MethylGenotyper: Accurate Estimation of SNP Genotypes and Genetic Relatedness from DNA Methylation Data.

Epigenome-wide association studies (EWAS) are susceptible to widespread confounding caused by population structure and genetic relatedness. Nevertheless, kinship estimation is challenging in EWAS without genotyping data. Here, we proposed MethylGenotyper, a method that for the first time enables accurate genotyping at thousands of single nucleotide polymorphisms (SNPs) directly from commercial DNA methylation microarrays. We modeled the intensities of methylation probes near SNPs with a mixture of three beta distributions corresponding to different genotypes and estimated parameters with an expectation-maximization algorithm. We conducted extensive simulations to demonstrate the performance of the method. When applying MethylGenotyper to the Infinium EPIC array data of 4662 Chinese samples, we obtained genotypes at 4319 SNPs with a concordance rate of 98.26%, enabling the identification of 255 pairs of close relatedness. Furthermore, we showed that MethylGenotyper allows for the estimation of both population structure and cryptic relatedness among 702 Australians of diverse ancestry. We also implemented MethylGenotyper in a publicly available R package (https://github.com/Yi-Jiang/MethylGenotyper) to facilitate future large-scale EWAS.

Expression, DNA methylation pattern and transcription factor EPB41L3 in gastric cancer: a study of 262 cases.

PURPOSE: DNA methylation prominently inactivates tumor suppressor genes and facilitates oncogenesis. Previously, we delineated a chromosome 18 deletion encompassing the erythrocyte membrane protein band 4.1-like 3 (EPB41L3) gene, a progenitor for the tumor suppressor that is differentially expressed in adenocarcinoma of the lung-1 (DAL-1) in gastric cancer (GC). METHODS: Our current investigation aimed to elucidate EPB41L3 expression and methylation in GC, identify regulatory transcription factors, and identify affected downstream pathways. Immunohistochemistry demonstrated that DAL-1 expression is markedly reduced in GC tissues, with its downregulation serving as an independent prognostic marker. RESULTS: High-throughput bisulfite sequencing of 70 GC patient tissue pairs revealed that higher methylation of non-CpGs in the EPB41L3 promoter was correlated with more malignant tumor progression and higher-grade tissue classification. Such hypermethylation was shown to diminish DAL-1 expression, thus contributing to the malignancy of GC phenotypes. The DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR) was found to partially restore DAL-1 expression. Moreover, direct binding of the transcription factor CDC5L to the upstream region of the EPB41L3 promoter was identified via chromosome immunoprecipitation (ChIP)-qPCR and luciferase reporter assays. Immunohistochemistry confirmed the positive correlation between CDC5L and DAL-1 protein levels. Subsequent RNA-seq analysis revealed that DAL-1 significantly influences the extracellular matrix and space-related pathways. GC cell RNA-seq post-5-Aza-CdR treatment and single-cell RNA-seq data of GC tissues confirmed the upregulation of AREG and COL17A1, pivotal tumor suppressors, in response to EPB41L3 demethylation or overexpression in GC epithelial cells. CONCLUSION: In conclusion, this study elucidates the association between non-CpG methylation of EPB41L3 and GC progression and identifies the key transcription factors and downstream molecules involved. These findings enhance our understanding of the role of EPB41L3 in gastric cancer and provide a solid theoretical foundation for future research and potential clinical applications.

The EPB41L3 gene, frequently exhibiting haplotype deletions and reduced expression in gastric cancer tissues, points to its potential role as a tumor suppressor. However, tumor suppressor genes are not only influenced by genomic deletions but also by their methylation status. Our study highlights the significantly lower expression of EPB41L3 in gastric cancer compared to adjacent non-cancerous tissues across 262 patients. We also discovered that elevated non-CpG island methylation of EPB41L3 correlates strongly with tumor malignancy progression, based on the analysis of 70 paired gastric cancer samples. Moreover, we identified CDC5L as a crucial transcription factor interacting with the EPB41L3 promoter. Integrative analyses of transcriptomic and single-cell sequencing data further revealed that AREG and COL17A1 are key downstream molecules regulated by DAL-1, with their expression tightly controlled by EPB41L3 methylation and expression levels. These insights enhance our understanding of EPB41L3's role in gastric cancer and could open new avenues for targeted therapies.

Dynamic structural remodeling of LINC01956 enhances temozolomide resistance in MGMT-methylated glioblastoma.

