially methylated genes in prostate tumors compared with normal adjacent tissues, and 141 differentially methylated genes between aggressive vs. indolent tumors. Additionally, 723 differentially methylated genes were found in therapy resistant cell lines compared to parental controls. Analysis of the methylated genes obtained from aggressive prostate tumors from Puerto Rican patients and therapy-resistant PCa cell lines revealed four overlapping methylated genes: RNF220, FAM65B, PRDM16, and DGKH. Further analysis of PCa cells resistant to androgen depletion, enzalutamide, or docetaxel demonstrated impaired cellular migration of resistant phenotypes upon DNA methyltransferase (DNMT) inhibition using 5-azacytidine or zebularine compared to parental cell lines. DNMT1 was associated with resistance in PCa cell lines, and future studies will include silencing of methyltransferases will evaluation of drug re-sensitization. These findings provide critical insight into the epigenetic landscape of aggressive prostate tumors among PR men and highlight several candidate methylation biomarkers for assessing risk in this population. Citation Format: Carlos J. Diaz Osterman, Anders Berglund, Shannalee Martinez, Camila Luis, Julie Dutil, Jaime Matta, Jarline Encarnacion-Medina, Carmen Ortiz-Sanchéz, Liang Wang, Jasreman Dhillon, Youngchul Kim, Hyun Y. Park, Gilbert Ruiz-Deya, Kosj Yamoah, Jong Y. Park. Epigenetic variations associated with therapy resistance among prostate tumors from Puerto Rican men [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 3675.

Abstract 5698: Genome-wide DNA methylation profiling for differentiating synchronous endometrial and ovarian cancer

Abstract Background: Synchronous endometrial and ovarian cancer (SEOC) refers to simultaneously discovered endometrial and ovarian tumours on the same patient. SEOC may represent double primary (DP) tumours that arise independently on the two sites, or clonally related (CR) tumours that arise either on the endometrium or ovary and metastasized to the other site. Accurate differential diagnosis between these two scenarios can implicate on the treatment options and prognostication of SEOC. However, it is difficult to achieve as the histopathological features of DP and CR tumours are often ambiguous. DNA methylation landscape of SEOC may provide markers for differentiating DP and CR tumours but remain largely unexplored. Methods: The archival tissues of six pairs of SEOC and their accompanied normal uterine tissues were retrieved. The tumours were accessed by conventional histopathological criteria and mitochondrial DNA (mtDNA) sequencing to categorize them into DP or CR. We then analyzed the methylation profiles of &amp;gt;850,000 CpG sites of the tumor samples using the Illumina Infinium MethylationEPIC BeadChip array. Downstream methylation analysis was performed in the R/Bioconductor package ChAMP. Gene ontology (GO) enrichment analysis on the methylation driven genes was done using PANTHER. Results: mtDNA sequencing revealed that among the 6 cases of SEOC, there are 3 DP and 3 CR. Clustering analysis of differentially methylated CpGs (DMPs) between tumours of the same tissue type effectively distinguished endometrial and ovarian tumours into DP or CR. Thus, DP and CR endometrial tumour showed 50 DMPs and 15 differentially methylated regions (DMRs), but no significantly enriched signaling pathway was identified. On the other hand, there were 1,105 DMPs and 44 DMRs that mapped to 601 known genes between DP and CR ovarian tumours. GO analysis revealed that the hypomethylated genes in CR ovarian tumour were enriched in several metastatic-related processes, including cellular response to stimulus, regulation of cell projection organisation, cell migration and negative regulation of voltage-gated potassium channel activity. Conclusions: Taken together, our findings demonstrated that DNA methylation profiling can be used for SEOC patient stratification and may enhance our understanding of epigenetic regulation of SEOC pathogenesis. Citation Format: Yusanne Yung Sing Chan, Oscar Gee Wan Wong, Annie Nga Yin Cheung. Genome-wide DNA methylation profiling for differentiating synchronous endometrial and ovarian cancer [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 5698.

Abstract 4020: Methylation analysis of matched primary and recurrent IDH-mutant astrocytoma; an update from the GLASS-NL consortium

Abstract INTRODUCTION. Diffuse gliomas are the most frequent and devastating primary CNS tumors in adults. Standard treatment has limited efficacy and without exception these gliomas recur. The evolutionary processes that drive progression in glioma of the IDH-mutant astrocytoma subgroup, remain unclear. The GLASS-NL consortium was initiated to gain insight into the molecular mechanisms underlying glioma evolution and to identify markers of progression in IDH-mutant astrocytomas. Such markers can ultimately assist clinical decision making. Here, we present the results of genome wide DNA-methylation profiling of samples included in the GLASS-NL study. METHODS. 110 adult patients were identified with an IDH-mutant, 1p19q non-codeleted, astrocytoma at first diagnosis. All patients underwent surgical resection of the tumor at least twice, separated by &amp;gt;6 months with a median of 41.9 months (IQR:26.5-65.9). After surgical resection of the initial tumor, 63% and 22% of the patients were treated with radiotherapy or chemotherapy respectively. DNA-methylation profiling was performed on 235 samples from 103 patients, using the Illumina Infinium MethylationEPIC BeadChip array. Copy number alterations (CNAs) were extracted from these data. Methylation subclasses were determined according to Capper et al. (Nature, 2018). Overall survival (OS) was measured from date of initial surgery. RESULTS. Of all primary tumors, 85 (87%) of the tumor samples were assigned to the A_IDH (‘low grade’) methylation subclass and 10 (10%) to the A_IDH_HG (‘high grade’) subclass. The relative proportion of high grade tumors increased ~three-fold at tumor recurrence (32/101, 32%) and even further in the second recurrence (15/29, 52%). The high grade subclass of the recurrent, but not the initial tumor sample, was negatively associated with OS (p &amp;lt; 0.0001). The overall DNA-methylation levels of recurrent samples were lower than that of initial samples. This difference is explained by the increased number of high grade samples at recurrence, since near identical DNA-methylation levels were observed in samples that remained low grade. Analysis of CNAs revealed several chromosomal arms and bands that were more frequently altered in samples of the high grade methylation subclass compared to low grade. In addition, gains and losses of specific genes, such as homozygous deletion (HD) of CDKN2A/B, were more frequent in high grade samples. Overall DNA-methylation levels of recurrent samples with CDKN2A/B HD were lower than that of samples without this deletion. However, CDKN2A/B HD alone does not fully explain DNA-demethylation at malignant progression and other molecular aberrations are likely to contribute as well. CONCLUSION. Longitudinal methylation profiling analysis of IDH-mutant astrocytoma reveals a shift towards a higher grade at tumor recurrence coinciding with reduced genome-wide DNA-methylation levels. Citation Format: Wies Rijan Vallentgoed, Anneke Niers, Karin van Garderen, Martin van den Bent, Erik van Dijk, Kaspar Draaisma, Paul van Eijk, Iris de Heer, Mathilde Kouwenhoven, Johan Kros, Wendy de Leng, Ivonne Martin, Pierre Robe, Marion Smits, Mircea Tesileanu, Hinke van Thuijl, Roel Verhaak, Bart Westerman, Mark van de Wiel, Bauke Ylstra, Pieter Wesseling, Pim French. Methylation analysis of matched primary and recurrent IDH-mutant astrocytoma; an update from the GLASS-NL consortium [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 4020.

HGG-14. Molecular characterization of unique biological subgroups among H3 wild type high-grade gliomas

INTRODUCTION: Paediatric high-grade gliomas (HGG) are characterised by the aggressive biological behaviour with dismal prognosis of long-term survival 10-15%. Current molecular-biological diagnostic approaches allow for more precise characterization and determination of new unique subgroups of HGG. Our aim was to identify novel and rare HGG subgroups within our institution cohort. PATIENTS AND METHODS: Our reference centre patients′ cohort consisted of 97 clinically annotated patients with HGG diagnosed between 2000 and 2021. Sanger sequencing was used for screening of the most common HGG-related oncogenic drivers; furthermore we employed whole genome methylation array (Illumina Infinium MethylationEPIC BeadChip) and for selected samples RNA sequencing and expression profiling. RESULTS: Based on H3 status and previous radiotherapy we separated our HGG cases into the RIG, H3mut and H3wt groups. In contrast to H3mut(n=35) and RIG(n=11) that were uniformly fatal, H3wt group contained a proportion of long-term survivors. In the H3wt group we found patients carrying driver mutations in IDH1/2 (n=2) and BRAFV600E (7). Five young patients (under 3) consisted of 3 infant hemispheric gliomas (with NTRK and ROS1 fusions), one gliomatosis cerebri and one brainstem anaplastic astrocytoma with MYB/QKI fusion. We also identified a rare EWSR1-PATZ1 gene fusion in one patient. Importantly, long-term survivors recruited from these subgroups. On the contrary, four cases of MYCN GBM with poor prognosis presented in various locations: one disseminated, one gliomatosis cerebri and two with hemispheric tumour. We identified one patient with “hypermutated” glioblastoma and used targeted therapy with Nivolumab. In three samples of our patients with thalamic glioblastomas, we detected “loss of H3K27-trimethylation” caused by EZHIP overexpression. These tumours proved to be very aggressive with early metastatic recurrence and dismal prognosis. SUMMARY: Detailed characterization of H3 wild-type HGG is very important for further understanding of their biological behaviour, diagnostics, prognostication and identification of therapeutic targets.

