Development and validation of a high-throughput screening method for two polymorphisms in the serotonin transporter gene.
OBJECTIVE
The human serotonin (5-hydroxytryptamine) transporter, encoded by the SLC6A4 gene on chromosome 17q11.1-q12, is the cellular reuptake site for serotonin and a site of action for several drugs with central nervous system effects, including both therapeutic agents (e.g. antidepressants) and drugs of abuse (e.g. cocaine). It is known that the serotonin transporter plays an important role in the metabolic cycle of a broad range of antidepressants, antipsychotics, anxiolytics, antiemetics, and antimigraine drugs. The identification and characterization of variations that increase the response to common medications is a challenging and increasingly important task with regard to prediction of drug response. Therefore, the aim of this study was to establish a high-throughput single nucleotide polymorphism (SNP) screening method for two polymorphisms in the serotonin transporter gene, focusing on the SLC6A4 variations rs140701 and rs2066713.


METHODS
We developed a classical restriction fragment length polymorphism (RFLP) PCR protocol as a reference, followed by a new protocol established for the LightCycler real-time PCR method. To validate the method, the allele frequencies in 169 individuals (112 women, 57 men) were determined and compared with published data. The population was divided into two groups: one group comprised 87 individuals with various mental disorders and the other consisted of 82 healthy persons.


RESULTS
No difference was found in the prevalence of the two SNPs between the two populations. Subsequently, the determined allele frequencies were compared with previously published data. We found that 68% of the whole study population (groups I and II) carried a mutated allele of the rs140701 variation. With regard to the rs2066713 polymorphism, we found an allele frequency of 61% in the population. Both results are consistent with published data.


CONCLUSIONS
The developed protocol for RT-PCR analysis of both variations turned out to be reliable and economical, and thus suitable for routine laboratory use.
Eidens
Moritz
M
PharmGenomics GmbH, Mainz, Germany. moritz.eidens@gmail.com
Weise
Alexander
A
Prause
Stefan
S
Dahmen
Norbert
N
Wunsch
Annette
A
Weber
Mathias Max
MM
Forst
Thomas
T
Pfützner
Andreas
A
D000328:Adult; D000483:Alleles; D005260:Female; D020022:Genetic Predisposition to Disease; D006801:Humans; D008297:Male; D008875:Middle Aged; D020641:Polymorphism, Single Nucleotide; D050486:Serotonin Plasma Membrane Transport Proteins / Q000235:genetics*
C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins