Our results implicate genetically driven NPY expression in emotional functioning at three levels of analysis. At the neural circuit level, we found that low-expression NPY genotypes were associated with greater hemodynamic responses in medial PFC and rostral ACC in healthy individuals viewing negative words. At the level of psychological experience, individuals with low-expression NPY genotypes reported more negative affect during a stressor involving sustained, moderate pain over 20 minutes. At the level of syndromal, categorical diagnosis, we found that low-expression NPY genotypes were more prevalent among patients with MDD. These convergent findings support a model in which genetically driven low NPY expression predisposes certain individuals to hyper-responsivity to negative stimuli within key affective circuit elements, including medial PFC, rostral ACC, and (based on prior work26,29
) the amygdala. The association of these same low-expression NPY genotypes with negative affect during stress and with MDD suggests that these NPY-associated neural response patterns may mediate risk for at least some forms of depression.
The association we found with activation of medial PFC and rostral ACC builds upon prior neuroimaging studies that have implicated NPY genotype in amygdala function. Using the same haplotype groupings that we employ here, Zhou and colleagues used fMRI with threat-related stimuli (fearful and angry faces) and reported that low-expression NPY genotypes were associated with increased hemodynamic responses in right amygdala and hippocampus.26
Domschke and colleagues used fMRI while subliminally presenting emotional faces to MDD patients.29
Analyzing a single-nucleotide polymorphism in the NPY gene (rs16147, −399T/C), they found that amygdala responses to angry faces (and to a lesser extent, sad faces) were greater among individuals with the CC genotype, which would include the low-expression group in our analyses.29
We detected no task or genotype effects in the amygdala. We attribute this result to our use of a different fMRI task, one that involves reading emotionally-valenced words and that does not generally engage the amygdala.27, 32, 34, 44, 45
Thus, we view our findings as complementary to (rather than in conflict with) previous studies of amygdala responses to threat-related facial stimuli. By using an emotion word task, we demonstrate for the first time that NPY genotype has effects on the function of medial PFC and rostral ACC, core circuit elements that have been multiply implicated in normal emotion processing, regulation of emotion, and MDD pathophysiology.1-3, 30-34
In particular, we found low- and high-expression genotypes were associated with activation and deactivation, respectively, in the rostral ACC. This cortical region has been consistently implicated in normal emotion processing and depression.3, 30, 46
Thus, our fMRI findings add substantially to previously described central effects of NPY genotype, to include key emotional circuits in the frontal cortex. These findings also suggest that NPY expression in frontal cortex5, 19, 23, 24
may have important functional consequences.
Our finding of associations between NPY genotype, affect under stress, and MDD diagnosis are consistent with growing evidence that implicates NPY in both normal emotion regulation and affective disorders.10, 47
Plasma NPY concentration has been positively associated with resilience to psychological stress14-17
and expression of NPY in the central nervous system has been suggested as a general resilience mechanism.48, 49
Conversely, low NPY levels have been implicated in affective illnesses. Low-expression NPY haplotypes were associated with greater trait anxiety and undifferentiated anxiety disorders.26
Low plasma NPY concentrations were found among currently depressed patients with MDD21
but not among patients with remitted MDD.20
Postmortem studies have variably reported low NPY levels in frontal cortex of patients with MDD and bipolar disorder.19, 23, 24
Early studies of cerebrospinal fluid NPY in MDD patients were discrepant,18, 25
but a more recent study reported robust reductions among patients with treatment-resistant MDD.22
Furthermore, the latter study found a greater prevalence of the −399C allele (rs16147) among those same MDD patients.22
Because our low-expression group includes individuals who are −399C/C homozygotes, our study represents a quasi-replication of that finding with a less treatment-resistant sample. Furthermore, our findings from healthy subjects during the pain-stress challenge suggest that NPY genotype influences an individual's affective experience under stress, even before the onset of illness. Taken together, the evidence suggests that genetic predisposition to low NPY expression increases risk for MDD (and possibly other affective disorders) by increasing sensitivity to negative stimuli at the psychological and neural-circuit levels, and possibly at the cell and molecular levels as well.
