scholarly journals Buffy Coat DNA Methylation Profile Is Representative of Methylation Patterns in White Blood Cell Types in Normal Pregnancy

2022 ◽  
Vol 9 ◽  
Author(s):  
Ranine Ghamrawi ◽  
Igor Velickovic ◽  
Ognjen Milicevic ◽  
Wendy M. White ◽  
Lillian Rosa Thistlethwaite ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Blood Cell ◽  
White Blood Cell ◽  
Enrichment Analysis ◽  
Normal Pregnancy ◽  
Buffy Coat ◽  
Pathway Enrichment Analysis ◽  
Bioinformatics Pipeline ◽  
Methylation Patterns

Background: We aimed to assess the extent to which the buffy coat DNA methylome is representative of methylation patterns in constitutive white blood cell (WBC) types in normal pregnancy.Methods: A comparison of differential methylation of buffy coat DNA vs DNA isolated from polymorphonuclear (PMN) and lymphocytic fractions was performed for each blood sample obtained within 24 h prior to delivery from 29 normotensive pregnant women. Methylation profiles were obtained using an Illumina Human Methylation 450 BeadChip and CHaMP bioinformatics pipeline. A subset of differentially methylated probes (DMPs) showing discordant methylation were further investigated using statistical modeling and enrichment analysis.Results: The smallest number of DMPs was found between the buffy coat and the PMN fraction (2.96%). Pathway enrichment analysis of the DMPs identified biological pathways involved in the particular leukocyte lineage, consistent with perturbations during isolation. The comparisons between the buffy coat and the isolated fractions as a group using linear modeling yielded a small number of probes (∼29,000) with discordant methylation. Demethylation of probes in the buffy coat compared to derived cell lines was more common and was prevalent in shelf and open sea regions.Conclusion: Buffy coat is representative of methylation patterns in WBC types in normal pregnancy. The differential methylations are consistent with perturbations during isolation of constituent cells and likely originate in vitro due to the physical stress during cell separation and are of no physiological relevance. These findings help the interpretation of DNA methylation profiling in pregnancy and numerous other conditions.

Characterization of Methylation Patterns in Diffuse Large B Cell Lymphoma By Genome-Wide Methylation Analysis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1243-1243
Author(s):  
Fengyi Zhao ◽  
Lei Zhang ◽  
Yan Qin ◽  
Ming-Zhe Han ◽  
Xiaohong Han ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Signaling Pathway ◽  
Methylation Level ◽  
Enrichment Analysis ◽  
B Cell Lymphoma ◽  
Mapk Signaling ◽  
Mapk Signaling Pathway ◽  
Pathway Enrichment Analysis ◽  
Ffpe Tissues ◽  
Methylation Patterns

Background: Diffuse large B cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma worldwide. Although the reference standard for identifying of the cell types is considered of gene expression profiling (GEP). But immunohistochemistry (IHC) is the most common method commercially available. The purpose of this study was to characterize the circulating cell-free DNA (cfDNA) methylation profile in DLBCL and to compare this profile with methylation observed in formalin fixed paraffin-embedded (FFPE) tissues. Additional efforts were made to correlate the observed methylation patterns with prognostic analysis and selected clinical features. Methods: The cfDNA and DNA of FFPE were extracted from 72 patients and 39 patients respectively. We assessed DNA methylation from plasma samples obtained from 29 individuals with GCB DLBCL at the time before treatment along with 43 samples of non-GCB DLBCL as controls. DNA from FFPE tissues were extracted from 11 individuals of GCB DLBCL and 28 individuals with non-GCB DLBCL. DNA methylation was analyzed with the Infinium MethylationEPIC BeadChip that quantitatively measures the methylation levels of more than 850,000 CpG sites across the genome. M values were used for visualization and intuitive interpretation of the results. Moreover, pathway enrichment analysis was performed with the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Database. Results: We found a total of 207 significant differentional differentially methylated positions (DMPs) of cfDNA between the GCB and non-GCB groups, identified with a p value of 0.001 (Fig. 1A). Of these, 65 presented at least 10% (|Δbeta| > 0.1) difference in the methylation level between GCB and non-GCB. 29 (44.6%) were found hypermethylated in GCB DLBCL, while 36 (55.4%) appeared hypomethylated (Fig. 1B). The distribution of the DMPs identified according to their location relative to CpG islands (CGI) were represented in Fig. 1C. Unsupervised clustering performed on DNA methylation values for the 207 DMPs identified is presented in Fig. 1D. These results highlight the differences between GCB and non-GCB samples. There are 1549 significant DMPs of DNA from FFPE between the GCB and non-GCB groups, identified with a p value of 0.001 (Fig. 1E). Of these, 1512 presented at least 10% (|Δbeta| > 0.1) difference in the methylation level between GCB and non-GCB . 1370 (90.6%) were found hypermethylated in GCB DLBCL, while 142 (9.4%) appeared hypomethylated (Fig. 1F). The distribution of the DMPs identified according to their location relative to CpG islands (CGI) were represented in Fig. 1G. Unsupervised clustering performed on DNA methylation values for the 1549 DMPs identified is presented in Fig. 1H. These results highlight the differences between GCB and non-GCB in FFPE samples which according with that in serum. The KEGG pathway enrichment analysis of DNA from FFPE tissue methylation revealed that the process "PI3K/Akt, Ras, MAPK signaling pathway" and "Human papillomavirus infection" are likely major contributors to Hans pathological type. In addition, the enrichment analysis of cfDNA methylation revealed that the process "MAPK signaling pathway" is likely the most important factor. Furthermore, we also have analyzed the methylation level between refractory or relapsed (R/R) DLBCL patients and individuals with a good prognosis. The differential methylation patterns were also found both in serums and FFPE tissues. Conclusions: The DNA methylation differs in GCB and non-GCB DLBCL patients. MAPK signaling pathway plays an important role in it. The mechanism needs to be further explored. Figure 1 Disclosures No relevant conflicts of interest to declare.

Differences in Residual White Blood Cell Subset Counts in Buffy Coat-Depleted Red Cell Concentrates Prepared with Bottom and Top or Quadruple-Bag Systems

Vox Sanguinis ◽  
1999 ◽  
Vol 77 (2) ◽  
pp. 97-102 ◽  
Author(s):  
J. Wildt-Eggen ◽  
J.G. Schrijver ◽  
P.A. Kuiper-Kramer ◽  
M. Bins ◽  
H.C. Prooijen
Keyword(s):  
Blood Cell ◽  
White Blood Cell ◽  
Cell Subset ◽  
Buffy Coat ◽  
Red Cell

scholarly journals Cord blood buffy coat DNA methylation is comparable to whole cord blood methylation

2017 ◽  
Author(s):  
John Dou ◽  
Rebecca J. Schmidt ◽  
Kelly S. Benke ◽  
Craig Newschaffer ◽  
Irva Hertz-Picciotto ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Blood Cell ◽  
Cord Blood ◽  
Whole Blood ◽  
Cell Types ◽  
Buffy Coat ◽  
Sample Type ◽  
Blood Samples ◽  
Cell Composition ◽  
Cell Counts

AbstractBackgroundCord blood DNA methylation is associated with numerous health outcomes and environmental exposures. Whole cord blood DNA reflects all nucleated blood cell types, while centrifuging whole blood separates red blood cells by generating a white blood cell buffy coat. Both sample types are used in DNA methylation studies. Cell types have unique methylation patterns and processing can impact cell distributions, which may influence comparability.ObjectivesTo evaluate differences in cell composition and DNA methylation between buffy coat and whole cord blood samples.MethodsCord blood DNA methylation was measured with the Infinium EPIC BeadChip (Illumina) in 8 individuals, each contributing buffy coat and whole blood samples. We analyzed principal components (PC) of methylation, performed hierarchical clustering, and computed correlations of mean-centered methylation between pairs. We conducted moderated t-tests on single sites and estimated cell composition.ResultsDNA methylation PCs were associated with individual (PPC1=1.4x10-9; PPC2=2.9x10-5; PPC3=3.8x10-5; PPC4=4.2x10-6; PPC5=9.9x10-13), and not with sample type (PPC1-5>0.7). Samples hierarchically clustered by individual. Pearson correlations of mean-centered methylation between paired individual samples ranged from r=0.66 to r=0.87. No individual site significantly differed between buffy coat and whole cord blood when adjusting for multiple comparisons (5 sites had unadjusted P<10-5). Estimated cell type proportions did not differ by sample type (P=0.86), and estimated cell counts were highly correlated between paired samples (r=0.99).ConclusionsDifferences in methylation and cell composition between buffy coat and whole cord blood are much lower than inter-individual variation, demonstrating that both sample preparation types can be analytically combined and compared.

scholarly journals High expression of MCM10 is predictive of poor outcomes in lung adenocarcinoma

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e10560
Author(s):  
Mingrui Shao ◽  
Shize Yang ◽  
Siyuan Dong
Keyword(s):  
Lung Adenocarcinoma ◽  
Complex Disease ◽  
Enrichment Analysis ◽  
Integrated Approach ◽  
Replication Initiation ◽  
Prognostic Biomarkers ◽  
Normal Lung ◽  
Pathway Enrichment Analysis ◽  
Therapeutic Value

Backgrounds Lung adenocarcinoma is a complex disease that results in over 1.8 million deaths a year. Recent advancements in treating and managing lung adenocarcinoma have led to modest decreases in associated mortality rates, owing in part to the multifactorial etiology of the disease. Novel prognostic biomarkers are needed to accurately stage the disease and act as the basis of adjuvant treatments. Material and Methods The microarray datasets GSE75037, GSE31210 and GSE32863 were downloaded from the Gene Expression Omnibus (GEO) database to identify prognostic biomarkers for lung adenocarcinoma and therapy. The differentially expressed genes (DEGs) were identified by GEO2R. Functional and pathway enrichment analysis were performed by Kyoto Encyclopedia of Genes and Genomes and Gene Ontology (GO). Validation was performed based on 72 pairs of lung adenocarcinoma and adjacent normal lung tissues. Results Results showed that the DEGs were mainly focused on cell cycle and DNA replication initiation. Forty-one hub genes were identified and further analyzed by CytoScape. Here, we provide evidence which suggests MCM10 is a potential target with prognostic, diagnostic and therapeutic value. We base this on an integrated approach of comprehensive bioinformatics analysis and in vitro validation using the A549 lung adenocarcinoma cell line. We show that MCM10 overexpression correlates with a poor prognosis, while silencing of this gene decreases aberrant growth by 2-fold. Finally, evaluation of 72 clinical biopsy samples suggests that overexpression of MCM10 in the lung adenocarcinoma highly correlates with larger tumor size. Together, this work suggests that MCM10 may be a clinically relevant gene with both predictive and therapeutic value in lung adenocarcinoma.

scholarly journals Differences in DNA methylation of white blood cell types at birth and in adulthood reflect postnatal immune maturation and influence accuracy of cell type prediction

2018 ◽  
Author(s):  
Meaghan J Jones ◽  
Louie Dinh ◽  
Hamid Reza Razzaghian ◽  
Olivia de Goede ◽  
Julia L MacIsaac ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Immune System ◽  
Blood Cell ◽  
Cord Blood ◽  
White Blood Cell ◽  
Blood Cells ◽  
Cell Types ◽  
Cell Type ◽  
Cell Type Specific ◽  
The Impact

AbstractBackgroundDNA methylation profiling of peripheral blood leukocytes has many research applications, and characterizing the changes in DNA methylation of specific white blood cell types between newborn and adult could add insight into the maturation of the immune system. As a consequence of developmental changes, DNA methylation profiles derived from adult white blood cells are poor references for prediction of cord blood cell types from DNA methylation data. We thus examined cell-type specific differences in DNA methylation in leukocyte subsets between cord and adult blood, and assessed the impact of these differences on prediction of cell types in cord blood.ResultsThough all cell types showed differences between cord and adult blood, some specific patterns stood out that reflected how the immune system changes after birth. In cord blood, lymphoid cells showed less variability than in adult, potentially demonstrating their naïve status. In fact, cord CD4 and CD8 T cells were so similar that genetic effects on DNA methylation were greater than cell type effects in our analysis, and CD8 T cell frequencies remained difficult to predict, even after optimizing the library used for cord blood composition estimation. Myeloid cells showed fewer changes between cord and adult and also less variability, with monocytes showing the fewest sites of DNA methylation change between cord and adult. Finally, including nucleated red blood cells in the reference library was necessary for accurate cell type predictions in cord blood.ConclusionChanges in DNA methylation with age were highly cell type specific, and those differences paralleled what is known about the maturation of the postnatal immune system.