Accelerated epigenetic aging in suicide attempters uninfluenced by high intent-to-die and choice of lethal methods

Suicide attempts (SA) are associated with excess non-suicidal mortality, putatively mediated in part by premature cellular senescence. Epigenetic age (EA) estimators of biological age have been previously demonstrated to strongly predict physiological dysregulation and mortality risk. Herein, we investigate if violent SA with high intent-to-die is predictive of epigenetics-derived estimates of biological aging. The genome-wide methylation pattern was measured using the Illumina Infinium Methylation EPIC BeadChip in whole blood of 88 suicide attempters. Subjects were stratified into two groups based on the putative risk of later committed suicide (low- [n = 58] and high-risk [n = 30]) in dependency of SA method (violent or non-violent) and/or intent-to-die (high/low). Estimators of intrinsic and extrinsic EA acceleration, one marker optimized to predict physiological dysregulation (DNAmPhenoAge/AgeAccelPheno) and one optimized to predict lifespan (DNAmGrimAge/AgeAccelGrim) were investigated for associations to severity of SA, by univariate and multivariate analyses. The study was adequately powered to detect differences of 2.2 years in AgeAccelGrim in relation to SA severity. Baseline DNAmGrimAge exceeded chronological age by 7.3 years on average across all samples, conferring a mean 24.6% increase in relation to actual age. No individual EA acceleration marker was differentiated by suicidal risk group (p > 0.1). Thus, SA per se but not severity of SA is related to EA, implicating that excess non-suicidal mortality in SA is unrelated to risk of committed suicide. Preventative healthcare efforts aimed at curtailing excess mortality after SA may benefit from acting equally powerful to recognize somatic comorbidities irrespective of the severity inherent in the act itself.

Epigenetic age acceleration in U.S. testicular cancer survivors (TCS).

5033 Background: Given the young age at diagnosis and effective therapies (cisplatin-based chemotherapy and/or surgical approaches), most TCS gain many decades of life. Attention has been drawn to the potential downsides of these treatment successes, including the accelerated development of age-related diseases. Previous studies have suggested that cytotoxic treatment and carcinogenesis are associated with epigenetic changes, leading to premature biological aging processes. Methods: We recruited 24 TCS managed with surgical approaches alone and 24 cisplatin-treated TCS (all also treated with surgical removal of the cancerous testis) and who had histologic/serological germ cell tumor diagnosis before 55 years, and were disease-free at routine follow up. We also included 310 cancer-free race- and age-matched (+/- 5 years) males from a normative cohort, i.e., the Coronary Artery Risk Development in Young Adults (CARDIA) Study. We undertook genome-wide interrogation of blood DNA methylation (DNAm) using the Illumina Infinium Methylation EPIC BeadChip (EPIC array). We quantified aging using DNAm GrimAge, a validated, composite biomarker consisting of DNAm surrogate biomarkers of seven plasma proteins associated with various age-related conditions plus a DNAm surrogate of smoking pack-years. The comparison analysis was adjusted for age, race, smoking status, blood cell type proportions estimated using DNA methylation batch effect, and other laboratory-related technical factors. Results: The median chronological age of TCS was 28 years (range 19-64) with 25% smokers. Compared to their chronological age, TCS managed with surgical approaches alone had a mean GrimAge of 39.4 years (i.e., 9.2 years older, paired t-test P = 0.001), and cisplatin-treated TCS had a mean GrimAge of 45.2 years (i.e., 14.9 years older, paired t-test P &lt; 0.001). Compared to the matched CARDIA controls, TCS managed with surgical approaches alone were on average 3.0 years epigenetically older (P = 0.093), and cisplatin-treated TCS were on average 11.2 years epigenetically older (P &lt; 0.001), suggesting an increasing trend with treatment burden (P-trend &lt; 0.001). Conclusions: Our data showed a faster epigenetic aging process in TCS. Consistent with previous studies, our data is in line with the hypothesis that TC cytotoxic treatment may induce premature aging. Although TCS managed with surgical approaches alone had no exposure to cytotoxic drugs, their epigenetic age may also be accelerated due to TC development and/or risk factors that contributed to it as well as post-therapy factors.