We tested this model of NPY function in affective processing using a functional genomics strategy that differs from conventional approaches in important ways. Conventional molecular genetic association studies are more susceptible to false positives because the total number of statistical comparisons (and therefore, the extent to which type I error should be corrected) is not always apparent, leading to “hypothesis creep”.42, 43
Furthermore, a nonfunctional locus may be more prone to spurious replication because the direction of the effect is ambiguous.43
We have avoided these pitfalls by testing a single a priori
hypothesis using a haplotype-based classification previously validated with in vitro
and in vivo
NPY expression data.26
This functionally informed strategy increases statistical power by avoiding the multiple-comparison problem, and by targeting genetic variation that has functional impact. This functional genomics approach may also be compared to conventional measurements of peripheral NPY levels. Such measures may approximate the variables of most interest (e.g., synaptic NPY levels), but unlike genotype they are subject to other sources of variability, which could include peripheral sympathetic activation,22
clinical state (depressed versus remission),20
and random measurement error. Thus, our strategy improves on the classic statistical genetics approach by leveraging prior measurements of peripheral and central NPY levels. Our confidence in these results is further strengthened by the coherent directionality of the haplotype-driven effect across three levels of analysis. Nonetheless, independent, replication and meta-analyses of larger pooled samples will be essential to validate these findings.
Several limitations of the present study are noteworthy. First, we have interpreted these findings as supportive of a causative model in which (i) genetically driven variation in NPY expression causes neural hyper-responsiveness in key circuit elements and (ii) hyper-responsive circuits cause negative affect and increase risk of developing MDD. Given the correlative nature of these experiments, however, our findings can only suggest causality, and other models are certainly possible. Experimental interventions in animal models are needed to test causal mechanisms. Second, our subject sample was one of convenience and may not be representative of the general population or of MDD patients encountered in usual clinical practice. For example, our sample was limited to individuals who were willing to volunteer for neuroimaging experiments and genotyping, which could bias certain personality traits of the sample. Third, because definitive expression data was unavailable for minor NPY haplotypes, we were unable to include about 16% of subjects in our analyses. We felt that this limitation was outweighed by the benefits of functionally validated haplotype classification. The role of NPY genotype among those individuals will require characterization of in vivo and in vitro expression data for minor haplotypes. Fourth, about two-thirds of our subjects were of European ancestry, so the extent to which these findings apply to individuals of other genetic backgrounds remains to be seen. Similarly, because our MDD sample was 84% female, we were unable to test for association of low-expression NPY genotype among men. Control analyses indicated that the association with MDD survived (and actually strengthened) after controlling for sex, but sexual dimorphism in the NPY system deserves to be explored. Fifth, the design of this study did not allow us to characterize the degree to which NPY genotype might contribute differentially to risk of MDD versus anxiety. We favor a model of shared risk, but this remains to be tested. Sixth, the sample sizes employed here were limiting in some ways. For example, only 58 subjects were classified in the neuroimaging study, and only 8 had a low-expression genotype. Limited statistical power may have prevented us from detecting brain regions besides mPFC and rACC that are truly modulated by NPY genotype, and parametric statistical tests become less valid for sub-groups that contain smaller numbers of observations.
Our findings may eventually have clinical implications. The heterogeneity of MDD represents a major barrier to improving our understanding of its etiology, pathophysiology, and optimal treatment. Based on the NPY system's established role in anxiety and stress responses in experimental animals, and the increasing evidence for its dysregulation in affective disorders, the NPY system may be an excellent target for MDD subtyping and treatment selection. Along those lines, a recent report suggested that response to antidepressant medication varies with NPY genotype.29
The greatest potential for NPY-based biological markers may lie in guiding development of novel antidepressant agents for the many individuals who fail to respond to currently available treatments.