CpG DNA Methylation Research Articles

Common DNA sequence variation influences epigenetic aging in African populations.

Aging is associated with genome-wide changes in DNA methylation in humans, facilitating the development of epigenetic age prediction models. However, most of these models have been trained primarily on European-ancestry individuals, and none account for the impact of methylation quantitative trait loci (meQTL). To address these gaps, we analyzed the relationships between age, genotype, and CpG methylation in 3 understudied populations: central African Baka (n = 35), southern African ‡Khomani San (n = 52), and southern African Himba (n = 51). We find that published prediction methods yield higher mean errors in these cohorts compared to European-ancestry individuals, and find that unaccounted-for DNA sequence variation may be a significant factor underlying this loss of accuracy. We leverage information about the associations between DNA genotype and CpG methylation to develop an age predictor that is minimally influenced by meQTL, and show that this model remains accurate across a broad range of genetic backgrounds. Intriguingly, we also find that the older individuals and those exhibiting relatively lower epigenetic age acceleration in our cohorts tend to carry more epigenetic age-reducing genetic variants, suggesting a novel mechanism by which heritable factors can influence longevity.

Development of an epigenetic clock resistant to changes in immune cell composition

Epigenetic clocks are age predictors that use machine-learning models trained on DNA CpG methylation values to predict chronological or biological age. Increases in predicted epigenetic age relative to chronological age (epigenetic age acceleration) are connected to aging-associated pathologies, and changes in epigenetic age are linked to canonical aging hallmarks. However, epigenetic clocks rely on training data from bulk tissues whose cellular composition changes with age. Here, we found that human naive CD8+ T cells, which decrease in frequency during aging, exhibit an epigenetic age 15–20 years younger than effector memory CD8+ T cells from the same individual. Importantly, homogenous naive T cells isolated from individuals of different ages show a progressive increase in epigenetic age, indicating that current epigenetic clocks measure two independent variables, aging and immune cell composition. To isolate the age-associated cell intrinsic changes, we created an epigenetic clock, the IntrinClock, that did not change among 10 immune cell types tested. IntrinClock shows a robust predicted epigenetic age increase in a model of replicative senescence in vitro and age reversal during OSKM-mediated reprogramming.

Methylation marks in blood DNA reveal breast cancer risk in patients fulfilling hereditary disease criteria

Less than 15–20% of patients who meet the criteria for hereditary breast and ovarian cancer (HBOC) carry pathogenic coding genetic mutations, implying that other molecular mechanisms may contribute to the increased risk of this condition. DNA methylation in peripheral blood has been suggested as a potential epigenetic marker for the risk of breast cancer (BC). We aimed to discover methylation marks in peripheral blood associated with BC in 231 pre-treatment BC patients meeting HBOC criteria, testing negative for coding pathogenic variants, and 156 healthy controls, through methylation analysis by targeted bisulfite sequencing on 18 tumor suppressor gene promoters (330 CpG sites). We found i) hypermethylation in EPCAM (17 CpG sites; p = 0.017) and RAD51C (27 CpG sites; p = 0.048); ii) hypermethylation in 36 CpG-specific sites (FDR q < 0.05) in the BC patients; iii) four specific CpG sites were associated with a higher risk of BC (FDR q < 0.01, Bonferroni p < 0.001): cg89786999-FANCI (OR = 1.65; 95% CI:1.2–2.2), cg23652916-PALB2 (OR = 2.83; 95% CI:1.7–4.7), cg47630224-MSH2 (OR = 4.17; 95% CI:2.1–8.5), and cg47596828-EPCAM (OR = 1.84; 95% CI:1.5–2.3). Validation of cg47630224-MSH2 methylation in one Australian cohort showed an association with 3-fold increased BC risk (AUC: 0.929; 95% CI: 0.904–0.955). Our findings suggest that four DNA methylation CpG sites may be associated with a higher risk of BC, potentially serving as biomarkers in patients without detectable coding mutations.

Integrative analysis reveals associations between oral microbiota dysbiosis and host genetic and epigenetic aberrations in oral cavity squamous cell carcinoma.

Dysbiosis of the human oral microbiota has been reported to be associated with oral cavity squamous cell carcinoma (OSCC) while the host-microbiota interactions with respect to the potential impact of pathogenic bacteria on host genomic and epigenomic abnormalities remain poorly studied. In this study, the mucosal bacterial community, host genome-wide transcriptome and DNA CpG methylation were simultaneously profiled in tumors and their adjacent normal tissues of OSCC patients. Significant enrichment in the relative abundance of seven bacteria species (Fusobacterium nucleatum, Treponema medium, Peptostreptococcus stomatis, Gemella morbillorum, Catonella morbi, Peptoanaerobacter yurli and Peptococcus simiae) were observed in OSCC tumor microenvironment. These tumor-enriched bacteria formed 254 positive correlations with 206 up-regulated host genes, mainly involving signaling pathways related to cell adhesion, migration and proliferation. Integrative analysis of bacteria-transcriptome and bacteria-methylation correlations identified at least 20 dysregulated host genes with inverted CpG methylation in their promoter regions associated with enrichment of bacterial pathogens, implying a potential of pathogenic bacteria to regulate gene expression, in part, through epigenetic alterations. An in vitro model further confirmed that Fusobacterium nucleatum might contribute to cellular invasion via crosstalk with E-cadherin/β-catenin signaling, TNFα/NF-κB pathway and extracellular matrix remodeling by up-regulating SNAI2 gene, a key transcription factor of epithelial-mesenchymal transition (EMT). Our work using multi-omics approaches explored complex host-microbiota interactions and provided important insights into genetic and functional basis in OSCC tumorigenesis, which may serve as a precursor for hypothesis-driven study to better understand the causational relationship of pathogenic bacteria in this deadly cancer.