The mechanisms underlying stimuli-induced dynamic structural remodeling of RNAs for the maintenance of cellular physiological function and survival remain unclear. Here, we showed that in MGMT promoter-methylated glioblastoma (GBM), the RNA helicase DEAD-box helicase 46 (DDX46) is phosphorylated by temozolomide (TMZ)-activated checkpoint kinase 1 (CHK1), resulting in a dense-to-loose conformational change and an increase in DDX46 helicase activity. DDX46-mediated tertiary structural remodeling of LINC01956 exposes the binding motifs of LINC01956 to the 3' untranslated region of O6-methylguanine DNA methyltransferase (MGMT). This accelerates recruitment of MGMT mRNA to the RNA export machinery and transportation of MGMT mRNA from the nucleus to the cytoplasm, leading to increased MGMT abundance and TMZ resistance. Using patient-derived xenograft (PDX) and tumor organoid models, we found that treatment with the CHK1 inhibitor SRA737abolishes TMZ-induced structural remodeling of LINC01956 and subsequent MGMT up-regulation, resensitizing TMZ-resistant MGMT promoter-methylated GBM to TMZ. In conclusion, these findings highlight a mechanism underlying temozolomide-induced RNA structural remodeling and may represent a potential therapeutic strategy for patients with TMZ-resistant MGMT promoter-methylated GBM.

Mitochondrial dysfunction gene expression, DNA methylation, and inflammatory cytokines interaction activate Alzheimer's disease: a multi-omics Mendelian randomization study.

BACKGROUND: Mitochondrial dysfunction (MD) is increasingly recognized as a key pathophysiological contributor in Alzheimer disease (AD). As differential MD genes expression may serve as either a causative factor or a consequence in AD, and expression of these genes could be influenced by epigenetic modifications or interact with inflammatory cytokines, hence, the precise role of MD in AD remains uncertain. METHODS: Meta-analysis of brain transcriptome datasets was conducted to pinpoint differentially expressed genes (DEGs) associated with MD in AD. We utilized three-step SMR to analyze the AD genome-wide association study summaries with expression quantitative trait loci (eQTLs) and DNA methylation QTLs from the blood and brain tissues, respectively. Through SMR and colocalization analysis, we further explored the interactions between brain eQTLs and inflammatory cytokines. RESULTS: Five datasets were meta-analyzed to prioritize 825 DEGs in AD from 1339 MD-related genes. Among these, seven genes from blood samples such as NDUFS8 and SPG7 and thirty-two genes from brain tissue including CLU and MAPT were identified as candidate AD-causal MD genes and regulated by methylation level. Furthermore, we revealed 13 MD gene expression-inflammatory pathway pairs involving LDLR, ACE and PTPMT1 along with interleukin-17C, interleukin-18 and hepatocyte growth factor. CONCLUSIONS: This study highlighted that the AD-causal MD genes could be regulated by epigenetic changes and interact with inflammatory cytokines, providing evidence for AD prevention and intervention.

Epigenomic biomarkers insights in PBMCs for prognostic assessment of ECMO-treated cardiogenic shock patients.

OBJECTIVE: As the global use of extracorporeal membrane oxygenation (ECMO) treatment increases, survival rates have not correspondingly improved, emphasizing the need for refined patient selection to optimize resource allocation. Currently, prognostic markers at the molecular level are limited. METHODS: Thirty-four cardiogenic shock (CS) patients were prospectively enrolled, and peripheral blood mononuclear cells (PBMCs) were collected at the initiation of ECMO (t0), two-hour post-installation (t2), and upon removal of ECMO (tr). The PBMCs were analyzed by comprehensive epigenomic assays. Using the Wilcoxon signed-rank test and least absolute shrinkage and selection operator (LASSO) regression, 485,577 DNA methylation features were analyzed and selected from the t0 and tr datasets. A random forest classifier was developed using the t0 dataset and evaluated on the t2 dataset. Two models based on DNA methylation features were constructed and assessed using receiver operating characteristic (ROC) curves and Kaplan-Meier survival analyses. RESULTS: The ten-feature and four-feature models for predicting in-hospital mortality attained area under the curve (AUC) values of 0.78 and 0.72, respectively, with LASSO alpha values of 0.2 and 0.25. In contrast, clinical evaluation systems, including ICU scoring systems and the survival after venoarterial ECMO (SAVE) score, did not achieve statistical significance. Moreover, our models showed significant associations with in-hospital survival (p < 0.05, log-rank test). CONCLUSIONS: This study identifies DNA methylation features in PBMCs as potent prognostic markers for ECMO-treated CS patients. Demonstrating significant predictive accuracy for in-hospital mortality, these markers offer a substantial advancement in patient stratification and might improve treatment outcomes.

MethylSeqLogo: DNA methylation smart sequence logos.

BACKGROUND: Some transcription factors, MYC for example, bind sites of potentially methylated DNA. This may increase binding specificity as such sites are (1) highly under-represented in the genome, and (2) offer additional, tissue specific information in the form of hypo- or hyper-methylation. Fortunately, bisulfite sequencing data can be used to investigate this phenomenon. METHOD: We developed MethylSeqLogo, an extension of sequence logos which includes new elements to indicate DNA methylation and under-represented dimers in each position of a set binding sites. Our method displays information from both DNA strands, and takes into account the sequence context (CpG or other) and genome region (promoter versus whole genome) appropriate to properly assess the expected background dimer frequency and level of methylation. MethylSeqLogo preserves sequence logo semantics-the relative height of nucleotides within a column represents their proportion in the binding sites, while the absolute height of each column represents information (relative entropy) and the height of all columns added together represents total information RESULTS: We present figures illustrating the utility of using MethylSeqLogo to summarize data from several CpG binding transcription factors. The logos show that unmethylated CpG binding sites are a feature of transcription factors such as MYC and ZBTB33, while some other CpG binding transcription factors, such as CEBPB, appear methylation neutral. CONCLUSIONS: Our software enables users to explore bisulfite and ChIP sequencing data sets-and in the process obtain publication quality figures.

Differences in the DNA methylome of T cells in adults with asthma of varying severity.

BACKGROUND: DNA methylation plays a critical role in asthma development, but differences in DNA methylation among adults with varying asthma severity are less well-defined. OBJECTIVE: To examine how DNA methylomic patterns differ among adults with asthma based on asthma severity and airway inflammation. METHODS: Peripheral blood T cells from 35 adults with asthma in Beijing, China, were serially collected over time (130 samples total) and analyzed for global DNA methylation using the Illumina MethylationEPIC Array. Differential methylation was compared among subjects with varying airway inflammation and severity, as measured by fraction of exhaled nitric oxide, forced expiratory volume in one second (FEV1), and Asthma Control Test (ACT) scores. RESULTS: Significant differences in DNA methylation were noted among subjects with different degrees of airway inflammation and asthma severity. These differences in DNA methylation were annotated to genes that were enriched in pathways related to asthma or T cell function and included gene ontology categories related to MHC class II assembly, T cell activation, interleukin (IL)-1, and IL-12. Genes related to P450 drug metabolism, glutathione metabolism, and developmental pathways were also differentially methylated in comparisons between subjects with high vs low FEV1 and ACT. Notable genes that were differentially methylated based on asthma severity included RUNX3, several members of the HLA family, AGT, PTPRC, PTPRJ, and several genes downstream of the JAK2 and TNF signaling pathway. CONCLUSION: These findings demonstrate how adults with asthma of varying severity possess differences in peripheral blood T cell DNA methylation that contribute to differences in clinical indices of asthma.

Comprehensive molecular and clinical findings in 29 patients with multi-locus imprinting disturbance.

BACKGROUND: Multi-locus imprinting disturbance (MLID) with methylation defects in various differentially methylated regions (DMRs) has recently been identified in approximately 150 cases with imprinting disorders (IDs), and deleterious variants have been found in genes related to methylation maintenance of DMRs, such as those encoding proteins constructing the subcortical maternal complex (SCMC), in a small fraction of patients and/or their mothers. However, integrated methylation analysis for DMRs and sequence analysis for MLID-causative genes in MLID cases and their mothers have been performed only in a single study focusing on Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS) phenotypes. RESULTS: Of 783 patients with various IDs we have identified to date, we examined a total of 386 patients with confirmed epimutation and 71 patients with epimutation or uniparental disomy. Consequently, we identified MLID in 29 patients with epimutation confirmed by methylation analysis for multiple ID-associated DMRs using pyrosequencing and/or methylation-specific multiple ligation-dependent probe amplification. MLID was detected in approximately 12% of patients with BWS phenotype and approximately 5% of patients with SRS phenotype, but not in patients with Kagami-Ogata syndrome, Prader-Willi syndrome, or Angelman syndrome phenotypes. We next conducted array-based methylation analysis for 78 DMRs and whole-exome sequencing in the 29 patients, revealing hypomethylation-dominant aberrant methylation patterns in various DMRs of all the patients, eight probably deleterious variants in genes for SCMC in the mothers of patients, and one homozygous deleterious variant in ZNF445 in one patient. These variants did not show gene-specific methylation disturbance patterns. Clinically, neurodevelopmental delay and/or intellectual developmental disorder (ND/IDD) was observed in about half of the MLID patients, with no association with the identified methylation disturbance patterns and genetic variants. Notably, seven patients with BWS phenotype were conceived by assisted reproductive technology (ART). CONCLUSIONS: The frequency of MLID was 7.5% (29/386) in IDs caused by confirmed epimutation. Furthermore, we revealed diverse patterns of hypomethylation-dominant methylation defects, nine deleterious variants, ND/IDD complications in about half of the MLID patients, and a high frequency of MLID in ART-conceived patients.

Epigenetic Contributors to PTSD: a Comprehensive Review.

BACKGROUND: Post-traumatic stress disorder (PTSD) is a complex condition triggered by traumatic events. The molecular mechanisms underlying PTSD are not fully understood, but epigenetic modifications, particularly DNA methylation, may play a key role. The objective of this review was to identify the most significant epigenetic markers associated with PTSD. MATERIALS AND METHODS: Our search yielded 325 articles, of which 19 met our inclusion criteria for detailed analysis: published between 2018 and 2024, original research, containing molecular-genetic and statistical data, reporting diagnostic verification methods, PTSD as a primary condition, and a sample of at least 40 patients Results: the strongest correlation was found between PTSD and methylation changes in cg17057218, cg22324981, cg04755409 of BDNF, cg05656210, cg12169700, cg20756026 of MAD1L1, HLA-DPA1, HLA-DPB1 (chr6: 33047185 - 33049505) and SPATC1L (chr21: 47604052 - 47605174). The most works on associations of genetic clock with PTSD found significantly increased GrimAge acceleration in patients with PTSD. CONCLUSIONS: Epigenetic modifications, particularly DNA methylation, play a significant role in PTSD pathophysiology. While specific gene methylation changes are associated with PTSD, the link between PTSD and epigenetic aging remains unclear. Variability across studies suggests that trauma type, duration, and genetic factors may influence these epigenetic processes. Further research is essential to fully understand these relationships.

SMARTdb: An Integrated Database for Exploring Single-cell Multi-omics Data of Reproductive Medicine.

Single-cell multi-omics sequencing has greatly accelerated reproductive research in recent years, and the data are continually growing. However, utilizing these data resources is challenging for wet-lab researchers. A comprehensive platform for exploring single-cell multi-omics data related to reproduction is urgently needed. Here, we introduce the single-cell multi-omics atlas of reproduction (SMARTdb), an integrative and user-friendly platform for exploring molecular dynamics of reproductive development, aging, and disease, which covers multi-omics, multi-species, and multi-stage data. We curated and analyzed single-cell transcriptomic and epigenomic data of over 2.0 million cells from 6 species across the entire lifespan. A series of powerful functionalities are provided, such as "Query gene expression", "DIY expression plot", "DNA methylation plot", and "Epigenome browser". With SMARTdb, we found that the male germ cell-specific expression pattern of RPL39L and RPL10L is conserved between human and other model animals. Moreover, DNA hypomethylation and open chromatin may collectively regulate the specific expression pattern of RPL39L in both male and female germ cells. In summary, SMARTdb is a powerful platform for convenient data mining and gaining novel insights into reproductive development, aging, and disease. SMARTdb is publicly available at https://smart-db.cn.

Identification of SLC22A17 DNA methylation hotspot as a potential biomarker in cutaneous melanoma.

BACKGROUND: Cancer onset and progression are driven by genetic and epigenetic alterations leading to oncogene activation and the silencing of tumor suppressor genes. Among epigenetic mechanisms, DNA methylation (methDNA) is gaining growing interest in cancer. Promoter hypomethylation is associated with oncogene activation while intragenic methDNA can be involved in transcriptional elongation, alternative spicing, and the activation of cryptic start sites. Several genes involved in the modulation of the tumor microenvironment are regulated by methDNA, including the Solute Carrier Family 22 Member 17 (SLC22A17), which is involved in iron trafficking and extracellular matrix remodeling cooperating with the Gelatinase-Associated Lipocalin (NGAL) ligand. However, the exact role of intragenic methDNA in cancer has not been fully investigated. Therefore, the aim of the present study is to explore the role of methDNA in the regulation of SLC22A17 in cutaneous melanoma (CM), used as a tumor model. METHODS: Correlation and differential analyses between SLC22A17 expression and methDNA were performed using the data contained in The Cancer Genome Atlas and Gene Expression Omnibus databases. Functional studies on melanoma cell lines treated with 5-Azacytidine (5-Aza) were conducted to assess the correlation between methDNA and SLC22A17 expression. A validation study on the diagnostic potential of the in silico-identified SLC22A17 methDNA hotspot was finally performed by analyzing tissue samples obtained from CM patients and healthy controls. RESULTS: The computational analyses revealed that SLC22A17 was significantly downregulated in CM, and its expression was related to promoter hypomethylation and intragenic hypermethylation. Moreover, SLC22A17 overexpression and hypermethylation of two intragenic methDNA hotspots were associated with a better clinical outcome in CM patients. The correlation between SLC22A17 methDNA and expression was confirmed in 5-Aza-treated cells. In agreement with in silico analyses, the SLC22A17 promoter methylation hotspot showed higher methDNA levels in CM samples compared to nevi. In addition, the methDNA levels of this hotspot were positively correlated with advanced CM. CONCLUSIONS: The SLC22A17 methDNA hotspot could represent a promising biomarker for CM, highlighting the regulatory role of methDNA on SLC22A17 expression. These results pave the way for the identification of novel epigenetic biomarkers and therapeutic targets for the management of CM patients.

Enzymatic TET-1 inhibition highlights different epigenetic behaviours of IL-1ß and TNFα in tumour progression of OS cell lines.

Osteosarcoma (OS) is the most frequent primary malignant bone tumour, whose heterogeneity represents a major challenge for common antitumour therapies. Inflammatory cytokines are known to be necessary for OS progression. Therefore, to optimise therapy, it is important to discover reliable biomarkers by identifying the mechanism generating OS and investigating the inflammatory pathways that support the undifferentiated state. In this work, we highlight the differences of epigenetic activities of IL-1ß and TNFα, and the susceptibility of TET-1 enzymatic inhibition, in tumour progression of three different OS cell lines. Investigating DNA methylation of IL-6 promoter and determining its expression, we found that TET enzymatic inhibition influences proliferation induced by inflammatory cytokines in OS cell lines. Moreover, Bobcat 339 treatment blocks IL-1ß epigenetic action on IL-6 promoter, while only partially those of TNFα as well as inhibits IL-1ß-dependent epithelial-mesenchymal transition (EMT) process, but only partially those of TNFα. In conclusion, this work highlights that IL-1ß and TNFα have different effects on DNA demethylation in OS cell lines, making DNA methylation a potential biomarker of disease. Specifically, in IL-1ß treatment, TET-1 inhibition completely blocks tumour progression, while in TNFα actions, it is only partially effective. Given that these two inflammatory pathways can be therapeutic targets for treating these tumours, knowledge of their distinct epigenetic behaviours can be useful for developing precise and specific therapeutic strategies for this disease.

MGMT methylation and its prognostic significance in inoperable IDH-wildtype glioblastoma: the MGMT-GBM study.

INTRODUCTION: The methylation of the O6-Methylguanine-DNA Methyltransferase (MGMT) promoter is a valid biomarker for predicting response to therapy with alkylating agents and, independently, prognosis in IDH-wildtype(IDH-w) glioblastoma. We aim to study the impact of its methylation in overall survival of the unresectable IDH-w glioblastoma undergoing biopsy and systemic treatment. METHODS: We collected six-year retrospective (2017-2023) data at a quaternary neurosurgery center for patients undergoing biopsy as the only surgical procedure for an unresectable IDH wildtype glioblastoma. Data was collected from patient records including neuro-oncology multidisciplinary team meeting (MDT) documentation. Patients were grouped into categories according to different types of treatment received after biopsy (no treatment, chemotherapy (CT), radiotherapy (RT), chemoradiotherapy (CRT), chemoradiotherapy with adjuvant temozolomide (CRT with adjuvant TMZ), EORTC-NCIC protocol followed by second line treatment) and according to methylation status (unmethylated (< 5%), borderline methylated (5-15%) and strongly methylated (> 15%)). Survival analysis was performed. RESULTS: 166 glioblastoma IDH wildtype patients were included in the study with mean age of 62.5 years (M: F = 1.5: 1). 70 (49.3%) patients had unmethylated MGMT status (< 5%), 29 (20.4%) patients had borderline methylated MGMT status (5-15%) and 43 (30.2%) patients had methylated MGMT status (> 15%). 36 (25.3%) patients did not receive any treatment post biopsy, 13 (9.1%) received CT only, 27 (19%) RT only, 12 (8.4%) CRT, 33 (23.2%) CRT with adjuvant TMZ, whereas 21 (14.7%) received EORTC-NCIC protocol along with second line treatment. In biopsy only group, there was no notable difference in survival outcomes among the different methylation statuses. For biopsy and any-other-form-of-treatment methylated groups showed a distinct trend of better survival compared to the borderline or unmethylated groups. Overall, methylated patients had better survival as compared to unmethylated or borderline groups. CONCLUSION: Methylated MGMT status are predictors for better overall survival in unresectable IDH wildtype glioblastoma patients undergoing biopsy and treatment regardless of the treatment modality.

KLF4 regulates trophoblast function and associates with unexplained recurrent spontaneous abortion.

BACKGROUND: Recurrent spontaneous abortion (RSA) is defined as two or more consecutive spontaneous abortions before 20 weeks with the same spouse [1]. However, approximately 50% of RSA cases of unknown cause are classified as unexplained recurrent spontaneous abortion (URSA). Potential factors include decreased trophoblast cell migration and invasion, leading to impaired placental implantation and maintenance of the normal maternal-fetal interface. However, the mechanism of this pathogenesis remains unknown. In this study, we investigated the potential role and mechanism of KLF4 in regulating URSA by influencing the invasion and migration ability of trophoblast cells. METHODS: We firstly identified 817 differentially expressed genes by performing a difference analysis of the dataset GSE121950 [2] related to recurrent abortion, and intersected the top 10 genes obtained respectively by the three algorithms: DMNC, MNC, and EPC using Venn Diagram.To detect the expression levels of core genes, villi samples were obtained from normal pregnant women and patients with URSA. RT-qPCR analysis revealed a significant difference in KLF4 mRNA expression and KLF4 was then analyzed. Trophoblast cell lines HTR8 and JEG3 were used to investigate the effect of KLF4 on trophoblastic function. Wound healing and transwell assays was performed to detect the invasion and migration of trophoblast cells. The expression of epithelial-mesenchymal transition(EMT) molecules were detected by RT-qPCR and western blot. Promoter detection and epigenetic modification were detected by chromatin immunoprecipitation (ChIP) assay. Molecular nuclear localization was detected by immunofluorescence and subcellular fractionation. Miscarried mice model was used to study the effects of KLF4 on URSA induced by reduced trophoblast invasion and migration. RESULTS: KLF4 is highly expressed in the villi of patients with URSA. KLF4 inhibits the expression level of H3R2ME2a in trophoblast cells by regulating the transcriptional level and nuclear translocation of PRMT6, thereby inhibiting the possible regulatory mechanism of trophoblastic invasion and providing a potential treatment strategy for URSA in vivo. CONCLUSIONS: The KLF4/PRMT6/H3R2ME2a axis regulates mechanisms associated with unexplained recurrent spontaneous abortion by regulating trophoblast function.

Silencing of maternally expressed RNAs in Dlk1-Dio3 domain causes fatal vascular injury in the fetal liver.

The mammalian imprinted Dlk1-Dio3 domain contains multiple lncRNAs, mRNAs, the largest miRNA cluster in the genome and four differentially methylated regions (DMRs), and deletion of maternally expressed RNA within this locus results in embryonic lethality, but the mechanism by which this occurs is not clear. Here, we optimized the model of maternally expressed RNAs transcription termination in the domain and found that the cause of embryonic death was apoptosis in the embryo, particularly in the liver. We generated a mouse model of maternally expressed RNAs silencing in the Dlk1-Dio3 domain by inserting a 3 × polyA termination sequence into the Gtl2 locus. By analyzing RNA-seq data of mouse embryos combined with histological analysis, we found that silencing of maternally expressed RNAs in the domain activated apoptosis, causing vascular rupture of the fetal liver, resulting in hemorrhage and injury. Mechanistically, termination of Gtl2 transcription results in the silencing of maternally expressed RNAs and activation of paternally expressed genes in the interval, and it is the gene itself rather than the IG-DMR and Gtl2-DMR that causes the aforementioned phenotypes. In conclusion, these findings illuminate a novel mechanism by which the silencing of maternally expressed RNAs within Dlk1-Dio3 domain leads to hepatic hemorrhage and embryonic death through the activation of apoptosis.

