DNA methylation of cord blood cell types: Applications for mixed cell birth studies

<p>Epigenome-wide association studies of disease widely use DNA methylation measured in blood as a surrogate tissue. Cell proportions can vary between people and confound associations of exposure or outcome. An adequate reference panel for estimating cell proportions from adult whole blood for DNA methylation studies is available, but an analogous cord blood cell reference panel is not yet available. Cord blood has unique cell types and the epigenetic signatures of standard cell types may not be consistent throughout the life course. Using magnetic bead sorting, we isolated cord blood cell types (nucleated red blood cells, granulocytes, monocytes, natural killer cells, B cells, CD4⁺T cells, and CD8⁺T cells) from 17 live births at Johns Hopkins Hospital. We confirmed enrichment of the cell types using fluorescence assisted cell sorting and ran DNA from the separated cell types on the Illumina Infinium HumanMethylation450 BeadChip array. After filtering, the final analysis was on 104 samples at 429,794 probes. We compared cell type specific signatures in cord to each other and methylation at 49.2% of CpG sites on the array differed by cell type (F-test <i>P</i> < 10⁻⁸). Differences between nucleated red blood cells and the remainder of the cell types were most pronounced (36.9% of CpG sites at <i>P</i> < 10⁻⁸) and 99.5% of these sites were hypomethylated relative to the other cell types. We also compared the mean-centered sorted cord profiles to the available adult reference panel and observed high correlation between the overlapping cell types for granulocytes and monocytes (both r=0.74), and poor correlation for CD8⁺T cells and NK cells (both r=0.08). We further provide an algorithm for estimating cell proportions in cord blood using the newly developed cord reference panel, which estimates biologically plausible cell proportions in whole cord blood samples.</p

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

<p>Cord 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, 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. We evaluated differences in cell composition and DNA methylation between cord blood buffy coat and whole cord blood samples. Cord blood DNA methylation was measured with the Infinium EPIC BeadChip (Illumina) in eight 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. DNA methylation PCs were associated with individual (<i>P</i>_PC1 = 1.4 × 10⁻⁹; <i>P</i>_PC2 = 2.9 × 10⁻⁵; <i>P</i>_PC3 = 3.8 × 10^-5; <i>P</i>_PC4 = 4.2 × 10^-6; <i>P</i>_PC5 = 9.9 × 10^-13, <i>P</i>_PC6 = 1.3 × 10⁻¹¹) and not with sample type (<i>P</i>_PC1-6>0.7). Samples hierarchically clustered by individual. Pearson correlations of mean-centered methylation between paired 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 (five sites had unadjusted <i>P</i><10⁻⁵). Estimated cell type proportions did not differ by sample type (<i>P</i> = 0.46), and estimated proportions were highly correlated between paired samples (r = 0.99). Differences 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.</p

<em>BMP4</em> Was Associated with NSCL/P in an Asian Population

<div><h3>Background</h3><p>The Bone Morphogenetic Protein 4 gene (<em>BMP4</em>) is located in chromosome 14q22-q23 which has shown evidence of linkage for isolated nonsyndromic cleft lip with or without cleft palate (NSCL/P) in a genome wide linkage analysis of human multiplex families. <em>BMP4</em> has been shown to play crucial roles in lip and palatal development in animal models. Several candidate gene association analyses also supported its potential risk for NSCL/P, however, results across these association studies have been inconsistent. The aim of the current study was to test for possible association between markers in and around the <em>BMP4</em> gene and NSCL/P in Asian and Maryland trios.</p> <h3>Methodology/Principal Findings</h3><p>Family Based Association Test was used to test for deviation from Mendelian assortment for 12 SNPs in and around <em>BMP4</em>. Nominal significant evidence of linkage and association was seen for three SNPs (<em>rs10130587</em>, <em>rs2738265</em> and <em>rs2761887</em>) in 221 Asian trios and for one SNP (<em>rs762642</em>) in 76 Maryland trios. Statistical significance still held for <em>rs10130587</em> after Bonferroni correction (corrected p = 0.019) among the Asian group. Estimated odds ratio for carrying the apparent high risk allele at this SNP was 1.61 (95%CI = 1.20, 2.18).</p> <h3>Conclusions</h3><p>Our results provided further evidence of association between <em>BMP4</em> and NSCL/P.</p> </div

Additional file 1: of Elevated polygenic burden for autism is associated with differential DNA methylation at birth

Supplementary Figures S1–S23. (PDF 8163 kb

Schematic genomic structure of <i>BMP4</i> and coordinates of 13 genotyped SNPs.

<p>Three variants (NM_001202.3, NM_130850.2 and NM_130851.2) of the human <i>BMP4</i> gene are shown in the figure with 4 exons each aligned from left to right. Filled gray and black boxes represent the untranslated and translated exons respectively. All three variants encode an identical protein through translation of exons 3 and 4. Scale on top of the figure shows position of the gene and coordinates for the 13 genotyped SNPs basing on GRCh build 37.3. Minor allele frequency for one SNP (<i>rs2855527</i>) was less than 1% in both Asian and Maryland founders and was excluded from association analysis.</p

Additional file 4: of Elevated polygenic burden for autism is associated with differential DNA methylation at birth

iPSYCH-Broad ASD Group participants’ affiliations. (XLSX 14 kb

Gender of NSCL/P probands.

<p>Gender of NSCL/P probands.</p

TDT analysis for 12 SNPs in and around <i>BMP4</i> in Asian trios.

<p>FAM*: number of informative families, T: number of transmitted alleles, NT: number of un-transmitted alleles.</p

Estimated RR(case|G no E) and RR(case|G and E) from conditional logistic regression using cases and 3 pseudo-controls in 259 CP case-parent trios for 9 SNPs in <i>WDR1</i> considering maternal exposure to environmental tobacco smoke.

<p>TA: target allele and its frequency among parents of Asian ancestry.</p

Estimated RR(case|G no E) and RR(case|G and E) from conditional logistic regression using cases and 3 pseudo-controls in 259 Asian CP case-parent trios for 15 SNPs in <i>SLC2A9</i> considering GxE interaction between each SNP and maternal exposure to environmental tobacco smoke.

<p>TA: target allele and its frequency among parents of Asian ancestry.</p