Proteomic analysis of lymphoblastoid cells derived from monozygotic twins discordant for bipolar disorder: a preliminary study

Abstract

Bipolar disorder is a severe mental illness characterized by recurrent manic and depressive episodes. In bipolar disorder, family and twin studies suggest contributions from genetic and environmental factors; however, the detailed molecular pathogenesis is yet unknown. Thus, identification of biomarkers may contribute to the clinical diagnosis of bipolar disorder. Monozygotic twins discordant for bipolar disorder are relatively rare but have been reported. Here we performed a comparative proteomic analysis of whole cell lysate derived from lymphoblastoid cells of monozygotic twins discordant for bipolar disorder by using two-dimensional differential in-gel electrophoresis (2D-DIGE). We found approximately 200 protein spots to be significantly differentially expressed between the patient and the co-twin (t test, p<0.05). Some of the proteins were subsequently identified by liquid chromatography tandem mass spectrometry and included proteins involved in cell death and glycolysis. To examine whether these proteins could serve as biomarkers of bipolar disorder, we performed Western blot analysis using case-control samples. Expression of phosphoglycerate mutase 1 (PGAM1), which is involved in glycolysis, was significantly up-regulated in patients with bipolar disorder (t test, p<0.05). Although PGAM1 cannot be regarded as a qualified biomarker of bipolar disorder from this preliminary finding, it could be one of the candidates for further study to identify biomarkers of bipolar disorder.

Figure 1. Gene networks showing interrelationships between differentially expressed genes in the twin with bipolar disorder.

Figure 2. Protein expression validation by Western blot analysis with an internal standard, NM23A.

Proteins from each of the eight pairs of samples were separated by SDS-PAGE and transferred to PVDF membranes. Proteins were immunodetected using the respective primary antibodies and fluorescent secondary antibodies. Signals were captured with FX and signal intensity and shown by the. a) PGAM1, b) PSME1, c) WARS. Scatter plots show the ratio of each protein to an internal standard protein, NM23A, measured by densitometric scanning of the band intensities. The p values were calculated using a t test in all proteins. Number of the subjects is 8 for bipolar disorder and 8 for controls, respectively. The absolute band intensity for the PGAM1 was also significantly higher in patients with bipolar disorder (0.93+/−0.23 [mean +/− standard deviation] [arbitrary unit]) than control subjects (0.39+/−0.18, p<0.0005).

Figure 3. Verification of the alternation of PGAM1 using the other internal protein, tubulin alpha (TUBA).

The methods are similar to the Figure 2, except that tubulin alpha (TUBA) was used for the internal standard. a) PGAM1, b) PSME1, c) WARS. Scatter plots show the ratio of each protein to an internal standard protein, TUBA, measured by densitometric scanning of the band intensities. The PGAM1/TUBA ratio was significantly higher in patients with bipolar disorder compared with controls (p<0.05). Number of the subjects is 8 for bipolar disorder and 8 for controls, respectively.