Cytosine-phosphate-guanine Research Articles

MTORC1-GLUT1-mediated glucose metabolism drives hyperactivation of B cells in primary Sjogren's syndrome.

Primary Sjögren's syndrome (pSS) is a chronic systemic autoimmune disease characterized by B cell hyperactivation and hypergrammaglobulinemia. Currently, the role of metabolic pathways in the B cells of pSS patients is poorly defined. Here, we showed that upon cytosine phosphate-guanine (CpG)/sCD40L/IL-4 stimulation, B cells proportionally increased glycolysis and oxygen consumption, and compared with B cells from healthy controls (HCs), B cells from pSS patients exhibited higher glycolysis capacity and maximal oxidative respiration (OXPHOS). We also found that glucose transporter 1 (GLUT1) expression in B cells from pSS patients was significantly higher than that in B cells from HCs. Treatment with 2-deoxy-d-glucose (2-DG) inhibited the activation of B cells in pSS patients. Both 2-DG and Metformin inhibited the proliferation, formation of plasma/plasmablasts and decreased the IgG and IgM levels in the supernatants of B cells from pSS patients. Furthermore, inhibition of mTORC1 by rapamycin had an effect similar to that of 2-DG, suppressing B cell activation, proliferation and antibody production. Taken together, we demonstrated that B cells from pSS patients are more metabolically active than those from HCs and suggested that the mTORC1-GLUT1 glycolysis pathways were the major drivers of B cell hyperactivation and autoantibody production in pSS patients.

CD47KO/CRT dual-bioengineered cell membrane-coated nanovaccine combined with anti-PD-L1 antibody for boosting tumor immunotherapy.

Tumor vaccines trigger tumor-specific immune responses to prevent or treat tumors by activating the hosts’ immune systems, and therefore, these vaccines have potential clinical applications. However, the low immunogenicity of the tumor antigen itself and the low efficiency of the vaccine delivery system hinder the efficacy of tumor vaccines that cannot produce high-efficiency and long-lasting antitumor immune effects. Here, we constructed a nanovaccine by integrating CD47KO/CRT dual-bioengineered B16F10 cancer cell membranes and the unmethylated cytosine-phosphate-guanine (CpG) adjuvant. Hyperbranched PEI25k was used to load unmethylated cytosine-phosphate-guanine (CpG) through electrostatic adsorption to prepare PEI25k/CpG nanoparticles (PEI25k/CpG-NPs). CD47KO/CRT dual-bioengineered cells were obtained by CRISPR-Cas9 gene editing technology, followed by the cell surface translocation of calreticulin (CRT) to induce immunogenic cell death (ICD) in vitro. Finally, the extracted cell membranes were coextruded with PEI25k/CpG-NPs to construct the CD47KO/CRT dual-bioengineered cancer cell membrane-coated nanoparticles (DBE@CCNPs). DBE@CCNPs could promote endocytosis of antigens and adjuvants in murine bone marrow derived dendritic cells (BMDCs) and induce their maturation and antigen cross-presentation. To avoid immune checkpoint molecule-induced T cell dysfunction, the immune checkpoint inhibitor, the anti-PD-L1 antibody, was introduced to boost tumor immunotherapy through a combination with the DBE@CCNPs nanovaccine. This combination therapy strategy can significantly alleviate tumor growth and may open up a potential strategy for clinical tumor immunotherapy.

Locally correlated kinetics of post-replication DNA methylation reveals processivity and region specificity in DNA methylation maintenance.

DNA methylation occurs predominantly on cytosine-phosphate-guanine (CpG) dinucleotides in the mammalian genome, and the methylation landscape is maintained over mitotic cell division. It has been posited that coupling of maintenance methylation activity among neighbouring CpGs is critical to stability over cellular generations; however, the mechanism is unclear. We used mathematical models and stochastic simulation to analyse data from experiments that probe genome-wide methylation of nascent DNA post-replication in cells. We find that DNA methylation maintenance rates on individual CpGs are locally correlated, and the degree of this correlation varies by genomic regional context. By using theory of protein diffusion along DNA, we show that exponential decay of methylation rate correlation with genomic distance is consistent with enzyme processivity. Our results provide quantitative evidence of genome-wide methyltransferase processivity in vivo. We further developed a method to disentangle different mechanistic sources of kinetic correlations. From the experimental data, we estimate that an individual methyltransferase methylates neighbour CpGs processively if they are 36 basepairs apart, on average. But other mechanisms of coupling dominate for longer inter-CpG distances. Our study demonstrates that quantitative insights into enzymatic mechanisms can be obtained from replication-associated, cell-based genome-wide measurements, by combining data-driven statistical analyses with hypothesis-driven mathematical modelling.

