After 10-weeks of Ins treatment, there has been a significant decrease in seizure activity that may be related to increased brain Ins levels. Using candidate gene approach and complete exome sequencing, we identified in two unrelated patients with autism spectrum disorders (ASD) and isolated low serotonin in brain-as reflected by the serotonin endmetabolite 5-hydroxindolacetic acid in CSF-a combination of heterozygous non-synonymous mutations in serotonin-related and/or autismassociated genes, including SLC29A4, SLC6A4, ITGB3, and others. Besides the previously reported codon-alterations from association studies, we newly identified in the brain monoamine transporter gene SLC29A4, encoding a putative serotonin-reuptake transporter protein PMAT, the alterations p.A138T or p.D326E with reduced activity. DNA sequencing of SLC29A4/PMAT in 125 individuals with ASD and 300 unaffected (control) subjects revealed the non-synonymous heterozygous alteration p.M24L or p.D29G in 7 additional subjects with ASD (and not in 300 control subjects). According to a hypothetical "genetic accumulationmodel" where the affected subjects must differ from non-affected family members (parents and siblings) in the sum of all mutations, the two ASD patients showed additional alterations in mainly serotonin homeostasis, but also other candidate genes that have previously been linked to ASD and/or intellectual disability. Our findings link a combination of mutations in several serotonin-related genes to ASD and mental retardation concomitant to low serotonin in CNS. Metabolomics refers to the comprehensive analysis of small organic molecules from biological fluids or tissues. The two main techniques used in metabolomics are (i) proton NMR spectroscopy (H-NMRS) and (ii) mass spectrometry (MS) coupled to liquid chromatography (LC) or gas chromatography.