Increased methylation of glucocorticoid receptor gene promoter 1_F in peripheral blood of patients with generalized anxiety disorder

Highlights

• Basal serum cortisol elevates in generalized anxiety disorder (GAD).

• Peripheral glucocorticoid receptor (GR) sensitivity decreases in GAD.

• GR gene promoter methylation increased in peripheral blood in GAD.

• GR sensitivity correlates with its gene promoter methylation and gene expression.

• GR sensitivity and its gene promoter methylation correlates with anxiety symptom.

Abstract

Previous findings on the dysfunction of hypothalamic-pituitary-adrenal (HPA) axis in generalized anxiety disorder (GAD) are controversial, and the molecular mechanisms underlying such dysfunction remain unclear. We analyzed the methylation status of the NR3C1 1_F promoter and the expression of glucocorticoid receptor-α isoform (GRα) in peripheral blood mononuclear cells (PMBCs), the basal cortisol level in serum, and a functional neuroendocrine marker for GR sensitivity in the PMBCs in 64 patients with current GAD and 85 healthy controls. We found that patients with GAD had significantly elevated levels of morning basal serum cortisol (P < 0.0001) and diminished GR sensitivity in the PBMCs (P < 0.0001) compared with healthy controls. The overall methylation levels across NR3C1 1_F promoter (P < 0.0001) and percent methylation at each of the 5 CpG sites including CpG12, 21, 30, 31, and 32 (P < 0.001) significantly increased. Accordingly, the mRNA levels of GRα significantly decreased (P < 0.0001) in the PBMCs in patients with GAD compared with healthy controls, with the effects specific in patients without childhood traumatic experience. Moreover, both serum basal cortisol levels and GR sensitivity in the PBMCs were negatively correlated with the overall methylation levels of the NR3C1 1_F promoter (P < 0.0001) and positively correlated with GRα mRNA levels (P = 0.007) in the PBMCs. In sum, our study revealed the increased activity of the HPA axis and diminished peripheral glucocorticoid responsiveness of GR underlying episodes of GAD. Furthermore, such dysfunction of the HPA axis is associated with both increased DNA methylation of NR3C1 1_F promoter and decreased GRα expression.

Introduction

Generalized anxiety disorder (GAD) is a chronic and disabling syndrome with an estimated lifetime prevalence of 4–7% in the general population (Angst et al., 2009, Lieb, 2005). The core symptom of GAD is excessive, unreasonable anxiety and worrying that is difficult to control; however, its psychopathology is not well understood. Chronic over-activation of the hypothalamic-pituitary-adrenal (HPA) axis causes wear and tear of the brain because of the toxic effects of elevated cortisol levels (McEwen, 2008). HPA axis dysregulation is believed to play an important role in the pathophysiology of anxiety disorders.

Anxiety disorder (AD) has—although inconsistently—been linked to the hyperactivity of the HPA axis (Abelson et al., 2007, Arborelius et al., 1999). Several studies on panic disorder revealed elevated basal cortisol levels (Abelson and Curtis, 1996, Bandelow et al., 2000, Garcia-Leal et al., 2005) and diminished suppression of cortisol after a dexamethasone (DEX) suppression test (DST) (Abelson et al., 2007, Westberg et al., 1991) following current anxiety symptoms, whereas others report negative results (Okasha et al., 1994, Targum, 1992). For GAD, fewer studies have investigated its HPA axis activity, indicating increased (Lenze et al., 2011, Mantella et al., 2008) or normal cortisol levels (Chaudieu et al., 2008, Gerra et al., 2000) and weakened (Okasha et al., 1994) or normal (Schweizer et al., 1986, Tiller et al., 1988) suppression of dexamethasone on serum cortisol. In sum, the findings regarding anxiety-related HPA axis dysregulation in previous literature remain inconclusive.

Glucocorticoid receptor (GR) is a key regulator of HPA axis function. The activity of the HPA axis is controlled through the negative feedback regulation by GRs in the paraventricular nucleus, the pituitary gland, and the hippocampus (McEwen, 2008). Recent studies indicate that GR gene (NR3C1 in humans) is epigenetically modified by environmental factors such as prenatal traumatic experience and early life adversities to produce prolonged alterations in stress reactivity and feedback regulation of the HPA axis (Begum et al., 2013, McGowan et al., 2009, Yehuda et al., 2014, Zhang et al., 2013). The differential methylation modification of the NR3C1 promoter is involved in several types of psychiatric disorders including posttraumatic stress disorder (Vukojevic et al., 2014, Yehuda et al., 2015), bipolar disorder (BD) (Perroud et al., 2014), and major depressive disorder (MDD) (Na et al., 2014) and has become an important area of research in biological psychiatry (see review (Turecki and Meaney, 2016)). Moreover, a recent study reported the first evidence of reduced leukocyte methylation of NR3C1 in association with AD (Tyrka et al., 2016). With respect to GAD, however, there are insufficient studies to clarify the role of NR3C1 methylation in its pathophysiology.

Besides the methylation modification in the NR3C1 promoter, its expression is involved in psychiatric pathogenesis (Gola et al., 2014). There are 2 highly homologous isoforms of the GR—GRα and GRβ—in humans that are produced by alternative splicing of the NR3C1 transcript. GRα is a predominant and functionally active glucocorticoid receptor. More importantly, in mood disorders, GRα mRNA expression decreases in the peripheral blood cells (Gola et al., 2014). In the present study, we analyzed DNA methylation of the NR3C1 1_F promoter and GRα expression in the peripheral blood mononuclear cells (PMBCs), basal cortisol level in serum, and a functional neuroendocrine marker of the HPA axis—the lysozyme IC_50-DEX on dexamethasone-inhibited lysozyme activity test in PMBCs—in 64 patients with GAD and 85 healthy controls. We sought to answer 3 questions: 1) whether GR sensitivity and serum cortisol change following GAD development; 2) whether the methylation levels of NR3C1 1_F promoter and GRα expression in the peripheral blood is associated with GAD; 3) whether there are associations among NR3C1 1_F promoter methylation, GRα expression, GR sensitivity, serum cortisol level, and clinical symptoms.