Blood diagnostic biomarkers for major depressive disorder using multiplex DNA methylation profiles: discovery and validation

Abstract

Aberrant DNA methylation in the blood of patients with major depressive disorder (MDD) has been reported in several previous studies. However, no comprehensive studies using medication-free subjects with MDD have been conducted. Furthermore, the majority of these previous studies has been limited to the analysis of the CpG sites in CpG islands (CGIs) in the gene promoter regions. The main aim of the present study is to identify DNA methylation markers that distinguish patients with MDD from non-psychiatric controls. Genome-wide DNA methylation profiling of peripheral leukocytes was conducted in two set of samples, a discovery set (20 medication-free patients with MDD and 19 controls) and a replication set (12 medication-free patients with MDD and 12 controls), using Infinium HumanMethylation450 BeadChips. Significant diagnostic differences in DNA methylation were observed at 363 CpG sites in the discovery set. All of these loci demonstrated lower DNA methylation in patients with MDD than in the controls, and most of them (85.7%) were located in the CGIs in the gene promoter regions. We were able to distinguish patients with MDD from the control subjects with high accuracy in the discriminant analysis using the top DNA methylation markers. We also validated these selected DNA methylation markers in the replication set. Our results indicate that multiplex DNA methylation markers may be useful for distinguishing patients with MDD from non-psychiatric controls.

Keywords: DNA methylation; biomarkers; blood; epigenetic; major depressive disorder; microarray; multiplex.

Figure 1.

Diagnostic differences in DNA methylation. The X axis represents average β differences (Δβ value) between control subjects and patients with major depressive disorder (MDD). The Y axis represents –log₁₀ P values. Each dot represents an individual CpG site (a total of 431,489 CpG sites). Red dots represent 363 CpG sites that showed significant diagnostic differences between patients with MDD and the controls at 5% FDR correction. Δβ value > 0 corresponds to higher DNA methylation in patients with MDD than in the controls, whereas Δβ value < 0 corresponds to lower DNA methylation in patients with MDD than in the controls. Of these 363 CpG sites, all of them demonstrated lower DNA methylation in patients with MDD than in the controls.

Figure 2.

Plot of discriminate scores of patients with major depressive disorder and control subjects. The following color scale is used: black, major depressive disorder (MDD); white, control (CTRL). The x axis represents discriminant scores of samples. (A) Discriminant scores of 20 MDD patients and 19 CTRLs. We were able to segregate patients with MDD from the CTRLs using the top 18 MDD-associated DNA methylation markers of the Mann-Whitney U test comparison results in a discovery set of samples. (B) Discriminant scores of 12 MDD patients and 12 CTRLs. We were also able to obtain 100% sensitivity and 100% specificity using the same17 multiplex DNA methylation markers in a replication set of samples.

Figure 3.

GSK3B DNA methylation and expression. (A) GSK3B DNA methylation. The average DNA methylation level of CpG site (cg14472315) in the CGI in the promoter region of the GSK3B gene was significantly lower in patients with major depressive disorder (MDD) than that in the controls (CTRLs) in a discovery set (MDD, n = 20, and CTRL, n 19) (0.079 ± 0.014 and 0.106 ± 0.016, respectively, Mann-Whitney U-test, P = 9.7 × 10⁻⁶). (B) GSK3B expression. The average expression level of the GSK3B gene was significantly higher in patients with MDD than that in the CTRLs in a discovery set (MDD, n = 19, and CTRL, n = 19) (0.197 ± 0.168 and 0.098 ± 0.03, respectively, Student t test, P = 5.0 × 10⁻³). (C) Relationship between the GSK3B promoter DNA methylation and expression. A significant inverse correlation between the GSK3B promoter DNA methylation and expression was observed in the combined samples (MDD, n = 19, and CTRL, n = 19) (r = −0.319; P = 0.025, one-tailed).