Cytosine-phosphate-guanine Research Articles

Combination of immune checkpoint blockade and targeted gene regulation of angiogenesis for facilitating antitumor immunotherapy.

The rapid development of tumor immunotherapy has improved the management of patients with cancer. However, several key problems of tumor immunotherapy, including the insufficient activation of effector T cells, poor tumor invasion, and poor immune killing ability, lead to a low response rate. In the present study, a synergistic strategy was developed by combining in situ tumor vaccines, gene-mediated downregulation of tumor angiogenesis, and anti-PD-L1 therapy. In situ tumor vaccines and antitumor angiogenesis were achieved by codelivering unmethylated cytosine-phosphate-guanine (CpG) and vascular endothelial growth factor (VEGF)-silencing gene (shVEGF) via a hyaluronic acid (HA)-modified HA/PEI/shVEGF/CpG system. Necrotic tumor cells and CpG adjuvants formed in situ tumor vaccines and activated the host immune response. Moreover, VEGF silencing reduced tumor angiogenesis and prompted the homogeneous distribution of tumor blood vessels to facilitate immune cell infiltration. Meanwhile, anti-angiogenesis also improved the immunosuppressive tumor microenvironment. To further improve the specific tumor-killing effect, an anti-PD-L1 antibody was introduced for immune checkpoint blockade, thereby boosting antitumor immune responses. The combination therapy strategy presented in the present study could act in the multiple stages of the tumor immunotherapy cycle, which is expected to offer a new avenue for clinical tumor immunotherapy.

DNA methylation in cocaine use disorder-An epigenome-wide approach in the human prefrontal cortex.

Cocaine use disorder (CUD) is characterized by a loss of control over cocaine intake and is associated with structural, functional, and molecular alterations in the human brain. At the molecular level, epigenetic alterations are hypothesized to contribute to the higher-level functional and structural brain changes observed in CUD. Most evidence of cocaine-associated epigenetic changes comes from animal studies while only a few studies have been performed using human tissue. We investigated epigenome-wide DNA methylation (DNAm) signatures of CUD in human post-mortem brain tissue of Brodmann area 9 (BA9). A total of N = 42 BA9 brain samples were obtained from N = 21 individuals with CUD and N = 21 individuals without a CUD diagnosis. We performed an epigenome-wide association study (EWAS) and analyzed CUD-associated differentially methylated regions (DMRs). To assess the functional role of CUD-associated differential methylation, we performed Gene Ontology (GO) enrichment analyses and characterized co-methylation networks using a weighted correlation network analysis. We further investigated epigenetic age in CUD using epigenetic clocks for the assessment of biological age. While no cytosine-phosphate-guanine (CpG) site was associated with CUD at epigenome-wide significance in BA9, we detected a total of 20 CUD-associated DMRs. After annotation of DMRs to genes, we identified Neuropeptide FF Receptor 2 (NPFFR2) and Kalirin RhoGEF Kinase (KALRN) for which a previous role in the behavioral response to cocaine in rodents is known. Three of the four identified CUD-associated co-methylation modules were functionally related to neurotransmission and neuroplasticity. Protein-protein interaction (PPI) networks derived from module hub genes revealed several addiction-related genes as highly connected nodes such as Calcium Voltage-Gated Channel Subunit Alpha1 C (CACNA1C), Nuclear Receptor Subfamily 3 Group C Member 1 (NR3C1), and Jun Proto-Oncogene, AP-1 Transcription Factor Subunit (JUN). In BA9, we observed a trend toward epigenetic age acceleration (EAA) in individuals with CUD remaining stable even after adjustment for covariates. Results from our study highlight that CUD is associated with epigenome-wide differences in DNAm levels in BA9 particularly related to synaptic signaling and neuroplasticity. This supports findings from previous studies that report on the strong impact of cocaine on neurocircuits in the human prefrontal cortex (PFC). Further studies are needed to follow up on the role of epigenetic alterations in CUD focusing on the integration of epigenetic signatures with transcriptomic and proteomic data.

A functional genomics pipeline to identify high-value asthma and allergy CpGs in the human methylome

DNA methylation of cytosines at cytosine-phosphate-guanine (CpG) dinucleotides (CpGs) is a widespread epigenetic mark, but genome-wide variation has been relatively unexplored due to the limited representation of variable CpGs on commercial high-throughput arrays. To explore this hidden portion of the epigenome, this study combined whole-genome bisulfite sequencing with in silico evidence of gene regulatory regions to design a custom array of high-value CpGs. This study focused on airway epithelial cells from children with and without allergic asthma because these cells mediate the effects of inhaled microbes, pollution, and allergens on asthma and allergic disease risk. This study identified differentially methylated regions from whole-genome bisulfite sequencing in nasal epithelial cell DNA from a total of 39 children with and without allergic asthma of both European and African ancestries. This study selected CpGs from differentially methylated regions, previous allergy or asthma epigenome-wide association studies (EWAS), or genome-wide association study loci, and overlapped them with functional annotations for inclusion on a custom Asthma&Allergy array. This study used both the custom and EPIC arrays to perform EWAS of allergic sensitization (AS) in nasal epithelial cell DNA from children in the URECA (Urban Environment and Childhood Asthma) birth cohort and using the custom array in the INSPIRE [Infant Susceptibility to Pulmonary Infections and Asthma Following RSV Exposure] birth cohort. Each CpG on the arrays was assigned to its nearest gene and its promotor capture Hi-C interacting gene and performed expression quantitative trait methylation (eQTM) studies for both sets of genes. Custom array CpGs were enriched for intermediate methylation levels compared to EPIC CpGs. Intermediate methylation CpGs were further enriched among those associated with AS and for eQTMs on both arrays. This study revealed signature features of high-value CpGs and evidence for epigenetic regulation of genes at AS EWAS loci that are robust to race/ethnicity, ascertainment, age, and geography.

DNA Methylation Architecture Provides Insight into the Pathogenesis of Upper Tract Urothelial Carcinoma: A Systematic Review and Meta-Analysis.

Numerous studies suggested methylation modifications play an important role in upper tract urothelial carcinoma (UTUC), but few have depicted DNA methylation architecture on the pathological process of UTUC. We aimed to better understand the pathogenesis of UTUC and provide precision medicine references when managing UTUC patients. PubMed, Cochrane Library, EMBASE, and Scopus were searched for UTUC until December 31, 2020. Methodological quality assessment was conducted according to NIH recommendations. Meta-analysis was conducted to assess the prognostic effect of methylated genes. Kaplan-Meier survival analyses were performed to validate methylated genes and cytosine-phosphate-guanine (CpG) sites. Eleven studies (3619 patients) were eligible to investigate 12 methylated genes and 10 CpGs. The quality of all the studies was fair to good. Meta-analysis found the pooled effect of eligible methylated genes had a low risk of tumor recurrence (HR=0·67; 95% CI: 0·51-0·87; P=·003), but a high risk of tumor progression (HR=1·60; 95% CI: 1·17-2·18; P=·003) and cancer-specific mortality (HR=1·35; 95% CI: 1·06-1·72; P=·01). For individual methylation status of GDF15, HSPA2, RASSF1A, TMEFF2, and VIM, the pooled effect of each gene was found pleiotropic on both diagnosis and prognosis. Survival analysis suggested higher methylation of SPARCL1 had a better disease-specific survival (P=·048). We combined meta-analysis and Kaplan-Meier survival analysis using the most updated evidence on the methylation of UTUC. Candidate biomarkers with essential diagnosis and prognosis function might provide precision medicine references for personalized therapies.

DNA methylation analysis is used to identify novel genetic loci associated with circulating fibrinogen levels in blood

BackgroundFibrinogen plays an essential role in blood coagulation and inflammation. Circulating fibrinogen levels may be determined based on interindividual differences in DNA methylation at cytosine-phosphate-guanine (CpG) sites and vice versa. ObjectivesTo perform an EWAS to examine an association between blood DNA methylation levels and circulating fibrinogen levels to better understand its biological and pathophysiological actions. MethodsWe performed an epigenome-wide association study of circulating fibrinogen levels in 18 037 White, Black, American Indian, and Hispanic participants, representing 14 studies from the Cohorts for Heart and Aging Research in Genomic Epidemiology consortium. Circulating leukocyte DNA methylation was measured using the Illumina 450K array in 12 904 participants and using the EPIC array in 5133 participants. In each study, an epigenome-wide association study of fibrinogen was performed using linear mixed models adjusted for potential confounders. Study-specific results were combined using array-specific meta-analysis, followed by cross-replication of epigenome-wide significant associations. We compared models with and without CRP adjustment to examine the role of inflammation. ResultsWe identified 208 and 87 significant CpG sites associated with fibrinogen levels from the 450K (p < 1.03 × 10−7) and EPIC arrays (p < 5.78 × 10−8), respectively. There were 78 associations from the 450K array that replicated in the EPIC array and 26 vice versa. After accounting for overlapping sites, there were 83 replicated CpG sites located in 61 loci, of which only 4 have been previously reported for fibrinogen. The examples of genes located near these CpG sites were SOCS3 and AIM2, which are involved in inflammatory pathways. The associations of all 83 replicated CpG sites were attenuated after CRP adjustment, although many remained significant. ConclusionWe identified 83 CpG sites associated with circulating fibrinogen levels. These associations are partially driven by inflammatory pathways shared by both fibrinogen and CRP.

