Background: The serotonin transporter, the target of a group of antidepressant drugs, is involved in the regulation of the availability and reuptake of serotonin. A variable number of tandem repeats in the promoter region of the serotonin transporter gene, designated 5-HTTLPR, affects the transcription of this gene and appears to modulate the susceptibility to a variety of diseases including depression. Of importance, 5-HTTLPR alleles composed of the same number of basic units may differ at single nucleotide positions providing an additional source of variation.
Objective: To develop a procedure for detailed genotyping of 5-HTTLPR based upon simultaneous analysis of tandem repeat size variation and single nucleotide variations.
Methods: We elaborated a list of all known 5-HTTLPR alleles to provide an overview of the allele repertoire at this polymorphic locus. Fragments of 5-HTTLPR were PCR-amplified in reaction mixtures prepared with and without 7-deaza-dGTP. The amplified fragments were treated with NciI and NlaIII and subjected to agarose gel electrophoresis. Alleles were identified by comparison of the observed electrophoretic patterns with the predicted patterns. Two hundred samples of human genomic DNA representing a variety of different 5-HTTLPR alleles were included in the study.
Main results: We were able to amplify fragments of 5-HTTLPR, which are GC-rich, without the use of 7-deaza-dGTP. This is an advantage as modified nucleotides may inhibit restriction enzymes and interfere with allele determination. After having developed a 5-HTTLPR genotyping assay, we examined all samples of DNA in two separate rounds of analyses and found complete agreement between the results from these two rounds.
Conclusion: On the basis of simultaneous analysis of tandem repeat size variation and variation of single nucleotides we designed a reliable assay for the determination of the major alleles and several of the rare alleles at the polymorphic locus 5-HTTLPR.