Effect of carrier molecular weight on physicochemical properties and the in vitro immune-stimulatory activity of the CpG-dextran conjugates

The effect of dextran molecular weight on the in vitro physicochemical and immune properties of cytosine-phosphate-guanine (CpG) oligodeoxynucleotide-amino-dextran conjugates is investigated. CpG-1668 was conjugated at the 3′-end to amino-dextran of differing molecular weight (20, 40, 70 or 110-kDa) via a stable bis-aryl hydrazone linkage. Conjugate formation was confirmed by agarose gel electrophoresis and dynamic light scattering measured the size and surface charge of conjugates. Uptake and immune-stimulatory activity of CpG-dextran by antigen-presenting cells was evaluated by flow cytometry and confocal microscopy. Degradation by DNase I was monitored by loss of the fluorescent signal from labelled CpG and changes in size and zeta potential. Hydrazone bond formation (UV 354 nm) showed on average four CpG molecules conjugated per polymer. CpG-dextran prepared from 20 or 40-kDa dextran had a size of 17 nm while 70 or 110-kDa was 30 nm. CpG-dextran was preferentially taken up by dendritic cells, followed by macrophages and then B-cells. Only the 20-kDa dextran conjugate significantly enhanced uptake by bone-marrow derived dendritic cells (BMDCs) compared to free CpG. Confocal microscopy showed that CpG and CpG-dextran accumulates in the endo-lysosomal compartment of BMDCs at 24 h. All conjugates upregulated activation markers (CD40, CD80 or CD86) of BMDCs to a similar level as for free CpG. CpG-dextran 40-kDa produced highest levels of cytokines (TNF-α, IL-6, and IL-12p70) secreted by BMDCs. Enzymatic protection assays showed that the conjugate made from dextran 20-kDa provided no protection for CpG while the higher molecular weight conjugates reduced degradation by DNase I. The 40-kDa dextran conjugate produced the greatest in vitro immune activity, this was due to the conjugate being relatively small in size for cell uptake while sufficiently large enough to protect CpG from nuclease attack. These in vitro studies identify the need to consider the molecular weight of the carrier in bioconjugate design.

Versatile BP/Pd-FPEI-CpG nanocomposite for "three-in-one" multimodal tumor therapy

Multimodal nanocatalytic therapy has shown promising potential in cancer treatments. However, to design an effective multimodal nanocatalytic modality, with a deep understanding of synergistic therapy, remains a grand challenge. Herein, we propose an innovative “three-in-one” multimodal strategy with integrated photothermal/photodynamic/chemodynamic modalities for tumor-specific treatment, by rationally constructing a multifunctional two-dimensional (2D) nanoplatform based on the black phosphorous (BP)/Pd nanosheets (NSs) via in-situ growth. Interestingly, such a hybridization of different types of 2D NSs mediates formation of the Pd-P coordination bonds. With strong interfacial Pd-P interaction, the heterostructured NSs display enhanced cascade enzymatic activities in tumor microenvironment, leading to sufficient generation of reactive oxygen species (ROS) as therapeutic species including cytotoxic superoxide (O2•−) radicals by successive catalase-/oxidase-catalysis, and deleterious singlet oxygen (1O2) radicals under light irradiation. The incorporation of Pd also improved the photothermal activity of the material. Furthermore, by facile functionalization with fluoro-polyethyleneimine (FPEI) and loading of cytosine-phosphate-guanine (CpG) as an immune adjuvant, the versatilities of this unique 2D hetero-nanoplatform are further extended, strikingly improving therapeutic efficacy for tumor cells, and simultaneously avoiding significant damage to normal tissues. The work provides useful insights into the design of new 2D nanoplatforms for multimodal tumor treatment.

Epigenetic and integrative cross-omics analyses of cerebral white matter hyperintensities on MRI.

Cerebral white matter hyperintensities on MRI are markers of cerebral small vessel disease, a major risk factor for dementia and stroke. Despite the successful identification of multiple genetic variants associated with this highly heritable condition, its genetic architecture remains incompletely understood. More specifically, the role of DNA methylation has received little attention. We investigated the association between white matter hyperintensity burden and DNA methylation in blood at ∼450 000 cytosine-phosphate-guanine (CpG) sites in 9732 middle-aged to older adults from 14 community-based studies. Single CpG and region-based association analyses were carried out. Functional annotation and integrative cross-omics analyses were performed to identify novel genes underlying the relationship between DNA methylation and white matter hyperintensities. We identified 12 single CpG and 46 region-based DNA methylation associations with white matter hyperintensity burden. Our top discovery single CpG, cg24202936 (P = 7.6 × 10-8), was associated with F2 expression in blood (P = 6.4 × 10-5) and co-localized with FOLH1 expression in brain (posterior probability = 0.75). Our top differentially methylated regions were in PRMT1 and in CCDC144NL-AS1, which were also represented in single CpG associations (cg17417856 and cg06809326, respectively). Through Mendelian randomization analyses cg06809326 was putatively associated with white matter hyperintensity burden (P = 0.03) and expression of CCDC144NL-AS1 possibly mediated this association. Differentially methylated region analysis, joint epigenetic association analysis and multi-omics co-localization analysis consistently identified a role of DNA methylation near SH3PXD2A, a locus previously identified in genome-wide association studies of white matter hyperintensities. Gene set enrichment analyses revealed functions of the identified DNA methylation loci in the blood-brain barrier and in the immune response. Integrative cross-omics analysis identified 19 key regulatory genes in two networks related to extracellular matrix organization, and lipid and lipoprotein metabolism. A drug-repositioning analysis indicated antihyperlipidaemic agents, more specifically peroxisome proliferator-activated receptor-alpha, as possible target drugs for white matter hyperintensities. Our epigenome-wide association study and integrative cross-omics analyses implicate novel genes influencing white matter hyperintensity burden, which converged on pathways related to the immune response and to a compromised blood-brain barrier possibly due to disrupted cell-cell and cell-extracellular matrix interactions. The results also suggest that antihyperlipidaemic therapy may contribute to lowering risk for white matter hyperintensities possibly through protection against blood-brain barrier disruption.

DNA methylation patterns and gene expression from amygdala tissue of mature Brahman cows exposed to prenatal stress.

Prenatal stress can alter postnatal performance and temperament of cattle. These phenotypic effects may result from changes in gene expression caused by stress-induced epigenetic alterations. Specifically, shifts in gene expression caused by DNA methylation within the brain’s amygdala can result in altered behavior because it regulates fear, stress response and aggression in mammals Thus, the objective of this experiment was to identify DNA methylation and gene expression differences in the amygdala tissue of 5-year-old prenatally stressed (PNS) Brahman cows compared to control cows. Pregnant Brahman cows (n = 48) were transported for 2-h periods at 60 ± 5, 80 ± 5, 100 ± 5, 120 ± 5, and 140 ± 5 days of gestation. A non-transported group (n = 48) were controls (Control). Amygdala tissue was harvested from 6 PNS and 8 Control cows at 5 years of age. Overall methylation of gene body regions, promoter regions, and cytosine-phosphate-guanine (CpG) islands were compared between the two groups. In total, 202 genes, 134 promoter regions, and 133 CpG islands exhibited differential methylation (FDR ≤ 0.15). Following comparison of gene expression in the amygdala between the PNS and Control cows, 2 differentially expressed genes were identified (FDR ≤ 0.15). The minimal differences observed could be the result of natural changes of DNA methylation and gene expression as an animal ages, or because this degree of transportation stress was not severe enough to cause lasting effects on the offspring. A younger age may be a more appropriate time to assess methylation and gene expression differences produced by prenatal stress.

Integrated Analysis of Gene Expression and Methylation Data to Identify Potential Biomarkers Related to Atherosclerosis Onset.

Atherosclerosis is a kind of chronic inflammatory cardiovascular disease. Epigenetic regulation plays a crucial role in atherosclerosis. Our study was aimed at finding potential biomarkers associated with the occurrence of atherosclerosis. Two datasets were downloaded from the Gene Expression Omnibus (GEO) database. The epigenome-wide association study (EWAS) analysis was performed on methylation data using CpGassoc package. The differential expression analysis was conducted on mRNA data using limma package. The GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) functional enrichment was done in clusterProfiler package. Finally, the logistic regression model was constructed using generalized linear model (glm) function. Between atherosclerotic vs. nonatherosclerotic samples, totally 4980 cytosine-phosphate-guanine (CpG) sites (annotated to 2860 genes) and 132 differentially expressed genes (DEGs) related to atherosclerosis were identified. The annotated 2860 genes and 132 DEGs were significantly enriched in 9 and 4 KEGG pathways and 289 and 132 GO terms, respectively. After cross-analysis, 6 crucial CpG sites were screened to build the model, including cg01187920, cg03422911, cg08018825, cg10967350, cg14473924, and cg25313204. The diagnostic model could reliably separate the atherosclerosis samples from nonatherosclerotic samples. In conclusion, the 6 CpG sites are probably potential diagnostic biomarkers for atherosclerosis, including cg01187920, cg03422911, cg08018825, cg10967350, cg14473924, and cg25313204.

Comparative analysis of CpG islands in two genotypes of African swine fever virus.

African swine fever (ASF) is an acute, hemorrhagic, and devastating viral infectious disease that causes important economic losses to the swine industry. Currently, there are no effective vaccines or drugs available. Epigenetic mechanisms, especially cytosine methylation of cytosine- -phosphate-guanine (CpG) islands, have a significant impact on the life cycle of several viruses. Hence, drugs targeting DNA methylation may potentially be used for the treatment of ASF. Here, we selected the inner core, core shell, inner membrane, capsid, and external envelope membrane, to analyze the characteristics of CpG islands in the ASF virus (ASFV) genomes. Furthermore, we analyzed the promoters and CpG islands in the upstream regions of these genes. Results showed that the CpG islands of seven genes were conserved in the genomes of two genotype of ASFV strains, whereas the CpG islands of other genes were relatively conserved (ASFV strains differed mainly in the quantity of CpG islands). The different distribution of CpG islands in the genomes of different ASFV strains may affect their methylation status, which may in turn affect the regulation of viral gene expression, leading to different clinical outcomes. In addition, the predicted promoter regions based on the upstream sequences of most genes overlapped with CpG island positions. Methylation of the binding sites of the promoter regions inhibits the binding of the transcription factors to the promoters, thus inhibiting the activation of the promoters and limiting the synthesis of viral proteins. The results of this study provide a basis for exploring new antiviral therapeutic strategies from an epigenetic perspective.

Epigenetically-regulated miR-30a/c-5p directly target TWF1 and hamper ccRCC cell aggressiveness

Clear cell renal cell carcinoma (ccRCC) is highly prone to metastasize and displays an extremely low 5-year survival rate. Not only miRNAs (miRs) are key gene expression regulators but can also be epigenetically modified. Abnormal miR expression has been linked with epithelial-mesenchymal transition (EMT)-driven ccRCC progression. MiR-30a/c-5p were found downregulated in ccRCC and associated with aggressiveness. Herein, we sought to unravel miR-30a/c-5p mechanistic role in ccRCC. RNA sequencing and genome-wide methylome data of ccRCC and normal tissue samples from The Cancer Genome Atlas database were integrated to identify candidate miRs cytosine-phosphate-guanine (CpG) loci deregulated in ccRCC. TargetScan was searched to identify miR putative targets. MiR-30a/c-5p expression and promoter methylation was evaluated in vitro, by PCR. Western blot, functional and luciferase assays were performed after cell transfection with either pre-miR, antimiR, or siRNA against twinfilin-1 (TWF1). Immunohistochemistry (IHC) was performed in ccRCC tissues. We found miR-30c-5p downregulation and aberrant promoter methylation in ccRCC tissues. In vitro studies revealed concomitant miR-30a/c-5p downregulation and increased promoter methylation, as well as a significant re-expression following decitabine treatment. Functional assays demonstrated that both miRs significantly decreased cell aggressiveness and the protein levels of EMT-promoting players, while upregulating epithelial markers, namely Claudin-1 and ZO-1. Importantly, we confirmed TWF1 as a direct target of both miRs, and its potential involvement in epithelial-mesenchymal transition/mesenchymal-epithelial transition regulation. IHC analysis revealed higher TWF1 expression in primary tissues from patients that developed metastases, after surgical treatment. Our results implicate miR-30a/c-5p in ccRCC cells' aggressiveness attenuation by directly targeting TWF1 and hampering EMT.

0031 Sleep Regularity is Associated with DNA Methylation in Cognitive, Cardiovascular and Mood-related Genes: A GWAS-informed Study in Adolescents

Abstract Introduction Adolescence is associated with a delay in the circadian timing of sleep. However, social factors prevent adolescents from adapting to a later sleep-wake pattern, leading to different forms of circadian misalignment that may increase the risk of cardiovascular and mental health disorders. Several GWAS have identified genes associated with sleep and circadian phenotypes, however, little is known regarding the epigenetic basis and significance of sleep timing and its regularity in adolescence. Methods We analyzed data from 230 adolescents from the Penn State Child Cohort follow-up study who provided a blood sample for DNA extraction and had complete at-home 7-night (at least 3) actigraphy (ACT) data. ACT-measured sleep midpoint was calculated as the intra-individual mean of the 7-night midpoint (zeroed to midnight) of the sleep period. ACT-measured sleep regularity was calculated as the intra-individual standard deviation of the 7-night sleep midpoint. Epigenome-wide single nucleotide resolution of DNA methylation in cytosine-phosphate-guanine (CpG) sites and surrounding regions were obtained from peripheral leukocytes. This study focuses on methylation sites in GWAS-informed genes previously associated with sleep and circadian phenotypes. Linear regression assessed the association between sleep midpoint and sleep regularity with site-specific methylation levels, adjusting for sex, age, race/ethnicity, body mass index, and psychotropic medication use. Using the Benjamini & Hochberg method to adjust for a false discovery rate. Adjusted p-values are reported as q-values. Results Sleep midpoint was not associated with a significant change in methylation at any of the measured intragenic sites. Sleep regularity was significantly associated with differential methylation at 238 intragenic sites in 147 genes with an adjusted p<0.05, of which, three sites were within GWAS-informed sleep/circadian-related genes. Higher sleep irregularity was associated with hypermethylation in MAD1L1 (q=2.4x10-2) and with hypomethylation in CALN1 (q=3.8 x 10-4) and ZNF618 (q=3.8 x 10-2). Conclusion Sleep irregularity is associated with altered DNA methylation in genes previously identified in GWAS of sleep/circadian phenotypes. Our data provides evidence for a potential epigenetic link between sleep irregularity and genes involved in neurocognitive functioning (CALN1), internalizing disorders (MAD1L1) and blood pressure (ZNF618). Support (If Any) National Institutes of Health (R01HL136587, R01MH118308, UL1TR000127)

0032 Objective and Subjective Measures of Sleep Initiation are Differentially Associated with DNA Methylation in Adolescents

Abstract Introduction The onset of puberty is associated with a shift in the circadian timing of sleep leading to delayed sleep initiation [i.e., later sleep onset time (SOT)] driven by later bedtimes and/or longer sleep onset latency (SOL). Subjective sleep initiation, as per self-reports, and objective sleep initiation, as per actigraphy (ACT) or polysomnography (PSG), assess equally relevant but different domains of the same physiological process. Several GWAS have identified genes associated with sleep and circadian phenotypes; however, little is known regarding the epigenetic basis and significance of delayed sleep initiation in adolescence, a critical developmental period. Methods We analyzed data from 263 adolescents from the Penn State Child Cohort follow-up study who had complete subjective (self-reported questionnaires), at-home 7-night ACT, and in-lab 9-hour PSG data for bedtime, SOL and SOT. Epigenome-wide single-nucleotide resolution of DNA methylation in cytosine-phosphate-guanine (CpG) sites and surrounding regions were obtained from peripheral leukocytes. Linear regression assessed the association between bedtime, SOL and SOT with site-specific methylation levels, adjusting for sex, age, race/ethnicity, body mass index, and psychotropic medication use. P-values were adjusted using the Benjamini & Hochberg method to correct for false discovery rate and, thus, q-values are reported. Results Exome-wide analysis showed differential methylation in 1450 unique genes across the 9 sleep measurements, while GWAS-informed analysis yielded 57 genes. Gene hits were identified for bedtime (PSG), SOL (subjective, ACT and PSG) and SOT (subjective and PSG): 14 genes were associated with both subjective and PSG-measured SOL, 34 with both ACT- and PSG-measured SOL, and one (TBC1D22A) with subjective, ACT- and PSG-measured SOL. One gene (ABTB2) was associated with subjective and PSG-measured SOT. Conclusion Objective and subjective sleep initiation in adolescents is associated with altered DNA methylation in genes previously identified in adult GWAS of sleep and circadian phenotypes. Our data provides evidence for a potential epigenetic link between habitual (subjective and ACT) SOL and in-lab SOT and DNAm in genes involved in circadian regulation (i.e., RASD1, RAI1), metabolism (i.e., FADS1, WNK1, SLC5A6), and neuropsychiatric disorders (i.e., PRR7, SDK1, FAM172A). Support (If Any) National Institutes of Health (R01HL136587, R01MH118308, UL1TR000127)

Glutamic Acid Decarboxylase 1 Gene Methylation and Panic Disorder Severity: Making the Connection by Brain Gray Matter Volume.

ObjectiveThis study aimed to test the hypothesis that the relationship between glutamic acid decarboxylase (GAD) 1 gene methylation and severity of clinical symptoms of panic disorder (PD) is mediated by the effect of GAD1 gene methylation on gray matter volume (GMV) and the effect of GMV on PD.MethodsPanic disorder (n = 24) patients were recruited consecutively from the Affiliated Brain Hospital of Nanjing Medical University through outpatient and public advertising, eligible healthy controls (HCs) (n = 22) were recruited from public advertising. We compared GMV and GAD1 gene methylation in PD and HCs to estimate the differences, and on the basis of the relationship between gray matter volumes and GAD1 gene methylation in PD patients was evaluated, the role of GMV as a mediator of GAD1 gene methylation and PD clinical symptoms was analyzed.ResultsPanic disorder patients had significantly lower methylation in the GAD1 promoter region on Cytosine-phosphate-guanine (CPG) 7 than HCs (t = 2.380, p = 0.021). Pearson correlation analysis found a significant negative association between cg171674146 (cg12) site and clinical severity (n = 24, r = −0.456, p = 0.025). Compared to HCs, patients with PD had decreased gray matter volumes in several brain regions, which were also associated with PD severity. Left postcentral gyrus (PoCG) GMV mediated the association between cg12 methylation and PD severity, and there was a significant mediation effect of right angular gyrus (ANG) gray matter volumes on the relationship between cg12 methylation and PD severity.LimitationNo direct results can be derived for methylation patterns in different brain regions; the study is cross-sectional; relatively small size.

A systematic review and metaanalysis of observational studies on the effects of epigenetic factors on serum triglycerides.

Epigenetic modifications might be associated with serum triglycerides (TG) levels. This study aims to systematically review the studies on the relationship between the methylation of specific cytosine-phosphate-guanine (CpG) sites and serum TG levels. This systematic review and meta-analysis study was conducted according to the PRISMA 2020 (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) statement. A systematic literature search was conducted in Medline database (PubMed), Scopus, and Cochrane library up to end of 2020. All observational studies (cross-sectional, case-control, and cohort) were included. Studies that assessed the effect of DNA methylation of different CpG sites of ABCG1, CPT1A, and SREBF1 genes on serum TG levels were selected. The National Institutes of Health (NIH) checklist was used to assess the quality of included articles. Among 2790 articles, ten studies were included in the quantitative analysis and fourteen studies were included in the systematic review. DNA methylation of ABCG1 gene had significant positive association with TG levels (β = 0.05, 95% CI = 0.04, 0.05, P heterogeneity < 0.001). There was significant inverse association between DNA methylation of CPT1A gene and serum TG levels (β = -0.03, 95% CI = -0.03, -0.02, P heterogeneity < 0.001). DNA methylation of SREBF1 gene was positively and significantly associated with serum TG levels (β = 0.03; 95% CI = 0.02-0.04, P heterogeneity < 0.001). DNA methylation of ABCG1 and SREBF1 genes has positive association with serum TG level, whereas this association is opposite for CPT1A gene. The role of epigenetic factors should be considered in some populations with high prevalence of hypertriglyceridemia.

DNA Methylation Patterns of Glucocorticoid Pathway Genes in Preterm Birth Among Black Women.

Preterm birth (PTB; <37weeks gestation) rates have increased for 5 of the last 6 consecutive years in the United States. These rates are particularly alarming for U.S. non-Hispanic Black women who give birth prematurely at 1.5 times the rate of non-Hispanic White women. Previous research suggests that psychological stress is associated with PTB in Black women. However, the biological pathways by which stress alters birth timing are not clear. We examined DNA methylation (DNAm) in peripheral blood leukocytes in 6 glucocorticoid, stress-related genes in 44 (22 PTB; 22 term birth) pregnant Black women. Four cytosine-phosphate-guanine (CpG) sites were identified as differentially methylated (p < 0.05) between women with PTB and women with term births. The ability to identify stress-related biological markers that are associated with PTB among Black women would provide a critical step toward decreasing the PTB disparity among these women. Future studies should include larger sample sizes and gene expression analyses of the stress-related biological pathways to PTB.

Cell type-specific abnormalities of central nervous system in myotonic dystrophy type 1.

Myotonic dystrophy type 1 is a multisystem genetic disorder involving the muscle, heart and CNS. It is caused by toxic RNA transcription from expanded CTG repeats in the 3′-untranslated region of DMPK, leading to dysregulated splicing of various genes and multisystemic symptoms. Although aberrant splicing of several genes has been identified as the cause of some muscular symptoms, the pathogenesis of CNS symptoms prevalent in patients with myotonic dystrophy type 1 remains unelucidated, possibly due to a limitation in studying a diverse mixture of different cell types, including neuronal cells and glial cells. Previous studies revealed neuronal loss in the cortex, myelin loss in the white matter and the presence of axonal neuropathy in patients with myotonic dystrophy type 1. To elucidate the CNS pathogenesis, we investigated cell type-specific abnormalities in cortical neurons, white matter glial cells and spinal motor neurons via laser-capture microdissection. We observed that the CTG repeat instability and cytosine–phosphate–guanine (CpG) methylation status varied among the CNS cell lineages; cortical neurons had more unstable and longer repeats with higher CpG methylation than white matter glial cells, and spinal motor neurons had more stable repeats with lower methylation status. We also identified splicing abnormalities in each CNS cell lineage, such as DLGAP1 in white matter glial cells and CAMKK2 in spinal motor neurons. Furthermore, we demonstrated that aberrant splicing of CAMKK2 is associated with abnormal neurite morphology in myotonic dystrophy type 1 motor neurons. Our laser-capture microdissection-based study revealed cell type-dependent genetic, epigenetic and splicing abnormalities in myotonic dystrophy type 1 CNS, indicating the significant potential of cell type-specific analysis in elucidating the CNS pathogenesis.

Gaseous air pollutants and DNA methylation in a methylome-wide association study of an ethnically and environmentally diverse population of U.S. adults

Epigenetic mechanisms may underlie air pollution-health outcome associations. We estimated gaseous air pollutant-DNA methylation (DNAm) associations using twelve subpopulations within Women's Health Initiative (WHI) and Atherosclerosis Risk in Communities (ARIC) cohorts (n = 8397; mean age 61.3 years; 83% female; 46% African-American, 46% European-American, 8% Hispanic/Latino). We used geocoded participant address-specific mean ambient carbon monoxide (CO), nitrogen oxides (NO2; NOx), ozone (O3), and sulfur dioxide (SO2) concentrations estimated over the 2-, 7-, 28-, and 365-day periods before collection of blood samples used to generate Illumina 450 k array leukocyte DNAm measurements. We estimated methylome-wide, subpopulation- and race/ethnicity-stratified pollutant-DNAm associations in multi-level, linear mixed-effects models adjusted for sociodemographic, behavioral, meteorological, and technical covariates. We combined stratum-specific estimates in inverse variance-weighted meta-analyses and characterized significant associations (false discovery rate; FDR<0.05) at Cytosine-phosphate-Guanine (CpG) sites without among-strata heterogeneity (PCochran's Q > 0.05). We attempted replication in the Cooperative Health Research in Region of Augsburg (KORA) study and Normative Aging Study (NAS). We observed a −0.3 (95% CI: −0.4, −0.2) unit decrease in percent DNAm per interquartile range (IQR, 7.3 ppb) increase in 28-day mean NO2 concentration at cg01885635 (chromosome 3; regulatory region 290 bp upstream from ZNF621; FDR = 0.03). At intragenic sites cg21849932 (chromosome 20; LIME1; intron 3) and cg05353869 (chromosome 11; KLHL35; exon 2), we observed a −0.3 (95% CI: −0.4, −0.2) unit decrease (FDR = 0.04) and a 1.2 (95% CI: 0.7, 1.7) unit increase (FDR = 0.04), respectively, in percent DNAm per IQR (17.6 ppb) increase in 7-day mean ozone concentration. Results were not fully replicated in KORA and NAS. We identified three CpG sites potentially susceptible to gaseous air pollution-induced DNAm changes near genes relevant for cardiovascular and lung disease. Further harmonized investigations with a range of gaseous pollutants and averaging durations are needed to determine the effect of gaseous air pollutants on DNA methylation and ultimately gene expression.

Genetic variability in exon 1 of the glucocorticoid receptor gene NR3C1 is associated with postoperative complications.

Patients undergoing major surgery experience postoperative inflammation, which may contribute to postoperative morbidity. Endogenous glucocorticoids (GCs) are an essential part of the stress response, but this response varies between individuals, which may in turn affect clinical outcome and specifically postoperative inflammation. Exon 1 of the NR3C1 gene, encoding the GC receptor (GR), contains an established region of differential regulation. DNA methylation patterns in this region have been found to differ between individuals. The present study investigated the methylation status and genotype in the cytosine-phosphate-guanine (CpG) island in exon 1 of NR3C1 in 24 patients [Median age 65.5 (range 42–81) years, 11 male, 13 female] who underwent major abdominal (12 pancreatic, 12 hepatic) surgery and explored its association with postoperative complications. DNA was extracted from peripheral blood leukocytes and underwent targeted bisulfite sequencing of the CpG island. Complications were graded according to the Clavien-Dindo classification and 14 out of 24 patients had postoperative complications. Multifactorial and partial least square analyses were used to analyse the data. A homogenous demethylated pattern was observed in all patients and no single CpG methylation was associated with postoperative complications. Four SNPs were significantly associated with higher Clavien-Dindo scores. Genetic variability in the chromosome 5:143,402,505–143,405,805 region of exon 1 of the GR gene NR3C1, but not DNA methylation, was associated with more severe postoperative complications in patients having major abdominal surgery. These results indicated that the patients' response to GCs may be of clinical importance for inflammatory conditions.

