Figure 2From: Autism spectrum disorder associated with low serotonin in CSF and mutations in the SLC29A4 plasma membrane monoamine transporter (PMAT) geneDiscovery and functional characterization of PMAT mutations from ASD patients. Out of 248 patients diagnosed with ASD and normal or isolated low 5HIAA in the CSF, 8 patients were carrying a heterozygous mutation in the SLC29A4 gene encoding for PMAT: c.86A > G/p.D29G in 2 subjects, c.412G > A/p.A138T in 5 subjects, and c.978 T > G/p.D326E in 1 subject, indicating a cumulative prevalence of 3.2% in our analyzed cohort of ASD patients (Additional file 4: Figure S1). (a) Cell surface biotinylation and Western blot analysis demonstrated comparable plasma membrane expression of PMAT-wildtype (wt), PMAT-D29G, PMAT-A138T, and PMAT-D326E proteins. Empty pEYFP-C1 vector was transfected into MDCK cells to generate the control cell line. (b) Confocal microscopy imaging of MDCK cells expressing PMAT-wt, PMAT-D29G, PMAT-A138T, PMAT-D326E, and empty pEYFP-C1 vector. The three mutated PMAT proteins revealed normal localization in the plasma membrane similar to PMAT-wt. Scale bars: 20 μm. (c) Serotonin (5-HT), dopamine and 1-methyl-4-phenylpyridinium (MPP+) transport activity were significantly reduced in MDCK cells transfected with PMAT-A138T and PMAT-D326E. For all the transport activity experiments, the values are indicated in mean ± SD from three independent experiments (n = 3) with different cell passages. For each experiment, uptake was carried out in triplicates on the same plate. Significant difference from the corresponding value for PMAT-wt (100% transport activity) is indicated by asterisks: *, P < 0.05 and **, P < 0.01 (Student’s t-test).Back to article page