To assess whether there may be a genetic association of PTSD with polymorphisms in either the PACAP (ADCYAP1
) or PAC1 receptor (ADCYAP1R1
) locus, we performed a tag-SNP analysis (r2
= 0.8; MAF = 0.1) across both genes with a total of 44 SNPs (14 ADCYAP1 and 30 ADCYAP1R1 SNPs)
. Using logistic regression, we examined whether each SNP was associated with PTSD diagnosis in this cohort of highly traumatized urban civilian subjects (n = 798)11,12,23
, in total, or stratified by gender (females: n = 503; males: n = 295). Only the ADCYAP1R1
receptor SNP rs2267735 (p
= 0.0002 in females; NS
in males) remained significant after experiment-wide multiple correction for sex and 44 independent tests ( and Suppl. Fig. 1
). No SNPs in the peptide ADCYAP1
gene met experiment-wide criteria for association (Suppl. Fig. 2
). Given these striking gender differences and recent data demonstrating that ADCYAP1R1
gene expression may be dynamically modulated by estrogen24
, the distribution of estrogen response elements (EREs) within ADCYAP1R1
gene was examined (Suppl. Table 4
). We found that rs2267735 was within a predicted ERE (, Genomatix; matrix similarity = 0.877, core similarity = 1.0). Because rs2267735 is positioned within the central variable region of the consensus sequence, in silico
analyses do not currently allow us to predict how the ‘C’ vs ‘G’ allele may differentially alter ERE function and further in vitro
analyses are warranted.
Genetic Association of PAC1 Receptor (ADCYAP1R1) with PTSD
We next determined if the association between rs2267735 and PTSD diagnosis could be replicated in an additional 439 subjects. These subjects were from the same overall study, but were interviewed and had DNA collected after the original discovery population. Thus they served as a replication source from the same population but distinct in time and with different interviewing staff. The table in shows the logistic regression results for males and females separately in the initial population described in the tag-SNP analysis, the replication sample from the same population, and the combined sample of 1237 individuals. The main effect of the SNP on PTSD diagnosis could be replicated in women (p<0.05) and combining both samples increased the significance of the association (N = 763, p< 0.00002). As in the discovery sample, no effects were observed in males (male combined sample N = 474, p = 0.7).
Association of ADCYAP1R1 rs2267735 with PTSD symptoms and physiological fear responses
To further examine ADCYAP1R1 rs2267735 SNP associations with continuous PTSD symptom levels in females, we analyzed both an additive and a dominant model with total PTSD symptoms and symptom subscales using the combined samples (). The ‘CC’ allele was most robustly associated with total PTSD symptoms and among subscales, hyperarousal symptoms were the most strongly associated with rs2267735. Notably, even after controlling for childhood trauma history and adult trauma, age and race, (which slightly reduces total N due to missing data) the rs2267735 ‘CC’ allele was associated with higher levels of PTSD hyperarousal symptoms compared to ‘G’ carriers in women (p = 0.0008, ), but not men (p=0.51).
We repeated the above analyses with Beck Depression Inventory (BDI) symptoms and history of lifetime substance abuse, and found no associations with these measures and rs2267735 (Suppl. Fig. 3
), suggesting that this association may be relatively specific to PTSD. To address whether rs2267735 might be associated with other severe psychiatric illnesses, we performed analyses using bipolar disorder, schizophrenia, and Alzheimer’s disease samples. From the data of the Genetic Association Information Network (GAIN) publicly accessible database (http://www.ncbi.nlm.nih.gov/projects/gap
), we analyzed the association of rs2267735 (included on the Affymetrix 6.0 SNP array) with bipolar disorder as well as schizophrenia. We did not observe a significant association of this SNP with these two disorders in subjects with African American (954 cases, 1195 controls) or European (1378 cases, 1351 controls) ancestry. Specifically, we found that all pre-computed p-values for associations of rs2267735 with schizophrenia or bipolar disorder were higher than the multiple-testing correction p-value of 0.01, indicating no major contribution of this variant.
Additionally, we examined the association of rs2267735 and Alzheimer’s in a previously characterized Alzheimer’s disease sample25
. In this cohort of 342 subjects, we found no association with rs226735 and Alzheimer’s disease diagnosis using either the additive genetic model (p=0.19
) or the dominant/recessive model (p=0.89
). These data suggest that we find robust associations with rs2267735 in women, but not men, with PTSD. In contrast, we find no association with depression symptoms, substance abuse, Alzheimer’s disease, bipolar disorder, and schizophrenia across different samples. Note that for all of these negative results, due to the limited sample sizes, we cannot rule out the possibility that rs2267735 may be associated with PTSD in men or with other disorders with a smaller effect size than we see with PTSD in women.
To parallel our results with plasma PACAP38 levels, we next examined whether physiological measures of fear are differentially associated with the ADCYAP1R1
rs2267735 SNP. In PTSD, but not depression18
, fear response to an inhibitory CS−, or ‘safety signal’, is exaggerated. The discrimination between CS+ and CS− improves across the training procedure in controls, but not in those with PTSD. We examined whether rs2267735 was associated with impaired fear discrimination late in conditioned acquisition, during the same period noted in . Notably, females with the ‘CC’ genotype were significantly less able to discriminate CS+ from CS− signals (, sex by genotype interaction, p<0.05
, and ‘CC’ vs ‘G’ carriers in females, p<0.05
We next examined whether a difference in dark-enhanced startle, a measure of increased anxiety in humans that is similar to light-enhanced startle in rodents26–28
, was differentially associated with rs2267735. Again, we found that females, but not males, with the ‘CC’ genotype showed significantly more startle in the dark compared to the light (, males, N=35, p=0.71
; females, N=53, p=0.02
). Together, these data suggest that the ADCYAP1R1
rs2267735 SNP may be relatively specific in its association with PTSD psychological and physiological phenotypes. Further, the robust association of rs2267735 with hyperarousal symptoms suggests that the role of PACAP/PAC1 may be specifically involved in the normalization of the stress response, a process which is particularly dysregulated in PTSD.