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Impact of red blood cells on genome-wide DNA methylation and expression analyses of cord blood hematopoietic cells

ABSTRACT: Genome-wide DNA methylation (DNAm) studies have been extremely useful to understand hematopoiesis in humans. For cell-specific epigenetic studies, fluorescence-activated cell sorting (FACS) is the gold standard technique to isolate homogeneous cell populations of interest. However, in cord blood, the DNAm signature of isolated hematopoietic cells may be significantly altered by heterotopic interactions with nucleated red blood cells (nRBCs) that go undetected during conventional cell sorting. Using the Illumina 450K array, genome-wide DNAm profiles of the following cell types were obtained: (1) T lymphocytes, monocytes, and nRBCs isolated using a “standard” strategy lacking exclusion for erythroid lineage markers; and (2) CD4 and CD8 T lymphocytes, B lymphocytes, Natural Killer (NK) cells, granulocytes, monocytes, and nRBCs isolated using a “stringent” strategy formally excluding erythroid lineage-specific markers. DNAm profiles of cord blood cells isolated by the standard sorting strategy showed significant heterotopic cross-contamination between cell populations, whereas the cord blood cells sorted by the stringent strategy displayed DNAm profiles more consistent with their expected hematopoietic lineage relationships.

ORGANISM(S): Homo sapiens

PROVIDER: GSE68456 | GEO | 2015/10/02

SECONDARY ACCESSION(S): PRJNA282766

REPOSITORIES: GEO

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Project description:All subjects were recruited at Centennial Women?s Hospital and the Perinatal Research Center in Nashville, TN beginning in 2003. Pregnant women were enrolled during their first clinical visit after obtaining informed consent as described previously. Demographic and clinical data specific to the fetus was collected from clinical records. Gestational age of the neonate was determined by maternal reporting of the last menstrual period and corroboration by ultrasound dating. Accurate knowledge of gestational age (GA) is essential for proper monitoring and care of neonates. However, accurate GA measures are often not available. DNA methylation has previously been shown to associate with GA, and has been used to accurately predict chronological age in adults. In the current study, we examine whether DNA methylation in cord blood can be used to predict gestational age at birth. Results: We found that GA can be accurately predicted from DNA methylation of neonatal cord blood and blood spot samples (DNAm GA), using 148 CpG sites selected through elastic net regression in six training datasets (N=207). We evaluated predictive accuracy in six testing datasets (N=1,202), and found that the accuracy of DNAm GA meets or exceeds accuracy of gestational age estimates based on established methods. We also found an increased DNAm GA, relative to clinical GA, was associated with increased birthweight percentile (p=.00057), adjusting for GA, sex, and ancestry, suggesting that DNAm GA could represent developmental age more accurately than clinical estimates of GA. Conclusions: Further development of this predictor could provide a method of accurate neonatal estimation of GA for use in resource-limited populations, or in cases where GA cannot be estimated clinically. When clinical estimates are available, the predictor can be used to test hypotheses related to developmental age and other early life circumstances, and may provide increased accuracy beyond clinical estimates. 36 Umbilical cord blood samples were collected in EDTA tubes soon after placental delivery. Blood samples were centrifuged at 3,000 RPM to separate plasma, and buffy coats were aliquoted and stored at -80oC. DNA was extracted using the DNeasy Kit (Qiagen). DNA methylation was interrogated for each sample using the HumanMethylation450 BeadChip (Illumina).

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Impact of red blood cells on genome-wide DNA methylation and expression analyses of cord blood hematopoietic cells

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