Epigenome-wide meta-analysis of BMI in nine cohorts: Examining the utility of epigenetically predicted BMI

This study sought to examine the association between DNA methylation and body mass index (BMI) and the potential of BMI-associated cytosine-phosphate-guanine (CpG) sites to provide information about metabolic health. We pooled summary statistics from six trans-ethnic epigenome-wide association studies (EWASs) of BMI representing nine cohorts (n= 17,034), replicated these findings in the Women's Health Initiative (WHI, n= 4,822), and developed an epigenetic prediction score of BMI. In the pooled EWASs, 1,265 CpG sites were associated with BMI (p<1E-7) and 1,238 replicated in the WHI (FDR<0.05). We performed several stratified analyses to examine whether these associations differed between individuals of European and African descent, as defined by self-reported race/ethnicity. We found that five CpG sites had a significant interaction with BMI by race/ethnicity. To examine the utility of the significant CpG sites in predicting BMI, we used elastic net regression to predict log-normalized BMI in the WHI (80% training/20% testing). This model found that 397 sites could explain 32% of the variance in BMI in the WHI test set. Individuals whose methylome-predicted BMI overestimated their BMI (high epigenetic BMI) had significantly higher glucose and triglycerides and lower HDL cholesterol and LDL cholesterol compared to accurately predicted BMI. Individuals whose methylome-predicted BMI underestimated their BMI (low epigenetic BMI) had significantly higher HDL cholesterol and lower glucose and triglycerides. This study confirmed 553 and identified 685 CpG sites associated with BMI. Participants with high epigenetic BMI had poorer metabolic health, suggesting that the overestimation may be driven in part by cardiometabolic derangements characteristic of metabolic syndrome.

A novel approach for predicting Alzheimer’s disease using machine learning on DNA methylation in blood

Alzheimer’s disease (AD) is the leading cause of dementia worldwide. Mild cognitive impairment (MCI) is an early stage of mental decline that can precede the development of AD. Oftentimes, the progression of MCI to AD is difficult to predict. An AD diagnosis can involve invasive brain scans and spinal fluid tests. However, one promising biomarker for the diagnosis of AD is epigenetic data, specifically the methylation level of cytosine-phosphate-guanine (CpG) sites in the DNA. AD is linked to environmental factors, with some factors causing changes in these DNA methylation levels. We hypothesized that machine learning models can use blood DNA methylation levels, sex, and age to predict between cognitive normality, MCI, and AD with at least 50% accuracy. In this paper, we generated four machine learning models and two dataset dimensionality reduction methods in order to test this hypothesis. We trained the models on data from CpG sites from whole blood. When predicting if a patient would be cognitively normal, have MCI, or have AD, we achieved an accuracy of 53.33%, which is 20% greater than random guessing. We achieved this accuracy using a gradient boosting decision trees model in combination with a logistic regression method for feature selection. While this accuracy is low, the feature selection method that we developed may be useful in future research, as it identifies CpG sites that are most correlated with AD. Since peripheral blood is easily accessible through blood draw, our model represents a practical way to assist in the diagnosis of AD.

Bioinspired immuno-radio-enhancers toward synergistic nanomedicine through radiation-induced abscopal effects and immunocheckpoint blockade therapies.

Local radio-therapy combined with immunotherapy has attracted great interest in controlling local tumors. In this study, we have developed membrane-cloaked manganese dioxide nanoparticles displaying anti-PD-L1 antibodies as targeted immuno-radio-enhancers. Mediated by anti-PD-L1 antibodies (aPD-L1) expressed on cell membranes, this kind of membrane-coated nanosystem can selectively deliver cytosine-phosphate-guanine (CpG)-loaded MnO2 nanoparticles (NPs) and induce systemic anti-tumor immunities, thereby achieving favorable immuno/radio-therapeutic outcomes. Through expressing various functional proteins onto cellular membranes, the new class of membrane-camouflaged nanovehicles can be endowed with a wide variety of artificial functionalities such as enzymatic catalytic capabilities and specific targeting. This versatile nanoplatform, in general, enables the targeted delivery of theranostics, opening a new avenue for personalized nanomedicine.

PYCR in Kidney Renal Papillary Cell Carcinoma: Expression, Prognosis, Gene Regulation Network, and Regulation Targets.

