Accurate, Large-Scale Genotyping of 5HTTLPR and Flanking Single Nucleotide Polymorphisms in an Association Study of Depression, Anxiety, and Personality Measures

Serotonergic neurotransmission impacts on a wide range of behaviors, including cognition and emotion (1,2), and drugs targeting serotonin reuptake are clinically effective antidepressants (3). As a result, one of the most studied polymorphisms for association with a broad range of psychiatric and personality phenotypes is the length polymorphism repeat (LPR) in the promoter region of the serotonin transporter gene (5HTT renamed SLC6A4) (5HTTLPR). The 5HTTLPR polymorphism comprises a 43-base pair (bp) (4–8) insertion or deletion (long, "L," with 16 repeat units or short, "S," with 14 repeat units, alleles, respectively). The S allele (frequency in Caucasians ~.45 [9]) reduces transcriptional efficiency, resulting in decreased SLC6A4expression and function (10). Association studies and subsequent meta-analyses (Table 1) (11–15) have shown conflicting results in support of an association between the S allele and anxiety, depression, and the personality trait neuroticism (a measure of emotional stability that is genetically correlated to both anxiety and depression [16–18]). Conflicting results have dogged candidate gene association studies for many complex disorders, attributable to small sample sizes of both primary and replication studies, heterogenous subject populations, association dependent on environmental conditions such as stressful life events (19), and differing instruments for assessment of phenotypic traits and statistical methods (e.g., [20,21]). However, an additional problem specific to 5HTTLPR relates to the genotyping assay, which has caused considerable bias toward S allele identification (22,23). Furthermore, association may have been compromised by the presence of an A/G single nucleotide polymorphism (SNP), rs25531, that lies within the L allele of 5HTTLPR (5,8); the L allele with the rarer G allele of rs25531 (denoted L) is functionally equivalent to the S allele because of changes to the activating protein 2 (AP2) transcription factor binding site altered by this SNP (5,8).