LETTER TO THE EDITOR The impact of CACNA1C allelic variation on effective connectivity during emotional processing in bipolar disorder Molecular Psychiatry (2013) 18, 526--527; doi:10.1038/mp.2012.61; published online 22 May 2012 The A allele of the CACNA1C rs1006737 polymorphism is consistently associated with bipolar disorder (BD). 1 No studies have examined the relationship between this polymorphism and brain connectivity, which is disrupted in BD. 2 We investigated the relationship between rs1006737 polymorphism and effective connectivity in BD families. During perception of fearful faces, the presence of the A risk allele was associated with decreased outflow of information from medial frontal gyrus (MFG), which was significantly more marked in patients than in their unaffected relatives and healthy controls. Participants in the study consisted of 20 remitted outpatients with BD1 (42 ± 12years, 10 females, intelligence quotient (IQ) ¼ 114±15), 20 of their unaffected first-degree relatives (42±11years, 11 females, IQ ¼ 120±6) and 20 unrelated healthy controls (43 ± 14years, 8 females, IQ ¼ 113 ± 14), who had previously participated in an event-related functional magnetic resonance imaging study on emotional face processing. 3 Before connectivity analysis, the raw functional images from the fearful faces task were motion-corrected, normalized and 8 mm- smoothed with SPM5. Voxelwise time series were then extracted from MFG, left putamen and left amygdala, that is, regions that displayed hyperactivity in patients and relatives. 3 CACNA1C is widely expressed in brain tissue including these regions. 4 Data were then included to cluster Granger analysis, 5 which tests whether a time series precedes and/or follows another time series, for example, whether increases of BOLD signal in MFG were followed by increases of BOLD signal in left putamen. Individual Granger connections were thresholded with Po0.05, and the statistical significance of each link at the group level was assessed by means of a binomial test. 6 False discovery rate was applied to correct for multiple comparisons at group level. Finally, connectivity of each individual was summarized by ‘out- degrees’ (number of regions of interests (ROIs) whose signals follow the signal of a given ROI) and ‘in-degrees’ (number of ROIs whose signals precede the signal of the ROI). 6 The out-degree represents functional connections departing from the ROI, whereas the in-degree represents functional connections arriving. All connectivity pathways in the network were statistically significant in both directions (highest corrected P ¼ 2e34), that is, the BOLD signals within each of the three ROIs were found to significantly and reciprocally influence each other. However, MFG showed statistically stronger out-degrees than in-degrees (out ¼ 2.7 vs in ¼ 2.3, Wilcoxon W ¼ 486.5, corrected P ¼ 0.001), whereas left putamen showed a trend to stronger in-degrees than out-degrees (in ¼ 2.5 vs out ¼ 2.2), that is, a net information flow from MFG to left putamen can be inferred (Figure 1a). MFG out-degree connectivity gradually decreased with the number of CACNA1C A risk alleles (GG ¼ 2.9 4AG ¼ 2.7 4AA ¼ 2.3; Spearman’s r ¼ 0.394, corrected P ¼ 0.011), mainly due to a decrease in the outflow to left putamen (present in 94% of G/G individuals, 78% of A/G individuals and 62% of the A/A individuals; r ¼ 0.300, uncorrected P ¼ 0.029). This genetic modulation was found to be particularly strong in patients (MFG out-degree r ¼ 0.706, P ¼ 0.001; outflow to left putamen r ¼ 0.594, P ¼ 0.007), while correlated to a weaker extent in their unaffected relatives and healthy controls (MFG out-degree rhop0.311; outflow to left putamen rp0.314; all PX0.05). This genotype  diagnosis interaction was statistically significant (Monte Carlo permutation tests randomly choosing 20 individuals to be labeled as ‘patients’: MFG out-degree P ¼ 0.027; outflow to left putamen P ¼ 0.023; Figure 1b). Figure 1. (a) Connectivity pathways between MFG, left amygdala and left putamen (in each pair of brain regions the stronger connection is shown as a thick arrow whereas the weaker connection as a broken arrow, thus a net information flow from MFG to left putamen can be inferred; (b) MFG out-degree in the fearful condition (y axis) associated with allelic variation in CACNA1C rs1006737 polymorphism in patients with BD, their unaffected relatives and controls. Molecular Psychiatry (2013) 18, 526 -- 527 & 2013 Macmillan Publishers Limited All rights reserved 1359-4184/13 www.nature.com/mp