Identifying DNA methylation signatures in high-grade serous ovarian cancer: Results vary by control tissue type.

e17559 Background: High grade serous ovarian cancer (HGSC) is the most common and fatal epithelial ovarian cancer and is often diagnosed in late stages. DNA methylation has emerged as a potential biomarker for the early detection of cancer, including ovarian cancer. Studies examining DNA methylation in ovarian tumor tissue have used adjacent non-tumor tissues or tissues from unaffected women as the control; however, few have used paired tissue and research is sparse on how results may vary by the control non-tumor tissue type. Therefore, we examined DNA methylation signatures in HGSC tumor tissue using different control non-tumor tissue types. Methods: We examined DNA methylation signatures using the Illumina Infinium MethylationEPIC BeadChip (850k) in formalin-fixed paraffin-embedded tissue. In women with HGSC, we compared DNA methylation patterns between paired adjacent non-tumor ovary (n = 74) and fallopian tube (n = 80) tissues. We compared DNA methylation patterns in these adjacent non-tumor tissues with non-tumor ovary (n = 8) and fallopian tube (n = 8) tissues from unaffected women without ovarian cancer carrying a pathogenic variant in BRCA1/2 ( BRCA1/2+). Lastly, we compared the overlap in differentially methylated CpGs identified in HGSC tumor tissue compared to paired adjacent non-tumor ovary (n = 50) and fallopian tube (n = 52) tissues. We processed the methylation data and used principal components analysis (PCA) and the adjusted Rand Index to compare the non-tumor tissue type methylation patterns. We used a paired t-test between individual-matched tumor and adjacent non-tumor tissue for each CpG site and then applied Bonferroni’s method to adjust the obtained p-values. Results: PCA comparing methylation patterns between paired adjacent non-tumor ovary and fallopian tube tissues from women with HGSC showed an adjusted Rand Index of 0.78, revealing separate clusters that cannot be combined. PCA comparing methylation patterns from the adjacent non-tumor tissues from women with HGSC and the non-tumor tissues from unaffected BRCA1/2+ women showed an adjusted Rand Index of -0.10 and 0.07 for the ovary and fallopian tube tissues, respectively, revealing overlapping clusters that can be combined. Lastly, comparison of the top differentially methylated CpGs identified in HGSC tumor tissue when using paired adjacent non-tumor ovary versus fallopian tube tissues as the control showed minimal overlap (6.8% and 4.0% for hypermethylated and hypomethylated CpGs, respectively). Conclusions: These results suggest that paired adjacent non-tumor ovary and fallopian tube tissues from women with HGSC have different DNA methylation patterns and result in different methylation signatures identified in HGSC tumor tissue. When comparing results across studies, including for validation, the type of non-tumor tissue control must be considered.

MATERNAL OBESITY AND GESTATIONAL DIABETES ARE ASSOCIATED WITH DNA METHYLATION ALTERATIONS IN THE OFFSPRING THROUGHOUT THE FIRST YEAR OF LIFE