Multi-omics cluster defines the subtypes of CRC with distinct prognosis and tumor microenvironment

BackgroundColorectal cancer (CRC) is a complex malignancy characterized by diverse molecular profiles, clinical outcomes, and limited precision in prognostic markers. Addressing these challenges, this study utilized multi-omics data to define consensus molecular subtypes in CRC and elucidate their association with clinical outcomes and underlying biological processes.MethodsConsensus molecular subtypes were obtained by applying ten integrated multi-omics clustering algorithms to analyze TCGA-CRC multi-omics data, including mRNA, lncRNA, miRNA, DNA methylation CpG sites, and somatic mutation data. The association of subtypes with prognoses, enrichment functions, immune status, and genomic alterations were further analyzed. Next, we conducted univariate Cox and Lasso regression analyses to investigate the potential prognostic application of biomarkers associated with multi-omics subtypes derived from weighted gene co-expression network analysis (WGCNA). The function of one of the biomarkers MID2 was validated in CRC cell lines.ResultsTwo CRC subtypes linked to distinct clinical outcomes were identified in TCGA-CRC cohort and validated with three external datasets. The CS1 subtype exhibited a poor prognosis and was characterized by higher tumor-related Hallmark pathway activity and lower metabolism pathway activity. In addition, the CS1 was predicted to have less immunotherapy responder and exhibited more genomic alteration compared to CS2. Then a prognostic model comprising five genes was established, with patients in the high-risk group showing substantial concordance with the CS1 subtype, and those in the low-risk group with the CS2 subtype. The gene MID2, included in the prognostic model, was found to be correlated with epithelial–mesenchymal transition (EMT) pathway and distinct DNA methylation patterns. Knockdown of MID2 in CRC cells resulted in reduced colony formation, migration, and invasion capacities.ConclusionThe integrative multi-omics subtypes proposed potential biomarkers for CRC and provided valuable knowledge for precision oncology.

Alcohol drinking, DNA methylation and psychiatric disorders: A multi-omics Mendelian randomization study to investigate causal pathways.

Whether alcohol-related DNA methylation has a causal effect on psychiatric disorders has not been investigated. Furthermore, a comprehensive investigation into the causal relationship and underlying mechanisms linking alcohol consumption and psychiatric disorders has been lacking. This study aimed to evaluate the causal effect of general alcohol intake and pathological drinking behaviors on psychiatric disorders, alcohol-associated DNA methylation on gene expression and psychiatric disorders, and gene expression on psychiatric disorders. Two-sample design Mendelian randomization (MR) analysis. Various sensitivity and validation analyses, including colocalization analysis, were conducted to test the robustness of the results. Genome-wide association study (GWAS) data mainly from GWAS and Sequencing Consortium of Alcohol and Nicotine use (GSCAN), Genetics of DNA Methylation Consortium (GoDMC) and Psychiatric Genomics Consortium (PGC) with European ancestry. The GWAS summary data on general alcohol intake (drinks per week, n = 941 280), pathological drinking behaviors (including alcohol use disorder [AUD, n = 313 959] and problematic alcohol use [PAU, n = 435 563]) and psychiatric disorders (including schizophrenia, major depressive disorder and bipolar disorder, n = 51 710-500 199) were included. Alcohol-related DNA methylation CpG sites (n = 9643) and mQTL data from blood (n = 27 750) and brain (n = 1160), BrainMeta v2 and GTEx V8 eQTL summary data (n = 73-2865) were also included. Genetic variants were selected as instrumental variables for exposures, including drinks per week, AUD, PAU, alcohol-related DNA methylation CpG sites (mQTL) and genes selected (eQTL). Pathological drinking behaviors were associated with an increased risk of psychiatric disorders after removing outliers or controlling for alcohol consumption. MR analysis identified 10 alcohol-related CpG sites with colocalization evidence that were causally associated with psychiatric disorders (P = 1.65 × 10-4-7.52 × 10-22). Furthermore, the expression of genes (RERE, PTK6, GATAD2B, COG8, PDF and GAS5) mapped to these CpG sites in the brain, led by the cortex, were significantly associated with psychiatric disorders (P = 1.19 × 10-2-3.51 × 10-7). Pathological drinking behavior and alcohol-related DNA methylation appear to have a causal effect on psychiatric disorders. The expression of genes regulated by the alcohol-related DNA methylation sites may underpin this association.