Pyrroline-5-carboxylate reductase (PYCR) includes three human genes encoding three isozymes, PYCR1, PYCR2, and PYCR3 (or PYCRL), which facilitate the final step in the conversion of glutamine to proline. These genes play important roles in regulating the cell cycle and redox homeostasis as well as promoting growth signaling pathways. Proline is abnormally upregulated in a variety of cancers, and as the last key enzyme in proline production, PYCR plays an integral role in promoting tumorigenesis and cancer progression. However, its role in patients with kidney renal papillary cell carcinoma (KIRP) has not been fully elucidated. In this study, we aimed to systematically analyze the expression, gene regulatory network, prognostic value, and target prediction of PYCR in patients with KIRP, elucidate the association between PYCR expression and KIRP, and identify potential new targets for the clinical treatment of KIRP. We systematically analyzed the expression, prognosis, gene regulatory network, and regulatory targets of PYCR1, PYCR2, and PYCRL in KIRP using multiple online databases including cBioPortal, STRING, MethSurv, GeneMANIA, Gene Expression Profiling Interactive Analysis (GEPIA), Metascape, UALCAN, LinkedOmics, and TIMER. The expression levels of PYCR1, PYCR2, and PYCRL were considerably upregulated in patients with KIRP based on sample type, sex, age, and individual cancer stage. PYCR1 and PYCR2 transcript levels were markedly upregulated in females than in males, and patients aged 21-40 years had higher PYCR1 and PYCR2 transcript levels than those in other age groups. Interestingly, PYCR2 transcript levels gradually decreased with age. In addition, the expressions of PYCR1 and PYCR2 were notably correlated with the pathological stage of KIRP. Patients with KIRP with low PYCR1 and PYCR2 expression had longer survival than those with high PYCR1 and PYCR2 expression. PYCR1, PYCR2, and PYCRL were altered by 4%, 7%, and 6%, respectively, in 280 patients with KIRP. The methylation levels of cytosine-phosphate-guanine (CpG) sites in PYCR were markedly correlated with the prognosis of patients with KIRP. PYCR1, PYCR2, PYCRL, and their neighboring genes form a complex network of interactions. The molecular functions of the genes, as demonstrated by their corresponding Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, included calcium channel activity, phospholipid binding, RNA polymerase II-specificity, and kinase and GTPase-regulatory activities. PYCR1, PYCR2, and PYCRL targeted miR-21, miR-221, and miR-222, resulting in a better prognosis of KIRP. We analyzed mRNA sequencing data from 290 patients with KIRP and found that ADA, NPM3, and TKT were positively associated with PYCR1 expression; PFDN2, JTB, and HAX1 were positively correlated with PYCR2 expression; SHARPIN, YDJC, and NUBP2 were positively correlated with PYCRL expression; PYCR1 was positively correlated with B cell and CD8+ T-cell infiltration levels; macrophage infiltration was negatively correlated with PYCR2 expression; and PYCRL expression was negatively correlated with B-cell, CD8+ T cell, and dendritic cell infiltration levels. PYCR1, PYCR2, and PYCRL may be potential therapeutic and prognostic biomarkers for patients with KIRP. The regulation of microRNAs (miRNAs), including miR-21, miR-221, and miR-222, may prove an important strategy for KIRP treatment.

Targeted Methylation Profiling of Single Laser-Capture Microdissected Post-Mortem Brain Cells by Adapted Limiting Dilution Bisulfite Pyrosequencing (LDBSP)

A reoccurring issue in neuroepigenomic studies, especially in the context of neurodegenerative disease, is the use of (heterogeneous) bulk tissue, which generates noise during epigenetic profiling. A workable solution to this issue is to quantify epigenetic patterns in individually isolated neuronal cells using laser capture microdissection (LCM). For this purpose, we established a novel approach for targeted DNA methylation profiling of individual genes that relies on a combination of LCM and limiting dilution bisulfite pyrosequencing (LDBSP). Using this approach, we determined cytosine-phosphate-guanine (CpG) methylation rates of single alleles derived from 50 neurons that were isolated from unfixed post-mortem brain tissue. In the present manuscript, we describe the general workflow and, as a showcase, demonstrate how targeted methylation analysis of various genes, in this case, RHBDF2, OXT, TNXB, DNAJB13, PGLYRP1, C3, and LMX1B, can be performed simultaneously. By doing so, we describe an adapted data analysis pipeline for LDBSP, allowing one to include and correct CpG methylation rates derived from multi-allele reactions. In addition, we show that the efficiency of LDBSP on DNA derived from LCM neurons is similar to the efficiency obtained in previously published studies using this technique on other cell types. Overall, the method described here provides the user with a more accurate estimation of the DNA methylation status of each target gene in the analyzed cell pools, thereby adding further validity to this approach.

CanMethdb: a database for genome-wide DNA methylation annotation in cancers.

DNA methylation within gene body and promoters in cancer cells is well documented. An increasing number of studies showed that cytosine-phosphate-guanine (CpG) sites falling within other regulatory elements could also regulate target gene activation, mainly by affecting transcription factors (TFs) binding in human cancers. This led to the urgent need for comprehensively and effectively collecting distinct cis-regulatory elements and TF-binding sites (TFBS) to annotate DNA methylation regulation. We developed a database (CanMethdb, http://meth.liclab.net/CanMethdb/) that focused on the upstream and downstream annotations for CpG-genes in cancers. This included upstream cis-regulatory elements, especially those involving distal regions to genes, and TFBS annotations for the CpGs and downstream functional annotations for the target genes, computed through integrating abundant DNA methylation and gene expression profiles in diverse cancers. Users could inquire CpG-target gene pairs for a cancer type through inputting a genomic region, a CpG, a gene name, or select hypo/hypermethylated CpG sets. The current version of CanMethdb documented a total of 38986060 CpG-target gene pairs (with 6769130 unique pairs), involving 385217 CpGs and 18044 target genes, abundant cis-regulatory elements and TFs for 33 TCGA cancer types. CanMethdb might help biologists perform in-depth studies of target gene regulations based on DNA methylations in cancer. The main program is available at https://github.com/chunquanlipathway/CanMethdb. Supplementary data are available at Bioinformatics online.

Blood MAPT expression and methylation status in Alzheimer's disease.

This study aimed to investigate the expression levels and methylation status of microtubule-associated protein tau (MAPT) in the blood of Alzheimer's disease (AD) patients and age- and sex-matched healthy controls. Fifty AD outpatients and 50 healthy contorls were enrolled. Blood samples were collected for processing of complementary DNA and genomic DNA. MAPT messenger ribonucleic acid (mRNA) expression was analyzed by real-time quantitative polymerase chain reaction. The methylation rates of four cytosine-phosphate-guanine (CpG) sites in the upstream region of MAPT exon1 were evaluated by the pyrosequencing method. No significant differences in MAPT mRNA expression levels were found between AD and control subjects (AD 0.97 ± 0.49 vs. control 1.0 ± 0.64, p = 0.62). MAPT mRNA expression levels were not correlated with any other clinical characteristics or results of psychological tests. MAPT mRNA expression levels were significantly higher in AD subjects treated with acetylcholinesterase inhibitors (AchEIs) (n = 25) than in subjects not treated with AChEIs (n = 25) (unmedicated 0.83 ± 0.33 vs. medicated 1.12 ± 0.59, p = 0.049). The AD subjects did not differ from the control subjects in methylation rates at selected CpG sites. MAPT methylation status were not correlated with clinical characteristics, the results of psychological tests, or MAPT mRNA expression. MAPT mRNA expression levels and methylation status in blood do not appear useful as biomarkers for AD or the examined CpG sites were not genetically significant for MAPT gene expression or AD pathology. However, AChEIs may alter MAPT mRNA expression. Further studies are needed to explore blood biomarkers that can discriminate AD patients from controls.

LINE-1 hypomethylation is associated with poor outcomes in locoregionally advanced oropharyngeal cancer

Background and purposeCurrently, human papillomavirus (HPV) positivity represents a strong prognostic factor for both reduced risk of relapse and improved survival in patients with oropharyngeal squamous cell carcinoma (OPSCC). However, a subset of HPV-positive OPSCC patients still experience poor outcomes. Furthermore, HPV-negative OPSCC patients, who have an even higher risk of relapse, are still lacking suitable prognostic biomarkers for clinical outcome. Here, we evaluated the prognostic value of LINE-1 methylation level in OPSCC patients and further addressed the relationship between LINE-1 methylation status and p53 protein expression as well as genome-wide/gene-specific DNA methylation.ResultsIn this study, DNA was extracted from 163 formalin-fixed paraffin-embedded tissue samples retrospectively collected from stage III-IVB OPSCC patients managed with curative intent with up-front treatment. Quantitative methylation-specific PCR revealed that LINE-1 hypomethylation was directly associated with poor prognosis (5-year overall survival—OS: 28.1% for LINE-1 methylation < 35% vs. 69.1% for ≥ 55%; p < 0.0001). When LINE-1 methylation was dichotomized as < 55% versus ≥ 55%, interaction with HPV16 emerged: compared with hypermethylated HPV16-positive patients, subjects with hypomethylated HPV16-negative OPSCC reported an adjusted higher risk of death (HR 4.83, 95% CI 2.24–10.38) and progression (HR 4.54, 95% CI 2.18–9.48). Tumor protein p53 (TP53) gene is often mutated and overexpressed in HPV-negative OPSCC. Since p53 has been reported to repress LINE-1 promoter, we then analyzed the association between p53 protein expression and LINE-1 methylation levels. Following p53 immunohistochemistry, results indicated that among HPV16-negative patients with p53 ≥ 50%, LINE-1 methylation levels declined and remained stable at approximately 43%; any HPV16-positive patient reported p53 ≥ 50%. Finally, DNA methylation analysis demonstrated that genome-wide average methylation level at cytosine–phosphate–guanine sites was significantly lower in HPV16-negative OPSCC patients who relapsed within two years. The subsequent integrative analysis of gene expression and DNA methylation identified 20 up-regulated/hypomethylated genes in relapsed patients, and most of them contained LINE-1 elements in their promoter sequences.ConclusionsEvaluation of the methylation level of LINE-1 may help in identifying the subset of OPSCC patients with bad prognosis regardless of their HPV status. Aberrant LINE-1 hypomethylation might occur along with TP53 mutations and lead to altered gene expression in OPSCC.

Genome-wide DNA methylation profiling of gastric cardia cancer.

Aberrant DNA methylation has been found in various cancer types including gastric cancer, yet the genome-wide DNA methylation profile of gastric cardia cancer (GCC) remains unclear. Therefore, we aimed to profile the DNA methylation pattern of GCC and identify promising diagnostic epigenetic biomarkers. We investigated the genome-wide DNA methylation pattern in eight pairs of GCC and adjacent normal tissues using Illumina 850K microarrays. Subsequently, bisulfite-pyrosequencing and quantitative real-time PCR were performed on eight pairs of GCC-adjacent normal tissues for validation. Finally, we performed immunohistochemistry to examine ADHFE1 expression on 126 pairs of GCC-adjacent normal samples. DNAmethylome analysis showed global hypomethylation and local hypermethylation of promoter cytosine-phosphate-guanine (CpG) islands (CGIs) in GCC tissues compared with gastric cardia normal mucosa (P<2.2×10-16 ). Differential methylation analysis identified a total of 91 723 differentially-methylated probes (DMPs), and the candidate gene with the largest average DNA methylation difference mapped to ADHFE1 (mean Δβ=0.53). Subsequently, three DMPs in the ADHFE1 promoter were validated by pyrosequencing. Notably,the mean methylation level of the three candidate DMPs (ADHFE1_cg08090772, ADHFE1_cg19283840, and ADHFE1_cg20295442) was negatively associated with ADHFE1 mRNA expression level (Spearman rho=-0.64, P=0.01). Moreover, both mRNA (P=0.0213) and protein (P<0.0001) expression of ADHFE1 were significantly decreased in GCCs compared with the adjacent normal tissues. Our results reveal DNA methylation aberrations in GCC and that ADHFE1 gene DNA methylation contributes to the risk of GCC, thus providing novel mechanistic insights into gastric cardia cancer carcinogenesis.

Epigenome-wide analysis of maternal exposure to green space during gestation and cord blood DNA methylation in the ENVIRONAGE cohort

BackgroundDNA methylation programming is sensitive to prenatal life environmental influences, but the impact of maternal exposure to green space on newborns DNA methylation has not been studied yet. MethodsWe conducted a meta-epigenome-wide association study (EWAS) of maternal exposure to green space during gestation with cord blood DNA methylation in two subsets of the ENVIRONAGE cohort (N = 538). Cord blood DNA methylation was measured by Illumina HumanMethylation 450K in one subset (N = 189) and EPICarray in another (N = 349). High (vegetation height>3 m (m)), low (vegetation height<3 m) and total (including both) high-resolution green space exposures during pregnancy were estimated within 100 m and 1000 m distance around maternal residence. In each subset, we sought cytosine-phosphate-guanine (CpG) sites via linear mixed models adjusted on newborns' sex, ethnicity, gestational age, season at delivery, sampling day, maternal parity, age, smoking, education, and estimated blood cell proportions. EWASs results were meta-analysed via fixed-effects meta-analyses. Differentially methylated regions (DMRs) were identified via ENmix-combp and DMRcate algorithms. Sensitivity analyses were additionally adjusted on PM2.5, distance to major roads, urbanicity and neighborhood income. In the 450K subset, cord blood expression of differentially methylated genes was measured by Agilent microarrays and associated with green space. Results147 DMRs were identified, 85 of which were still significant upon adjustment for PM2.5, distance to major roads, urbanicity and neighborhood income, including HLA-DRB5, RPTOR, KCNQ1DN, A1BG-AS1, HTR2A, ZNF274, COL11A1 and PRSS36 DMRs. One CpG reached genome-wide significance, while 54 CpGs were suggestive significant (p-values<1e-05). Among them, a CpG, hypermethylated with 100 m buffer total green space, was annotated to PAQR9, whose expression decreased with 1000 m buffer low green space (p-value = 1.45e-05). ConclusionsOur results demonstrate that maternal exposure to green space during pregnancy is associated with cord blood DNA methylation, mainly at loci organized in regions, in genes playing important roles in neurological development (e.g., HTR2A).

