With an understanding of our candidate gene's structure and dHPLC methods in place to efficiently evaluate sequence variation, the task now turns to defining the subject pool. In this final section, we discuss the findings that have arisen from studies of SERT knockout mice, as well as provide a short review of leads apparent in studies of the hSERT 5HTTL-PR and 3rd intron VNTR.
The cloning of the mouse SERT gene (Bengel et al., 1997) identified genomic sequences suitable for the creation of targeting vectors and the production of SERT knockout mice. Homozygous targeted mice were found to lack SERT mRNA and antidepres-sant-sensitive 5-HT transport and antagonist binding, as assessed in membranes and via autoradiography, and to have significantly lowered tissue stores of 5-HT (Bengel et al., 1998). Although heterozygous animals exhibit a reduction in mRNA and binding sites compared with wild-type controls, they retained full transport function. We speculate that retention of function may arise from altered trafficking to redistribute pools of transporters from intracellular pools to the cell surface. On the one hand, this may be a fortu itous demonstration of plasticity in the system and could indicate that CNS-based pheno-types may be hard to identify because the most common human variants are likely to be carried as heterozygous alleles. On the other hand, these changes arise from a null allele. Changes arising from an altered protein sequence that corrupts transporter maturation or function could have dominant-negative attributes, corrupting expression of the wild-type allele, as recent studies have suggested homomultimerism of SERT subunits (Kilic and Rudnick, 2000). Full loss of SERT function leads to significant changes in 5HT levels, receptor expression, and receptor signaling (Fabre et al., 2000; Li et al., 2000; Gobbi et al., 2001; Mossner et al., 2001). The density of 5-HT1A receptors in the dorsal raphe was reduced in both male and female SERT knockout mice, but this reduction was more significant in females than in males (Li et al., 2000). Insensitivity to pharmacologic challenges or stressors that require intact autoreceptor function might represent useful phenotypes indicative of novel SERT polymorphisms. Further exploration of cognitive and somatosensory phenotypes are needed in these animals, given the disruptions of the barrel field formation evident in the homozygous knockouts (Salichon et al., 2001).
Two recent efforts to characterize the peripheral phenotypes of SERT knockout mice have revealed interesting facets of SERT biology that may translate to human conditions enriched for SERT sequence variants. Chen and co-workers (2001) have examined the impact of the SERT KO on GI function. As noted, SERT is richly expressed in the enteric nervous system, where it functions to terminate the actions of 5HT released by intrinsic myenteric and submucosal neurons (Wade et al., 1996; Gershon, 1999). Functional studies with SERT antagonists reveal a biphasic impact on colonic motility that is consistent with hyperactivation of 5HT receptors followed by their desensitization at higher concentrations of extracellular 5-HT (Wade et al., 1996). SERT is also found in the crypt epithelium, where it may limit effects on fluid secretion triggered by enterochromaffin cell-derived 5-HT. In the SERT knockouts, colonic motility, and stool water were altered, along with the increase in motility (diarrhea) that alternated irregularly with decreased motility (constipation), a phenotype often seen in human subjects suffering from IBS (Manning et al., 1978; Thompson, 1993). The knockout mouse studies also demonstrate that 5-HT levels in the gut are unaffected by SERT loss of function, unlike the CNS where a compensatory reduction is seen. This lack of compensation is in keeping with the physical separation of sites of primary production (enterochromaffin cells) and sites of clearance (crypt epithelium). Thus, loss of SERT function could establish a situation in the gut where exaggerated levels of extracellular 5-HT could occur, suggesting that a GI phenotype may be a penetrant phenotype for loss of function SERT polymorphisms. Also of note in the study on GI phenotypes in SERT knockout mice is the absence of 5HT from the blood. Presumably, with a lack of platelet transporters to sequester 5-HT from the blood, the 5HT that enters the blood cannot be protected from degradation or elimination. Screening subjects for hyposerotonemia may thus reveal novel loss of function alleles of hSERT.
In addition to the gut, pulmonary function has been explored recently in SERT knockout mice (Eddahibi et al., 2001). As expected from the notion that a single gene encodes SERT proteins in the brain and periphery, the homozygous SERT knockout eliminates SERT immunoreactivity in pulmonary smooth muscle cells and 5-HT uptake capacity. Interestingly, 5HT and SERT have been implicated in the proliferation of pulmonary smooth muscle cells arising from hypoxia (Lee et al., 1989; Eddahibi et al., 2001). Loss of SERT in the knockouts eliminates the 5-HT-triggered smooth muscle cell proliferation. Conceivably, elevated SERT expression, as provided by a gain-of-function variant, could increase risk for 5-HT dependent pulmonary hypertension (Eddahibi and Adnot, 2002).
