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, 137 (Pt 4), 1107-19

Dopamine Transporter Deficiency Syndrome: Phenotypic Spectrum From Infancy to Adulthood

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Dopamine Transporter Deficiency Syndrome: Phenotypic Spectrum From Infancy to Adulthood

Joanne Ng et al. Brain.

Abstract

Dopamine transporter deficiency syndrome due to SLC6A3 mutations is the first inherited dopamine 'transportopathy' to be described, with a classical presentation of early infantile-onset progressive parkinsonism dystonia. In this study we have identified a new cohort of patients with dopamine transporter deficiency syndrome, including, most significantly, atypical presentation later in childhood with a milder disease course. We report the detailed clinical features, molecular genetic findings and in vitro functional investigations undertaken for adult and paediatric cases. Patients presenting with parkinsonism dystonia or a neurotransmitter profile characteristic of dopamine transporter deficiency syndrome were recruited for study. SLC6A3 mutational analysis was undertaken in all patients. The functional consequences of missense variants on the dopamine transporter were evaluated by determining the effect of mutant dopamine transporter on dopamine uptake, protein expression and amphetamine-mediated dopamine efflux using an in vitro cellular heterologous expression system. We identified eight new patients from five unrelated families with dopamine transporter deficiency syndrome. The median age at diagnosis was 13 years (range 1.5-34 years). Most significantly, the case series included three adolescent males with atypical dopamine transporter deficiency syndrome of juvenile onset (outside infancy) and progressive parkinsonism dystonia. The other five patients in the cohort presented with classical infantile-onset parkinsonism dystonia, with one surviving into adulthood (currently aged 34 years) and labelled as having 'juvenile parkinsonism'. All eight patients harboured homozygous or compound heterozygous mutations in SLC6A3, of which the majority are previously unreported variants. In vitro studies of mutant dopamine transporter demonstrated multifaceted loss of dopamine transporter function. Impaired dopamine uptake was universally present, and more severely impacted in dopamine transporter mutants causing infantile-onset rather than juvenile-onset disease. Dopamine transporter mutants also showed diminished dopamine binding affinity, reduced cell surface transporter, loss of post-translational dopamine transporter glycosylation and failure of amphetamine-mediated dopamine efflux. Our data series expands the clinical phenotypic continuum of dopamine transporter deficiency syndrome and indicates that there is a phenotypic spectrum from infancy (early onset, rapidly progressive disease) to childhood/adolescence and adulthood (later onset, slower disease progression). Genotype-phenotype analysis in this cohort suggests that higher residual dopamine transporter activity is likely to contribute to postponing disease presentation in these later-onset adult cases. Dopamine transporter deficiency syndrome remains under-recognized and our data highlights that dopamine transporter deficiency syndrome should be considered as a differential diagnosis for both infantile- and juvenile-onset movement disorders, including cerebral palsy and juvenile parkinsonism.

Keywords: SLC6A3; dopamine; dopamine transporter (DAT); dystonia; juvenile; parkinsonism.

Figures

Figure 1
Figure 1
Family tree for Cases 1–3. Four generation family tree representing family members and ancestral relatives of Patients 1 (IV:8), 2 (IV:5) and 3 (IV:4). These brothers are three of eight children from Pakistani consanguineous first cousin parents. They have five healthy unaffected sisters. The parents’ (III:3 and III:4) fathers (II:2 and II:3) are brothers. Females are represented by circles and males by squares. DTDS disease status is indicated by black shading. Consanguinity is represented by a horizontal parallel double bar. Diagonal lines indicate deceased individuals (I:1 and I:2). Genotypes are indicated for SLC6A3 mutation c.914C>T (p. Ala314Val) and indicate that parents (III:3 and III:4) are both heterozygous carriers (CT), the affected children (IV:4 IV:5 IV:6) are homozygous for the mutation (TT) and the unaffected siblings are either wild-type (CC) (IV:6 and IV:7) or heterozygous carriers (CT) (IV:1 IV:2 and IV:3).
Figure 2
Figure 2
Immunoblotting studies of mutant human DAT. LLC-PK1 cells transient transfected with indicated human DAT constructs were subjected to cell surface biotinylation. Biotinylated proteins (A, top) and whole cell lysates (A, bottom) were analysed by western blotting with antibodies against DAT (A) and β-actin (B). Equal amounts of total lysate protein were loaded for each mutated human DAT as for wild-type examined in the same experiment. There were four sets of experiments with their own wild-type (WT) controls, performed at different points in time, on Tyr470Ser, Gly386Arg, Arg445Cys/Arg85Leu/Arg445Cys-Arg85Leu, and Ala314Val.
Figure 3
Figure 3
Amperometry studies for human DAT variant Arg445Cys. (A) HEK293 cells expressing human DAT or human DATArg445Cys were loaded with dopamine (2 mM) by a whole-cell patch clamp pipette. Amphetamine (10 µM) induces a robust dopamine release from human DAT cells (quantified by amperometric current). Amphetamine fails to induce dopamine release from human DAT Arg445Cys cells (*P < 0.05 Student’s t-test human DAT versus human DAT Arg445Cys, n = 4). (B) The DAT inhibitor cocaine (COC; 10 µM) does not alter the amperometric current signal in human DAT nor human DAT Arg445Cys (n = 3), indicating that there is not a constitutive DAT-mediated dopamine efflux.
Figure 4
Figure 4
Schematic representation of the dopamine transporter. The DAT consists of 12 transmembrane domains (TM1–12) connected by extracellular (EC) and intracellular (IC) loops, some of which include helical portions (e2, e3, e4a, e4b, and i1, i5, respectively). Amino acids are indicated by white circles and mutated amino acids by red circles. All amino acids subject to missense mutation are identified: Arg85 (R) is shown at transmembrane domain 1 b, Gly386 (G), in the e4b portion of EC loop 4, Ala314 (A) at domain 6 a, Arg445 (R) at the bottom of TM9 and Try470 (Y) at the top of TM10.
Figure 5
Figure 5
Structural homology modelling of DAT based on LeuT. Transmembrane domains and loops are in grey; mutated amino acids affected by missense mutations in yellow and bound ions in purple (Na+) and green (Cl).

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