A multi-dimensional characterization of anxiety in monozygotic twin pairs reveals susceptibility loci in humans

Abstract

The etiology of individual differences in human anxiousness is complex and includes contributions from genetic, epigenetic (i.e., DNA methylation) and environmental factors. Past genomic approaches have been limited in their ability to detect human anxiety-related differences in these factors. To overcome these limitations, we employed both a multi-dimensional characterization method, to select monozygotic twin pairs discordant for anxiety, and whole genome DNA methylation sequencing. This approach revealed 230 anxiety-related differentially methylated loci that were annotated to 183 genes, including several known stress-related genes such as NAV1, IGF2, GNAS, and CRTC1. As an initial validation of these findings, we tested the significance of an overlap of these data with anxiety-related differentially methylated loci that we previously reported from a key neural circuit of anxiety (i.e., the central nucleus of the amygdala) in young monkeys and found a significant overlap (P-value < 0.05) of anxiety-related differentially methylated genes, including GNAS, SYN3, and JAG2. Finally, sequence motif predictions of all the human differentially methylated regions indicated an enrichment of five transcription factor binding motifs, suggesting that DNA methylation may regulate gene expression by mediating transcription factor binding of these transcripts. Together, these data demonstrate environmentally sensitive factors that may underlie the development of human anxiety.

Fig. 1. Intrapair differences in childhood afternoon cortisol, amygdala function and behavior

a The correlations between the intrapair differences in childhood cortisol (x axis) and amygdala-modulated recovery (y axis) are shown. Note: Twin pairs selected for whole genome epigenetic analysis are circled in black. b–f The distribution of the twin differences for the full sample is shown for HPA activity (cortisol level; b; N = 299) and anxious behaviors (c–f; N = 184 to 215). The selected pairs are indicated by color showing their relative twin difference compared to the full sample (pair A = green; B = red; C = green)

Fig. 2. Representative regions of differential methylation in human anxiety

A smoothing plot that shows the relative location of differentially methylated (y axis) CpG dinucleotides (x axis; black tick marks) in GNAS (a), NAV1 (b), IGF2 (c), and CRTC1 (d). The DNA methylation profiles for the anxious (red) and unaffected (control; blue) twin-pairs are shown and the genomic region of significance between twin-pairs is highlighted (peach). Each corresponding co-twin is indicated by a different line pattern (pair A = solid; B = dashed; C = dash + dot)

Fig. 3. Characterization of a potential role(s) of DMRs in gene expression

Identification of DMR-associated transcription factor sequence motifs that were predicted by the DREME suite (E-value < 10e−3). The putative transcription binding factors were predicted using SpaMo directly from the DREME suite and are shown next to each sequence motifs