The variable number of tandem repeats element in DAT1 regulates in vitro dopamine transporter density.
BACKGROUND
A 40-bp variable number of tandem repeats (VNTR) polymorphism exists in the 15th exon of DAT1, the gene encoding the human dopamine transporter (DAT). Though the VNTR resides in a region encoding the 3' untranslated region (UTR) and does not alter the protein's amino acid sequence, the prevalent 10-repeat variant has shown both linkage and association to Attention Deficit Hyperactivity Disorder (ADHD). In this study, we examined the effects of the DAT1 VNTR on measures of in vitro DAT expression and pharmacology. A series of four DAT1 constructs, each containing the DAT1 coding region, but varying with respect to the downstream presence or content of the 3'UTR, were engineered and stably transfected into an HEK-293 variant using Flp-In integration, an enzyme-mediated, site-specific recombination technology.


RESULTS
[3H] Win 35,428 saturation binding assays and DAT immunoblots revealed statistically significant differences in DAT expression attributable to DAT1 genotype. Cells harboring the 10-repeat DAT1 variant were characterized by a Bmax approximately 50% greater than cells with the 9-repeat VNTR; those containing only the DAT1 coding region or the coding region flanked by a truncated 3' UTR resulted in greater DAT density than either of the naturalistic 9- and 10-repeat variants. Competition binding assays showed no statistically significant DAT1 genotype effects on the DAT affinity for methylphenidate, a finding consistent with the positional location of the VNTR.


CONCLUSIONS
This study identified the DAT1 VNTR as a functional polymorphism and provides an interpretive framework for its association with behavioral phenotypes.
VanNess
Sidney H
SH
Laboratory of Biological Psychopathology, Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Atlanta, Georgia, USA. svannes@emory.edu
Owens
Michael J
MJ
Kilts
Clinton D
CD
D001289:Attention Deficit Disorder with Hyperactivity / Q000235:genetics; D001667:Binding, Competitive; D002460:Cell Line; D004254:DNA Nucleotidyltransferases; D050483:Dopamine Plasma Membrane Transport Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D005786:Gene Expression Regulation / Q000235:genetics; D005818:Genetic Engineering; D005822:Genetic Vectors; D005838:Genotype; D006801:Humans; D018598:Minisatellite Repeats / Q000502:physiology*; D010641:Phenotype; D011110:Polymorphism, Genetic; D014162:Transfection
D050483:Dopamine Plasma Membrane Transport Proteins; C493214:SLC6A3 protein, human; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase