Detection of average methylation level of specific genes by binary-probe hybridization

Abstract Abstract 599 Aberrant epigenetic silencing of genes through aberrant promoter hypermethylation, as occurs with tumor suppressor genes (TSG), has been implicated in the pathogenesis of MDS and other myeloid malignancies and has been clinically targeted by hypomethylating agents. To date, most of the studies investigating hypermethylation of TSG in hematologic malignancies targeted empirically selected gene promoters but with the advent of methylation arrays, global analysis of methylation pattern became technically possible. We applied methylation arrays (Illumina ®) allowing for simultaneous analysis of 25K CpG sites, focusing on the WHO defined subentity of MDS/MPD, CMML, because of possible efficacy of hypomethylating agents in this disease and the need to identify diagnostic markers and predictors of response. We hypothesized that by comparing CMML patients to patients with similar monocytoid entities, we would be able to establish an epigenetic signature that was consistent across these diagnoses. We studied patients with CMML (N=26), JMML (N=22) and monocytoid forms primary AMLs (N=16; M4 N=9 and M5 N=7) to controls (N=28). In addition we studied 35 patients with advanced and 37 low risk MDS and 9 with MDS/MPN. We developed an analytic algorithm that included establishment of the methylome of normal marrow to define normal/physiologic methylation status for each of CpG islands. These parameters were used as a reference for analysis of concordantly hypermethylated genes in patients, using methylation status as either a continuous (β, where β is proportional to the percentage of cells with methylated status at the locus) or dichotomized variable (where hypermethylation was defined as a β-value greater than the 97th percentile of controls). Each disease was individually compared with controls in order to establish genes aberrantly hypermethylated within the specific entity and the established methylome of each entity was compared with that of other entities. As expected, comparison of the average methylation level across all genes showed no significant differences between groups. Among all subgroups, there were only 58 genes that were consistently hypermethylated; the majority of genes were uniquely hypermethylated in each of the disease subgroups. When CMML and JMML were examined as exemplary conditions, global methylation analysis demonstrated that there was concordant hypermethylation in 25%, 50% and 75% of CMML patients in 1086, 13 and 0 CpG sites, respectively. In contrast, there was a great deal of concordant methylation in JMML with 3796, 1006, 176 of methylated promoters concordant in 25%, 50% and 75% of patients, respectively. The genes that were the most consistently hypermethylated in each entity were selected for further analysis. In JMML, the most consistently hypermethyated genes included LHX6, CDK10, ITGA2B and RAP1GA1. These genes were differentially hypermethylated in JMML compared to CMML (p<.0001 for each gene). In CMML, examples of the most consistently hypermethylated promoters included RPL36, BCORL, GPR171 and HAPLN1; hypermethylation of GPR171 and HAPLN1 clearly distinguished CMML from JMML (p<.0001, p=.0075, respectively.) GPR171 was hypermethylated in significantly more CMML patients than patients with M4 and M5 (p<.0001). In contrast, HAPLN1 was hypermethylated in more patients with M4 and M5 than with CMML (87% of patients). This finding led us to speculate that methylation of HAPLN1 may be a marker associated with disease progression. In fact, HAPLN1 was hypermethylated in 67% of patients with CMML1, compared with 78% of patients with CMML2. We also compared the whole epigenome profile of each subentity to each other. We selected genes whose average methylation level in a disease entity was greater than the cutoff of 2 standard deviations above the mean of controls. This resulted in selection of 550 genes in CMML patients, of which 230 were also part of the conserved epigenetic pattern of M4, while only 146 were part of the conserved epigenetic pattern of M5. M4 and M5 showed more similarity with each other, sharing 355 genes within their epigenetic profiles. In conclusion there are few shared epigenetic changes among the monocytoid/ myelomonocytoid malignancies; however, epigenetic changes in these entities are largely unique to each entity. These data suggest that methylation analysis may be useful to supplement histomorphologic diagnostic criteria in distinguishing between these monocytoid malignancies. Disclosures: No relevant conflicts of interest to declare.

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Transcriptome and DNA Methylome Reveal Insights Into Phytoplasma Infection Responses in Mulberry (Morus multicaulis Perr.)

Frontiers in Plant Science ◽

10.3389/fpls.2021.697702 ◽

2021 ◽

Vol 12 ◽

Author(s):

Chaorui Liu ◽

Xiaonan Dong ◽

Yuqi Xu ◽

Qing Dong ◽

Yuqi Wang ◽

...

Keyword(s):

Dna Methylation ◽

Methylation Level ◽

Differentially Expressed ◽

Kinase Gene ◽

Differentially Methylated Genes ◽

Pathogenesis Related Protein ◽

Significant Difference ◽

Average Methylation ◽

Phytoplasma Infection ◽

Average Methylation Level

To reveal whether the response of mulberry to phytoplasma infection is associated with genome-wide DNA methylation changes, the methylome and transcriptome patterns of mulberry in response to phytoplasma infection were explored. Though the average methylation level of the infected leaves showed no significant difference from that of healthy leaves, there were 1,253 differentially methylated genes (DMGs) and 1,168 differentially expressed genes (DEGs) in the infected leaves, and 51 genes were found simultaneously to be differently methylated and expressed. It was found that the expression of G-type lectin S-receptor-like serine/threonine protein kinase gene (Mu-GsSRK) was increased, but its methylation level was decreased in the pathogen-infected or salicylic acid (SA)-treated leaves. Overexpression of Mu-GsSRK in Arabidopsis and in the hairy roots of mulberry enhanced transgenic plant resistance to the phytoplasma. Moreover, overexpression of Mu-GsSRK enhanced the expressions of pathogenesis-related protein 1, plant defensin, and cytochrome P450 protein CYP82C2 genes in transgenic plants inoculated with pathogens, which may contribute to the enhanced disease resistance against various pathogens. Finally, the DNA methylation dynamic patterns and functions of the differentially expressed and methylated genes were discussed. The results suggested that DNA methylation has important roles in mulberry responses to phytoplasma infection.

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Abstract MP32: Epigenome-Wide DNA Methylation Profiling in a CVD Cohort: The ARIC Study

Circulation ◽

10.1161/circ.127.suppl_12.amp32 ◽

2013 ◽

Vol 127 (suppl_12) ◽

Author(s):

James S Pankow ◽

Ellen W Demerath ◽

Weihua Guan ◽

Myriam Fornage ◽

Thomas H Mosley ◽

...

Keyword(s):

Dna Methylation ◽

X Chromosome ◽

Methylation Level ◽

Association Studies ◽

Univariate Analysis ◽

European Ancestry ◽

Genome Wide Association Studies ◽

Stage Of Development ◽

Average Methylation ◽

Aric Study

DNA methylation is mitotically heritable modification in chromatin structure that impacts transcriptional control of genes and cellular function. Recent technological advances provide opportunities to systematically interrogate variation in DNA methylation across the genome in large epidemiologic studies. However, unlike inherited changes to the genetic sequence, variation in site-specific methylation varies by tissue, stage of development, disease state, and may be affected by gender, aging and exposure to environmental factors. As a result, there is likely a greater threat of confounding in epigenome-wide methylation studies compared to genome-wide association studies of SNPs. The Illumina Infinium HumanMethylation450 BeadChip was used to measure DNA methylation in peripheral blood obtained from African American participants from the Jackson, Mississippi and Forsyth County, North Carolina field centers of the Atherosclerosis Risk in Communities (ARIC) Study, a population-based cohort of middle-aged men and women. After excluding outlier samples and CpG sites using quality control filters, we analyzed 473,687 sites in 2873 subjects who were between 47-71 years of age at the time of DNA collection. We used linear regression with robust standard errors to examine cross-sectional associations of demographic factors with the beta value, an estimate of the average methylation level at each locus, and applied a Bonferroni correction to account for multiple testing. In univariate analysis, 91% of sites on the X chromosome and 10% of sites on the autosomes exhibited statistically significant gender differences in methylation level (p<1x10-7). Average methylation was higher in women than men for most of the significant sites (63% and 89% on the X chromosome and autosomes, respectively). Percent European ancestry estimated from ancestry informative markers was significantly associated with methylation level at 4% of sites. Age was also significantly associated with methylation at 4% of sites; average methylation was lower in older subjects compared to younger subjects for the majority (58%) of these sites. As we begin to implement epigenome-wide studies of DNA methylation and CVD outcomes, these results indicate that such studies will require careful consideration of adjustment techniques to avoid confounding by gender, age, and other covariates.

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Inference about causation between body mass index and DNA methylation in blood from a twin family study

10.1101/223040 ◽

2017 ◽

Author(s):

Shuai Li ◽

Ee Ming Wong ◽

Minh Bui ◽

Tuong L Nguyen ◽

Ji-Hoon Eric Joo ◽

...

