scholarly journals Methotrexate Treatment of Newly Diagnosed RA Patients Is Associated With DNA Methylation Differences at Genes Relevant for Disease Pathogenesis and Pharmacological Action

2021 ◽  
Vol 12 ◽  
Author(s):  
Kari Guderud ◽  
Line H. Sunde ◽  
Siri T. Flåm ◽  
Marthe T. Mæhlen ◽  
Maria D. Mjaavatten ◽  
...  
Keyword(s):  
Dna Methylation ◽  
T Cells ◽  
Treatment Outcome ◽  
Pharmacological Action ◽  
Newly Diagnosed ◽  
Reduced Representation ◽  
Cpg Sites ◽  
Reduced Representation Bisulfite Sequencing ◽  
Before And After ◽  
Cell Type Specific

BackgroundMethotrexate (MTX) is the first line treatment of rheumatoid arthritis (RA), and methylation changes in bulk T cells have been reported after treatment with MTX. We have investigated cell-type specific DNA methylation changes across the genome in naïve and memory CD4+ T cells before and after MTX treatment of RA patients. DNA methylation profiles of newly diagnosed RA patients (N=9) were assessed by reduced representation bisulfite sequencing.ResultsWe found that MTX treatment significantly influenced DNA methylation levels at multiple CpG sites in both cell populations. Interestingly, we identified differentially methylated sites annotated to two genes; TRIM15 and SORC2, previously reported to predict treatment outcome in RA patients when measured in bulk T cells. Furthermore, several of the genes, including STAT3, annotated to the significant CpG sites are relevant for RA susceptibility or the action of MTX.ConclusionWe detected CpG sites that were associated with MTX treatment in CD4+ naïve and memory T cells isolated from RA patients. Several of these sites overlap genetic regions previously associated with RA risk and MTX treatment outcome.

Abstract 40: Transcriptome and DNA Methylation Changes in Patients with Subarachnoid Hemorrhage Undergoing Remote Ischemic Preconditioning

Stroke ◽  
2015 ◽  
Vol 46 (suppl_1) ◽  
Author(s):  
Elina Nikkola ◽  
Arthur Ko ◽  
Mark J Connolly ◽  
Yinn Cher Ooi ◽  
Päivi Pajukanta ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Expression Profiles ◽  
Methylation Status ◽  
Gene Expression Profiles ◽  
Reduced Representation ◽  
Ischemic Conditioning ◽  
Genome Wide ◽  
Reduced Representation Bisulfite Sequencing ◽  
Before And After

Background: Remote ischemic conditioning (RIC) is a phenomenon by which brief periods of sublethal ischemia in one tissue confers protection from ischemia to distant tissues. We hypothesize that RIC triggers a cascade of integrated gene expression and methylation changes, leading to neuroprotection in subarachnoidal hemorrhage (SAH) patients. Our goal was to identify and compare changes in DNA methylation and gene expression profiles before and after RIC. Methods: Patients enrolled in a clinical trial of RIC after SAH, receiving RIC by limb cuff transient ischemia sessions. Fourteen SAH patients (64% female, mean age 51) underwent 3-4 RIC sessions and gave a blood sample before and after RIC, seven days apart. The transcriptome analysis of whole blood was performed using paired-end, 100-bp RNA-sequencing. We employed STAR and HTSeq to align and count reads; EdgeR to normalize the counts and detect differential expression (DE); and David to search for functional categories of the DE genes. Genome-wide DNA methylation profiles were assessed using reduced representation bisulfite sequencing (RRBS); Bismark with Bowtie to align the RRBS data, and the differential methylation analysis package (DMAP) to call the methylation status of CpG sites. Bedtools was used to overlap the DE genes with differentially methylated regions. Results: Of the 12,411 genes passing QC, 168 genes were differentially expressed after RIC (FDR<0.05). These genes were enriched for pathways involving mitosis and nuclear division (P50% after RIC in at least one individual. Of the 8,069 sites, 723 were differentially methylated (Bonferroni P<0.05). Our overlap analysis showed that 88 of the significantly altered methylation sites resided in 39 DE genes, including CEACAM8 and CRISP3, both implicated previously for stroke. Conclusions: Our data suggest that RIC alters expression of a specific set of genes involved in stroke via changes in regional DNA methylation. Further studies are warranted to replicate these pilot results.

