In this report we have identified a molecular phenotype for rs3813034 and demonstrated that it is associated with PD. We have determined that the T allele of rs3813034 leads to more efficient usage of the distal polyadenylation signal in which it occurs than the G allele. This is consistent with in vitro
studies of polyadenylation that have shown that a T in the position of rs3813034 within the canonical polyadenylation signal (AAUAAA) leads to more efficient polyadenylation than a G (50
). Our observation that rs3813034 affects the balance of SERT polyadenylation forms in brain, lymphoblast cultures, and heterologous expression experiments provides confidence that the molecular phenotype of rs3813034 is both reliable and potentially relevant to behavioral variability.
In contrast to the polymorphic effects of rs3813034, the gender difference in SERT polyadenylation was only present in brain and not lymphoblasts. This is likely because lymphoblast cultures are grown under identical conditions regardless of the gender of the individual from whom the cells were derived and that gender-specific hormones influence the SERT DPF. This possibility will require further studies which we are currently conducting. To provide additional confidence that this gender effect was not spurious we tested brains from Swiss Webster mice and found a similar gender effect on the SERT DPF.
Our biological characterization of SERT polyadenylation allowed us to refine the reported association between markers in SERT and PD in this sample (32
). We made a directional prediction that the G allele of rs3813034 would be the risk allele for PD because the G allele mimics the effects of female gender which is itself a risk factor for PD. We confirmed this prediction demonstrating that the G allele of rs3813034 confers increased risk for PD. The gender difference in the SERT DPF in brain also led us to predict that the risk effect of the G allele of rs3813034 would be greater in women than men because females display a lower SERT DPF in brain independent of genotype at rs381034 and therefore risk alleles of rs3813034 would represent second
and/or third hits
conferring greater risk on females than males. This prediction is somewhat supported by our data because we did identify a greater apparent risk effect of the G allele in women than men () but the estimation of the risk effect in men is tentative because the overall association in males was not significant. We note that the association we report is not an independent replication of the previously reported association between SERT and PD using the sample we tested (32
). Rather we propose that rs3813034 is the risk factor that explains the reported association to markers with which it is in high linkage disequilibrium.
Although we do not know the all of the different biological attributes of SERT mRNA molecules that are proximally versus distally polyadenylated we did find that the SERT DPF is correlated with total SERT expression suggesting that the distal sequence stabilizes SERT mRNA either directly through effects on the secondary structure of the message or through interactions with RNA binding proteins or microRNA's. Bioinformatic analysis of the distal sequence identifies putative targets for RNA binding proteins and/or microRNA's that can affect message stability, translational activity, and subcellular localization but these algorithms are not adequately reliable to make confident predictions. Nonetheless, the high degree of sequence similarity of the proximal and distal polyadenylation forms of SERT between human and mouse (Figure S4 in Supplement 1
) is de facto
evidence that both forms have important evolutionarily conserved functions (51
). Mechanistic studies to identify the biological functions of the distal sequence are under way and may help further guide association studies of the role of rs3813034 in psychopathology.
Dysregulation of serotonin neurotransmission has been implicated in the pathophysiology of PD (7
). SSRI's are effective treatments for PD and selectively target SERT (52
). Serotonin turnover which is in part related to SERT activity is increased the brain of PD subjects and normalizes with SSRI treatment (15
). SERT binding to platelets from PD subjects is reduced and also normalizes with SSRI or tricyclic antidepressant treatment (55
). Similarly, SERT binding in the brains of subjects with current PD is decreased in raphe, temporal cortex, and thalamus and normalizes in PD subjects in remission in the raphe and temporal cortex (56
). These findings might be explained at least in part by our finding that a low SERT DPF appears to be a risk factor for PD and is also correlated with lower expression of SERT mRNA. Further studies will be required to test this possibility directly as mRNA levels do not necessarily predict protein levels. There have been reports that a polymorphism adjacent to rs3813034 (rs1042173) is functional and this polymorphism is one of those previously reported to be associated with PD in this sample (32
). rs3813034 and rs1042173 are in absolute LD in Caucasians and therefore any independent PD risk effects of these two polymorphisms cannot be addressed in this study. We note however that the reported functional studies of rs1042173 used only in vitro
analyses to identify a molecular phenotype and found conflicting effects of the alleles of rs10422173 on SERT expression (57
). The problem of identifying the specific risk factor amongst highly correlated polymorphisms is an unavoidable challenge in complex trait genetics. We believe that intensive characterization of the molecular and cellular effects of candidate risk factors using the most biologically representative approaches is one way to address this problem.
A limitation of the PD association study is that the controls came from two sources and were recruited and ascertained using different methods. The cases were also recruited and ascertained differently than the bulk of the controls. We believe that the impact of this possible source of heterogeneity is minimal because the genotype distribution of rs3813034 in the two control samples is similar and the case/control sample was empirically determined to have balanced population structure. The present association of rs3813034 with PD will require replication before it can be considered a risk factor for PD and/or other serotonin transporter-related behavioral disorders but several aspects of this study suggest the association is valid: 1) The molecular characterization of rs3813034 demonstrates that it is a functional polymorphism; 2) The directional prediction we made based on the molecular characterization of rs3813034 was confirmed by association testing; 3) This sample has a balanced population structure between cases and controls that was determined empirically minimizing the risk that the observed association is due to population stratification; 4) The association displays a dose effect of risk alleles at rs3813034; 5) The case-control sample has been rigorously ascertained and is well powered to identify an association of the observed effect size to a common polymorphism such as rs3813034.