Candidate gene and genome-wide association studies (GWAS) have identified genetic variants that modulate risk for human disease; many of these associations require further study to replicate the results. Here we report the first large-scale application of the phenome-wide association study (PheWAS) paradigm within electronic medical records (EMRs), an unbiased approach to replication and discovery that interrogates relationships between targeted genotypes and multiple phenotypes. We scanned for associations between 3,144 single-nucleotide polymorphisms (previously implicated by GWAS as mediators of human traits) and 1,358 EMR-derived phenotypes in 13,835 individuals of European ancestry. This PheWAS replicated 66% (51/77) of sufficiently powered prior GWAS associations and revealed 63 potentially pleiotropic associations with P < 4.6 × 10−6 (false discovery rate < 0.1); the strongest of these novel associations were replicated in an independent cohort (n = 7,406). These findings validate PheWAS as a tool to allow unbiased interrogation across multiple phenotypes in EMR-based cohorts and to enhance analysis of the genomic basis of human disease.

The promise of “personalized medicine” guided by an understanding of each individual’s genome has been fostered by increasingly powerful and economical methods to acquire clinically relevant features. We describe operational implementation of prospective genotyping linked to an advanced clinical decision support system to guide individualized healthcare in a large academic health center. This approach to personalized medicine includes patient and healthcare provider engagement, identifying relevant genetic variation for implementation, assay reliability, point-of-care decision support, and necessary institutional investments. In one year, approximately 3,000 patients, most scheduled for cardiac catheterization, were genotyped on a multiplexed platform including CYP2C19 variants that modulate response to the widely-used antiplatelet drug clopidogrel. These data are deposited into the Electronic Medical Record and point-of-care decision support is deployed when clopidogrel is prescribed for those with variant genotypes. The establishment of programs such as this is a first step toward implementing and evaluating strategies for personalized medicine.

Efflux of dopamine through the dopamine transporter (DAT) is critical for the psychostimulatory properties of amphetamines, but the underlying mechanism is unclear. Here we show that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) plays a key role in this efflux. CaMKIIalpha bound to the distal C terminus of DAT and colocalized with DAT in dopaminergic neurons. CaMKIIalpha stimulated dopamine efflux via DAT in response to amphetamine in heterologous cells and in dopaminergic neurons. CaMKIIalpha phosphorylated serines in the distal N terminus of DAT in vitro, and mutation of these serines eliminated the stimulatory effects of CaMKIIalpha. A mutation of the DAT C terminus impairing CaMKIIalpha binding also impaired amphetamine-induced dopamine efflux. An in vivo role for CaMKII was supported by chronoamperometry measurements showing reduced amphetamine-induced dopamine efflux in response to the CaMKII inhibitor KN93. Our data suggest that CaMKIIalpha binding to the DAT C terminus facilitates phosphorylation of the DAT N terminus and mediates amphetamine-induced dopamine efflux.

Plasmalemmal neurotransmitter transporters (NTTs) regulate the level of neurotransmitters, such as dopamine (DA) and glutamate, following their release at brain synapses. Stimuli including protein kinase C (PKC) activation can lead to the internalization of some NTTs and a reduction in neurotransmitter clearance capacity. We find that the protein Flotillin-1/Reggie-2 (Flot1) is required for PKC-regulated internalization of members of two different NTT families, the DA transporter (DAT) and the glial glutamate transporter EAAT2, and we have identified a conserved serine residue in Flot1 that is essential for transporter internalization. Further analysis revealed that Flot1 is also required to localize DAT within plasma membrane microdomains in stable cell lines, and is essential for amphetamine-induced reverse transport of DA in neurons but not for DA uptake. In sum, our findings provide evidence for a critical role of Flot1-enriched membrane microdomains in PKC-triggered DAT endocytosis and the actions of amphetamine.