Epigenetic landscape reorganisation and reactivation of embryonic development genes are associated with malignancy in IDH-mutant astrocytoma.

Accurate grading of IDH-mutant gliomas defines patient prognosis and guides the treatment path. Histological grading is challenging, and aside from CDKN2A/B homozygous deletions in IDH-mutant astrocytomas, there are no other objective molecular markers used for grading. RNA-sequencing was conducted on primary IDH-mutant astrocytomas (n = 138) included in the prospective CATNON trial, which was performed to assess the prognostic effect of adjuvant and concurrent temozolomide. We integrated the RNA-sequencing data with matched DNA-methylation and NGS data. We also used multi-omics data from IDH-mutant astrocytomas included in the TCGA dataset and validated results on matched primary and recurrent samples from the GLASS-NL study. Since discrete classes do not adequately capture grading of these tumours, we utilised DNA-methylation profiles to generate a Continuous Grading Coefficient (CGC) based on classification scores from a CNS-tumour classifier. CGC was an independent predictor of survival outperforming current WHO-CNS5 and methylation-based classification. Our RNA-sequencing analysis revealed four distinct transcription clusters that were associated with (i) upregulation of cell cycling genes; (ii) downregulation of glial differentiation genes; (iii) upregulation of embryonic development genes (e.g. HOX, PAX, and TBX) and (iv) upregulation of extracellular matrix genes. The upregulation of embryonic development genes was associated with a specific increase of CpG island methylation near these genes. Higher grade IDH-mutant astrocytomas have DNA-methylation signatures that, on the RNA level, are associated with increased cell cycling, tumour cell de-differentiation and extracellular matrix remodelling. These combined molecular signatures can serve as an objective marker for grading of IDH-mutant astrocytomas.

Causal effects of cardiovascular health on five epigenetic clocks.

BACKGROUND: This work delves into the relationship between cardiovascular health (CVH) and aging. Previous studies have shown an association of ideal CVH with a slower aging rate, measured by epigenetic age acceleration (EAA). However, the causal relationship between CVH and EAA has remained unexplored. METHODS AND RESULTS: We performed genome-wide association studies (GWAS) on the (12-point) CVH score and its components using the Taiwan Biobank data, in which weighted genetic risk scores were treated as instrumental variables. Subsequently, we conducted a one-sample Mendelian Randomization (MR) analysis with the two-stage least-squares method on 2383 participants to examine the causal relationship between the (12-point) CVH score and EAA. As a result, we observed a significant causal effect of the CVH score on GrimAge acceleration (GrimEAA) (ß [SE]: - 0.993 [0.363] year; p = 0.0063) and DNA methylation-based plasminogen activator inhibitor-1 (DNAmPAI-1) (ß [SE]: - 0.294 [0.099] standard deviation (sd) of DNAmPAI-1; p = 0.0030). Digging individual CVH components in depth, the ideal total cholesterol score (0 [poor], 1 [intermediate], or 2 [ideal]) was causally associated with DNAmPAI-1 (ß [SE]: - 0.452 [0.150] sd of DNAmPAI-1; false discovery rate [FDR] q = 0.0102). The ideal body mass index (BMI) score was causally associated with GrimEAA (ß [SE]: - 2.382 [0.952] years; FDR q = 0.0498) and DunedinPACE (ß [SE]: - 0.097 [0.030]; FDR q = 0.0044). We also performed a two-sample MR analysis using the summary statistics from European GWAS. We observed that the (12-point) CVH score exhibits a significant causal effect on Horvath's intrinsic epigenetic age acceleration (ß [SE]: - 0.389 [0.186] years; p = 0.036) and GrimEAA (ß [SE]: - 0.526 [0.244] years; p = 0.031). Furthermore, we detected causal effects of BMI (ß [SE]: 0.599 [0.081] years; q = 2.91E-12), never smoking (ß [SE]: - 2.981 [0.524] years; q = 1.63E-7), walking (ß [SE]: - 4.313 [1.236] years; q = 0.004), and dried fruit intake (ß [SE]: - 1.523 [0.504] years; q = 0.013) on GrimEAA in the European population. CONCLUSIONS: Our research confirms the causal link between maintaining an ideal CVH and epigenetic age. It provides a tangible pathway for individuals to improve their health and potentially slow aging.

