Figure 2

Case–control analysis of DNA methylation and gene expression in LCLs. (a) DNA methylation levels of five CpG sites shown in the Figure 1 in LCLs (bipolar disorder (BD), n=20, controls, n=20) using pyrosequencing. Two CpG sites (3 and 4) showed significantly higher DNA methylation in BD patients. Values indicate mean±s.d. Asterisk indicates significance; P=0.008 and P=0.035 for CpG3 and CpG4, respectively, by Mann–Whitney U test. (b) Relationship between expression level of SLC6A4 and HTTLPR genotype (BD, n=19, controls, n=19). One datum in each diagnostic group was turned out to be at outlier by Smirnov–Grubbs test and removed from data analysis. Among the all samples (n=38), the mRNA level of SLC6A4 tended to be lower in BD patients compared with controls (P=0.08; Mann–Whitney U-test). The mRNA expression level was significantly lower in BD patients compared with controls only in subjects with the S/S genotype (P=0.007; Mann–Whitney U-test) and not in those with the S/L genotype (P=0.867; Mann–Whitney U-test). Values indicate mean±s.d. An asterisk indicates significant difference (P<0.05). NS, not statistically significant. (c) Relationship between the DNA methylation of CpG3 and HTTLPR genotype (BD, n=20, controls, n=20). The DNA methylation of the CpG3 site was significantly higher in BD patients compared with controls only in the subjects with the S/S genotype (P=0.020, Mann–Whitney U-test) but not in those with the S/L genotype (P=0.105, Mann–Whitney U-test). The DNA methylation of the CpG4 site also tended to be higher in BD patients compared with controls only in the subjects with the S/S genotype (P=0.052, Mann–Whitney U-test) and not in those with the S/L genotype (P=0.279, Mann–Whitney U-test). Values indicate mean±s.d. (d, e) Correlation between the DNA methylation at the CpG3 site and mRNA expression level of SLC6A4 (BD, n=19, controls, n=19). There was a significant correlation in the subjects with the S/S genotype (r=−0.425, P=0.043, n=23); (d), whereas no significant correlation was found in the subjects with the S/L genotype (r=−0.262, P=0.346, n=15); (e). Closed circles represent patients with BD and open circles represent control subjects.