Epigenome-wide DNA methylation in leukocytes and toenail metals: The normative aging study

BackgroundEnvironmental metal exposures have been associated with multiple deleterious health endpoints. DNA methylation (DNAm) may provide insight into the mechanisms underlying these relationships. Toenail metals are non-invasive biomarkers, reflecting a medium-term time exposure window. ObjectivesThis study examined variation in leukocyte DNAm and toenail arsenic (As), cadmium (Cd), lead (Pb), manganese (Mn), and mercury (Hg) among elderly men in the Normative Aging Study, a longitudinal cohort. MethodsWe repeatedly collected samples of blood and toenail clippings. We measured DNAm in leukocytes with the Illumina HumanMethylation450 K BeadChip. We first performed median regression to evaluate the effects of each individual toenail metal on DNAm at three levels: individual cytosine-phosphate-guanine (CpG) sites, regions, and pathways. Then, we applied a Bayesian kernel machine regression (BKMR) to assess the joint and individual effects of metal mixtures on DNAm. Significant CpGs were identified using a multiple testing correction based on the independent degrees of freedom approach for correlated outcomes. The approach considers the effective degrees of freedom in the DNAm data using the principal components that explain >95% variation of the data. ResultsWe included 564 subjects (754 visits) between 1999 and 2013. The numbers of significantly differentially methylated CpG sites, regions, and pathways varied by metals. For example, we found six significant pathways for As, three for Cd, and one for Mn. The As-associated pathways were associated with cancer (e.g., skin cancer) and cardiovascular disease, whereas the Cd-associated pathways were related to lung cancer. Metal mixtures were also associated with 47 significant CpG sites, as well as pathways, mainly related to cancer and cardiovascular disease. ConclusionsThis study provides an approach to understanding the potential epigenetic mechanisms underlying observed relations between toenail metals and adverse health endpoints.

A correlation map of genome-wide DNA methylation patterns between paired human brain and buccal samples

Epigenome-wide association studies (EWAS) assessing the link between DNA methylation (DNAm) and phenotypes related to structural brain measures, cognitive function, and neurodegenerative diseases are becoming increasingly more popular. Due to the inaccessibility of brain tissue in humans, several studies use peripheral tissues such as blood, buccal swabs, and saliva as surrogates. To aid the functional interpretation of EWAS findings in such settings, there is a need to assess the correlation of DNAm variability across tissues in the same individuals. In this study, we performed a correlation analysis between DNAm data of a total of n = 120 matched post-mortem buccal and prefrontal cortex samples. We identified nearly 25,000 (3% of approximately 730,000) cytosine-phosphate-guanine (CpG) sites showing significant (false discovery rate q < 0.05) correlations between buccal and PFC samples. Correlated CpG sites showed a preponderance to being located in promoter regions and showed a significant enrichment of being determined by genetic factors, i.e. methylation quantitative trait loci (mQTL), based on buccal and dorsolateral prefrontal cortex mQTL databases. Our novel buccal–brain DNAm correlation map will provide a valuable resource for future EWAS using buccal samples for studying DNAm effects on phenotypes relating to the brain. All correlation results are made freely available to the public online.

Genetic and pharmacological targeting of GSDMD ameliorates systemic inflammation in macrophage activation syndrome

