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De Novo Mutation of the Dopamine Transporter (DAT) Gene in Autism Reveals a Novel Component of ASD Pathogenesis

Friday, 3 May 2013: 09:00-13:00
Banquet Hall (Kursaal Centre)
N. G. Campbell, P. J. Hamilton, K. B. Erreger, A. N. Belovich, H. Matthies, A. Galli and J. S. Sutcliffe, Molecular Physiology & Biophysics and Vanderbilt Brain Institute, Vanderbilt University, Nashville, TN
Background:  Risk for ASD is largely genetically determined, however, ASD genetic architecture is highly complex. Studies focusing on rare DNA copy number variation (CNV) point to de novo mutation as one important class of genetic liability. Recently, whole exome sequencing (WES) has become a focus of genetic studies in ASD, has implicated numerous genes and strongly underscored the extreme heterogeneity of ASD risk. Several groups have conducted WES on large case-control samples and families. Among the first de novo coding mutations (DNMs) identified by the NIH ARRA Autism Sequencing Consortium was a missense substitution (T356M) in the SLC6A3gene encoding the dopamine (DA) transporter (DAT). DAT functions presynaptically to uptake DA released into the synapse, thus regulating synaptic [DA] and signaling to postsynaptic receptors. Previous studies have documented association of a rare, functional DAT variant with ADHD, which co-occurs in ~40% of people with ASD.

Objectives:  To characterize functional impact caused by T356M, we sought to use both in vitro and in vivo systems to evaluate its effect on DAT function and regulation.  

Methods:  Wildtype and T356M mutant DAT expression constructs were transfected into heterologous Chinese hamster ovary (CHO) cells to measure DA uptake and, using the electrophysiological technique amperometry with patch-clamped cells, the ability of DAT to efflux DA into the extracellular space when treated with amphetamine (AMPH); DA efflux is a property of the wildtype transporter. In vivo analysis of wildtype and T356M DAT employed a “humanized” Drosophila harboring the WT (or knock-in mutant) human gene replacing the endogenous gene. Initial studies focused on locomotion in these models.

Results:  Significant cross-species conservation of the T356 residue and use of prediction algorithms suggested that this missense variant would have a damaging effect on the protein. Expression of T356M DAT in CHO cells revealed a substantial reduction, near absence, of DAT-dependent DA uptake relative to wildtype (p<0.001). Remarkably, application of 10 µM Zn2+ partially restored reuptake activity of T356M DAT compared with mutant DAT expressing cells without Zn2+ supplementation. 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. Lastly, Drosophila containing the T356M knock-in mutation, compared with those containing wildtype DAT, show significantly increased basal locomotion across the 12-hour light cycle (p<0.05). This behavior is similar to the hyperactivity observed in DAT null Drosophila.

Conclusions:  We have characterized a novel DNM affecting 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 DNM 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. 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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