Although functional coding variants have yet to be identified in hSERTs, functional variants in promoter and intronic regions have been investigated for their relationship to clinical syndromes. As noted above, the s alleles of the 5HTTLPR have been found to associate with reduced transcriptional activity of the SERT promoter and with neuroticism and anxiety traits (Lesch et al., 1996). However, the degree to which the 5-HTTLPR influences SERT expression is at present controversial, with both supportive (Little et al., 1998; Heinz et al., 2000) and contradictory (Willeit et al., 2001) evidence. Recently, Du and co-workers (Du et al., 2000) were able to replicate the finding of Lesch of an association between 5-HTTLPR s alleles and neuroticism, but only in a male population, the gender of the original Lesch studies. These findings suggest that gender-specific expression of phenotypes may need to be considered in evaluation of SERT variants and that neuroti-cism and anxiety continue to represent behavioral traits that may enrich for novel SERT alleles. Although an extensive review of the investigation of the 5HTTLPR in clinical studies is beyond the scope of the present study (for a recent comprehensive review, see Hahn et al., 2002), polymorphic status at the 5HTTLPR has been connected to autism (Cook et al., 1997; Klauck et al., 1997; Tordjman et al., 2001), affective disorders (Green-berg, 1998), drug abuse and alcoholism (Little et al., 1998; Twitchell et al., 2001), and OCD (McDougle et al., 1998; Bengel et al., 1999; Camarena et al., 2001). In a search focusing on loss-of-function SERT alleles, it may seem counterintuitive to utilize disorders that effectively respond to serotonin-selective reuptake inhibitors (SSRIs), such as anxiety, major depression, or OCD. Possibly, gain-of-function alleles may lurk in these syndromes. However, it should also be remembered that the mouse knockout studies show us that altered SERT expression is likely to trigger a series of compensatory changes leading to diminished serotonergic signaling. Diminished serotonergic tone might then be elevated by a pharmacologic reduction in residual 5-HT clearance, establishing 5HT levels high enough to desensitize somatic autoreceptors and thereby eliminate autoreceptor-based inhibition of raphe firing. Certainly this is a complex scenario that will only become clearer with clinical evaluation of subjects with bona fide transporter mutations. Until then, understanding whether SSRIs ameliorate or exacerbate changes in heterozygous knockout mice could be informative.
Given that SERTs are the major targets for SSRIs (Fuller and Wong, 1990; Barker and Blakely, 1995), phenotypes linked to SSRI responsiveness might be considered in the hunt for novel SERT alleles. In this regard, recent evidence suggests that the l allele of the 5-HTTLPR may be associated with a favorable response to SSRIs. Billett (1997) initially found no association between overall SSRI response and the 5-HTTLPR polymorphisms in 72 OCD patients. In contrast, Smeraldi and colleagues (1998) found an association between the 5HTTLPR l allele and response to fluvoxamine in 102 depressed patients with delusional depression (Smeraldi, 1998). Kim and co-workers (2000) evaluated 120 depressed patients and 252 controls and found that an s/s genotype in 5HTTLPR was associated with better response to fluoxetine or paroxetine. Zanardi (2000) found an association between the l allele of the 5-HTTLPR and response to paroxetine. An association between an l/l genotype and faster rate of paroxetine response was found by Pollock and collaborators (Pollock et al., 2000). Finally, Mundo and colleagues (2001) have reported that the 5-HTTLPR s allele predicts risk for mania in bipolar subjects treated with antidepressants, which suggests that drug-targeted phenotypes could be an important area for future analyses. The 3rd intron VNTR of SLC6A4 has also received attention in association and phar-macogenomic studies, although the number of studies is not as extensive as those involving the 5HTTLPR. Kim and colleagues (2000) found that a VNTR 12/12 genotype was associated with a better response to fluoxetine or paroxetine. The lack of a 12-bp repeat s allele in the VNTR most powerfully predicted nonresponse. One possibility for incorporating these findings in the search for novel SERT alleles would be to target pharmacologically defined subjects bearing the "inappropriate" 5HTTLPR genotype. For example, one could target mania-exhibiting bipolar subjects expressing the l/l genotype as a population that can be enriched for novel alleles that could phenocopy the s/s genotype. By analogy with OI subjects who have a NET loss of function mutation and who have blunted responses to tyramine (Shannon et al., 2000), future studies might also account for blunted 5HT efflux triggered by a fenfluramine challenge as an indication of loss of function in SERT that could be determined genetically.
Overall, the existing studies with SERT promoter polymorphisms point to anxiety and affective disorders, alcoholism, autism, and OCD as important syndromes for which novel SERT polymorphisms might increase risk and thus be study populations for polymorphism discovery. We note that as SERT expression is not limited to the brain, comorbidi-ties with peripheral syndromes, such as IBS or pulmonary hypertension, should also be considered (Blakely, 2001). Finally, inclusion of antidepressant responsiveness or fenflu-ramine challenges to query the functional status of SERTs in vivo could further narrow the study population to provide the best opportunity for success. We certainly have no reason to expect that SERT would be excluded from genetic variation. Although the SERT gene structure features distributed, small exons, which do not favor a high frequency of exonic mutations, such arguments could be made for other genes, including ion channels and other transporters where we have clear evidence of functional variants. More likely, we have only just begun to understand how to define the appropriate clinical populations. The leads provided by SERT transgenic studies and the existing association studies with common SERT variants establish initial syndromes to explore for SERT variants. High-throughput polymorphism discovery techniques that allow for the cost-effective analysis of DNA samples without recourse to sequencing are essential because the hunt is for rare variants and much "normal" DNA will need to be evaluated before informative variants are identified. Finding the first few variants may be anxiety-provoking and gut-wrenching, but hopefully the phenotypic lessons learned will translate into more effective diagnoses and therapies.
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