Objective: Obesity is a chronic metabolic disease of increasing prevalence and health importance in modern societies, which represents an added risk for the development of multiple pathologies. Moreover, epidemiological studies have shown how maternal obesity or gestational diabetes during pregnancy constitutes an important risk factor related to the appearance of cardiovascular and metabolic diseases in the offspring. Furthermore, this period of life is especially sensitive to extrinsic and intrinsic influence, so epigenetic remodelling may help explain the molecular mechanisms that underlie epidemiological findings. The main objective of this work was to determine the DNA methylation landscape of children born to mothers with obesity and gestational diabetes across their first year of life. Design and method: We have characterized the methylome of blood samples from a paediatric cohort of 39 subjects consisting of children born to mothers who suffered from obesity or obesity with gestational diabetes during pregnancy and healthy controls. We performed longitudinal measurements throughout the first year of life (0, 6 and 12 months; total N = 92) using Illumina Infinium MethylationEPIC BeadChip arrays to profile more than 770,000 CpG sites. The design allowed us to carry out both cross-sectional and longitudinal analyses in order to derive DNA methylation alterations associated with developmental and pathological-related epigenomics. Results: We have identified abundant DNA methylation alterations during child development from birth to 6 months and, to a lesser extent, until 12 months of age. At the same time, using cross-sectional analyses, we have identified DNA methylation biomarkers that discriminate children born to mothers who suffered from obesity or obesity with gestational diabetes during pregnancy compared to their healthy counterparts. Moreover, our longitudinal design allowed us to determine that these alterations are at least maintained during the first year of life. In addition, gene enrichment analyses revealed that these DNA methylation alterations are associated with regions and genes related to feeding behaviour, appetite, metabolism and cell signalling. Conclusions: Our results support the existence of an intrauterine-mediated maternal obesity effect with an impact on the childhood methylome and possible functional implications.

The mediating effect of DNA methylation in the association between maternal sleep during pregnancy and offspring adiposity status: a prospective cohort study

BackgroundChildhood overweight/obesity is a global public health concern. It is important to identify its early-life risk factors. Maternal poor sleep is common in late pregnancy, and previous studies indicated that poor sleep may influence the offspring’s adiposity status. However, very few studies in humans investigated the effect of the different sleep parameters (sleep quantity, quality, and timing) on the offspring’s adiposity indicators, and long-term studies are even more scarce. In addition, the underlying mechanism remains unclear. The present study therefore aimed to examine the association between the three maternal sleep dimensions in the late pregnancy and the offspring adiposity indicators and to explore the potential mediating effect of the cord blood DNA methylation in the above association.MethodsIncluded participants in the current study were 2211 healthy pregnant women with singleton gestation from the Shanghai Birth Cohort (SBC) and Shanghai Sleep Birth Cohort (SSBC). Maternal nighttime sleep duration, quality, and midpoint (an indicator of circadian rhythm) were assessed by the same instrument in both cohorts during late pregnancy, and the offspring’s body mass index (BMI) and subcutaneous fat (SF) were measured at 2 years old. Additionally, in 231 SSBC samples, the genome-wide DNA methylation levels were measured using the Illumina Infinium Methylation EPIC BeadChip. The multivariate linear regression was used to determine the associations between the maternal sleep parameters and the offspring adiposity indicators. The epigenome-wide association study was conducted to identify the maternal sleep-related CpG sites. The mediation analysis was performed to evaluate the potential intermediate role of DNA methylation in the association between maternal sleep and offspring adiposity indicators.ResultsThe mean maternal nighttime sleep duration and the sleep midpoint for combined cohorts were 9.24 ± 1.13 h and 3.02 ± 0.82, respectively, and 24.5% of pregnant women experienced poor sleep quality in late pregnancy. After adjusting for the covariates, the maternal later sleep midpoint was associated with the increased SF in offspring (Coef. = 0.62, 95% CI 0.37–0.87, p < 0.001) at 2 years old. However, no significant associations of the nighttime sleep duration or sleep quality with the offspring adiposity indicators were found. In the SSBC sample, 45 differential methylated probes (DMPs) were associated with the maternal sleep midpoint, and then, we observed 10 and 3 DMPs that were also associated with the offspring’s SF and BMI at 2 years, of which cg04351668 (MARCH9) and cg12232388 significantly mediated the relationship of sleep midpoint and SF and cg12232388 and cg12225226 mediated the sleep midpoint–BMI association, respectively.ConclusionsMaternal later sleep timing in late pregnancy was associated with higher childhood adiposity in the offspring. Cord blood DNA methylation may play a mediation role in that relationship.

Role and Diagnostic Performance of Host Epigenome in Respiratory Morbidity after RSV Infection: The EPIRESVi Study.

