Single CpG Research Articles

Perfluorooctanesulfonic acid (PFOS) induced cancer related DNA methylation alterations in human breast cells: A whole genome methylome study

DNA methylation plays a pivotal role in cancer. The ubiquitous contaminant perfluorooctanesulfonic acid (PFOS) has been epidemiologically associated with breast cancer, and can induce proliferation and malignant transformation of normal human breast epithelial cells (MCF-10A), but the information about its effect on DNA methylation is sparse. The aim of this study was to characterize the whole-genome methylome effects of PFOS in our breast cell model and compare the findings with previously demonstrated DNA methylation alterations in breast tumor tissues. The DNA methylation profile was assessed at single CpG resolution in MCF-10A cells treated with 1 μM PFOS for 72 h by using Enzymatic Methyl sequencing (EM-seq). We found 12,591 differentially methylated CpG-sites and 13,360 differentially methylated 100 bp tiles in the PFOS exposed breast cells. These differentially methylated regions (DMRs) overlapped with 2406 genes of which 494 were long non-coding RNA and 1841 protein coding genes. We identified 339 affected genes that have been shown to display altered DNA methylation in breast cancer tissue and several other genes related to cancer development. This includes hypermethylation of GACAT3, DELEC1, CASC2, LCIIAR, MUC16, SYNE1 and hypomethylation of TTN and KMT2C. DMRs were also found in estrogen receptor genes (ESR1, ESR2, ESRRG, ESRRB, GREB1) and estrogen responsive genes (GPER1, EEIG1, RERG). The gene ontology analysis revealed pathways related to cancer phenotypes such as cell adhesion and growth. These findings improve the understanding of PFOS's potential role in breast cancer and illustrate the value of whole-genome methylome analysis in uncovering mechanisms of chemical effects, identifying biomarker candidates, and strengthening epidemiological associations, potentially impacting risk assessment.

Prenatal opioid exposure and the early life epigenome: results from ECHO

ABSTRACT Background Prenatal opioid exposure has been associated with adverse child health outcomes. Changes to the epigenome provide a plausible mechanism through which effects may be elicited. We investigated whether prenatal opioid exposure was associated with locus-specific changes in umbilical cord blood DNA methylation (DNAm) and gestational epigenetic age. Methods We leveraged data from the Environmental influences on Child Health Outcomes cohort. Prenatal opioid data was obtained from maternal self-report and/or medical record data. DNAm measures were generated from blood biospecimens collected at birth. Linear regression models tested associations between prenatal maternal opioid exposure and epigenetic outcomes in crude and fully adjusted models. Results We tested the association between prenatal opioid exposure and cord blood DNAm at 15 CpG sites in 385 (n = 25 exposed, n = 360 unexposed) individuals from three cohorts. We identified a single CpG site (cg14303187) that was nominally associated with prenatal opioid exposure (p = 0.02, β = −0.012; 95% CI, −0.023 to −0.0012). No significant associations between exposure and gestational epigenetic age were found (n = 716 individuals from eight cohorts; n = 29 exposed, n = 687 unexposed). Conclusions We identified a nominally significant association between prenatal opioid exposure and DNAm at one CpG site. Future studies should continue investigating the effect of this exposure on the epigenome.

Epigenetics of the non-coding RNA nc886 across blood, adipose tissue and skeletal muscle in offspring exposed to diabetes in pregnancy

BackgroundDiabetes in pregnancy is associated with increased risk of long-term metabolic disease in the offspring, potentially mediated by in utero epigenetic variation. Previously, we identified multiple differentially methylated single CpG sites in offspring of women with gestational diabetes mellitus (GDM), but whether stretches of differentially methylated regions (DMRs) can also be identified in adolescent GDM offspring is unknown. Here, we investigate which DNA regions in adolescent offspring are differentially methylated in blood by exposure to diabetes in pregnancy. The secondary aim was to characterize the RNA expression of the identified DMR, which contained the nc886 non-coding RNA.MethodsTo identify DMRs, we employed the bump hunter method in samples from young (9–16 yr, n = 92) offspring of women with GDM (O-GDM) and control offspring (n = 94). Validation by pyrosequencing was performed in an adult offspring cohort (age 28–33 years) consisting of O-GDM (n = 82), offspring exposed to maternal type 1 diabetes (O-T1D, n = 67) and control offspring (O-BP, n = 57). RNA-expression was measured using RT-qPCR in subcutaneous adipose tissue and skeletal muscle.ResultsOne significant DMR represented by 10 CpGs with a bimodal methylation pattern was identified, located in the nc886/VTRNA2-1 non-coding RNA gene. Low methylation status across all CpGs of the nc886 in the young offspring was associated with maternal GDM. While low methylation degree in adult offspring in blood, adipose tissue, and skeletal muscle was not associated with maternal GDM, adipose tissue nc886 expression was increased in O-GDM compared to O-BP, but not in O-T1D. In addition, adipose tissue nc886 expression levels were positively associated with maternal pre-pregnancy BMI (p = 0.006), but not with the offspring’s own adiposity.ConclusionsOur results highlight that nc886 is a metastable epiallele, whose methylation in young offspring is negatively correlated with maternal obesity and GDM status. The physiological effect of nc886 may be more important in adipose tissue than in skeletal muscle. Further research should aim to investigate how nc886 regulation in adipose tissue by exposure to GDM may contribute to development of metabolic disease.

Abstract LB422: Specific activation of the Epstein-Barr Virus BGLF4 locus via lowered DNA methylation in EBV-associated CNS lymphoproliferative disease

Abstract Introduction: Epstein-Barr virus positive (EBV+) central nervous system lymphoproliferative diseases (CNS-LPD) are aggressive clinical conditions with poor prognosis. We have previously reported use of a ganciclovir (GCV), zidovudine (AZT), rituximab and dexamethasone regimen (GARD) that induced complete and durable responses in patients with primary CNS post-transplant lymphoproliferative disease (PTLD). Responses were associated with detection of two GCV/AZT viral drug targets, the EBV protein kinases BGLF4 and BXLF1. These viral proteins are normally expressed in lytic phase EBV, and the molecular basis for expression in latently infected EBV+ CNS-LPD is unknown. Methods: RNA expression of EBV genes LMP1, BZLF1, BXLF1, and BGLF4 was measured in 12 CNS-LPD biopsies (n=8/12 DLBCL-type) via qRT-PCR. Mass spectrometry-based EpiTYPER assay was used to quantify DNA methylation in EBV gene promoters. Biopsies from 25 patients with systemic PTLD were used for comparison. The BGLF4 locus was investigated in detail using luciferase reporters and 5’RACE mapping of transcription start sites in 3 EBV-infected cell lines and 4 CNS-LPD biopsy samples. Overall EBV methylation was assessed in a screening assay covering 31 EBV loci. Mechanistic involvement of active demethylation via TET2 was studied in a HEK293 CRISPR-Cas9 gene knockout model under EBV (strain M81) infection. Results: Gene expression analysis of biopsy specimens showed expression of LMP1, BXLF1, and BGLF4, but not BZLF1. Expression of BXLF1 and BGLF4 was significantly higher in CNS-LPD when compared to systemic EBV+ PTLD (p=0.043 and p=0.00022, respectively). EpiTYPER data showed significantly decreased promoter methylation at BGLF4 (p=0.00013). Luciferase reporter analysis of the BGLF4 upstream sequence revealed 3 regions of promoter activity which were silenced by in vitro methylation of reporters. 5’RACE identified transcription start sites at all 3 BGLF4 promoters and detected their activity in EBV+ cell lines and CNS-LPD biopsies. By overlaying transcriptional activity, 5’RACE and DNA methylation data, we identified DNA methylation loss at single CpG dinucleotides correlated with BGLF4 expression (r2=0.73). These sites were specifically demethylated in CNS-LPD, while surrounding EBV methylation remained high. Knockout of TET2 followed by EBV infection revealed that active demethylation is required for demethylation at BGLF4 promoters. Conclusions: The molecular determinant for success of the GARD regimen in CNS-LPD has remained poorly understood, with immunohistochemical detection of BGLF4/BXLF1 as the only predictor of treatment response. Our results provide the molecular basis for expression of BGLF4 in CNS-LPD by showing decreased promotor DNA methylation. We identified specific BGLF4 methylation sites that hold promise as potential DNA biomarkers for EBV+ CNS-LPD. Citation Format: Christoph Weigel, Haley L. Klimaszewski, Selamawit Addissie, Sarah Schlotter, Fode Tounkara, James P. Dugan, Bradley M. Haverkos, Lynda Villagomez, Mark Lustberg, Pierluigi Porcu, Timothy Voorhees, Michael A. Caligiuri, Ginny Bumgardner, Christopher C. Oakes, Robert Baiocchi. Specific activation of the Epstein-Barr Virus BGLF4 locus via lowered DNA methylation in EBV-associated CNS lymphoproliferative disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB422.

7 Flexible probabilistic methods to unlock the clinical potential of liquid biopsy sampling

OBJECTIVES/GOALS: Decoding the origins of cell-free DNA (cfDNA) released from dying cells in a liquid biopsy sample (e.g. blood) offers the potential to provide insight into the dynamic, organism-wide changes reflective of health and disease. Thus, making cfDNA an ideal target for serial, minimally invasive monitoring of disease-related changes. METHODS/STUDY POPULATION: We develop a probabilistic method that leverages the co-regulation of neighboring CpG sites on individual methylome-wide sequencing (WGBS) reads to more flexibly model cell-specific methylation compared to prior methods that focus on the methylation rate of a single CpG site. We then extend our cross-sectional model to account for sequential sampling within the same subject. The increased sampling frequency is critical to identifying the evolutionary dynamics of disease progression influencing treatment response and resistance, and disease recurrence. We utilize Bayesian inference techniques to model patient-specific longitudinal profiles of cell-type turnover in simulated serial samples. RESULTS/ANTICIPATED RESULTS: We found our model more effective at capturing a range of methylation patterns on cfDNA fragments with lower Root Mean Square Error across simulations compared to a single CpG model. We apply our model to detect significant (p < 0.05, Friedman’s test) increases in cellular contributions from lung and cardiac tissue in breast cancer patients (n=15) undergoing radiation therapy compared to baseline. We also identify signals of radiation induced toxicity to the liver in right-sided breast cancer patients (n=8) receiving radiation treatment compared to left-sided breast cancer patients (n=7). Finally, we show our extended model results in more efficient estimates of simulated cell-type turnover profiles compared to analyzing serial samples cross-sectionally, ignoring the longitudinal nature of the data. DISCUSSION/SIGNIFICANCE: Here we address an unmet need in developing novel statistical methodologies to decode the origins of methylated cfDNA obtained from liquid biopsy samples. We demonstrate the far-ranging clinical utility of serial liquid biopsy sampling to complement and advance the standards of clinical care in oncology and other pathologies.

A high-throughput droplet digital PCR system aiming eight DNA methylation targets for age prediction

The droplet digital Polymerase Chain Reaction (ddPCR) has garnered recognition for its distinctive attribute of absolute quantification. And it has found practical utility in age prediction through DNA methylation profiles. However, a prevalent limitation in current ddPCR methodologies is the restricted capacity to detect only two targets concurrently in most instruments, leading to high costs, sample wastage, and labor-intensive procedures. To address the limitations, a novel high-throughput ddPCR system allowing for the simultaneous detection of eight targets was developed. Through the implementation of a new 8-plex ddPCR assay, coupled with comprehensive linear regression analyses involving primers and probes ratios, diverse inputs of single CpG sites with distinct primers and probes, and varying plex assay configurations, stable DNA methylation values for four CpGs and stable measurement precisions for distinct multiplex systems were consistently observed. These findings pave the way for advancing the field of chemistry science by enabling more efficient and cost-effective methods. Furthermore, the comparative validation of ddPCR and SNaPshot demonstrated a remarkable concordance in results, and the system also displayed well in the field of various aspects, including species specificity, DNA input, and aged samples. In this study, the recommended input of bisulfite-converted DNA was determined to be 10-50ng due to the double-positive droplets. Notably, the Pearson correlation coefficient squared values of four CpGs were 0.4878 (ASPA), 0.4832 (IGSF1), 0.6881 (COL1A1), and 0.6475 (MEIS1-AS3). And the testing set exhibited a mean absolute error of 4.5923 years, indicating the robustness and accuracy of the age-predictive model.

Association between CORIN promoter methylation and hypertensive disorders of pregnancy – A nested case-control study

IntroductionCorin protein and its coding gene variants have been associated with hypertensive disorders of pregnancy (HDP), but the underlying mechanisms are unclear. As a mediator linking fixed genome with the dynamic environment, DNA methylation at the CORIN gene may link corin with HDP but not has been studied. This study aimed to examine whether CORIN promoter methylation and HDP in Chinese pregnant women. MethodsBased on a cohort of Chinese pregnant women, we designed a nested case-control study including 196 cases with HDP and 200 healthy controls. DNA methylation levels in the CORIN promoter were quantified by pyrosequencing using peripheral blood before 20 gestational weeks. The association between DNA methylation in CORIN promoter and HDP was systemically examined by single CpG association analysis, followed by gene-based analysis. Multiple testing was controlled by the false discovery rate (FDR) method. ResultsThe single CpG association analysis found that, among the 5 CpG sites assayed, hypermethylation at one CpG site (Chr4:47839945) was significantly associated with HDP (OR = 1.94, raw P = 0.020), but the significance did not survive for multiple testing correction (FDR-P = 0.100). The gene-based association analysis found that DNA methylation of the 5 CpG sites was jointly associated with HDP (raw P = 0.003). In addition to HDP, CORIN promoter methylation was also significantly associated with dynamic blood pressure during pregnancy (raw P < 0.05). DiscussionHypermethylation in CORIN promoter at early pregnancy was associated with the risk of HDP during late pregnancy in Chinese women. However, further evidence is required to establish the causality between CORIN promoter methylation and HDP.

Methylation Patterns of the FKBP5 Gene in Association with Childhood Maltreatment and Depressive Disorders.

Childhood maltreatment is an important risk factor for adult depression and has been associated with changes in the hypothalamic pituitary adrenal (HPA) axis, including cortisol secretion and methylation of the FKBP5 gene. Furthermore, associations between depression and HPA changes have been reported. This study investigated the associations of whole-blood FKBP5 mRNA levels, serum cortisol levels, childhood maltreatment, and depressive symptoms with the whole-blood methylation status (assessed via target bisulfite sequencing) of 105 CpGs at the FKBP5 locus using data from the general population-based Study of Health in Pomerania (SHIP) (N = 203). Both direct and interaction effects with the rs1360780 single-nucleotide polymorphism were investigated. Nominally significant associations of main effects on methylation of a single CpG site were observed at intron 3, intron 7, and the 3'-end of the gene. Additionally, methylation at two clusters at the 3'-end and intron 7 were nominally associated with childhood maltreatment × rs1360780 and depressive symptoms × rs1360780, respectively. The results add to the understanding of molecular mechanisms underlying the emergence of depression and could aid the development of personalised depression therapy and drug development.

Epigenetic Peculiarity of Alphapapillomavirus Genus Based on the CpG Dinucleotides’ Island Variability

Background: Methylation plays a crucial role in genome regulation, serving as an essential epigenetic mechanism. CpG islands, which are regions of DNA rich in CpG dinucleotides, are ubiquitous epigenetic regulatory features that can modulate the functional dynamics of genes, thereby influencing the pathophysiology of pathogenic organisms within a host. Objectives: In this study, we aimed to investigate the distribution of CpG islands on Papillomavirus genomes and assess their potential impact on the neoplastic progression of different Alphapapillomavirus genera within host cells. Methods: We conducted an analysis of dinucleotide frequencies in DNA sequences from various Alphapapillomavirus genera. The sequences for our analysis were sourced from the specialized HPV (human papillomavirus) database (pave.niaid.nih.gov). Following a comprehensive comparative examination of entire genomes from different HPV species, we specifically focused on 63 genomes belonging to the Alphapapillomavirus genus to identify internal CpG island profiles. These investigations were conducted using online bioinformatics software (DBCAT), and statistical analysis was performed using GraphPad Prism v. 8.0.1. Results: Our preliminary findings revealed that 76.1% of the viruses in our study had fewer than 3 CpG islands but multiple CpG sites, while fewer than 25% of the viruses possessed at least 3 CpG islands. High-risk viruses were identified in 68.42% of genotypes with fewer than 3 CpG islands, whereas low-risk genotypes were observed in 15.7% of cases. Notably, some oncogenic viruses lacked CpG islands entirely, and this pattern was also observed in viruses with a single CpG island, occurring in 50% of cases. Based on the absence of CpG islands, genotypes such as HPV 67, HPV 97, and HPV 34 could potentially be classified as carcinogenic. Additionally, the distribution of CpG islands across HPV genes did not appear to be random. Genes like L2 and E2 contained a CpG island in 50% of cases, whereas oncogenes E6 and E7 consistently had a CpG island in 100% of cases in high-risk genotypes. Conclusions: Our findings suggest that the loss of 1 or more CpG islands could potentially transform an HPV genotype into a high-risk genotype. This process may be accelerated in the presence of host-related risk factors. Further research is required to validate whether CpG island distribution can aid in identifying oncogenic viruses.

Use of Multiple Machine Learning Approaches for Selecting Urothelial Cancer-Specific DNA Methylation Biomarkers in Urine.

Diagnosing urothelial cancer (UCa) via invasive cystoscopy is painful, specifically in men, and can cause infection and bleeding. Because the UCa risk is higher for male patients, urinary non-invasive UCa biomarkers are highly desired to stratify men for invasive cystoscopy. We previously identified multiple DNA methylation sites in urine samples that detect UCa with a high sensitivity and specificity in men. Here, we identified the most relevant markers by employing multiple statistical approaches and machine learning (random forest, boosted trees, LASSO) using a dataset of 251 male UCa patients and 111 controls. Three CpG sites located in ALOX5, TRPS1 and an intergenic region on chromosome 16 have been concordantly selected by all approaches, and their combination in a single decision matrix for clinical use was tested based on their respective thresholds of the individual CpGs. The combination of ALOX5 and TRPS1 yielded the best overall sensitivity (61%) at a pre-set specificity of 95%. This combination exceeded both the diagnostic performance of the most sensitive bioinformatic approach and that of the best single CpG. In summary, we showed that overlap analysis of multiple statistical approaches identifies the most reliable biomarkers for UCa in a male collective. The results may assist in stratifying men for cystoscopy.

DNA methylation alterations in Lewy Body Dementia

AbstractBackgroundThe epigenetic mechanism of DNA methylation has been implicated in the pathophysiology of neurodegenerative disorders such as Lewy body dementia (LBD). DNA methylation profiling may help to identify novel treatment targets and early disease markers. We hypothesized that DNA methylation alterations are detectable in blood due the influence of genetic and environmental factors as well as immune system changes associated with LBD.MethodGenome‐wide DNA methylation profiling was carried out using reduced representation bisulfite sequencing in blood samples from 89 LBD patients and 67 controls. Fifty five LBD patients had amyloid PET imaging. Data processing was performed using FastQC, TrimGalore, Bismark and MethylKit. DNA methylation alterations at specific CpG sites as well as at promoter, CpG island and CpG shore regions were compared between LBD cases and controls. A log odds logistic regression was used to identify differential DNA methylation after adjusting for age, sex and smoking status. The false discovery rate (FDR) was used for correction for multiple testing. We also tested the diagnostic classification performance of the differentially methylated regions using receiver operating characteristic (ROC) curve analysis and examined the potential of DNA methylation as a predictor of amyloid PET positivity in LBD.ResultTwo single CpG sites at TNFRSF1B and EMC6 and three CpG shore regions at ABCG5, ETAA1 and TENM4 reached genome‐wide significance in the differential DNA methylation analysis comparing LBD cases and controls. The combined panel of the top three differentially methylated CpG shores reached an area under the curve (AUC) of 0.91 for the diagnosis of LBD compared to controls (sensitivity 0.89, specificity 0.85). DNA methylation at TENM4 was associated with amyloid PET positivity in LBD with an AUC of 0.84 (sensitivity 0.99, specificity 0.55).ConclusionOur data suggest that DNA methylation alterations in blood are observed in a cohort of LBD patients compared to controls. Our findings need to be replicated in larger cohorts and tested for specificity in LBD in order to explore their future potential as diagnostic markers and/or targets for novel therapeutics.