Abstract 2417: Detection and quantification of site-specific DNA methylation from liquid biopsies as a pharmacodynamic biomarker of OTX-2002, a novel MYC-targeting epigenomic mRNA therapeutic

Abstract Omega Therapeutics has developed a novel platform of programmable epigenomic mRNA medicines capable of modifying chromatin state to specifically tune gene expression at the pre-transcriptional level. Epigenomic controllers (ECs) unlock targets that have been historically considered “undruggable,” with one of the most elusive being the MYC oncogene. A direct MYC-targeting anti-cancer agent has previously remained intangible, largely due to the absence of a drug binding pocket and tight autoregulation. A clinical trial is underway (MYCHELANGELO, NCT05497453) to investigate pre-transcriptional inhibition of MYC with OTX-2002 in patients with hepatocellular carcinoma (HCC). OTX-2002, a first-in-class mRNA therapeutic delivered via lipid nanoparticles (LNP), encodes two proteins that durably modify chromatin, in part, through CpG DNA methylation at the MYC locus. Using both liquid and solid biopsy sampling from in vivo studies, we investigated whether target engagement for OTX-2002 could be assessed by MYC methylation. Detection of DNA methylation has long held promise as an oncology biomarker given its functional roles in various cancer types and the potential signal afforded by methylated CpGs. Indeed, DNA methylation has been shown to serve as a robust analyte in liquid biopsy-derived multi-cancer early detection (MCED) and minimal residual disease (MRD) tests that have recently been utilized in the clinic. Many of these platforms are founded on complex models that leverage methylation signals across &amp;gt;1 million CpGs that can span tens of megabases of genomic space. We faced a distinct challenge when compared to these MCED/MRD tests in developing a pharmacodynamic methylation assay for OTX-2002 as the target region consists of just a few kilobases. With a tissue specific LNP delivery system, ultra-high sensitivity was required to identify rare ctDNA events from the larger cfDNA population. To this end, we designed a minimal hybridization/capture panel that targeted ~50 kb of genomic space, effectively allowing for ultra-deep methylation sequencing of MYC. When this technique was paired with enzymatic (EM) conversion for methylation detection and supported by an analysis pipeline focused on epiallele identification, this assay was able to detect methylation down to the theoretical limit in a dilution series of control genomic DNA, 1 in 104 copies of MYC. This degree of sensitivity translated to successful preclinical detection of on-target methylation by OTX-2002 from DNA extracted from plasma samples collected from mice-bearing human HCC xenografts. Overall, we present a non-invasive and exquisitely sensitive method of assessing target engagement and site-specific pharmacodynamic activity of a novel epigenomic medicine that can be directly translated to the clinical setting. Citation Format: Justin Chen, William Senapedis, Stephen Siecinski, Elmer Figueroa, Adam Katz, Samuel Mildrum, Yaoyu E. Wang, Houda Belaghzal, Kayleigh Gallagher, Graeme Hodgson, Charles W. O'Donnell, Thomas G. McCauley. Detection and quantification of site-specific DNA methylation from liquid biopsies as a pharmacodynamic biomarker of OTX-2002, a novel MYC-targeting epigenomic mRNA therapeutic [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2417.

Identification of a CpG-based signature coupled with gene expression as prognostic indicators for melanoma: a preliminary study

DNA methylation is an important part of the genomic biology, which recently allowed the identification of key biomarkers for a variety of cancers, including cutaneous melanoma. Despite the current knowledge in cutaneous melanoma, there is a clear need for new efficient biomarkers in clinical application of detection. We use The Cancer Genome Atlas data as a training set and a multi-stage screening strategy to identify prognostic characteristics of melanoma based on DNA methylation. Three DNA methylation CpG sites were identified to be related to the overall survival in the skin cutaneous melanoma cohort. This signature was validated in two independent datasets from Gene Expression Omnibus. The stratified analysis by clinical stage, age, gender, and grade retained the statistical significance. The methylation signature was significantly correlated with immune cells and anti-tumor immune response. Moreover, gene expression corresponding to the candidate CpG locus was also significantly correlated with the survival rate of the patient. About 49% of the prognostic effects of methylation are mediated by affecting the expression of the corresponding genes. The prognostic characteristics of DNA methylation combined with clinical information provide a better prediction value tool for melanoma patients than the clinical information alone. However, more experiments are required to validate these findings. Overall, this signature presents a prospect of novel and wide-ranging applications for appropriate clinical adjuvant trails.

Strengths and limitations of reduced representation bisulfite sequencing (RRBS) in the perspective of DNA methylation analysis in fish: a case-study on rainbow trout spermatozoa.

