Novel procedure for genotyping of the human serotonin transporter gene-linked polymorphic region (5-HTTLPR)--a region with a high level of allele diversity.
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.


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.


CONCLUSIONS
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.
Rasmussen
Henrik B
HB
Research Institute of Biological Psychiatry, H:S Sct. Hans Hospital, Copenhagen University Hospitals, 2 Boserupvej, DK-4000 Roskilde, Denmark. henrik.berg.rasmussen@shh.hosp.dk
Werge
Thomas M
TM
D004247:DNA / Q000097:blood / Q000235:genetics / Q000302:isolation & purification; D014644:Genetic Variation; D005838:Genotype; D006801:Humans; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length; D015183:Restriction Mapping; D050486:Serotonin Plasma Membrane Transport Proteins / Q000235:genetics*
C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; D004247:DNA