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Nucleated red blood cells impact DNA methylation and expression analyses of cord blood hematopoietic cells.

ABSTRACT: Genome-wide DNA methylation (DNAm) studies have proven extremely useful to understand human hematopoiesis. Due to their active DNA content, nucleated red blood cells (nRBCs) contribute to epigenetic and transcriptomic studies derived from whole cord blood. Genomic studies of cord blood hematopoietic cells isolated by fluorescence-activated cell sorting (FACS) may be significantly altered by heterotopic interactions with nRBCs during conventional cell sorting.We report that cord blood T cells, and to a lesser extent monocytes and B cells, physically engage with nRBCs during FACS. These heterotopic interactions resulted in significant cross-contamination of genome-wide epigenetic and transcriptomic data. Formal exclusion of erythroid lineage-specific markers yielded DNAm profiles (measured by the Illumina 450K array) of cord blood CD4 and CD8 T lymphocytes, B lymphocytes, natural killer (NK) cells, granulocytes, monocytes, and nRBCs that were more consistent with expected hematopoietic lineage relationships. Additionally, we identified eight highly differentially methylated CpG sites in nRBCs (false detection rate <5 %, |??| >0.50) that can be used to detect nRBC contamination of purified hematopoietic cells or to assess the impact of nRBCs on whole cord blood DNAm profiles. Several of these erythroid markers are located in or near genes involved in erythropoiesis (ZFPM1, HDAC4) or immune function (MAP3K14, IFIT1B), reinforcing a possible immune regulatory role for nRBCs in early life.Heterotopic interactions between erythroid cells and white blood cells can result in contaminated cell populations if not properly excluded during cell sorting. Cord blood nRBCs have a distinct DNAm profile that can significantly skew epigenetic studies. Our findings have major implications for the design and interpretation of genome-wide epigenetic and transcriptomic studies using human cord blood.

SUBMITTER: de Goede OM

PROVIDER: S-EPMC4567832 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Nucleated red blood cells impact DNA methylation and expression analyses of cord blood hematopoietic cells.

de Goede Olivia M OM Razzaghian Hamid R HR Price E Magda EM Jones Meaghan J MJ Kobor Michael S MS Robinson Wendy P WP Lavoie Pascal M PM

Clinical epigenetics 20150911

<h4>Background</h4>Genome-wide DNA methylation (DNAm) studies have proven extremely useful to understand human hematopoiesis. Due to their active DNA content, nucleated red blood cells (nRBCs) contribute to epigenetic and transcriptomic studies derived from whole cord blood. Genomic studies of cord blood hematopoietic cells isolated by fluorescence-activated cell sorting (FACS) may be significantly altered by heterotopic interactions with nRBCs during conventional cell sorting.<h4>Results</h4>We ...[more]

PMID: 26366232

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Project description:BackgroundNumerous studies have demonstrated that DNA methylation levels in the aryl hydrocarbon receptor repressor (AHRR) gene measured in cord blood are significantly associated with prenatal tobacco smoke exposure and can be used as a fetal exposure biomarker. The mechanism driving this demethylation has not been determined and it is unclear if all cord blood cell types are impacted. Nucleated red blood cells (nRBCs/CD235a+ cells) are developmentally immature RBCs that display genome-wide hypomethylation and are observed at increased frequency in the cord blood of smoking mothers. We tested if AHRR methylation levels in CD235a+ nRBCs or nRBC counts influenced AHRR methylation in whole cord blood.MethodsCord blood was collected from smoking (n = 34) and nonsmoking (n = 19) mothers and DNA was prepared from whole cord blood, isolated CD235a+ nRBCs, and CD14+ monocytes. AHRR methylation in cord blood DNA was measured using Illumina 850K arrays (cg05575921, chr5:373378). Pyrosequencing was used to compare methylation levels among cord blood, CD235a+, and CD14+ cells. We measured nRBC percentages using conventional complete blood counts and estimated percent nRBCs by a deconvolution model.ResultsMethylation levels in AHRR were significantly lower in nRBCs relative to whole cord blood and CD14+ monocytes. While AHRR methylation levels in the cell types were significantly correlated across all subjects, methylation values at the chr5:373378 CpG averaged 14.6% lower in nRBCs (range 0.4 to 24.8%; p = 3.8E-13) relative to CD14+, with nonsmokers showing a significantly greater hypomethylation (- 4.1%, p = 1.8E-02). Methylation level at the AHRR chr5:373378 CpG was strongly associated with self-reported smoking in both CD14+ monocytes (t test p = 5.7E-09) and nRBCs (p = 4.8E-08), as well as cotinine levels (regression p = 1.1E-07 and p = 3.6E-04, respectively). For subjects with whole blood 850K data, robust linear regression models adjusting for estimated cell type composition, either including nRBCs counts or estimates, modestly increased the association between smoking and cg05575921 methylation.ConclusionsPrenatal smoke exposure was highly significantly associated with AHRR methylation in cord blood, CD14+ monocytes, and CD235a+ nRBCs. AHRR methylation levels in nRBCs and nRBC counts had minimal effect on cord blood methylation measurements. However, regression models using estimated nRBCs or actual nRBC counts outperformed those lacking these covariates.