DNA methylation in CpG dinucleotides is an important epigenetic mark in fish spermatozoa since it has been shown that some sperm methylome features are transmitted to the offspring. Reduced representation bisulfite sequencing (RRBS) is one genome-scale methods developed to assess DNA methylation at CpG sites. It allows the sequencing of a reduced fraction of the genome expected to be enriched in CpGs. The aim of this study is to characterize the extent of the CpG sites that can be identified in the RRBS-reduced sequenced fraction of rainbow trout spermatozoa, in order to evaluate the potential of RRBS for sperm DNA methylation studies. We observed that RRBS did provide a reduced amount of genomic data, the sum of the CpGs analyzed on 12 males spanning 9% of the total genomic CpGs. CpGs were only slightly enriched in the RRBS data (×1.7 times the sequenced nucleotides), the possible causes being linked to trout genome structure and sequenced fragments size. All genomic functional features were represented in our CpG dataset, with a noticeable enrichment in exons but, strikingly, not in promoters. The number of CpGs shared between biological replicates was low, but this proportion reached workable values from six biological replicates (46% of the analyzed cytosines) on. The choices that are to be made regarding fragment size selection and the options during bioinformatic data processing are discussed. In all, RRBS is a relevant first-approach method to scan the CpG DNA methylation status of spermatozoa along rainbow trout genome, although in a very reduced pattern among biological replicates.

Variants at the Interleukin 1 Gene Locus and Pericarditis

Recurrent pericarditis is a treatment challenge and often a debilitating condition. Drugs inhibiting interleukin 1 cytokines are a promising new treatment option, but their use is based on scarce biological evidence and clinical trials of modest sizes, and the contributions of innate and adaptive immune processes to the pathophysiology are incompletely understood. To use human genomics, transcriptomics, and proteomics to shed light on the pathogenesis of pericarditis. This was a meta-analysis of genome-wide association studies of pericarditis from 5 countries. Associations were examined between the pericarditis-associated variants and pericarditis subtypes (including recurrent pericarditis) and secondary phenotypes. To explore mechanisms, associations with messenger RNA expression (cis-eQTL), plasma protein levels (pQTL), and CpG methylation of DNA (ASM-QTL) were assessed. Data from Iceland (deCODE genetics, 1983-2020), Denmark (Copenhagen Hospital Biobank/Danish Blood Donor Study, 1977-2022), the UK (UK Biobank, 1953-2021), the US (Intermountain, 1996-2022), and Finland (FinnGen, 1970-2022) were included. Data were analyzed from September 2022 to August 2023. Genotype. Pericarditis. In this genome-wide association study of 4894 individuals with pericarditis (mean [SD] age at diagnosis, 51.4 [17.9] years, 2734 [67.6%] male, excluding the FinnGen cohort), associations were identified with 2 independent common intergenic variants at the interleukin 1 locus on chromosome 2q14. The lead variant was rs12992780 (T) (effect allele frequency [EAF], 31%-40%; odds ratio [OR], 0.83; 95% CI, 0.79-0.87; P = 6.67 × 10-16), downstream of IL1B and the secondary variant rs7575402 (A or T) (EAF, 45%-55%; adjusted OR, 0.89; 95% CI, 0.85-0.93; adjusted P = 9.6 × 10-8). The lead variant rs12992780 had a smaller odds ratio for recurrent pericarditis (0.76) than the acute form (0.86) (P for heterogeneity = .03) and rs7575402 was associated with CpG methylation overlapping binding sites of 4 transcription factors known to regulate interleukin 1 production: PU.1 (encoded by SPI1), STAT1, STAT3, and CCAAT/enhancer-binding protein β (encoded by CEBPB). This study found an association between pericarditis and 2 independent sequence variants at the interleukin 1 gene locus. This finding has the potential to contribute to development of more targeted and personalized therapy of pericarditis with interleukin 1-blocking drugs.

Effect of Demethylation on the Presence of DNA Methyltransferases in Complex with Histone Methylation Markers in Two Myeloma Cell Lines

