Oxytocin receptor gene methylation: converging multilevel evidence for a role in social anxiety

Abstract

Social anxiety disorder (SAD) is a commonly occurring and highly disabling disorder. The neuropeptide oxytocin and its receptor (OXTR) have been implicated in social cognition and behavior. This study-for the first time applying a multilevel epigenetic approach-investigates the role of OXTR gene methylation in categorical, dimensional, and intermediate neuroendocrinological/neural network phenotypes of social anxiety. A total of 110 unmedicated patients with SAD and matched 110 controls were analyzed for OXTR methylation by direct sequencing of sodium bisulfite-converted DNA extracted from whole blood. Furthermore, OXTR methylation was investigated regarding SAD-related traits (Social Phobia Scale (SPS) and Social Interaction Anxiety Scale (SIAS)), salivary cortisol response during the Trier social stress test (TSST), and amygdala responsiveness to social phobia related verbal stimuli using fMRI. Significantly decreased OXTR methylation particularly at CpG Chr3: 8 809 437 was associated with (1) the categorical phenotype of SAD (p<0.001, Cohen's d=0.535), (2) increased SPS and SIAS scores (p<0.001), (3) increased cortisol response to the TSST (p=0.02), and (4) increased amygdala responsiveness during social phobia-related word processing (right: p(corr)<0.001; left: p(corr)=0.005). Assuming that decreased OXTR methylation confers increased OXTR expression, the present finding may reflect a compensatory upregulation for pathologically reduced oxytocin levels or a causally relevant increased OXTR activation in SAD and related traits. OXTR methylation patterns might thus serve as peripheral surrogates of oxytocin tone and aid in establishing accessible biomarkers of SAD risk allowing for indicated preventive interventions and personalized treatment approaches targeting the oxytocin system.

Figure 1

(a) The OXTR gene on chromosome 3p25-3p26.2 consists of four exons with the translation start site (ATG (+1)) in exon 3 and the stop codon (TGA) in exon 4. OXTR SNP rs53576 is localized in the third intronic region (not to scale). The presently analyzed amplicon (not to scale) is located within OXTR exon 3 downstream to the translation start site (Kumsta et al, 2013; Mamrut et al, 2013; Unternaehrer et al, 2012). (b) The sequence is displayed as per GRCh37 build, National Center for Biotechnology Information (NCBI) reference sequence NC_000017.10 (Chr3:8 809 281–8 809 534), and encompasses the presently analyzed amplicon with primer pair binding sites double underlined. CpG sites 1–12 analyzed in the present study are boxed and numbered (CpG1=Unternaehrer (U) CpG2 (Unternaehrer et al, 2012)=Chr3: 8 809 464 (NC_000017.10), CpG2=(U) CpG3=Chr3: 8 809 442, CpG3=(U) CpG4=Chr3: 8 809 437, CpG4=(U) CpG5=Chr3: 8 809 433, CpG5=(U) CpG6=Chr3: 8 809 428, CpG6=(U) CpG7=Chr3: 8 809 425, CpG7=(U) CpG8=Chr3: 8 809 422, CpG8=(U) CpG9=Chr3: 8 809 417, CpG9=(U) CpG10=Chr3: 8 809 413, CpG10=(U) CpG11=Chr3: 8 809 399, CpG11=(U) CpG12=Chr3: 8 809 394, CpG12=(U) CpG13=Chr3: 8 809 387), CpGs contained in the amplicon, but not readable because of technical difficulties particularly at the 3′ and 5′ ends of the amplicon, are underlined.

Figure 2

Mean methylation at OXTR CpG3 (Chr3: 8 809 437) in healthy controls (N=110; white bar, mean=0.273) and patients with social anxiety disorder (SAD; N=110; black bar, mean=0.183). Error bar±SE. ***Significant at p<0.001.

Figure 3

(a) Correlation of decreased methylation at OXTR CpG3 (Chr3: 8 809 437) with increased Social Phobia Scale (SPS; Stangier et al, 1999) scores (r=−0.39, p<0.001) and (b) Social Interaction Anxiety Scale (SIAS; Stangier et al, 1999) scores (r=−0.40, p<0.001) in the combined sample of 110 patients with social anxiety disorder and 108 healthy controls (data missing for two controls).

Figure 4

Correlation of decreased mean OXTR methylation across all 12 CpGs with increased cortisol salivary cortisol response after the Trier social stress test (TSST) in healthy subjects (N=16) (r=−0.56, p=0.03).

Figure 5

Association of mean OXTR methylation across all 12 CpGs and amygdala responsiveness to social phobia-related words vs negative words in patients with social anxiety disorder (N=25). (Left) Coronal view depicting the negative correlation thresholded at p<0.05, k=38, yielding a cluster-corrected p<0.05. Color bar, correlation coefficient r. (Right) Scatter plot depicting the correlation of mean OXTR methylation rate across all 12 CpGs and the fMRI contrast value extracted from x=26, y=−2, z=−14 (r=−0.663, p=0.0003).