scholarly journals DNA Methylation Profiling of Sorted Peripheral Blood Cells Using Microarray and Next Generation Sequencing Reveals Distinct Molecular Signatures in the Polymorphonuclear and Mononuclear Cells of Patients with Essential Thrombocythemia, Polycythemia Vera and Primary Myelofibrosis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5204-5204
Author(s):  
Chieh Lee Wong ◽  
Baoshan Ma ◽  
Martyna Adamowicz-Brice ◽  
Gareth Gerrard ◽  
Zainul Abidin Norziha ◽  
...  
Keyword(s):  
Dna Methylation ◽  
T Cells ◽  
Next Generation Sequencing ◽  
Next Generation ◽  
Cell Type ◽  
Cpg Sites ◽  
Dna Methylation Profiling ◽  
Cell Type Specific ◽  
Generation Sequencing ◽  
Methylation Profiling

Abstract Background The past decade has witnessed a significant progress in the understanding of the molecular pathogenesis of myeloproliferative neoplasms (MPN). Mutations in a large number of genes have now been implicated in the pathogenesis of MPN but these do not yet explain the differentiation into the separate MPN syndromes and do not give full prediction of the wide variation in prognosis. We hypothesized that epigenetic mechanisms may help explain these phenomena at a cell-type specific level. Aim The aim of this study was to perform DNA methylation profiling on different cell types from patients with MPN in order to identify regulatory loci adjacent to genes whose differential expression could elucidate the pathogenesis and predict survival in patients with MPN in a multiracial country. Methods We performed DNA methylation profiling on normal controls (NC) and patients with MPN from 3 different races (Malay, Chinese and Indian) in Malaysia who were diagnosed with essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF) according to the 2008 WHO diagnostic criteria for MPN. Two cohorts of patients, the patient and validation cohorts, from 3 tertiary-level hospitals were recruited prospectively over 3 years and informed consents were obtained. Peripheral blood samples were taken and sorted into polymorphonuclear cells (PMNs), mononuclear cells (MNCs) and T cells. DNA was extracted from each cell population. DNA methylation profiling was performed using the Illumina HumanMethylation450 Beadchip for microarray and subsequent confirmation was performed using the Fluidigm Access Array/Illumina Miseq next generation sequencing platform on the patient and validation cohorts respectively. Results Twenty-nine patients (11 ET, 11 PV and 7 PMF) and 11 NC were recruited into the patient cohort. Twelve patients (4 ET, 4 PV and 4 PMF) and 4 NC were recruited into the validation cohort. Methylation levels of the CpG sites for each cell type in each disease were compared with NC. In the patient cohort, the number of differentially methylated CpG sites in ET, PV and PMF was 1889, 6545 and 11,372 respectively for PMNs (p < 0.0001) and 732, 7700 and 49,219 respectively for MNCs (p < 0.0001). For T cells, the number of differentially methylated CpG sites in ET, PV and PMF were significantly less with 297, 1091 and 987 CpG sites respectively (p < 0.0001). Quantile-quantile plots showed a continuum of progressive skewness from ET to PV to MF for both PMNs and MNCs. However, this appearance was not seen in all 3 diseases for T cells. A total of 43 CpG sites showing the most significant difference in degrees of methylation from the different categories above were selected and all successfully validated using the Fluidigm/Miseq next generation sequencing platform on the validation cohort. For PMNs, 11 of the 14 CpG sites were associated with genes primarily involved in cell signaling pathways. For MNCs, 9 of the 15 CpG sites were associated with genes primarily involved in metabolic pathways and transcription regulation. Remarkably, there was no overlap between the validated PMN and MNC differentially methylated CpG sites or between disease subtypes. Fourteen differentially methylated CpG sites were validated in T cells. Conclusion This is the first study to use microarray and next generation sequencing platforms to compare cell type-specific methylation of CpG sites between different subtypes of MPN. The significantly lower differential methylation and the lack of skewness in the quantile-quantile plot in T cells validate the techniques used and indicate that they are not part of the neoplastic clone. The continuum of increasing number of differentially methylated CpG sites from ET to PV to MF in both PMNs and MNCs may be related to the increasing severity of the disease phenotypes. Differential methylation was greatest in PMF and was most markedly seen in MNCs which may be related to their more severe phenotype. The pattern of cell type-specific differentially methylated CpG sites and the lack of overlap between cell types and diseases provide further insight into the pathogenesis of MPN and into the mechanisms giving rise to the different disease subtypes. Differentially methylated CpG sites and the linked genes also indicate further routes of investigation and possible disease-specific targets for therapy not identified by mutation or gene expression analyses. Disclosures Aitman: Illumina: Honoraria.