2-Pyridin-4-yl-methylene-beta-boswellic Acid-A Potential Candidate for Targeting O6-Methylguanine-DNA Methyltransferase Epi-transcriptional Reprogramming in KRAS G13D-Microsatellite Stable, G12V-Microsatellite Instable Mutant Colon Cancer.

PMBA (2-Pyridin-4-yl-methylene-beta-boswellic acid), screened from among the 21 novel series of semisynthetic analogues of β-boswellic acid, is being presented as a lead compound for integrative management of KRAS mutant colorectal cancer (CRC), upon testing and analysis for its anticancerous activity on a panel of NCI-60 cancer cell lines and in vivo models of the disease. PMBA (1.7-29 μM) exhibited potent proliferation inhibition on the cell lines and showed sensitivity in microsatellite instability and microsatellite stable (GSE39582 and GSE92921) subsets of KRAS gene (Kirsten rat sarcoma viral oncogene homolog)-mutated colon cell lines, as revealed via flow cytometry analysis. A considerable decrease in mitogen-activated protein kinase pathway downstream effectors was observed in the treated cell lines via the western blot and STRING (Search tool for the retrieval of interacting genes/proteins) analysis. PMBA was further found to target KRAS at its guanosine diphosphate site. Treatment of the cell lines with PMBA showed significant reduction in MGMT promoter methylation but restored MGMT (O6-methylguanine-DNA methyltransferase) messenger ribonucleic acid expression via significant demethylation of the hypermethylated CpG (Cytosine phosphate guanine) sites in the MGMT promoter. A significant decrease in dimethylated H3K9 (Dimethylation of lysine 9 on histone 3) levels in the MGMT promoter in DNA hypo- and hypermethylated HCT-116G13D and SW-620G12V cells was observed after treatment. In the MNU (N-methyl-N-nitrosourea)-induced CRC in vivo model, PMBA instillation restricted and repressed polyp formation, suppressed tumor proliferation marker Ki67 (Marker of proliferation), ablated KRAS-associated cytokine signaling, and decreased mortality. Clinical trial data for the parent molecule revealed its effectiveness against the disease, oral bioavailability, and system tolerance. Comprehensively, PMBA represents a new class of KRAS inhibitors having a therapeutic window in the scope of a drug candidate. The findings suggest that the PMBA analogue could inhibit the growth of human CRC in vivo through downregulation of cancer-associated biomarkers as well as reactivate expression of the MGMT gene associated with increased H3K9 acetylation and H3K4 methylation with facilitated transcriptional activation, which might be important in silencing of genes associated with upregulation in the activity of KRAS.

High-Dimensional DNA Methylation Mediates the Effect of Smoking on Crohn's Disease.

Epigenome-wide mediation analysis aims to identify high-dimensional DNA methylation at cytosine–phosphate–guanine (CpG) sites that mediate the causal effect of linking smoking with Crohn’s disease (CD) outcome. Studies have shown that smoking has significant detrimental effects on the course of CD. So we assessed whether DNA methylation mediates the association between smoking and CD. Among 103 CD cases and 174 controls, we estimated whether the effects of smoking on CD are mediated through DNA methylation CpG sites, which we referred to as causal mediation effect. Based on the causal diagram, we first implemented sure independence screening (SIS) to reduce the pool of potential mediator CpGs from a very large to a moderate number; then, we implemented variable selection with de-sparsifying the LASSO regression. Finally, we carried out a comprehensive mediation analysis and conducted sensitivity analysis, which was adjusted for potential confounders of age, sex, and blood cell type proportions to estimate the mediation effects. Smoking was significantly associated with CD under odds ratio (OR) of 2.319 (95% CI: 1.603, 3.485, p < 0.001) after adjustment for confounders. Ninety-nine mediator CpGs were selected from SIS, and then, seven candidate CpGs were obtained by de-sparsifying the LASSO regression. Four of these CpGs showed statistical significance, and the average causal mediation effects (ACME) were attenuated from 0.066 to 0.126. Notably, three significant mediator CpGs had absolute sensitivity parameters of 0.40, indicating that these mediation effects were robust even when the assumptions were slightly violated. Genes (BCL3 and FKBP5) harboring these four CpGs were related to CD. These findings suggest that changes in methylation are involved in the mechanism by which smoking increases risk of CD.