Discovery 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).