Macrophage activation syndrome (MAS), a potentially life-threatening complication of autoimmune/autoinflammatory diseases, is characterized by the excessive expansion and activation of macrophages and cytotoxic T lymphocytes in multiple organs. Most commonly, MAS occurs in patients with systemic juvenile idiopathic arthritis and in its adult equivalent, adult-onset Still's disease (AOSD). Gasdermin D (GSDMD) is a critical pore-forming effector protein that mediates pro-inflammatory cytokine secretion via releasing its N terminal fragments to form transmembrane pores. GSDMD has been implicated in various inflammatory diseases, however, its role in MAS remains elusive. Here, we unveiled that the serum levels of GSDMD-N were elevated in patients with AOSD compared to heathy controls. In addition, the emergence of MAS features in AOSD patients resulted in further elevation. The serum levels of GSDMD were positively correlated with ferritin and interleukin-18 (IL-18). Repeated toll-like receptor 9 stimulation with unmethylated cytosine-phosphate-guanine (CpG) induced MAS symptoms in wild-type mice, including body weight loss, pancytopenia and hepatosplenomegaly. Genetic deletion and pharmacological inhibition of GSDMD ameliorated MAS symptoms in mice with the concomitant reduction of splenic and hepatic macrophage infiltration and IL-18 production. Consistent with these in vivo results, bone marrow-derived macrophages obtained from GSDMD−/− mice or treated with GSDMD inhibitor disulfiram exhibited attenuated IL-18 expression after CpG stimulation. Collectively, our findings identified GSDMD as a novel marker for MAS complication and a promising target for MAS treatment.

Epigenetic analysis of cell-free DNA by fragmentomic profiling.

Cell-free DNA (cfDNA) fragmentation patterns contain important molecular information linked to tissues of origin. We explored the possibility of using fragmentation patterns to predict cytosine-phosphate-guanine (CpG) methylation of cfDNA, obviating the use of bisulfite treatment and associated risks of DNA degradation. This study investigated the cfDNA cleavage profile surrounding a CpG (i.e., within an 11-nucleotide [nt] window) to analyze cfDNA methylation. The cfDNA cleavage proportion across positions within the window appeared nonrandom and exhibited correlation with methylation status. The mean cleavage proportion was ∼twofold higher at the cytosine of methylated CpGs than unmethylated ones in healthy controls. In contrast, the mean cleavage proportion rapidly decreased at the 1-nt position immediately preceding methylated CpGs. Such differential cleavages resulted in a characteristic change in relative presentations of CGN and NCG motifs at 5' ends, where N represented any nucleotide. CGN/NCG motif ratios were correlated with methylation levels at tissue-specific methylated CpGs (e.g., placenta or liver) (Pearson's absolute r > 0.86). cfDNA cleavage profiles were thus informative for cfDNA methylation and tissue-of-origin analyses. Using CG-containing end motifs, we achieved an area under a receiver operating characteristic curve (AUC) of 0.98 in differentiating patients with and without hepatocellular carcinoma and enhanced the positive predictive value of nasopharyngeal carcinoma screening (from 19.6 to 26.8%). Furthermore, we elucidated the feasibility of using cfDNA cleavage patterns to deduce CpG methylation at single CpG resolution using a deep learning algorithm and achieved an AUC of 0.93. FRAGmentomics-based Methylation Analysis (FRAGMA) presents many possibilities for noninvasive prenatal, cancer, and organ transplantation assessment.

Amplified cancer immunotherapy of PD-L1 blockade by sequential tumor microenvironment reshaping and DC maturation

The immune checkpoint blockade (ICB) of programmed death ligand-1 (PD-L1) or programmed death-1 (PD-1) has gained promising performances for triggering durable response and manageable toxicity of the host’s antitumor immunity. However, ICB therapy is still unsatisfactory considering the restrained tumor-infiltrating lymphocytes (TILs), poor accumulation and penetration of antibodies and immunosuppressive tumor microenvironment (TME). Herein, we developed a two-step sequential delivery strategy for potent immune cocktail therapy to amplify anti-PD-L1 immunotherapy. Quercetin (Que) was coated by bovine serum albumin (BSA) to form nanoparticles (QB NPs) for remodeling tumor microenvironment (TME) to increase the penetration of the subsequent nanoparticles (NPs) in tumor issues and promote T cell infiltration. Consequently, the nucleotide drug carrier, PF180, coated with pH-sensitive polyethyleneglycol (PEG) was fabricated to co-deliver unmethylated cytosine-phosphate-guanine (CpG) and plasmid DNA encoding small hairpin RNA of PD-L1 (pshPD-L1). Such nanoparticles (NPs) with high efficiency and low toxicity could significantly induce dendritic cell maturation and PD-L1 downregulation on tumor cell surface. This sequential delivery strategy effectively ameliorated the harmful extracellular matrix (ECM) via reducing the activity of tumor-associated fibroblasts (TAF). Together with the reduced expression of collagen and ɑ-SMA in tumor tissue, the silenced PD-L1 gene can strongly inhibit tumor growth. The infiltration of immune cells was also enhanced, favoring the reversal of the immunosuppressive tumor microenvironment. This combination treatment provided a feasible strategy to improve the antitumor efficacy of anti-PD-L1 cancer therapy.