Project description:BackgroundPremature infants are highly vulnerable to infection. This is partly attributable to the preterm immune system, which differs from that of the term neonate in cell composition and function. Multiple studies have found differential DNA methylation (DNAm) between preterm and term infants' cord blood; however, interpretation of these studies is limited by the confounding factor of blood cell composition. This study evaluates the epigenetic impact of preterm birth in isolated hematopoietic cell populations, reducing the concern of cell composition differences.MethodsGenome-wide DNAm was measured using the Illumina 450K array in T cells, monocytes, granulocytes, and nucleated red blood cells (nRBCs) isolated from cord blood of 5 term and 5 preterm (<31 weeks gestational age) newborns. DNAm of hematopoietic cells was compared globally across the 450K array and through site-specific linear modeling.ResultsNucleated red blood cells (nRBCs) showed the most extensive changes in DNAm, with 9258 differentially methylated (DM) sites (FDR < 5%, |Δβ| > 0.10) discovered between preterm and term infants compared to the <1000 prematurity-DM sites identified in white blood cell populations. The direction of DNAm change with gestational age at these prematurity-DM sites followed known patterns of hematopoietic differentiation, suggesting that term hematopoietic cell populations are more epigenetically mature than their preterm counterparts. Consistent shifts in DNAm between preterm and term cells were observed at 25 CpG sites, with many of these sites located in genes involved in growth and proliferation, hematopoietic lineage commitment, and the cytoskeleton. DNAm in preterm and term hematopoietic cells conformed to previously identified DNAm signatures of fetal liver and bone marrow, respectively.ConclusionsThis study presents the first genome-wide mapping of epigenetic differences in hematopoietic cells across the late gestational period. DNAm differences in hematopoietic cells between term and <31 weeks were consistent with the hematopoietic origin of these cells during ontogeny, reflecting an important role of DNAm in their regulation. Due to the limited sample size and the high coincidence of prematurity and multiple births, the relationship between cause of preterm birth and DNAm could not be evaluated. These findings highlight gene regulatory mechanisms at both cell-specific and systemic levels that may be involved in fetal immune system maturation.

Project description:BackgroundLittle is known about risk factors associated with out-of-hospital outcomes in survivors of critical illness. We hypothesized that the presence of nucleated red blood cells in patients who survived critical care would be associated with adverse outcomes following hospital discharge.MethodsWe performed a two-center observational cohort study of patients treated in medical and surgical intensive care units in Boston, Massachusetts. All data were obtained from the Research Patient Data Registry at Partners HealthCare. We studied 2878 patients, age???18 years, who received critical care between 2011 and 2015 and survived hospitalization. The exposure of interest was nucleated red blood cells occurring from 2 days prior to 7 days after critical care initiation. The primary outcome was mortality in the 90 days following hospital discharge. Secondary outcome was unplanned 30-day hospital readmission. Adjusted odds ratios were estimated by multivariable logistic regression models with inclusion of covariate terms thought to plausibly interact with both nucleated red blood cells and outcome. Adjustment included age, race (white versus nonwhite), gender, Deyo-Charlson Index, patient type (medical versus surgical), sepsis and acute organ failure.ResultsIn patients who received critical care and survived hospitalization, the absolute risk of 90-day postdischarge mortality was 5.9%, 11.7%, 15.8% and 21.9% in patients with 0/?l, 1-100/?l, 101-200/?l and more than 200/?l nucleated red blood cells respectively. Nucleated red blood cells were a robust predictor of postdischarge mortality and remained so following multivariable adjustment. The fully adjusted odds of 90-day postdischarge mortality in patients with 1-100/?l, 101-200/?l and more than 200/?l nucleated red blood cells were 1.77 (95% CI, 1.23-2.54), 2.51 (95% CI, 1.36-4.62) and 3.72 (95% CI, 2.16-6.39) respectively, relative to patients without nucleated red blood cells. Further, the presence of nucleated red blood cells is a significant predictor of the odds of unplanned 30-day hospital readmission.ConclusionIn critically ill patients who survive hospitalization, the presence of nucleated red blood cells is a robust predictor of postdischarge mortality and unplanned hospital readmission.

Project description:Exposure to phthalates has been shown to impede the human endocrine system, resulting in deleterious effects on pregnant women and their children. Phthalates modify DNA methylation patterns in infant cord blood. We examined the association between prenatal phthalate exposure and DNA methylation patterns in cord blood in a Korean birth cohort. Phthalate levels were measured in 274 maternal urine samples obtained during late pregnancy and 102 neonatal urine samples obtained at birth, and DNA methylation levels were measured in cord blood samples. For each infant in the cohort, associations between CpG methylation and both maternal and neonate phthalate levels were analyzed using linear mixed models. The results were combined with those from a meta-analysis of the levels of phthalates in maternal and neonatal urine samples, which were also analyzed for MEOHP, MEHHP, MnBP, and DEHP. This meta-analysis revealed significant associations between the methylation levels of CpG sites near the CHN2 and CUL3 genes, which were also associated with MEOHP and MnBP in neonatal urine. When the data were stratified by the sex of the infant, MnBP concentration was found to be associated with one CpG site near the OR2A2 and MEGF11 genes in female infants. In contrast, the concentrations of the three maternal phthalates showed no significant association with CpG site methylation. Furthermore, the data identified distinct differentially methylated regions in maternal and neonatal urine samples following exposure to phthalates. The CpGs with methylation levels that were positively associated with phthalate levels (particularly MEOHP and MnBP) were found to be enriched genes and related pathways. These results indicate that prenatal phthalate exposure is significantly associated with DNA methylation at multiple CpG sites. These alterations in DNA methylation may serve as biomarkers of maternal exposure to phthalates in infants and are potential candidates for investigating the mechanisms by which phthalates impact maternal and neonatal health.

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Nucleated red blood cells impact DNA methylation and expression analyses of cord blood hematopoietic cells.

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Nucleated red blood cells impact DNA methylation and expression analyses of cord blood hematopoietic cells.

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