Association of a polyadenylation polymorphism in the serotonin transporter and panic disorder

Abstract

Background: Genetic markers in the serotonin transporter are associated with panic disorder (PD). The associated polymorphisms do not include the serotonin transporter-linked polymorphic region and display no obvious functional attributes. A common polymorphism (rs3813034) occurs in one of the two reported polyadenylation signals for the serotonin transporter and is in linkage disequilibrium with the PD-associated markers. If functional, rs3813034 might be the risk factor that explains the association of the serotonin transporter and PD.

Methods: Quantitative polymerase chain reaction on human brain samples (n = 65) and lymphoblast cultures (n = 71) was used to test rs3813034 for effects on expression of the polyadenylation forms of the serotonin transporter. rs3813034 was also tested for association in a sample of PD cases (n = 307) and a control sample (n = 542) that has similar population structure.

Results: The balance of the two polyadenylation forms of the serotonin transporter is associated with rs3813034 in brain (p < .001) and lymphoblasts (p < .001). The balance of the polyadenylation forms is also associated with gender in brain only (p < .05). Association testing of rs3813034 in PD identified a significant association (p = .0068) with a relative risk of 1.56 and 1.81 for the heterozygous and homozygous variant genotypes, respectively.

Conclusions: rs3813034 is a functional polymorphism in the serotonin transporter that alters the balance of the two polyadenylation forms of the serotonin transporter. rs3813034 is a putative risk factor for PD and other behavioral disorders that involve dysregulation of serotonergic neurotransmission.

Figure 1

Schematic representation of the serotonin transporter message (mRNA) and qPCR assays. 5′ and 3′ UTR's are presented as narrow boxes. The protein-coding region is presented as a wide box. Primers and probes for the qPCR assays are placed in the location of their hybridization positions. Probes are represented on the strand to which they hybridize.

Figure 2

Distal polyadenylation fraction stratified by rs3813034 genotype. Values are normalized to the median value for the G/G genotype separately for brain and lymphoblasts. The center line of each plot represents the median value, boxes represent the 25% and 75% percentiles, and whiskers extend to the 10% and 90% percentiles. *p<0.05, **p<0.01, ***p<0.001.

Figure 3

Confirmation of allelic effects of rs3813034 in reporter constructs heterologously expressed in HEK293 cells. SERT DPF is plotted with values normalized to the median value for the G allele-containing construct. The center line of each plot represents the median value, boxes represent the 25% and 75% percentiles, and whiskers extend to the 10% and 90% percentiles. *p<0.05.

Figure 4

Distal polyadenylation fraction stratified by gender. Values are normalized to the median value for females separately for brain and lymphoblasts. The center line of each plot represents the median value, boxes represent the 25% and 75% percentiles, and whiskers extend to the 10% and 90% percentiles. *p<0.05.

Figure 5

Confirmation of the gender effect on SERT polyadenylation in mouse brain. SERT DPF in male and female brain from Swiss Webster mice (error bars represent the standard error of the mean). SERT DPF in males are normalized to the mean value for females. *p<0.05.

Figure 6

Correlation between the SERT DPF and total expression of SERT. Total expression of SERT is normalized to expression of GAPDH as an endogenous control gene. Dashed line is the least squares fit for the data (r=0.35, p=0.006).