Regions of variable DNA methylation in human placenta associated with newborn neurobehavior

Abstract

The placenta regulates the in utero environment and functionally impacts fetal development. Candidate gene studies identified variation in placental DNA methylation is associated with newborn neurologic and behavioral outcomes including movement quality, lethargic behavior, attention, and arousal. We sought to identify novel regions of variable DNA methylation associated with newborn attention, lethargy, quality of movement, and arousal by performing an epigenome-wide association study in 335 infants from a US birth cohort. Methylation status was quantified using the Illumina HumanMethylation450 BeadChip array and associations to newborn outcomes assessed by the NICU Network Neurobehavioral Scales (NNNS) were identified while incorporating established bioinformatics algorithms to control for confounding by cell type composition. Methylation of CpGs within FHIT (cg15970800) and ANKRD11 (cg16710656) demonstrated genome-wide significance (P < 1.8 × 10(-7)) in specific associations with infant attention. CpGs whose differential methylation was associated with all 4 neurobehavioral outcomes were common to 50 genes involved in biological processes relating to cellular adhesion and nervous system development. Comprehensive methylation profiling identified relationships between methylation of FHIT and ANKRD11, which have been previously linked to neurodevelopment and behavioral outcomes in genetic association studies. Subtle changes in DNA methylation of these genes within the placenta may impact normal variation of a newborn's ability to alter and track visual and auditory stimuli. Gene ontology analysis suggested that those genes with variable methylation related to these outcomes are over-represented in biological pathways involved in brain development and placental physiology, supportive of our hypothesis for a key role of the placenta in neurobehavioral outcomes.

Keywords: ANKRD11; FHIT; epigenetics; methylation; neurobehavior; placenta; prenatal programming.

Figure 1.

Volcano plot displaying results of adjusted reference free models for arousal (A), Attention (B), Lethargy (C) and Quality of Movement (D), where each dot represents 1 CpG site. The red line represents a significance level of P < 0.05, and a blue line represents a significance level of P < 0.005. CpGs that are highly significant after correction for multiple comparisons are shown by red asterisks.

Figure 2.

Volcano plot displaying the changes in β between adjusted and unadjusted model and associated P values for arousal (A), Attention (B), Lethargy (C) and Quality of Movement (D), where dot represents 1 CpG site. The red line represents a significance level of unadjusted P < 0.05 in association with delta value. CpG sites with an unadjusted P value of <0 .005 in association with NNNS outcome, which are above the blue line in Fig. 1, are shaded gray. No CpGs were significant after correction for multiple comparisons.

Figure 3.

Scatter plots of unadjusted model between (A) methylation of cg15970800 (FHIT 5′ UTR) and normalized infant attention scores, and (B) methylation of cg16710656 (ANKRD11 gene body) and normalized infant attention scores.

Figure 4.

Summary of the top 10 GO terms enriched among genes from the CpGs with highly significant correlations and NNNS scores, as visualized within cytoscape using REVIGO. GO terms are scaled to the log₂ enrichment of that term. Terms in pink text represent those associated with attention, purple terms are associated with arousal, blue terms are associated with lethargy, and green terms are associated with quality of movement.

Figure 5.

(A) Venn diagrams of similarities between CpGs with highly significant correlations (P < 0.005) between multiple NNNS scores. (B) Similarities from genes from the CpGs with highly significant correlations and NNNS scores. (C) Word cloud of top 15 GO terms enriched among genes from the CpGs with highly significant correlations and NNNS scores across all 4 GO terms (51 genes). GO terms are scaled to the log₂ enrichment of that term.