Background: Enzymes that catalyze CpG methylation in DNA, including the DNA methyltransferases 1 (DNMT1), 3A (DNMT3A) and 3B (DNMT3B) are also implicated in human developmental disorders and cancers, supporting the critical role of DNA methylation. Posttranslational modifications of histones influence the representation of DNMTs in protein complexes including specific histone markers. H3K4me3, H3K36me3 are histone markers of active gene transcription, while for example H3K9me3, H3K27me3 are histone markers of inactive chromatin associated with repressive transcription of the respective genes. Objective: The aim of the study was to find whether demethylation of myeloma cells can affect 1) the formation of a complex of the repressive histone marker - H3K9me3 with DNMT3A and/or DNMT3B; and 2) association of the transcriptionally active histone marker H3K4me3 with DNMT1. Material &amp; Methods: Chromatin lysate was isolated from two myeloma cell lines - KMS12-BM and KMS12-PE, after their 72 hours treatment with 5-Azacytidine (AZA) and/or 5-Aza-2´-deoxycitidine (DAC). Precipitation was performed with antibodies against H3K9me3 and/or H3K4me3. The occurrence of DNMT3A and/or DNMT3B in precipitated complex with H3K9me3 was quantified by qPCR (SYBR Green I) with primers specific for both promoter or coding regions of the respective DNMT genes. The assesment of DNMT1 in complex precipitated with H3K4me3 was quantified by qPCR (SYBR Green I) with primers specific for DNMT1. For result calculation, the 2-delta Ct values of immunoprecipitated samples were normalized to the 2-delta Ct value of IgG respective antibodies, and then the obtained 2(-(delta delta Ct)) value of analyzed samples was normalized to the 2(-(delta delta Ct)) value of control untreated cells (DMSO). Results: In KMS12-PE myeloma cells, we determined the 2(-(delta delta Ct)) values of DNMT3B in precipitated complex with H3K9me3 - 1.52 and 1.47, in cells treated with 0.5 µM AZA and 0.5 µM DAC, respectively. The obtained 2(-(delta delta Ct) values were higher than that of control untreated (DMSO) myeloma cells [with the of 2(-(delta delta Ct)) value of 0.85]. Increased occurrence of DNMT3A was found in H3K9me3 complex of all analyzed samples in comparison to untreated control cells [with the of 2(-(delta delta Ct)) value of 0.89]; the highest 2(-(delta delta Ct) values - 1.07 and 1.21 were determined after DAC treatment. The obtained 2(-(delta delta Ct)) values of DNMT1 from analyzed precipitated samples with H3K4me3 were lower than that of DMSO treated KMS12-PE cells [with the of 2(-(delta delta Ct)) value of 0.85]. In KMS12-BM, we did not detect any changes in the abundance of DNMT3B in precipitated complexes including H3K4me3. Conclusion: In KMS12-PE, but not in KMS12-BM, both demethylation agents used, 5-Aza-2´-deoxycitidine and 5-Azacytidine, affected the appearance of DNMT3A and DNMT3B in a protein complex containing the repressive histone marker H3K9me3 with possible potential to inactivate gene transcription.

Targeting EBV Encoded Viral Kinases with GCV, AZT, Rituximab and Dexamethasone (GARD) Results in Durable Responses in Patients with CNS Lymphoproliferative Disease

Introduction: Epstein-Barr virus positive (EBV+) central nervous system lymphoproliferative diseases (CNS-LPD) are aggressive clinical conditions with poor prognosis. EBV+ CNS-LPD often occurs in the setting of immune suppression and comorbidities (solid organ transplant, autoimmunity). Treatment with high-dose methotrexate has increased survival but is not a viable option for all patients, highlighting the need for alternative treatments. Previous research has reported EBV+ CNS-LPD exhibits unique genetic and immunobiological signatures, however, few studies have reported on the epigenetic landscape. We have previously reported use of ganciclovir (GCV), zidovudine (AZT), rituximab and dexamethasone (GARD) regimen induced complete and durable responses in a cohort of 13 patients with primary CNS post-transplant lymphoproliferative disease (PCNS-PTLD). Here we present 24 patients (10 from prior cohort and 14 new) with EBV+ CNS-LPD treated with GARD. We extend follow-up time for the previous cohort and add molecular context to clinical observations. We performed DNA methylation and expression analysis of EBV viral kinases in available tumor samples and revealed unique viral epigenetic states leading to expression of antiviral drug targets in an array of EBV+ CNS-LPD. Methods: We conducted an IRB-approved, retrospective review of patients with EBV+ CNS-LPD treated with GARD at The Ohio State University Wexner Medical Center between 1998-2022. Patient information was extracted from the electronic medical record. Treatment consisted of two-week induction of twice daily IV GCV/AZT (5mg/kg and 1,500 mg) IV dexamethasone (10-40mg), and weekly rituximab (375mg/m 2). After induction, dexamethasone was tapered, and GCV/AZT was switched to maintenance oral dosing of 450mg valganciclovir twice daily and 300mg of AZT twice daily. Rituximab was administered on days 1, 8, 15, and 22. Two-year overall survival (OS), five-year OS, and overall response rate were assessed. Available CNS-LPD biopsy samples were obtained, and expression of EBV lytic genes BZLF1, BXLF1 (thymidine kinase), and BGLF4 (protein kinase) were measured via qRT-PCR. Mass spectrometry based EpiTYPER assay was used to report the percentage of DNA CpG methylation in the gene promoters for viral BZLF1, BXLF1, BGLF4 and LMP1 genes . Biopsies from 17 patients with systemic PTLD with no CNS involvement were used for comparison. Results: 24 patients with EBV+ CNS-LPD were identified. 21 (87.5%) patients were diagnosed with a primary CNS-LPD (PCNS-LPD). Of the PCNS-LPD, 14 patients were PCNS-PTLD, 4 were receiving immunosuppression for autoimmune conditions, 1 patient had type 2 diabetes and two had no known history of immunosuppression. The remaining three patients had systemic PTLD with secondary CNS involvement. Diffuse large B-cell lymphoma was the most common histology (11; 45.8%) followed by grade 3 lymphomatoid granulomatosis (5; 20.8%), B-cell LPD (5;20.8%), polymorphic PTLD (2;8.3%) and one large cell lymphoma of ambiguous lineage. All patients were HIV negative. EBER was positive in all biopsies and EBV PCR of cerebrospinal fluid was positive in patients without biopsy. Sixteen patients (66.7%) achieved a completed response, with an overall response rate of 83%. Two-year OS was 62.5% (95% CI, 45.8-85.2%) and five-year OS was 52.8% (95% CI, 35.7%-77.9%). Five CNS-LPD brain biopsies were available for analysis. qRT-PCR of biopsy specimens showed expression of LMP1, BXLF1, and BGLF4, but not BZLF1. Expression of BGLF4 was significantly higher (p=1.35e-5) in CNS-LPD when compared to systemic EBV+ PTLD. EpiTYPER data showed significantly decreased promoter methylation for viral kinases BXLF1 and BGLF4 (p=0.0281 p=0.0089). Conclusions: We have shown that GARD regimen shows efficacy in the treatment of EBV+ CNS-LPD, with 62.5% two-year OS and 52.8% five-year OS. Our results provide the molecular basis for expression of lytic viral kinases BXLF1 and BGLF4 in CNS-LPD by showing that both genes exhibit decreased promotor methylation. This expression of viral kinases in CNS-LPD supports the mechanistic rationale of this antiviral approach and could be further investigated as a predictive biomarker. Overall, this research highlights unique epigenetic features of EBV in CNS-LPD that support the use of antiviral, specifically the GARD regimen, in EBV+ CNS-LPD.