Keyword(s):

Body Mass Index ◽

Dna Methylation ◽

Body Mass ◽

Peripheral Blood ◽

Methylation Level ◽

Causal Effect ◽

False Discovery ◽

Average Methylation ◽

Twin Families ◽

Genomic Locations

AbstractBackgroundSeveral studies have reported DNA methylation in blood to be associated with body mass index (BMI), but only a few have investigated causal aspects of the association. We used a twin family design to assess this association at two life points and applied a novel analytical approach to investigate the evidence for causality.MethodsThe methylation profile of DNA from peripheral blood was measured for 479 Australian women (mean age 56 years) from 130 twin families. Linear regression was used to estimate the associations of methylation at ~410 000 cytosine-guanine dinucleotides (CpG), and of the average methylation at ~20 000 genes, with current BMI, BMI at age 18-21 years, and the change between the two (BMI change). A novel regression-based methodology for twins, Inference about Causation through Examination of Familial Confounding (ICE FALCON), was used to assess causation.ResultsAt 5% false discovery rate, nine, six and 12 CpGs at 24 loci were associated with current BMI, BMI at age 18-21 years and BMI change, respectively. The average methylation of BHLHE40 and SOCS3 loci was associated with current BMI, and of PHGDH locus was associated with BMI change. From the ICE FALCON analyses with BMI as the predictor and methylation as the outcome, a woman’s methylation level was associated with her co-twin’s BMI, and the association disappeared conditioning on her own BMI, consistent with BMI causing methylation. To the contrary, using methylation as the predictor and BMI as the outcome, a woman’s BMI was not associated with her co-twin’s methylation level, consistent with methylation not causing BMI.ConclusionFor middle-aged women, peripheral blood DNA methylation at several genomic locations is associated with current BMI, BMI at age 18-21 years and BMI change. Our study suggests that BMI has a causal effect on peripheral blood DNA methylation.

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Global methylation level in human adipose tissue is related to fat distribution

Diabetologie und Stoffwechsel ◽

10.1055/s-0033-1341831 ◽

2013 ◽

Vol 8 (S 01) ◽

Author(s):

M Keller ◽

D Schleinitz ◽

I Müller ◽

M Stumvoll ◽

P Kovacs ◽

...

Keyword(s):

Adipose Tissue ◽

Methylation Level ◽

Human Adipose Tissue ◽

Fat Distribution ◽

Global Methylation ◽

Global Methylation Level

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Detection of enteric adenoviruses in south African waters using gene probes

Water Science & Technology ◽

10.2166/wst.1995.0638 ◽

1995 ◽

Vol 31 (5-6) ◽

pp. 345-350 ◽

Cited By ~ 2

Author(s):

B. Genthe ◽

M. Gericke ◽

B. Bateman ◽

N. Mjoli ◽

R. Kfir

Keyword(s):

South African ◽

Water Samples ◽

Cellulose Fibre ◽

Gene Probe ◽

Viral Dna ◽

Virus Particles ◽

Gene Probes ◽

Probe Hybridization ◽

Enteric Adenoviruses ◽

Effect Of Chlorine

Gene probes developed locally for both enteric Adenoviruses 40 and 41 were used to determine whether these viruses were present in both raw and treated waters. Approximately sixty water samples were concentrated by ultrafiltration and analysed directly for the presence of enteric adenoviruses. Three pretreatment techniques, namely sephadex columns, cellulose fibre and GenecleanTM were tested for the removal of inhibitory substances from concentrated water samples. The effect of chlorine treatment on viral detection using gene probe hybridization was also examined by exposing adenoviruses to chlorine concentrations of up to 20mg/l for 1 hour. Enteric adenoviruses were detected in up to 59% of both raw and treated waters analysed. Cellulose fibre and GenecleanTM were found to successfully remove inhibitory substances from concentrated raw waters. Viral DNA was detected after exposure to a range of chlorine concentrations indicating that the viruses detected in the treated waters may have been inactivated virus particles.

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Clustering Count-based RNA Methylation Data Using a Nonparametric Generative Model

Current Bioinformatics ◽

10.2174/1574893613666180601080008 ◽

2018 ◽

Vol 14 (1) ◽

pp. 11-23 ◽

Cited By ~ 3

Author(s):

Lin Zhang ◽

Yanling He ◽

Huaizhi Wang ◽

Hui Liu ◽

Yufei Huang ◽

...