scholarly journals Norepinephrine exposure restrains endometrial decidualization in early pregnancy

2021 ◽  
Author(s):  
Jiju Wang ◽  
Yuhui Tang ◽  
Songcun Wang ◽  
Liyuan Cui ◽  
Da-Jin Li ◽  
...  
Keyword(s):  
Stromal Cells ◽  
Adrenergic Receptor ◽  
Enrichment Analysis ◽  
Normal Pregnancy ◽  
Receptor Expression ◽  
Gene Set Enrichment Analysis ◽  
Endometrial Stromal Cells ◽  
Significant Enrichment

Previous studies have focused on the role of norepinephrine on arrhythmias, generalized anxiety disorder, and cancer. This study aimed to investigate the effect of norepinephrine on endometrial decidualization. Artificial decidualization and norepinephrine-treated mice were established in vivo. In vitro, human endometrial stromal cells were treated with MPA and cAMP to induce decidualization. Decidual markers and important signaling molecules during decidualization were detected using quantitative real-time polymerase chain reaction and Western blot. RNA sequencing was performed to determine related signaling pathways. Exposure of excess norepinephrine significantly restricted the induced expression of decidualized markers Dtprp, BMP2, WNT4, and Hand2 in mice. In vitro, 10 µM norepinephrine markedly downregulated the expressions of prolactin, IGFBP1, and PLZF, which are the specifical markers of decidual stromal cells during decidualization. The gene set enrichment analysis showed that a significant enrichment in neuroactive ligand–receptor interactions of norepinephrine treatment group. The α1b-adrenergic receptor expression was upregulated by norepinephrine. Interestingly, norepinephrine did not inhibit the expression of IGFBP1 in endometrial stromal cells after silencing α1b-adrenergic receptor, while significantly suppressed the induced decidualization with overexpression of α1b-adrenergic receptor. When α1b-adrenergic receptor was activated, endometrial p-PKC was significantly increased under post-treatment with norepinephrine in vivo and in vitro. In addition, norepinephrine treatment inhibited embryo and fetal development using a normal pregnancy model. Therefore, norepinephrine exposure inhibited endometrial decidualization through the activation of the PKC signaling pathway by upregulating α1b-adrenergic receptor. Our study could explain some female reproductive problems due to stress and provide some novel strategies for this disorder.

scholarly journals DNA methylation and gene expression changes derived from assisted reproductive technologies can be decreased by reproductive fluids

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Sebastian Canovas ◽  
Elena Ivanova ◽  
Raquel Romar ◽  
Soledad García-Martínez ◽  
Cristina Soriano-Úbeda ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Cell Number ◽  
Rna Seq ◽  
Methylation Analysis ◽  
Number Of Children ◽  
Methylation Patterns

The number of children born since the origin of Assisted Reproductive Technologies (ART) exceeds 5 million. The majority seem healthy, but a higher frequency of defects has been reported among ART-conceived infants, suggesting an epigenetic cost. We report the first whole-genome DNA methylation datasets from single pig blastocysts showing differences between in vivo and in vitro produced embryos. Blastocysts were produced in vitro either without (C-IVF) or in the presence of natural reproductive fluids (Natur-IVF). Natur-IVF embryos were of higher quality than C-IVF in terms of cell number and hatching ability. RNA-Seq and DNA methylation analyses showed that Natur-IVF embryos have expression and methylation patterns closer to in vivo blastocysts. Genes involved in reprogramming, imprinting and development were affected by culture, with fewer aberrations in Natur-IVF embryos. Methylation analysis detected methylated changes in C-IVF, but not in Natur-IVF, at genes whose methylation could be critical, such as IGF2R and NNAT.

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