Accelerated epigenetic clock aging in maternal peripheral blood and preterm birth

Epigenetic clocks use CpG DNA methylation to estimate biological age. Acceleration is associated with cancer, heart disease, and shorter life span. Few studies evaluate DNA methylation age and pregnancy outcomes. AgeAccelGrim is a novel epigenetic clock that combines 7 DNA methylation components. This study aimed to determine whether maternal biological aging (via AgeAccelGrim) is associated with early preterm birth. A prospective cohort of patients with singleton pregnancies and at high risk of spontaneous preterm birth delivering at a tertiary university hospital were included in this study. Genome-wide CpG methylation was measured using the Illumina EPIC BeadChip (Illumina, Inc, San Diego, CA) from maternal blood samples obtained at <28 weeks of gestation. AgeAccelGrim and its 7 DNA methylation components were estimated by the Horvath DNA methylation age online tool. Positive values are associated with accelerated biological aging, whereas negative values are associated with slower biological aging relative to each subject's age. The primary outcome was preterm birth at <34 weeks of gestation (any indication). The secondary outcomes were preterm birth at <37 and <28 weeks of gestation. AgeAccelGrim was analyzed as a continuous variable and in quartiles. Exploratory analyses evaluated each of the 7 DNA methylation components included in the composite AgeAccelGrim. Data were analyzed by chi-square test, t test, rank-sum test, logistic regression (controlling a priori for maternal age, cell counts, low socioeconomic status, and gestational age at the time of sample collection), and Kaplan-Meier survival analyses. The log-rank test was used to test the equality of the survival functions. Overall, 163 patients met the inclusion criteria. Of the patients, 48%, 39%, and 21% delivered at <37, <34, and <28 weeks of gestation, respectively. The median AgeAccelGrim was-0.35 years (interquartile range,-2.24 to 1.31) for those delivering at term. Those delivering preterm had higher AgeAccelGrim values that were inversely proportional to delivery gestational age (preterm birth at <37 weeks of gestation:+0.40 years [interquartile range:-1.21 to+2.28]; preterm birth at <34 weeks of gestation:+0.51 years [interquartile range:-1.05 to+2.67]; preterm birth at <28 weeks of gestation:+1.05 years [interquartile range:-0.72 to+2.72]). Estimated DNA methylation of the 7 epigenetic clock component values was increased among those with preterm birth at <34 weeks of gestation, although the differences were only significant for DNA methylation of plasminogen activation inhibitor 1. In regression models, AgeAcccelGrim was associated with an elevated risk of preterm birth with increasing magnitude for increasing severity of preterm birth. For each 1-year increase in the AgeAccelGrim value (ie, each 1-year increase in biological age compared with chronologic age), the adjusted odds of preterm birth were 11% (adjusted odds ratio, 1.11; 95% confidence interval, 1.00-1.24), 13% (adjusted odds ratio, 1.13; 95% confidence interval, 1.01-1.26), and 18% (adjusted odds ratio, 1.18; 95% confidence interval, 1.04-1.35) higher for preterm birth at <37, <34, and <28 weeks of gestation, respectively. Similarly, individuals with accelerated biological aging (≥75th percentile AgeAccelGrim) had more than double the odds of preterm birth at <34 weeks of gestation (adjusted odds ratio, 2.36; 95% confidence interval, 1.10-5.08) and more than triple the odds of preterm birth at <28 weeks of gestation (adjusted odds ratio, 3.89; 95% confidence interval, 1.61-9.38). The adjusted odds ratio for preterm birth at <37 weeks of gestation was 1.73 but spanned the null (adjusted odds ratio, 1.73; 95% confidence interval, 0.81-3.69). In Kaplan-Meier survival analyses, those in the highest AgeAccelGrim quartile delivered the earliest (log-rank Pvalue of <.001). Accelerated biological aging was associated with preterm birth among high-risk patients. Future research confirming these findings and elucidating factors that slow biological aging may improve birth outcomes.

Causal Mediation Analysis for an Ordinal Outcome with Multiple Mediators

Causal mediation analysis is a popular approach for investigating whether the effect of an exposure on an outcome is through a mediator to better understand the underlying causal mechanism. In recent literature, mediation analysis with multiple mediators has been proposed for continuous and dichotomous outcomes. In contrast, methods for mediation analysis for an ordinal outcome are still underdeveloped. In this paper, we first review mediation analysis methods with a continuous mediator for an ordinal outcome and then develop mediation analysis with a binary mediator for an ordinal outcome. We further consider multiple mediators for an ordinal outcome in the counterfactual framework and provide identification assumptions for identifying the mediation effects. Under the identification assumptions, we propose a regression-based method to estimate the mediation effects through multiple mediators while allowing the presence of exposure-mediator interactions. The closed-form expressions of mediation effects are also obtained for three scenarios: multiple continuous mediators, multiple binary mediators, and multiple mixed mediators. We conduct simulation studies to assess the finite sample performance of our new methods and present the biases, standard errors, and confidence intervals to demonstrate that our proposed estimators perform well in a wide range of practical settings. Finally, we apply our proposed methods to assess the mediation effects of candidate DNA methylation CpG sites in the causal pathway from socioeconomic index to body mass index.

DNA methylation profile discriminates sporadic giant cell granulomas of the jaws and cherubism from their giant cell-rich histological mimics.

Sporadic giant cell granulomas (GCGs) of the jaws and cherubism-associated giant cell lesions share histopathological features and microscopic diagnosis alone can be challenging. Additionally, GCG can morphologically closely resemble other giant cell-rich lesions, including non-ossifying fibroma (NOF), aneurysmal bone cyst (ABC), giant cell tumour of bone (GCTB), and chondroblastoma. The epigenetic basis of these giant cell-rich tumours is unclear and DNA methylation profiling has been shown to be clinically useful for the diagnosis of other tumour types. Therefore, we aimed to assess the DNA methylation profile of central and peripheral sporadic GCG and cherubism to test whether DNA methylation patterns can help to distinguish them. Additionally, we compared the DNA methylation profile of these lesions with those of other giant cell-rich mimics to investigate if the microscopic similarities extend to the epigenetic level. DNA methylation analysis was performed for central (n = 10) and peripheral (n = 10) GCG, cherubism (n = 6), NOF (n = 10), ABC (n = 16), GCTB (n = 9), and chondroblastoma (n = 10) using the Infinium Human Methylation EPIC Chip. Central and peripheral sporadic GCG and cherubism share a related DNA methylation pattern, with those of peripheral GCG and cherubism appearing slightly distinct, while central GCG shows overlap with both of the former. NOF, ABC, GCTB, and chondroblastoma, on the other hand, have distinct methylation patterns. The global and enhancer-associated CpG DNA methylation values showed a similar distribution pattern among central and peripheral GCG and cherubism, with cherubism showing the lowest and peripheral GCG having the highest median values. By contrast, promoter regions showed a different methylation distribution pattern, with cherubism showing the highest median values. In conclusion, DNA methylation profiling is currently not capable of clearly distinguishing sporadic and cherubism-associated giant cell lesions. Conversely, it could discriminate sporadic GCG of the jaws from their giant cell-rich mimics (NOF, ABC, GCTB, and chondroblastoma).

Efficiently quantifying DNA methylation for bulk- and single-cell bisulfite data.

DNA CpG methylation (CpGm) has proven to be a crucial epigenetic factor in the mammalian gene regulatory system. Assessment of DNA CpG methylation values via whole-genome bisulfite sequencing (WGBS) is, however, computationally extremely demanding. We present FAst MEthylation calling (FAME), the first approach to quantify CpGm values directly from bulk or single-cell WGBS reads without intermediate output files. FAME is very fast but as accurate as standard methods, which first produce BS alignment files before computing CpGm values. We present experiments on bulk and single-cell bisulfite datasets in which we show that data analysis can be significantly sped-up and help addressing the current WGBS analysis bottleneck for large-scale datasets without compromising accuracy. An implementation of FAME is open source and licensed under GPL-3.0 at https://github.com/FischerJo/FAME.

