Functional genetic variation in the Rev-Erbα pathway and lithium response in the treatment of bipolar disorder

Abstract

Bipolar disorder (BD) is characterized by disruptions in circadian rhythms such as sleep and daily activity that often normalize after lithium treatment in responsive patients. As lithium is known to interact with the circadian clock, we hypothesized that variation in circadian 'clock genes' would be associated with lithium response in BD. We determined genotype for 16 variants in seven circadian clock genes and conducted a candidate gene association study of these in 282 Caucasian patients with BD who were previously treated with lithium. We found that a variant in the promoter of NR1D1 encoding Rev-Erbα (rs2071427) and a second variant in CRY1 (rs8192440) were nominally associated with good treatment response. Previous studies have shown that lithium regulates Rev-Erbα protein stability by inhibiting glycogen synthase kinase 3β (GSK3β). We found that GSK3β genotype was also suggestive of a lithium response association, but not statistically significant. However, when GSK3β and NR1D1 genotypes were considered together, they predicted lithium response robustly and additively in proportion to the number of response-associated alleles. Using lymphoblastoid cell lines from patients with BD, we found that both the NR1D1 and GSK3β variants are associated with functional differences in gene expression. Our findings support a role for Rev-Erbα in the therapeutic mechanism of lithium and suggest that the interaction between Rev-Erbα and GSK3β may warrant further study.

Figure 1

Additive effects of GSK3β and NR1D1 alleles on lithium response. A) All possible genotype combinations at rs2071427 and rs6438552 were determined for all Li-R and Li-NR subjects and scored (0-4) for the number of alleles associated with favorable lithium outcomes. Dashed lines indicate 95% confidence intervals for the regression line. Rates of response for each allelic combination are the following: 4 alleles: 75% (N=4), 3 alleles: 69% (N=26), 2 alleles: 57% (N=95), 1 allele: 44% (N=108), 0 alleles: 47% (N=48). B) Odds ratios for lithium response calculated for each of 2×2 GSK3β and NR1D1 genotype combinations using dominant and recessive models. Sample sizes for each subgroup are as follows: AA-AG:CC N=17, AA-AG:TT-TC N=116, GG:TT-TC N=119, GG:CC N= 29 C) Odds ratios for lithium response calculated for each of 3×3 GSK3β and NR1D1 genotype combinations. Sample sizes for each subgroup are as follows: AA:CC N=4, AA:TC N=13, AA:TT N=7, AG:CC N=13, AG:TC N=59, AG:TT N=37, GG:CC N=29, GG:TC N=71, GG:TT N=48.

Figure 2

Time course of NR1D1 expression. A) Expression in LCLs (N=12) over 24 hrs after serum shock varies in a non-linear manner suggestive of a ~24 hr periodic rhythm. Expression is similar when analyzed by B) Genotype (N= 6/ group) or C) Li response history (N= 5 Li-R and 7 Li-NR). In all cases data are presented as % baseline expression ± SEM.

Figure 3

NR1D1 expression differs by genotype after Li exposure. A) Full length transcript expression decreases after Li preferentially in AA homozygotes at the rs2071427 locus. B) Expression of full length + truncated NR1D1 transcript trends toward an increase after Li preferentially in AA homozygotes. C) Ratio of full length/(full+truncated) NR1D1 transcript is significantly and specifically decreased among AA homozygotes at rs2071427. NR1D1 expression among GG homozygotes does not change significantly after Li. N=10 LCLs/ group. Data reflect normalized averages ± SEM. (*) indicates significance of p<0.05

Figure 4

GSK3β expression varies by genotype in LCLs from BD patients. Expression was measured in LCLs who were identified as CC (N=2), TC (N= 9) or TT (N= 18) genotypes at rs6438552. Data were normalized to TT genotype. Post-hoc tests showed that the differences between CC vs. TT and TT vs. TC were significant (p<0.05), but that the differences between CC and TC were not.

Figure 5

Proposed model for the putative additive NR1D1-GSK3β genetic contribution to Li response. A) Variant rs2071427 is associated with altered NR1D1 transcript ratios in the presence of Li. This may lead to an increase in the truncated Rev-Erbα protein isoform which is more stable, but unable to serve as a substrate for GSK3β phosphorylation. B) Variant rs6438552 is associated with lower GSK3β gene expression, which may subsequently reduce GSK3β protein levels. C) These mechanistically independent processes could additively oppose Rev-Erbα phosphorylation, altering protein stability and/or limiting Rev-Erbα’s role in processes requiring this modification.