BackgroundRespiratory syncytial virus (RSV) infection has been associated with the subsequent development of recurrent wheezing and asthma, although the mechanisms involved are still unknown. We investigate the role of epigenetics in the respiratory morbidity after infection by comparing methylation patterns from children who develop recurrent wheezing (RW-RSV), subsequent asthma (AS-RVS), and those experiencing complete recovery (CR-RSV).MethodsProspective, observational study of infants aged < 2 years with RSV respiratory infection admitted to hospital and followed-up after discharge for at least three years. According to their clinical course, patients were categorized into subgroups: RW-RSV (n = 36), AS-RSV (n = 9), and CR-RSV (n = 32). The DNA genome-wide methylation pattern was analyzed in whole blood samples, collected during the acute phase of the infection, using the Illumina Infinium Methylation EPIC BeadChip (850K CpG sites). Differences in methylation were determined through a linear regression model adjusted for age, gender and cell composition.ResultsPatients who developed respiratory sequelae showed a statistically significant higher proportion of NK and CD8T cells (inferred through a deconvolution approach) than those with complete recovery. We identified 5,097 significant differentially methylated positions (DMPs) when comparing RW-RSV and AS-RVS together against CR-RSV. Methylation profiles affect several genes involved in airway inflammation processes. The most significant DMPs were found to be hypomethylated in cases and therefore generally leading to overexpression of affected genes. The lead CpG position (cg24509398) falls at the gene body of EYA3 (P-value = 2.77×10-10), a tyrosine phosphatase connected with pulmonary vascular remodeling, a key process in the asthma pathology. Logistic regression analysis resulted in a diagnostic epigenetic signature of 3-DMPs (involving genes ZNF2698, LOC102723354 and RPL15/NKIRAS1) that allows to efficiently differentiate sequelae cases from CR-RSV patients (AUC = 1.00). Enrichment pathway analysis reveals the role of the cell cycle checkpoint (FDR P-value = 4.71×10-2), DNA damage (FDP-value = 2.53×10-2), and DNA integrity checkpoint (FDR P-value = 2.56×10-2) in differentiating sequelae from CR-RSV patients.ConclusionsEpigenetic mechanisms might play a fundamental role in the long-term sequelae after RSV infection, contributing to explain the different phenotypes observed.

Investigating the relationship between melatonin patterns and methylation in circadian genes among day shift and night shift workers

ObjectivesMechanisms underlying the carcinogenicity of night shift work remain uncertain. One compelling yet understudied cancer mechanism may involve altered DNA methylation in circadian genes due to melatonin secretion patterns. The...

HPA-axis dysregulation is not associated with accelerated epigenetic aging in patients with hypersexual disorder

BackgroundHypersexual disorder (HD) - a nonparaphilic sexual desire disorder with impulsivity component - was evaluated for inclusion as a diagnosis in the DSM-5 and the diagnosis compulsive sexual behavior disorder is included as an impulse control disorder in the ICD-11. Hypothalamic-pituitary-adrenal (HPA)-axis hyperactivity is believed to affect cellular senescence and has been implicated in HD. No previous study investigated HD or HPA-axis dysregulation in relation to measures of epigenetic age (EA) acceleration. MethodsThis study reports on a case-control study set-up from a well-characterized cohort, contrasting EA predictors in relation to 60 HD patients and 33 healthy volunteers (HV) and 19 mixed HD/HV exhibiting dexamethasone suppression test (DST) non-suppression to 73 mixed HD/HV DST controls. The genome-wide methylation pattern was measured in whole blood from 94 subjects using the Illumina Infinium Methylation EPIC BeadChip and preprocessed according to specialized protocols suitable for epigenetic age estimation. The online DNAm Age Calculator (https://dnamage.genetics.ucla.edu/) was implemented to retrieve various EA predictors, which were compared between the in-silico generated subgroups. ResultsQuality control analyses indicated strong correlations between the EA measure DNA methylation GrimAge (DNAm GrimAge – the EA clock most reliably associated with mortality risk) and chronological age in all sub-groups. The study was adequately powered to detect differences of 2.5 and 3.0 years in DNAm GrimAge minus age in relation to both HD and HPA-axis dysregulation, respectively. Baseline DNAm GrimAge exceeded chronological age by 2.8 years on average across all samples. No EA acceleration marker was associated with HD or DST suppression status (p > 0.05). ConclusionEA acceleration markers shown to be strongly predictive of physiological dysregulation and mortality-risk, are not related to HD or DST non-suppression status (measured after 0.5 mg dexamethasone). The independency of HPA-axis dysregulation to EA acceleration does not support the biological relevance of this dosage-regimen when applied to patients with HD. These findings do not support the notion of accelerated cellular senescence in HD. Studies stratifying DST non-suppressors according to established dosage-regimens in somatic settings are needed to fully elucidate the putative contribution of HPA-axis dysregulation to EA.