De novo mutation in the dopamine transporter gene associates dopamine dysfunction with autism spectrum disorder. NG. Campbell1, PJ. Hamilton1, S. Sharma2, K. Erreger2, FH. Herborg3, C. Saunders4, AN. Belovich4, EH. Cook5, U. Gether3, HS. Mchaourab2, HJ. Matthies2, A. Galli1,2,4, JS. Sutcliffe1,2,6 1) Center for Molecular Neuroscience, Vanderbilt Univ, Nashville, TN; 2) Mol. Physiol. and Biophysics, Vanderbilt Univ, Nashville, TN; 3) Neuroscience and Pharmacology, Univ. of Copenhagen, Copenhagen, Denamrk; 4) Pharmacology, Vanderbilt Univ, Nashville, TN; 5) Psychiatry, Univ. of Illinois at Chicago, Chicago, IL; 6) Psychiatry, Vanderbilt Univ, Nashville, TN.
Risk for autism spectrum disorder (ASD) is largely genetically determined, however, its etiology is highly complex. Studies focusing on rare DNA copy number variation (CNV) point to de novo mutation as one significant class of genetic liability and recent whole exome sequencing studies has supported a role for coding de novo mutations (DNMs). Among the first DNMs identified by the NIH ARRA Autism Sequencing Consortium was a missense substitution (T356M) in the SLC6A3 gene encoding the dopamine (DA) transporter (DAT). DAT is a presynaptic regulator of dopaminergic tone in the central nervous system by mediating the high-affinity re-uptake of synaptically released DA. Here, we report the first functional, structural, and behavioral characterization of an ASD associated DNM in the dopamine transporter. Expression of T356M DAT in CHO cells revealed a near absence of DAT-dependent DA uptake relative to wildtype (p<0.001). Patch-clamp experiments pre-loading cells with DA revealed significantly diminished AMPH-induced DA efflux by T356M DAT relative to wildtype (p<0.01). Most notably, amperometry experiments revealed that mutant DAT constitutively leaks DA from the cell under basal conditions, contrary to wildtype transporter. In the bacterial homolog leucine transporter, substitution of A289 (the homologous site to T356) with a Met, promotes an outward-facing conformation upon substrate binding, a conformation required for substrate efflux. Lastly, Drosophila containing the T356M knock-in mutation demonstrates significantly increased basal locomotion, a trait associated with DA dysfunction and ASD (p<0.05). In conclusion, we have characterized a novel de novo mutation in DAT that demonstrates profound functional abnormalities. Given the powerful constitutive efflux of DA and virtual absence of DA uptake activity caused by the mutation, we consider it likely that this variant is a significant ASD risk factor. Taken together with prior association between abnormal DAT function and ADHD, these observations may provide a link between ASD risk and pathophysiology and ADHD, which co-occurs in ~40 percent of subjects with ASD. These studies also more broadly implicate altered regulation of DA homeostasis as a potential mechanism underlying part of the overall liability to ASD.
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