scholarly journals Testing cell-type-specific mediation effects in genome-wide epigenetic studies

2020 ◽  
Author(s):  
Xiangyu Luo ◽  
Joel Schwartz ◽  
Andrea Baccarelli ◽  
Zhonghua Liu
Keyword(s):  
Dna Methylation ◽  
Mediation Analysis ◽  
Methylation Data ◽  
Cell Type ◽  
Multiple Testing Procedure ◽  
Mediation Effects ◽  
Cpg Sites ◽  
Genome Wide ◽  
A Cell ◽  
Cell Type Specific

Abstract Epigenome-wide mediation analysis aims to identify DNA methylation CpG sites that mediate the causal effects of genetic/environmental exposures on health outcomes. However, DNA methylations in the peripheral blood tissues are usually measured at the bulk level based on a heterogeneous population of white blood cells. Using the bulk level DNA methylation data in mediation analysis might cause confounding bias and reduce study power. Therefore, it is crucial to get fine-grained results by detecting mediation CpG sites in a cell-type-specific way. However, there is a lack of methods and software to achieve this goal. We propose a novel method (Mediation In a Cell-type-Specific fashion, MICS) to identify cell-type-specific mediation effects in genome-wide epigenetic studies using only the bulk-level DNA methylation data. MICS follows the standard mediation analysis paradigm and consists of three key steps. In step1, we assess the exposure-mediator association for each cell type; in step 2, we assess the mediator-outcome association for each cell type; in step 3, we combine the cell-type-specific exposure-mediator and mediator-outcome associations using a multiple testing procedure named MultiMed [Sampson JN, Boca SM, Moore SC, et al. FWER and FDR control when testing multiple mediators. Bioinformatics 2018;34:2418–24] to identify significant CpGs with cell-type-specific mediation effects. We conduct simulation studies to demonstrate that our method has correct FDR control. We also apply the MICS procedure to the Normative Aging Study and identify nine DNA methylation CpG sites in the lymphocytes that might mediate the effect of cigarette smoking on the lung function.

scholarly journals Characterization of Global DNA Methylation in Different Gene Regions Reveals Candidate Biomarkers in Pigs with High and Low Levels of Boar Taint

2020 ◽  
Vol 7 (2) ◽  
pp. 77 ◽  
Author(s):  
Xiao Wang ◽  
Haja N. Kadarmideen
Keyword(s):  
Dna Methylation ◽  
Cpg Island ◽  
Cpg Islands ◽  
Gene Interaction ◽  
Boar Taint ◽  
Reduced Representation ◽  
Gene Interaction Networks ◽  
Reduced Representation Bisulfite Sequencing ◽  
Pathways Analysis ◽  
Porcine Gene

DNA methylation of different gene components, including different exons and introns, or different lengths of exons and introns is associated with differences in gene expression. To investigate the methylation of porcine gene components associated with the boar taint (BT) trait, this study used reduced representation bisulfite sequencing (RRBS) data from nine porcine testis samples in three BT groups (low, medium and high BT). The results showed that the methylation levels of the first exons and first introns were lower than those of the other exons and introns. The first exons/introns of CpG island regions had even lower levels of methylation. A total of 123 differentially methylated promoters (DMPs), 194 differentially methylated exons (DMEs) and 402 differentially methylated introns (DMIs) were identified, of which 80 DMPs (DMP-CpGis), 112 DMEs (DME-CpGis) and 166 DMIs (DMI-CpGis) were discovered in CpG islands. Importantly, GPX1 contained one each of DMP, DME, DMI, DMP-CpGi, DME-CpGi and DMI-CpGi. Gene-GO term relationships and pathways analysis showed DMP-CpGi-related genes are mainly involved in methylation-related biological functions. In addition, gene–gene interaction networks consisted of nodes that were hypo-methylated GPX1, hypo-methylated APP, hypo-methylated ATOX1, hyper-methylated ADRB2, hyper-methylated RPS6KA1 and hyper-methylated PNMT. They could be used as candidate biomarkers for reducing boar taint in pigs, after further validation in large cohorts.

