Serotonin transporter polymorphisms and panic disorder

Panic disorder (PD) is the most common anxiety disorder. Although PD seems to occur unprovoked and the underlying etiology is not well understood, studies have consistently shown that genetic factors explain approximately 48% of the variance. Moreover, family and twin studies support the view that the majority of PD cases have a complex genetic basis. Promising findings have most recently implicated the polymorphisms at the 3' end of the serotonin transporter gene SLC6A4 as PD risk variants. If independent studies can replicate the observed association with the SLC6A4 variants and their functional effects on gene expression, this would have a great impact on our understanding of the disease pathophysiology and would provide opportunities to investigate genotype-phenotype correlations.

According to the American Psychiatric Association, PD is defined as an episode of abrupt, intense fear accom panied by additional physiological or cognitive symp toms. Other anxiety disorders and also mood and substanceuse disorders are frequently observed as co morbidities [3,4]. Family and twin studies have consis tently shown that genetic factors explain approximately 48% of the variance in the disease [5], and segregation analyses support the view that the majority of PD cases have a complex genetic basis. This is also highlighted by several animal breeding experiments, which reveal that anxiety or emotional activity analogous to panic and anxiety is controlled by multiple genes, possibly in varying combinations [6]. However, the genetic archi tec ture underlying PD is heterogeneous and differs between cases. For example, the degree of genetic complexity and the pattern of genes involved might be different in familial versus nonfamilial, early versus lateonset cases or when different comorbid conditions, gender and potential intermediate or subphenotypes are considered.
On the molecular genetic level, linkage and candidate gene studies have been used for the genetic analysis of PD, and several potential linkage loci and tentative asso ciations with candidate genes have been found [7]. For several reasons, serotonergic neurotransmission, and especially the serotonin transporter gene SLC6A4, has attracted attention in the PD research field. Selective serotonin reuptake inhibitors (SSRIs) that target SLC6A4 are commonly used and effective treatments for PD [8]. In addition, mouse experiments have shown that SLC6A4 underexpression leads to anxietylike behavior [9], which would be in accordance with human studies that have found decreased SLC6A4 expression in brains of PD patients [10].
On the genomic level, SLC6A4 is located on chromo some 17q11 and consists of 15 exons. A large amount of genetic variation has been observed in SLC6A4. An insertiondeletion polymorphism in the promoter region of SLC6A4, called 5HTTLPR, has attracted particular attention because it has been shown that this poly morphism alters gene and protein expression and the lowexpressing short variant has been associated with anxiety [11]. Moreover, an association has been found, in healthy individuals as well as in patients with major depression, between 5HTTLPR and increased amygdala activation in response to fearful stimuli [1214]. However, most PD genetic association studies have failed to find an association between 5HTTLPR variants or amygdala activation and panic disorder [1517].

The role of a 3' SLC6A4 polymorphism in PD
A recently published study by Gyawali et al. [18] reports evidence that SLC6A4 might contribute to the develop ment of PD by a mutation other than 5HTTLPR. Their study [18] followed findings [19] of an association between PD and polymorphisms located in the 3' untranslated

Abstract
Panic disorder (PD) is the most common anxiety disorder. Although PD seems to occur unprovoked and the underlying etiology is not well understood, studies have consistently shown that genetic factors explain approximately 48% of the variance. Moreover, family and twin studies support the view that the majority of PD cases have a complex genetic basis. Promising findings have most recently implicated the polymorphisms at the 3' end of the serotonin transporter gene SLC6A4 as PD risk variants. If independent studies can replicate the observed association with the SLC6A4 variants and their functional effects on gene expression, this would have a great impact on our understanding of the disease pathophysiology and would provide opportunities to investigate genotype-phenotype correlations. region (UTR) of SLC6A4. None of these 3' UTR associated variants showed linkage disequilibrium to 5HTTLPR, suggesting an independent SLC6A4 locus at the 3' end of the gene. It is known that the SLC6A4 3' UTR is expressed in two alternative forms that differ by the presence or absence of a 123bp element [20] and the more 3' (distal) form contains an additional polyadeny lation signal. Gyawali et al. [18] hypothesized that one particular SNP rs3813034 located within this signal would alter the usage of this form relative to the more 5' (proximal) form. To test this hypothesis, the authors [18] analyzed 65 post mortem human brain samples and found that in brains expressing one of the rs3813034 alleles coding for G the relative expression of the distal to the proximal SLC6A4 form was significantly lower than that of brain samples carrying the alternative (T) allele. The same effect was seen in 71 human lymphoblast cultures. The authors [18] also found evidence that genderspecific effects contributed to the observed allele specific expression differences. The distal form of SLC6A4 was less expressed in brain samples from females than in those from males. To ensure that the gender specific association is a true positive finding, the authors [18] analyzed both expressed SLC6A4 isoforms in brains of male and female mice. In this dataset they also observed gender differences similar to those seen in humans, with a lower expression of the distal SLC6A4 isoform in female mouse brains.
Gyawali et al. [18] also examined whether rs3813034 is itself the variant causing the observed SLC6A4 expres sion differences. Using a functional approach, they cloned both forms of the 3' SLC6A4 UTR into plasmids; one construct encoded allele G and the other one allele T of rs3813034, and the remaining sequence was identical. The relative expression of the two polyadenylation forms was then quantified and the authors [18] observed that the G allele of rs3813034 caused significantly lower usage of the distal poly adenylation form than allele T.
Finally, rs3813034 was tested for PD association in a large casecontrol study (n = 307 PD patients and 544 controls) [18]. The G allele associated with lower expres sion of the distal SLC6A4 isoform was signifi cantly more frequent in patients (51%) than in controls (44%; P = 0.002) and thereby found to be the PD risk allele. This effect became stronger when the participants were stratified by gender. The risk allele was significantly more frequent in female PD patients (51%) than female controls (42%) (P = 0.003), whereas no Gallele associa tion was observed in males (P = 0.233) [18]. The finding was in accordance with the expression experiments, in which lower expression levels of the distal SLC6A4 form were observed in female brain samples from both humans and mice.

Conclusions and perspectives
These results are encouraging and are shedding new light on the role of SLC6A4 variation in panic disorder. Nevertheless, some questions remain. In particular, it has yet to be shown how a lower expression level of the distal SLC6A4 isoform affects protein function quantitatively and qualitatively, for example in a gender and/or cell typespecific manner. This is especially important because the short 5HTTLPR, with an obvious lower protein expression, has consistently been shown not to be associated with panic disorder. Given that SLC6A4 has never been tested systematically for association and the gene might harbor several potential risk variants, possible explanations for the discrepant findings may be that the interaction between different polymorphisms has not been controlled for in previous studies or that it has gender or cell typespecific consequences. Studies on large PD datasets with sufficient marker coverage for extensive haplotype analyses and additional functional studies are now required.
Although these two recent reports [18,19] are evidence that candidategene studies can still provide some surprises, this approach has obvious limitations. In contrast, as with other disorders, modern genomewide association studies of sufficiently large sample size will most probably lead to the identification of novel PD risk genes in the coming years and will contribute to our understanding of the underlying neurobiology of anxiety related disorders and behaviors [21,22]. This will increase our understanding of anxiety disorders and aid the development of better prevention strategies.