Rare coding variants of the adenosine A3 receptor are increased in autism: on the trail of the serotonin transporter regulome

Abstract

Background: Recent discoveries highlight rare genetic variation as an important class of autism spectrum disorder (ASD) risk factors, and that such variants can implicate biological networks for further investigation. Altered serotonin (5-HT) signaling has been implicated in ASD for over 50 years, and we and others have identified multiple, rare, ASD-associated variants in the 5-HT transporter (SERT, SLC6A4) gene that lead to elevated 5-HT re-uptake and perturbed regulation. We hypothesized that loci encoding SERT regulatory proteins harbor genetic variants that impact SERT function and/or regulation and therefore could contribute to ASD risk. The adenosine A3 receptor (A3AR) regulates SERT via protein kinase G (PKG) and p38 mitogen-activated protein kinase (p38 MAPK) signaling pathways leading to enhanced SERT surface expression and catalytic activity, respectively. Methods: To test our SERT network hypothesis, we asked whether a relative increase of rare functional variants in the A3AR gene (ADORA3) was present in cases vs. controls. Discovery Sanger sequencing of ADORA3 exons and flanking DNA in a case-control sample, and subsequent analysis of a comparison sample using whole exome sequence data were conducted to test for increased functional variants in cases. We evaluated the functional impact of two variants from the discovery sample on A3AR signaling and SERT activity. Results: Sequencing discovery showed an overall increase in rare coding variants in cases vs. controls (P=0.013). While a comparison sample from exome sequence did not show a significant enrichment (P=0.071), combined analysis strengthened evidence for association of rare, functional variants in ASD (P=0.0025). Two variants discovered in ASD cases (Leu90Val and Val171Ile) lie in or near the ligand-binding pocket, and Leu90Val was enriched individually in cases (P=0.040). In vitro analysis of cells expressing Val90 A3AR revealed elevated basal cGMP levels compared with cells expressing the wildtype receptor. Additionally, the specific A3AR agonist N6-(3-iodobenzyl)-N-methyl-5'-carbamoyladenosine (IB-MECA) induced increased cGMP levels across the full time course studied in Val90 A3AR cells, as compared with the wildtype receptor expressing cells. In Val90 A3AR/SERT co-transfected cells, IB-MECA stimulation elevated SERT activity over that seen with the wildtype receptor, with a delayed recovery of 5-HT uptake activity to baseline levels. By comparison, the Ile171 A3AR variant was unable to support IB-MECA stimulation of SERT. Although both Val90 and Ile171 were present in greater numbers in these ASD cases, segregation analysis in carrier families showed incomplete penetrance, consistent with other documented rare ASD risk alleles. Conclusions: Our results validate the hypothesis that the SERT regulatory network harbors rare, functional variants that impact SERT activity and regulation in ASD, and encourages further investigation of this network as a site for additional functional variation that may impact ASD risk.