Patients with SAD and healthy volunteers exhibited higher activation in the left amygdala in response to angry compared with neutral facial expressions but, contrary to our initial expectation, we did not observe a significant differential amygdala response between the 2 groups. In line with our expectation, increased amygdala activation to angry faces within the SAD group was associated with 5-HT–related high-response alleles: the s allele of the 5-HT transporter promoter polymorphism (5-HTTLPR) and the T allele of the TPH2 polymorphism.35–37,39,40
Patients carrying either s or T alleles displayed greater amygdala reactivity (angry > neutral) compared with patients being homozygous for the corresponding low-response alleles (ll or GG), and amygdala reactivity was particularly elevated in patients with both high-response alleles. Genetic influences on amygdala responsiveness were statistically less robust in controls, although analyses confirmed that healthy individuals carrying a high-response allele (s or T) showed significantly elevated amygdala reactivity (angry > neutral) compared with the low-response (ll+GG) subgroup.
We found a significant group difference in amygdala responsiveness between patients and controls only when we compared patients who had high-response alleles with controls who had low-response alleles. Intriguingly, we also found suggestive evidence for greater amygdala activation in response to angry faces among controls carrying high-response alleles compared with patients carrying low-response variants. Taken together, these results indicate that serotonin-related allelic variation, particularly in the TPH2 gene, is important for amygdala responsiveness to emotionally salient stimuli and that the serotonergic polymorphisms are stronger predictors of amygdala reactivity than a diagnosis of SAD. This was confirmed by multiple regression analysis in which the TPH2 polymorphism alone and the presence of both high-response alleles emerged as significant predictors of left amygdala responsiveness. Diagnosis and behavioural measures, on the other hand, accounted for small and insignificant portions of variance in amygdala reactivity.
The absence of a robust diagnosis-related effect on amygdala reactivity was somewhat surprising considering the respectable number of studies that have reported such an effect.7
At a liberal statistical threshold, there was a difference in the expected direction (SAD > control) in the right amygdala, partly driven by decreased regional cerebral blood flow (angry < neutral) among controls, especially in carriers of low-response alleles. Although other imaging paradigms could be more sensitive to detect small group differences in neural responding, our data suggest that 5-HT–related polymorphisms explain much more of the variance in amygdala reactivity than a diagnosis of SAD. The robustness and generalizability of this finding could be explored in future studies of SAD and other disorders associated with amygdala dysfunction.
Our results are consistent with our previous PET study of patients with SAD in which we noted that the 5-HTTLPR polymorphism modulated amygdala activation during a stressful public speaking task,41
even though the lateralization patterns differed. The results also add to a growing corpus of imaging data demonstrating serotonergic genetic influence on the amygdala response to social-emotional stimuli.35–37,39,40
Gene–gene analyses in the current study suggested a synergistic effect between the 5-HTTLPR and TPH2 polymorphisms such that patients with SAD carrying both high-response alleles (s+T) showed markedly increased left amygdala response (angry > neutral) relative to subgroups of patients carrying only 1 high-response allele and relative to low-response (ll+GG) patients and controls. Other investigators have noted additive effects of the s and T alleles on neural responding to emotional stimuli.55,56
In line with previous association studies failing to demonstrate a link between SAD and the 5-HT transporter or other monoamine-related genes,57,58
the allelic distribution in the current study did not differ between patients and controls, although the statistical power to find such a difference was low. It should be noted that one-third of the patients were homozygous for both low-response alleles and did not show elevated amygdala reactivity to angry faces, whereas two-thirds of the controls carried at least 1 high-response allele and did show exaggerated amygdala reactivity. Thus, it seems that 5-HT–related high-response alleles and amygdala hyperresponsivity are neither necessary nor sufficient to cause SAD to develop.
