Genetic variation in the human dopamine transporter gene and its role in attention deficit hyperactivity disorder

Multiple lines of evidence suggest that alteration of dopaminergic signaling may contribute to Attention Deficit Hyperactivity Disorder (ADHD). While a variable number tandem repeat (VNTR) in the 3' untranslated region of the human dopamine transporter (hDAT) gene has been found to be associated with ADHD, the functional relevance of the VNTR is uncertain. Although the hDAT coding region has been screened in a limited manner leading to the identification of 5 nonsynonymous mutations (V55A, R237Q, V382A, A559V, and E602G), no large-scale screen had been conducted on the hDAT gene for genetic variation within the coding region in ADHD subjects. To determine whether ADHD is a disorder enriched for functional hDAT coding variants, I established methods to identify genetic variation in hDAT exons and screened DNA from over 100 children with ADHD. In a screen of almost 425,000 base pairs of genomic DNA, I identified 22 different variants, including 5 novel variants and one nonsynonymous mutation. The nonsynonymous mutation, A559V, was identified in siblings that both suffered from ADHD. Interestingly, an independent group had previously identified A559V in a subject with bipolar disorder, a disorder known to exhibit comorbidity with ADHD. To investigate whether A559V, or the four previously identified nonsynonymous mutations in hDAT (R237Q, V55A, V382A, E602G) impact hDAT protein function, wild-type and mutant hDAT proteins were expressed in heterologous cell models. With the exception of V382A, the hDAT mutants exhibited equivalent transport, membrane protein expression, sensitivity to inhibitors, and modulation by signaling pathways as wild-type hDAT. V382A, on the other hand, exhibited decreased dopamine and norepinephrine transport, membrane protein expression, and altered regulation by the phorbol ester, PMA. Results from experiments with V382A suggest that this mutant may be stabilized in a transport inactive state at the plasma membrane following PMA treatment. Overall, the data suggests that rare hDAT genetic variants can impact protein function and perhaps contribute to psychiatric disorders such as ADHD.