Objectives: To identify ASD genetic risk factors and that subset acting to influence development and regulation of the serotonin system as evidenced by high levels of 5HT in ~35% of cases.
Methods: We assembled a wide range of genetic and statistical techniques to analyze our ACE cohort, in addition to larger existing samples that permit identification of risk loci using novel approaches with greater power. We recruited, phenotyped and determined blood [5HT] for more than 250 families. Genotyping and Sanger sequencing was conducted for loci most central to serotonin regulation. Select families with hyperserotonemic probands were used for whole exome sequencing (WES) to reveal novel de novo and inherited variants that in turn point to networks or pathway relationships, and specific molecules that contribute to both hyperserotonemia and ASD risk. Novel SERT coding variants were functionally assayed to test for effects on activity and regulation. We used larger datasets to test for potential enrichment of (expression) eQTLs and (methylation) mQTLs amongst the most associated loci, along with recent efforts to test for the ability of common variation to account globally for ASD liability and eventually variance in blood serotonin levels. CNV analyses were conducted by genotyping probands on high-density Illumina SNP arrays, and CNV prediction conducted using CNVision, which combines multiple algorithjms to increase predictive value of CNV calls. Putative CNVs were validated by qPCR in the entire family to determine de novo or inherited status.
Results: We detected two novel functional coding variants in SERT, one of which displayed elevated activity and related phosphorylation and constitutive activation of p38MAP kinase, a critical regulator of SERT. Another variant showed hypomorphic function. Analysis of de novo and inherited variants from WES of hyperserotonemic families has putatively identified molecular pathways such as integrin signaling, extracellular matrix (ECM)-receptor interactions, and focal adhesion as important functions contributing to serotonin-related risk factors in ASD. Numerous CNVs have been identified, and these both reinforce known risk loci and nominate new genes as being ASD risk factors. Using large ASD datasets, we observed a significant enrichment of brain eQTLs amongst the most ASD-associated genes.
Conclusions: The sum of our findings continues to highlight the 5HT system as being an important in harboring ASD risk loci, and that discovery from exome sequence and other data will permit us to better understand ASD risk factors, their interrelationships and that subset that relates to the role of dysregulated 5HT signaling in ASD risk.