As Rev-Erbα is a substrate for GSK3β phosphorylation, and Li has been shown to inhibit this interaction (Yin et al. 2006), we examined whether multiple genetic variants within this known signaling pathway could interact to better predict Li response outcomes. Considering the NR1D1 and GSK3β variants together, the total number of Li response-associated alleles (range 0–4) was highly correlated with clinical outcome (Fig. 1a; r2 = 0.97, F1,3 = 98, P < 0.0025). Subjects who were doubly homozygous for the favorable A allele at rs2071427 and C allele at rs6438552 had a 75% chance of being in the Li-R group. In comparison, those who were doubly homozygous for the unfavorable G allele at rs2071427 and T allele at rs6438552 had only a 44% response rate. Those with three, two or one favorable alleles were intermediate with progressively lower chances of favorable clinical outcomes. Two additional analyses were conducted to evaluate the Li response rates of specific genotype combinations. The first, a 2 × 2 analysis using only the best fitting transmission models (dominant for NR1D1 and recessive for GSK3β) maximized statistical power. The second, a 3 × 3 analysis using each of the nine possible genotypic combinations allowed for higher resolution comparisons of the each genotypic combination (Fig. 1b,c). In the 2 × 2 analysis, those with NR1D1:GSK3β genotypes of AA-AG:CC responded significantly more often than did the comparison group [χ2(1) = 9.36, OR = 7.5, 95% CI 1.68–33.46, P < 0.0025]. Conversely, those with GG:TT-TC genotypes responded significantly less often [χ2(1) = 7.40, OR = 0.52, 95% CI 0.32–0.83, P < 0.0075]. The more detailed 3 × 3 analysis suggested that the rs6438552 CC genotype at GSK3β nominally favored Li response, regardless of NR1D1 genotype. The statistical analysis showed that subjects with NR1D1:GSK3β genotype of AG:CC fared best [χ2(1) = 8.75, OR 11.87, 95% CI 1.52–92.25, P < 0.003], whereas those with GG:TC genotype fared worst [χ2(1) = 3.85, OR 0.58, 95% CI 0.33–1.00, P < 0.05]. Only four subjects had the doubly rare AA:CC genotype. While this combination showed a trend toward favorable Li response its OR was not significantly different from unity [χ2(1) = 0.84, OR 2.77, 95% CI 0.28–26.98, P < 0.36]. Similarly, while the Li response rate of the AG:CC genotype was nominally higher than the AA:CC genotype, these groups had overlapping confidence intervals and could not be statistically discriminated. None of the other genotype combinations differed significantly from unity in the post hoc OR analyses (range 0.66–2.77).
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) and no allele, 47% (N = 48). (b) OR 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 and GG:CC, N = 29. (c) OR 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 and GG:TT, N = 48.
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