In contrast to our previous PET study,41
carriers of the s and ll alleles in either the patient or control group did not differ on any behavioural measure. In large sample studies of nonpsychiatric populations, the serotonin transporter gene has been associated with anxiety-related traits,59–62
although there have been some inconsistent findings.60
The only significant influence of genotype on behavioural measures in the present study was a higher level of trait anxiety in patients carrying T alleles compared with GG homozygotes. Other investigators have also reported that the TPH2 polymorphism was associated with personality disorders and with traits characterized by emotional instability; however, the nature of the association was not always the same.63,64
Neither trait nor state anxiety predicted amygdala response to angry faces in the present study, although some imaging data suggest that these affective components can modulate the amygdala response to emotionally salient stimuli.20,65,66
Moreover, we could not replicate the findings of 2 previous imaging studies demonstrating significant positive correlation between right amygdala activation to negative faces and severity of SAD as measured by the Liebowitz Social Anxiety Scale.27,29
In fact, we noted that lower values on the scale were predictive of elevated left amygdala response in the SAD group. Despite having significantly higher levels of trait and state anxiety, patients with SAD did not exhibit higher baseline amygdala activity during the neutral face condition compared with controls, and amygdala responsiveness (angry v. neutral) did not correlate with activity during the neutral baseline.
There are several methodologic factors that may affect assessments of amygdala response during emotional face processing. For example, the magnitude of the amygdala activation may be influenced by attention demands67
and by different qualities of the facial stimuli such as valence of the emotional expression,3,4
emotional intensity level,68
degree of novelty/familiarity,69
and use of schematic or photographic stimuli.28
In within-group designs, it remains to be tested how these putative amygdala-modulating variables interact with 5-HT–related genotypes. There are also issues with the selection of participants related to characteristics such as age,71,72
personality and temperamental traits75,76
that may be of importance for group differences in amygdala responsiveness. Studies using between-group designs could benefit from taking genetic variation into account before attributing the source of differential amygdala response to the group factor of interest.
Our study had some limitations. First, sample sizes were modest when comparing genetic subgroups. The small number of participants prevented us from properly evaluating gene–gene interactions in controls and from demonstrating possible group differences of small effect sizes. Because we were not able to match participants on genotype beforehand, we were restricted to a post-hoc analytic approach and unbalanced groups. Second, other imaging techniques or designs may uncover additional effects of emotional stimuli on the magnitude and time course of amygdala activation. For example, Campbell and colleagues77
noted that the early and late temporal components of the amygdala fMRI response to negative faces differed among patients with SAD and controls. Third, although perception of emotional faces does not take place in a single brain region78
the present study focused on the amygdala response only, a decision motivated by the central role of the amygdala in emotional processing1–5
and by the complexity generated by the large number of genetic subgroups and contrasts. It should also be noted that we based some analyses and plots on voxel values extracted from the whole amygdala volume; however, it is possible that only certain subregions of the amygdala are activated by face processing tasks.
Future imaging genetic studies of emotional processing could explore other serotonergic and nonserotonergic polymorphisms and their interactions. For example, amygdala reactivity appears to be modulated by polymorphisms in the 5-HT1A
receptor gene (5-HT1A 1019C/G
and the catechol-O
-methyltransferase gene (COMT Val158Met
Functional connectivity analyses could be used to evaluate how genes affect not only the amygdala response but also the dynamic interplay between relevant nodes in a larger affective processing network. For instance, the 5-HTTLPR has been demonstrated to influence an amygdala–anterior cingulate cortex feedback circuit putatively involved in the regulation of emotion.81
Future studies could also evaluate effects of genotype on treatment response and concomitant neurofunctional changes. Interestingly, Stein and colleagues82
noted that the 5-HTTLPR s allele was associated with poorer response to selective serotonin reuptake inhibitors in patients with SAD. Treatment may modulate amygdala activity differentially owing to genetic makeup.
In conclusion, we demonstrate that variation in 2 genes of major importance for central serotonergic function influence amygdala responsiveness during affective processing to a larger degree than a diagnosis of SAD. Further research is needed to determine the relevance of serotonergic genotypes and amygdala function in the etiology of anxiety disorders. Meanwhile, the present study underscores the importance of accounting for serotonergic polymorphisms when studying group differences in amygdala responsiveness.