Keyword(s):

Clustering Analysis ◽

Methylation Level ◽

Optimal Number ◽

Generative Model ◽

Methylation Data ◽

Sequencing Data ◽

Number Of Clusters ◽

Rna Methylation ◽

Clustering Effect ◽

Optimal Number Of Clusters

Background: RNA methylome has been discovered as an important layer of gene regulation and can be profiled directly with count-based measurements from high-throughput sequencing data. Although the detailed regulatory circuit of the epitranscriptome remains uncharted, clustering effect in methylation status among different RNA methylation sites can be identified from transcriptome-wide RNA methylation profiles and may reflect the epitranscriptomic regulation. Count-based RNA methylation sequencing data has unique features, such as low reads coverage, which calls for novel clustering approaches. Objective: Besides the low reads coverage, it is also necessary to keep the integer property to approach clustering analysis of count-based RNA methylation sequencing data. Method: We proposed a nonparametric generative model together with its Gibbs sampling solution for clustering analysis. The proposed approach implements a beta-binomial mixture model to capture the clustering effect in methylation level with the original count-based measurements rather than an estimated continuous methylation level. Besides, it adopts a nonparametric Dirichlet process to automatically determine an optimal number of clusters so as to avoid the common model selection problem in clustering analysis. Results: When tested on the simulated system, the method demonstrated improved clustering performance over hierarchical clustering, K-means, MClust, NMF and EMclust. It also revealed on real dataset two novel RNA N6-methyladenosine (m6A) co-methylation patterns that may be induced directly by METTL14 and WTAP, which are two known regulatory components of the RNA m6A methyltransferase complex. Conclusion: Our proposed DPBBM method not only properly handles the count-based measurements of RNA methylation data from sites of very low reads coverage, but also learns an optimal number of clusters adaptively from the data analyzed. Availability: The source code and documents of DPBBM R package are freely available through the Comprehensive R Archive Network (CRAN): https://cran.r-project.org/web/packages/DPBBM/.

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ATF4, DLX3, FRA1, MSX2, C/EBP-ζ, and C/EBP-α Shape the Molecular Basis of Therapeutic Effects of Zoledronic Acid in Bone Disorders

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520620666200721101904 ◽

2020 ◽

Vol 20 (18) ◽

pp. 2274-2284

Author(s):

Faroogh Marofi ◽

Jalal Choupani ◽

Saeed Solali ◽

Ghasem Vahedi ◽

Ali Hassanzadeh ◽

...

Keyword(s):

Gene Expression ◽

Zoledronic Acid ◽

Mrna Expression ◽

Promoter Methylation ◽

Methylation Level ◽

Target Genes ◽

Osteoblastic Differentiation ◽

Therapeutic Effects ◽

Significant Difference ◽

Scheduled Time

Objective: Zoledronic Acid (ZA) is one of the common treatment choices used in various boneassociated conditions. Also, many studies have investigated the effect of ZA on Osteoblastic-Differentiation (OSD) of Mesenchymal Stem Cells (MSCs), but its clear molecular mechanism(s) has remained to be understood. It seems that the methylation of the promoter region of key genes might be an important factor involved in the regulation of genes responsible for OSD. The present study aimed to evaluate the changes in the mRNA expression and promoter methylation of central Transcription Factors (TFs) during OSD of MSCs under treatment with ZA. Materials and Methods: MSCs were induced to be differentiated into the osteoblastic cell lineage using routine protocols. MSCs received ZA during OSD and then the methylation and mRNA expression levels of target genes were measured by Methylation Specific-quantitative Polymerase Chain Reaction (MS-qPCR) and real.time PCR, respectively. The osteoblastic differentiation was confirmed by Alizarin Red Staining and the related markers to this stage. Results: Gene expression and promoter methylation level for DLX3, FRA1, ATF4, MSX2, C/EBPζ, and C/EBPa were up or down-regulated in both ZA-treated and untreated cells during the osteodifferentiation process on days 0 to 21. ATF4, DLX3, and FRA1 genes were significantly up-regulated during the OSD processes, while the result for MSX2, C/EBPζ, and C/EBPa was reverse. On the other hand, ATF4 and DLX3 methylation levels gradually reduced in both ZA-treated and untreated cells during the osteodifferentiation process on days 0 to 21, while the pattern was increasing for MSX2 and C/EBPa. The methylation pattern of C/EBPζ was upward in untreated groups while it had a downward pattern in ZA-treated groups at the same scheduled time. The result for FRA1 was not significant in both groups at the same scheduled time (days 0-21). Conclusion: The results indicated that promoter-hypomethylation of ATF4, DLX3, and FRA1 genes might be one of the mechanism(s) controlling their gene expression. Moreover, we found that promoter-hypermethylation led to the down-regulation of MSX2, C/EBP-ζ and C/EBP-α. The results implicate that ATF4, DLX3 and FRA1 may act as inducers of OSD while MSX2, C/EBP-ζ and C/EBP-α could act as the inhibitor ones. We also determined that promoter-methylation is an important process in the regulation of OSD. However, yet there was no significant difference in the promoter-methylation level of selected TFs in ZA-treated and control cells, a methylation- independent pathway might be involved in the regulation of target genes during OSD of MSCs.

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Profile of copper-associated DNA methylation and its association with incident acute coronary syndrome

Clinical Epigenetics ◽

10.1186/s13148-021-01004-w ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

Pinpin Long ◽

Qiuhong Wang ◽

Yizhi Zhang ◽

Xiaoyan Zhu ◽

Kuai Yu ◽

...

Keyword(s):

Dna Methylation ◽

Acute Coronary Syndrome ◽

Methylation Level ◽

Meta Analysis ◽

Regulation Of Gene Expression ◽

Density Lipoprotein ◽

Blood Leukocytes ◽

Coronary Syndrome ◽

A Genome ◽

Increased Risk

Abstract Background Acute coronary syndrome (ACS) is a cardiac emergency with high mortality. Exposure to high copper (Cu) concentration has been linked to ACS. However, whether DNA methylation contributes to the association between Cu and ACS is unclear. Methods We measured methylation level at > 485,000 cytosine-phosphoguanine sites (CpGs) of blood leukocytes using Human Methylation 450 Bead Chip and conducted a genome-wide meta-analysis of plasma Cu in a total of 1243 Chinese individuals. For plasma Cu-related CpGs, we evaluated their associations with the expression of nearby genes as well as major cardiovascular risk factors. Furthermore, we examined their longitudinal associations with incident ACS in the nested case-control study. Results We identified four novel Cu-associated CpGs (cg20995564, cg18608055, cg26470501 and cg05825244) within a 5% false discovery rate (FDR). DNA methylation level of cg18608055, cg26470501, and cg05825244 also showed significant correlations with expressions of SBNO2, BCL3, and EBF4 gene, respectively. Higher DNA methylation level at cg05825244 locus was associated with lower high-density lipoprotein cholesterol level and higher C-reactive protein level. Furthermore, we demonstrated that higher cg05825244 methylation level was associated with increased risk of ACS (odds ratio [OR], 1.23; 95% CI 1.02–1.48; P = 0.03). Conclusions We identified novel DNA methylation alterations associated with plasma Cu in Chinese populations and linked these loci to risk of ACS, providing new insights into the regulation of gene expression by Cu-related DNA methylation and suggesting a role for DNA methylation in the association between copper and ACS.

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Associations among phthalate exposure, DNA methylation of TSLP, and childhood allergy

Clinical Epigenetics ◽

10.1186/s13148-021-01061-1 ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

Wan-Ru Wang ◽

Nai-Tzu Chen ◽

Nai-Yun Hsu ◽

I-Ying Kuo ◽

Hsin-Wen Chang ◽

...

Keyword(s):

Dna Methylation ◽

Methylation Level ◽

Respiratory Symptoms ◽

Smoking Status ◽

Phthalate Esters ◽

Allergic Diseases ◽

Percentage Increase ◽

Phthalate Exposure ◽

Settled Dust ◽

Butyl Phthalate

Abstract Background Dysregulation of thymic stromal lymphopoietin (TSLP) expressions is linked to asthma and allergic disease. Exposure to phthalate esters, a widely used plasticizer, is associated with respiratory and allergic morbidity. Dibutyl phthalate (DBP) causes TSLP upregulation in the skin. In addition, phthalate exposure is associated with changes in environmentally induced DNA methylation, which might cause phenotypic heterogeneity. This study examined the DNA methylation of the TSLP gene to determine the potential mechanism between phthalate exposure and allergic diseases. Results Among all evaluated, only benzyl butyl phthalate (BBzP) in the settled dusts were negatively correlated with the methylation levels of TSLP and positively associated with children’s respiratory symptoms. The results revealed that every unit increase in BBzP concentration in the settled dust was associated with a 1.75% decrease in the methylation level on upstream 775 bp from the transcription start site (TSS) of TSLP (β = − 1.75, p = 0.015) after adjustment for child’s sex, age, BMI, parents’ smoking status, allergic history, and education levels, PM2.5, formaldehyde, temperature; and relative humidity. Moreover, every percentage increase in the methylation level was associated with a 20% decrease in the risk of morning respiratory symptoms in the children (OR 0.80, 95% CI 0.65–0.99). Conclusions Exposure to BBzP in settled dust might increase children’s respiratory symptoms in the morning through decreasing TSLP methylation. Therefore, the exposure to BBzP should be reduced especially for the children already having allergic diseases.

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