Effects of multisuperovulation on the transcription and genomic methylation of oocytes and offspring.

BACKGROUND: Controlled ovarian stimulation is a common skill of assisted reproductive technologies (ARTs). In the clinic, some females would undergo more than one controlled ovarian stimulation cycle. However, few studies have focused on the influence of multi-superovulation on oocytes and offspring. RESULTS: Here, we found that multi-superovulation disrupted the transcriptome of oocytes and that the differentially expressed genes (DEGs) were associated mainly with metabolism and fertilization. The disruption of mRNA degradation via poly (A) size and metabolism might be a reason for the reduced oocyte maturation rate induced by repeated superovulation. Multi-superovulation results in hypo-genomic methylation in oocytes. However, there was an increase in the methylation level of CGIs. The DMRs are not randomly distributed in genome elements. Genes with differentially methylated regions (DMRs) in promoters are enriched in metabolic pathways. With increasing of superovulation cycles, the glucose and insulin tolerance of offspring is also disturbed. CONCLUSIONS: These results suggest that multi-superovulation has adverse effects on oocyte quality and offspring health.

Phylo-Epigenetics in Phylogeny Analyses and Evolution.

Long-standing, continuous blurring and controversies in the field of phylogenetic interspecies relations, associated with insufficient explanations for dynamics and variability of speeds of evolution in mammals, hint at a crucial missing link. It has been suggested that transgenerational epigenetic inheritance and the concealed mechanisms behind play a distinct role in mammalian evolution. Here, a comprehensive sequence alignment approach in hominid species, i.e., Homo sapiens, Homo neanderthalensis, Denisovan human, Pan troglodytes, Pan paniscus, Gorilla gorilla, and Pongo pygmaeus, comprising conserved CpG islands of housekeeping genes, uncover evidence for a distinct variability of CpG dinucleotides. Applying solely these evolutionary consistent and inconsistent CpG sites in a classic phylogenetic analysis, calibrated by the divergence time point of the common chimpanzee (P. troglodytes) and the bonobo or pygmy chimpanzee (P. paniscus), a "phylo-epigenetic" tree has been generated, which precisely recapitulates branch points and branch lengths, i.e., divergence events and relations, as they have been broadly suggested in the current literature, based on comprehensive molecular phylogenomics and fossil records of many decades. It is suggested here that CpG dinucleotide changes at CpG islands are of superior importance for evolutionary developments. These changes are successfully inherited through the germ line, determining emerging methylation profiles, and they are a central component of transgenerational epigenetic inheritance. It is hidden in the DNA, what will happen on it later.

Genome-wide characterization of dynamic DNA 5-hydroxymethylcytosine and TET2-related DNA demethylation during breast tumorigenesis.

BACKGROUND: Breast tumorigenesis is a complex and multistep process accompanied by both genetic and epigenetic dysregulation. In contrast to the extensive studies on DNA epigenetic modifications 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) in malignant breast tumors, their roles in the early phases of breast tumorigenesis remain ambiguous. RESULTS: DNA 5hmC and 5mC exhibited a consistent and significant decrease from usual ductal hyperplasia to atypical ductal hyperplasia and subsequently to ductal carcinoma in situ (DCIS). However, 5hmC showed a modest increase in invasive ductal breast cancer compared to DCIS. Genomic analyses showed that the changes in 5hmC and 5mC levels occurred around the transcription start sites (TSSs), and the modification levels were strongly correlated with gene expression levels. Meanwhile, it was found that differentially hydroxymethylated regions (DhMRs) and differentially methylated regions (DMRs) were overlapped in the early phases and accompanied by the enrichment of active histone marks. In addition, TET2-related DNA demethylation was found to be involved in breast tumorigenesis, and four transcription factor binding sites (TFs: ESR1, FOXA1, GATA3, FOS) were enriched in TET2-related DhMRs/DMRs. Intriguingly, we also identified a certain number of common DhMRs between tumor samples and cell-free DNA (cfDNA). CONCLUSIONS: Our study reveals that dynamic changes in DNA 5hmC and 5mC play a vital role in propelling breast tumorigenesis. Both TFs and active histone marks are involved in TET2-related DNA demethylation. Concurrent changes in 5hmC signals in primary breast tumors and cfDNA may play a promising role in breast cancer screening.