Comparison of methylation estimates obtained via MinION nanopore sequencing and sanger bisulfite sequencing in the TRPA1 promoter region

BackgroundBisulfite sequencing has long been considered the gold standard for measuring DNA methylation at single CpG resolution. However, in recent years several new approaches like nanopore sequencing have been developed due to hints for a partial error-proneness of bisulfite sequencing. Since these errors were shown to be sequence-specific, we aimed to verify the methylation data of a particular region of the TRPA1 promoter from our previous studies obtained by bisulfite sequencing.MethodsWe compared methylation rates determined by direct bisulfite sequencing and nanopore sequencing following Cas9-mediated PCR-free enrichment.ResultsWe could show that CpG methylation levels above 20% corroborate well with our previous data. Within the range between 0 and 20% methylation, however, Sanger sequencing data have to be interpreted cautiously, at least in the investigated region of interest (TRPA1 promotor region).ConclusionBased on the investigation of the TRPA1- region as an example, the present work can help in choosing the right method out of the two current main approaches for methylation analysis for different individual settings regarding many factors like cohort size, costs and prerequisites that should be fulfilled for each method. All in all, both methods have their raison d’être. Furthermore, the present paper contains and illustrates some important basic information and explanation of how guide RNAs should be located for an optimal outcome in Cas9 mediated PCR free target enrichment.

Tuning Methylation-Dependent Silencing Dynamics by Synthetic Modulation of CpG Density.

Methylation of cytosines in CG dinucleotides (CpGs) within promoters has been shown to lead to gene silencing in mammals in natural contexts. Recently, engineered recruitment of methyltransferases (DNMTs) at specific loci was shown to be sufficient to silence synthetic and endogenous gene expression through this mechanism. A critical parameter for DNA methylation-based silencing is the distribution of CpGs within the target promoter. However, how the number or density of CpGs in the target promoter affects the dynamics of silencing by DNMT recruitment has remained unclear. Here, we constructed a library of promoters with systematically varying CpG content, and analyzed the rate of silencing in response to recruitment of DNMT. We observed a tight correlation between silencing rate and CpG content. Further, methylation-specific analysis revealed a constant accumulation rate of methylation at the promoter after DNMT recruitment. We identified a single CpG site between TATA box and transcription start site (TSS) that accounted for a substantial part of the difference in silencing rates between promoters with differing CpG content, indicating that certain residues play disproportionate roles in controlling silencing. Together, these results provide a library of promoters for synthetic epigenetic and gene regulation applications, as well as insights into the regulatory link between CpG content and silencing rate.

Epigenetic profiling of the D4Z4 locus: Optimization of the protocol for studying DNA methylation at single CpG site level.

The alteration of epigenetic modifications, including DNA methylation, can contribute to the etiopathogenesis and progression of many diseases. Among them, facioscapulohumeral dystrophy (FSHD) is a muscular disorder characterized by the loss of repressive epigenetic features affecting the D4Z4 locus (4q35). As a consequence, these alterations are responsible for DNA hypomethylation and a transcriptional-active chromatin conformation change that, in turn, lead to the aberrant expression of DUX4 in muscle cells. In the present study, methylation levels of 29 CpG sites of the DR1 region (within each repeat unit of the D4Z4 macrosatellite) were assessed on 335 subjects by employing primers designed for enhancing the performance of the assay. First, the DR1 original primers were optimized by adding M13 oligonucleotide tails. Moreover, the DR1 reverse primer was replaced with a degenerate one. As a result, the protocol optimization allowed a better sequencing resolution and a more accurate evaluation of DR1 methylation levels. Moreover, the assessment of the repeatability of measurements proved the reliability and robustness of the assay. The optimized protocol emerges as an excellent method to detect methylation levels compatible with FSHD.

The landscape of PBMC methylome in canine mammary tumors reveals the epigenetic regulation of immune marker genes and its potential application in predicting tumor malignancy

BackgroundGenome-wide dysregulation of CpG methylation accompanies tumor progression and characteristic states of cancer cells, prompting a rationale for biomarker development. Understanding how the archetypic epigenetic modification determines systemic contributions of immune cell types is the key to further clinical benefits.ResultsIn this study, we characterized the differential DNA methylome landscapes of peripheral blood mononuclear cells (PBMCs) from 76 canines using methylated CpG-binding domain sequencing (MBD-seq). Through gene set enrichment analysis, we discovered that genes involved in the growth and differentiation of T- and B-cells are highly methylated in tumor PBMCs. We also revealed the increased methylation at single CpG resolution and reversed expression in representative marker genes regulating immune cell proliferation (BACH2, SH2D1A, TXK, UHRF1). Furthermore, we utilized the PBMC methylome to effectively differentiate between benign and malignant tumors and the presence of mammary gland tumors through a machine-learning approach.ConclusionsThis research contributes to a better knowledge of the comprehensive epigenetic regulation of circulating immune cells responding to tumors and suggests a new framework for identifying benign and malignant cancers using genome-wide methylome.

A high-throughput test enables specific detection of hepatocellular carcinoma

High-throughput tests for early cancer detection can revolutionize public health and reduce cancer morbidity and mortality. Here we show a DNA methylation signature for hepatocellular carcinoma (HCC) detection in liquid biopsies, distinct from normal tissues and blood profiles. We developed a classifier using four CpG sites, validated in TCGA HCC data. A single F12 gene CpG site effectively differentiates HCC samples from other blood samples, normal tissues, and non-HCC tumors in TCGA and GEO data repositories. The markers were validated in a separate plasma sample dataset from HCC patients and controls. We designed a high-throughput assay using next-generation sequencing and multiplexing techniques, analyzing plasma samples from 554 clinical study participants, including HCC patients, non-HCC cancers, chronic hepatitis B, and healthy controls. HCC detection sensitivity was 84.5% at 95% specificity and 0.94 AUC. Implementing this assay for high-risk individuals could significantly decrease HCC morbidity and mortality.

Novel insights into systemic sclerosis using a sensitive computational method to analyze whole-genome bisulfite sequencing data

BackgroundAbnormal DNA methylation is thought to contribute to the onset and progression of systemic sclerosis. Currently, the most comprehensive assay for profiling DNA methylation is whole-genome bisulfite sequencing (WGBS), but its precision depends on read depth and it may be subject to sequencing errors. SOMNiBUS, a method for regional analysis, attempts to overcome some of these limitations. Using SOMNiBUS, we re-analyzed WGBS data previously analyzed using bumphunter, an approach that initially fits single CpG associations, to contrast DNA methylation estimates by both methods.MethodsPurified CD4+ T lymphocytes of 9 SSc and 4 control females were sequenced using WGBS. We separated the resulting sequencing data into regions with dense CpG data, and differentially methylated regions (DMRs) were inferred with the SOMNiBUS region-level test, adjusted for age. Pathway enrichment analysis was performed with ingenuity pathway analysis (IPA). We compared the results obtained by SOMNiBUS and bumphunter.ResultsOf 8268 CpG regions of ≥ 60 CpGs eligible for analysis with SOMNiBUS, we identified 131 DMRs and 125 differentially methylated genes (DMGs; p-values less than Bonferroni-corrected threshold of 6.05–06 controlling family-wise error rate at 0.05; 1.6% of the regions). In comparison, bumphunter identified 821,929 CpG regions, 599 DMRs (of which none had ≥ 60 CpGs) and 340 DMGs (q-value of 0.05; 0.04% of all regions). The top ranked gene identified by SOMNiBUS was FLT4, a lymphangiogenic orchestrator, and the top ranked gene on chromosome X was CHST7, known to catalyze the sulfation of glycosaminoglycans in the extracellular matrix. The top networks identified by IPA included connective tissue disorders.ConclusionsSOMNiBUS is a complementary method of analyzing WGBS data that enhances biological insights into SSc and provides novel avenues of investigation into its pathogenesis.