Preparation of reduced representation bisulfite sequencing libraries for genome-scale DNA methylation profiling

2011 ◽  
Vol 6 (4) ◽  
pp. 468-481 ◽  
Author(s):  
Hongcang Gu ◽  
Zachary D Smith ◽  
Christoph Bock ◽  
Patrick Boyle ◽  
Andreas Gnirke ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bisulfite Sequencing ◽  
Reduced Representation ◽  
Reduced Representation Bisulfite Sequencing ◽  
Dna Methylation Profiling ◽  
Genome Scale ◽  
Methylation Profiling

scholarly journals DNA methylation landscapes of 1538 breast cancers reveal a replication-linked clock, epigenomic instability and cis-regulation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Rajbir Nath Batra ◽  
Aviezer Lifshitz ◽  
Ana Tufegdzic Vidakovic ◽  
Suet-Feung Chin ◽  
Ankita Sati-Batra ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Cpg Island ◽  
Cpg Islands ◽  
Tumor Grade ◽  
Normal Breast ◽  
Breast Cancers ◽  
Reduced Representation ◽  
Reduced Representation Bisulfite Sequencing ◽  
Transcriptional Changes ◽  
In Cis

AbstractDNA methylation is aberrant in cancer, but the dynamics, regulatory role and clinical implications of such epigenetic changes are still poorly understood. Here, reduced representation bisulfite sequencing (RRBS) profiles of 1538 breast tumors and 244 normal breast tissues from the METABRIC cohort are reported, facilitating detailed analysis of DNA methylation within a rich context of genomic, transcriptional, and clinical data. Tumor methylation from immune and stromal signatures are deconvoluted leading to the discovery of a tumor replication-linked clock with genome-wide methylation loss in non-CpG island sites. Unexpectedly, methylation in most tumor CpG islands follows two replication-independent processes of gain (MG) or loss (ML) that we term epigenomic instability. Epigenomic instability is correlated with tumor grade and stage, TP53 mutations and poorer prognosis. After controlling for these global trans-acting trends, as well as for X-linked dosage compensation effects, cis-specific methylation and expression correlations are uncovered at hundreds of promoters and over a thousand distal elements. Some of these targeted known tumor suppressors and oncogenes. In conclusion, this study demonstrates that global epigenetic instability can erode cancer methylomes and expose them to localized methylation aberrations in-cis resulting in transcriptional changes seen in tumors.

5 Gene expression analysis and DNA methylation patterns of porcine somatic cell nuclear transfer blastocysts with high and low incidence of apoptosis

2019 ◽  
Vol 31 (1) ◽  
pp. 128
Author(s):  
L. Moley ◽  
R. Jones ◽  
R. Kaundal ◽  
A. Thomas ◽  
A. Benninghoff ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Enrichment Analysis ◽  
Epigenetic Reprogramming ◽  
Reduced Representation ◽  
False Discovery ◽  
Reduced Representation Bisulfite Sequencing ◽  
Methylation Patterns ◽  
Dna Methylation Analysis ◽  
Go Terms

Somatic cell NT (SCNT) efficiency remains poor, preventing the technology from being regularly used in the agricultural industry. It is believed that faulty epigenetic reprogramming of SCNT embryos leads to the low overall success. A clear apoptotic signature is associated with inappropriate gene expression and epigenomic aberrancies in many experimental cell culture systems, and we hypothesised that an apoptosis biomarker could be used to effectively separate properly reprogrammed porcine SCNT embryos from those that are destined to fail due to incomplete reprogramming. Therefore, our objective was to evaluate global gene expression and DNA methylation patterns in high- and low-apoptosis individual embryos in an effort to characterise the extent of genomic reprogramming that had taken place. Porcine SCNT blastocysts on Day 6 of development were stained with a nontoxic, noninvasive caspase activity reporter, and the top and bottom 20% of detected caspase activity were classified as high and low apoptosis, respectively (3 replicate cloning sessions; n=13 embryos per group). Genomic DNA and total RNA were isolated from each individual blastocyst. The RNA sequencing libraries were prepared using the Ovation SoLo RNA-Seq system (NuGen, San Carlos, CA, USA). Reduced representation bisulfite sequencing libraries were prepared for DNA methylation analysis using a modification of the single-cell reduced representation bisulfite sequencing global DNA methylation analysis approach detailed by Guo et al. (2015 Nat. Protoc. 10, 645-59). The RNA sequencing analysis using EdgeR (https://bioconductor.org/packages/release/bioc/html/edgeR.html) revealed 175 total differentially expressed genes (fold change ≥1.5; false discovery rate ≤0.05) between the high- and low-apoptosis SCNT embryos. This list of differentially expressed genes was used to perform enrichment analysis to identify overrepresented Gene Ontology (GO) terms or Kyoto Encyclopedia of Genes and Genomes pathways (DAVID Ease version 6.8 (https://david.ncifcrf.gov/) against the Sus scrofa background genome). However, no significantly enriched GO terms or pathways were identified (false discovery rate P&gt;0.05). Analysis of global DNA methylation patterns between high- and low-apoptosis SCNT embryos using MethylKit (Akalin et al. 2012Genome Biol. 13, R87) revealed 335 differentially methylated 100-bp regions with at least 25% difference in methylation (adjusted P ≤ 0.01). Gene transcription start sites associated with these regions were used for enrichment analysis; again, no significant enrichment of GO terms or Kyoto Encyclopedia of Genes and Genomes pathways was identified. Principal component analysis of CpG methylation showed the low-apoptosis embryos clustering more tightly than the high-apoptosis embryos, which were highly scattered. Ongoing comparisons of high- and low-apoptosis cloned embryos with naturally fertilized embryos produced invivo may provide more information about which embryos were properly reprogrammed. Although we are still pursuing a link between reprogramming and gene expression in high- and low-apoptosis embryos, we conclude that these data support a model of stochastic epigenetic reprogramming following SCNT and reinforce the necessity of identifying embryos most likely to be successful due to proper epigenetic reprogramming in order to increase SCNT efficiency.

Abstract P175: Discovery And Validation Of Unique Associations Of DNA Methylation Regions With 24-hour Blood Pressure Phenotypes In African Americans

Hypertension ◽  
2021 ◽  
Vol 78 (Suppl_1) ◽  
Author(s):  
Michelle L Roberts ◽  
Theodore A Kotchen ◽  
Xiaoqing Pan ◽  
Yingchuan Li ◽  
Chun Yang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
African Americans ◽  
Cumulative Effect ◽  
Epigenetic Mark ◽  
Discovery Cohort ◽  
Reduced Representation ◽  
Specific Pcr ◽  
Reduced Representation Bisulfite Sequencing ◽  
Sequencing Method ◽  
Independent Cohort

DNA methylation, an epigenetic mark, may reflect the interactions between DNA, environment, and lifestyle. It has been implicated in the development and progression of hypertension, a risk factor for cardiovascular disease. We hypothesize that regions of DNA methylation in blood cells can explain 24h BP phenotypes in African Americans. We performed Reduced Representation Bisulfite Sequencing (RRBS) in a discovery cohort of 281 African Americans. Several DNA methylation regions (MRs) were significantly associated with continuously monitored 24-h, daytime, or nighttime SBP, DBP, PP, and MAP after adjustments for covariates age, sex, and body mass index (False Discovery Rate (FDR) = 0.013 - 0.050). Each of these MRs explained a substantial portion of 24h BP variance, ranging from 6.5% - 9.4%. After FDR adjustment, there were no MRs significantly associated with clinic BPs (FDR > 0.1374), calculated by the average of 4 resting measurements (2 per arm) by sphygmomanometer. To interrogate specific regions of DNA methylation, our lab developed a potentially clinically applicable, deep, and targeted methylation sequencing method called Bisulfite-Specific PCR ULtrapLEx Targeted Sequencing (BULLET-Seq), and tested it in two reference samples for three MRs of interest. BULLET-Seq is able to accurately quantify the 10% changes in the dilution series when methylation rates ranged from ~40% to 90% (a chr19 methylation region; R 2 = 0.95 - 0.97) and can modestly measure these changes when rates range from ~2% to 4% (a chr5 region, R 2 = 0.82), and is questionable when methylation rates are below 2% (a chr13 region, R 2 = 0.03 - 0.27). Validation of the chr19 MR in an independent cohort (n=117) was performed in a single BULLET-Seq run. After covariate adjustment, the chr19 region was significantly associated with 24h BPs (SBP, DBP, and MAP; FDR < 0.05), confirming the findings from the discovery cohort. The MR accounted for up to 1.75% of BP variance in the 24h phenotypes. In conclusion, the reported DNA MRs have potential to be excellent markers for the cumulative effect of factors that influence 24h BPs and the BULLET-Seq workflow can be applied in clinical and population settings to screen up to thousands of patients.

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