Abstract
Specific Objective: SLC1A2 encoding excitatory amino acid transporter 2 (EAAT2), the principal transporter clearing synaptic glutamate, is a candidate gene for bipolar disorder (BD). The aim of this study was to examine the differences in methylation of the SLC1A2 promoter between BD and non-psychiatric subjects (NPS).
Methods: 150 BD subjects from the Mayo Clinic Bipolar Biobank were equally divided into five groups: (1) BD without comorbid alcohol use disorder (AD), nicotine use disorder (ND), or binge eating disorder (BE); (2) BD+AD; (3) BD+ND; (4) BD+ND+AD; and (5) BD+BE. Mayo Clinic Community Biobank provided NPS samples (n=32). First, we performed bisulfite conversion and methylation-sensitive high-resolution melt (HRM) PCR to examine methylation status of the two SLC1A2 promoter regions. To validate our findings, we employed TA cloning and DNA sequencing to map the whole 156 CpG sites in the approximately 2kb promoter region of the SLC1A2. One-way analysis of variance (ANOVA) and general linear regression model were used for the statistical analysis.
Summary of Results: HRM analysis in the CpG island between -1759 and -1468 revealed difference between the groups (p=0.0003). BD with comorbidities were hypomethylated compared to BD, while females showed hypermethylation (p=0.036). HRM PCR in CpG island between -785 and -654 revealed hypomethylation in BD with comorbidities in comparison to BD (p<0.0001). BD was a predictor of hypermethylation (p=0.035). In our complete sequencing study, we identified that CpG site #6 was hypermethylated in BD compared to NPS (p=0.05). CpG sites #3 (p=0.05) and #156 (p=0.04) were hypomethylated in BD+AD+ND compared to NPS. CpG sites #6 (p=0.01) and #48 (p=0.03) were hypomethylated in BD+AD+ND compared to BD.
Conclusions: DNA methylation is altered in two distinct regions of SLC1A2 promoter of BD patients. Addiction comorbidities have a significant impact on the level of promoter hypomethylation. HRM analysis was validated through bisulfite sequencing.