CpG DNA methylation changes during epididymal sperm maturation in bulls

BackgroundDuring epididymal transit spermatozoa acquire specific morphological features which enhance their ability to swim in a progressive manner and interact with the oocytes. At the same time, sperm cells undergo specific molecular rearrangements essential for the fertilizing sperm to drive a correct embryo development. To assess epigenetic sperm changes during epididymal maturation, the caput, corpus and cauda epididymis sperm tracts were isolated from eight bulls and characterized for different sperm quality parameters and for CpG DNA methylation using Reduced Representation Bisulfite Sequencing (RRBS) able to identify differentially methylated regions (DMRs) in higher CpG density regions.ResultsCaput sperm showed significant variation in motility and sperm kinetics variables, whereas spermatozoa collected from the corpus presented morphology variation and significant alterations in variables related to acrosome integrity. A total of 57,583 methylated regions were identified across the eight bulls, showing a significantly diverse distribution for sperm collected in the three epididymal regions. Differential methylation was observed between caput vs corpus (n = 11,434), corpus vs cauda (n = 12,372) and caput vs cauda (n = 2790). During epididymal transit a high proportion of the epigenome was remodeled, showing several regions in which methylation decreases from caput to corpus and increases from corpus to cauda.ConclusionsSpecific CpG DNA methylation changes in sperm isolated from the caput, corpus, and cauda epididymis tracts are likely to refine the sperm epigenome during sperm maturation, potentially impacting sperm fertilization ability and spatial organization of the genome during early embryo development.

Nc886, an RNA Polymerase III-Transcribed Noncoding RNA Whose Expression Is Dynamic and Regulated by Intriguing Mechanisms.

nc886 is a medium-sized non-coding RNA that is transcribed by RNA polymerase III (Pol III) and plays diverse roles in tumorigenesis, innate immunity, and other cellular processes. Although Pol III-transcribed ncRNAs were previously thought to be expressed constitutively, this concept is evolving, and nc886 is the most notable example. The transcription of nc886 in a cell, as well as in human individuals, is controlled by multiple mechanisms, including its promoter CpG DNA methylation and transcription factor activity. Additionally, the RNA instability of nc886 contributes to its highly variable steady-state expression levels in a given situation. This comprehensive review discusses nc886's variable expression in physiological and pathological conditions and critically examines the regulatory factors that determine its expression levels.

Differential CpG DNA methylation of peripheral B cells, CD4+ T cells, and salivary gland tissues in IgG4-related disease

ObjectivesImmunoglobulin-G4-related disease (IgG4-RD) is a distinct systemic autoimmune-mediated disease manifesting as chronic inflammation and tissue fibrosis. Since the role of DNA methylation in the pathogenesis of IgG4-RD is still unclear, we conduct this study to investigate epigenetic modifications in IgG4-RD.MethodsA genome-wide DNA methylation study was conducted with B cells, CD4+ T cells, and salivary gland tissues from IgG4-RD patients and matched controls by using the Illumina HumanMethylation 850K BeadChip. We further performed pyrosequencing and immunohistochemistry assays to validate the methylation status of some targets of interest.ResultsWe identified differentially methylated CpG sites including 44 hypomethylated and 166 hypermethylated differentially methylated probes (DMPs) in B cells and 260 hypomethylated and 112 hypermethylated DMPs in CD4+ T cells from 10 IgG4-RD patients compared with 10 healthy controls. We also identified 36945 hypomethylated and 78380 hypermethylated DMPs in salivary gland tissues of 4 IgG4-RD patients compared with 4 controls. DPM2 (cg21181453), IQCK (cg10266221), and ABCC13 (cg05699681, cg04985582) were hypermethylated and MBP (cg18455083) was hypomethylated in B cells, CD4+ T cells, and salivary gland tissues of IgG4-RD patients. We also observed the hypomethylated HLA-DQB2 in CD4+ T cells from IgG4-RD patients. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of DMPs in salivary gland tissues of IgG4-RD patients revealed enrichment of pathways involved in the regulation of immune cell responses and fibrosis.ConclusionThis is the first DNA methylation study in peripheral B cells, CD4+ T cells, and salivary gland tissues from IgG4-RD patients. Our findings highlighted the role of epigenetic modification of DNA methylation and identified several genes and pathways possibly involved in IgG4-RD pathogenesis.

Accelerated epigenetic clock aging in peripheral blood is associated with obstetric metabolic syndrome-related outcomes

Epigenetic clocks use CpG DNA methylation (DNAm) to estimate biologic age; acceleration is associated with diabetes mellitus (DM), heart disease, and shorter life. It is unknown if DNAm age is also related to cardiometabolic outcomes in pregnancy. AgeAccelGrim is a novel epigenetic clock that combines 7 DNAm components. We sought to determine if AgeAccelGrim is associated with obstetric metabolic syndrome.