On the accuracy of the epigenetic copy machine: comprehensive specificity analysis of the DNMT1 DNA methyltransferase.

The specificity of DNMT1 for hemimethylated DNA is a central feature for the inheritance of DNA methylation. We investigated this property in competitive methylation kinetics using hemimethylated (HM), hemihydroxymethylated (OH) and unmethylated (UM) substrates with single CpG sites in a randomized sequence context. DNMT1 shows a strong flanking sequence dependent HM/UM specificity of 80-fold on average, which is slightly enhanced on long hemimethylated DNA substrates. To explain this strong effect of a single methyl group, we propose a novel model in which the presence of the 5mC methyl group changes the conformation of the DNMT1-DNA complex into an active conformation by steric repulsion. The HM/OH preference is flanking sequence dependent and on average only 13-fold, indicating that passive DNA demethylation by 5hmC generation is not efficient in many flanking contexts. The CXXC domain of DNMT1 has a moderate flanking sequence dependent contribution to HM/UM specificity during DNA association to DNMT1, but not if DNMT1 methylates long DNA molecules in processive methylation mode. Comparison of genomic methylation patterns from mouse ES cell lines with various deletions of DNMTs and TETs with our data revealed that the UM specificity profile is most related to cellular methylation patterns, indicating that de novo methylation activity of DNMT1 shapes the DNA methylome in these cells.

Saliva biopsy: Detecting the difference of EBV DNA methylation in the diagnosis of nasopharyngeal carcinoma.

Saliva sampling is a non-invasive method, and could be performed by donors themselves. However, there are few studies reporting biomarkers in saliva in the diagnosis of NPC. A total of 987 salivary samples were used in this study. First, EBV DNA methylation was profiled by capture sequencing in the discovery cohort (n = 36). Second, a q-PCR based method was developed and five representative EBV DNA CpG sites (11 029 bp, 45 849 bp, 57 945 bp, 66 226 bp and 128 102 bp) were selected and quantified to obtain the methylated density in the validation cohort1 (n = 801). Third, a validation cohort2 (n = 108) was used to further verify the differences of EBV methylation in saliva. A significant increase of EBV methylation was found in NPC patients compared with controls. The methylated score of EBV genome obtained by capture sequencing could distinguish NPC from controls (sensitivity 90%, specificity 100%). Further, the methylated density of EBV DNA CpG sites revealed by q-PCR showed a good diagnostic performance. The sensitivity and specificity of detecting a single CpG site (11 029 bp) could reach 75.4% and 99.7% in the validation cohort1, and 78.2% and 100% in the validation cohort2. Besides, the methylated density of the CpG site was found to decrease below the COV in NPC patients after therapy, and increase above the COV after recurrence. Our study provides an appealing alternative for the non-invasive detection of NPC without clinical setting. It paves the way for conducting a home-based large-scale screening in the future.

High‐resolution transcriptomic and epigenetic profiling identifies novel regulators of COPD

Patients with chronic obstructive pulmonary disease (COPD) are still waiting for curative treatments. Considering its environmental cause, we hypothesized that COPD will be associated with altered epigenetic signaling in lung cells. We generated genome‐wide DNA methylation maps at single CpG resolution of primary human lung fibroblasts (HLFs) across COPD stages. We show that the epigenetic landscape is changed early in COPD, with DNA methylation changes occurring predominantly in regulatory regions. RNA sequencing of matched fibroblasts demonstrated dysregulation of genes involved in proliferation, DNA repair, and extracellular matrix organization. Data integration identified 110 candidate regulators of disease phenotypes that were linked to fibroblast repair processes using phenotypic screens. Our study provides high‐resolution multi‐omic maps of HLFs across COPD stages. We reveal novel transcriptomic and epigenetic signatures associated with COPD onset and progression and identify new candidate regulators involved in the pathogenesis of chronic lung diseases. The presence of various epigenetic factors among the candidates demonstrates that epigenetic regulation in COPD is an exciting research field that holds promise for novel therapeutic avenues for patients.