Epigenome-wide analysis of neonatal CD4(+) T-cell DNA methylation sites potentially affected by maternal fish oil supplementation

Abstract

Supplementation of fish oil rich in omega-3 polyunsaturated fatty acids (n-3 PUFA) during pregnancy has been shown to confer favorable health outcomes in the offspring. In a randomized controlled trial, we have previously shown that n-3 PUFA supplementation in pregnancy was associated with modified immune responses and some markers of immune maturation. However, the molecular mechanisms underlying these heritable effects are unclear. To determine whether the biological effects of maternal n-3 PUFA supplementation are mediated through DNA methylation, we analyzed CD4(+) T-cells purified from cryo-banked cord blood samples from a previously conducted clinical trial. Of the 80 mother-infant pairs that completed the initial trial, cord blood samples of 70 neonates were available for genome-wide DNA methylation profiling. Comparison of purified total CD4(+) T-cell DNA methylation profiles between the supplement and control groups did not reveal any statistically significant differences in CpG methylation, at the single-CpG or regional level. Effect sizes among top-ranked probes were lower than 5% and did not warrant further validation. Tests for association between methylation levels and key n-3 PUFA parameters, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), or total n-3 PUFAs were suggestive of dose-dependent effects, but these did not reach genome-wide significance. Our analysis of the microarray data did not suggest strong modifying effects of in utero n-3 PUFA exposure on CD4(+) T-cell methylation profiles, and no probes on the array met our criteria for further validation. Other epigenetic mechanisms may be more relevant mediators of functional effects induced by n-3 PUFA in early life.

Keywords: CBMC, cord blood mononuclear cells.; CD4+ T-cells; DHA; DHA, docosahexaenoic acid; DNA methylation; EPA; EPA, eicosapentaenoic acid; Fish oil; PUFA, polyunsaturated fatty acids; cord blood; maternal supplementation; n-3, omega 3; neonates; omega-3 fatty acids.

Figure 1.

Cord red blood cell fatty acid measurements in fish oil and control groups. Boxplots represent median and range. Statistical analysis by Man-Whitney U-test. EPA: eicosapentaenoic acid; DHA: docosahexaenoic acid; AA: arachidonic acid; total n-3 PUFAs (Sum 20:5, 22:5, 22:3); total n-6 PUFAs (Sum 18:2, 20:3, 20:4, 22:3, 22:4).

Figure 2.

Regression analysis of CpG sites that co-associate with cord red cell total n-3 fatty acid levels. (A) qq plot of P-values from the association test between CpGs and total n-3 fatty acids did not deviate from the null hypothesis. (B) Scatterplots of the top 4 CpGs ranked by P-value for the association with total n-3 fatty acids suggested a dose-response relationship. (C) Histogram of the coefficients from the regression model indicated a small effect size.

Figure 3.

Comparative analysis of DNA methylation profiles between treatment and intervention groups. (A) MDS scaling analysis did not discriminate treatment groups. Samples are indicated by open circles and projected along axes of the first 2 principal components of the top 5000 most variable CpG sites. (B) qq plot of P-values from the moderated t-test between intervention and control groups did not deviate from the null hypothesis. (C) Boxplots of data from 6 top-ranked CpG sites for the between-group comparison did not indicate substantive differences in methylation levels. Boxplots show median and range.