The main findings of this study are as follows. (i) Triallelic non-synonymous variation (rs35062132, c.1126C>G/T; R376G/C) is observed in both ASD patients and healthy controls in a Japanese population; the frequencies of the G allele are significantly higher in the ASD patients than in healthy controls. (ii) In HEK-293 cells expressing hOXTR-376G, the agonist-induced internalization and recycling of the OXTR are faster than that in the cells expressing hOXTR-376C or hOXTR-376R. (iii) In both HEK-293 cells and NG108-15 neuronal cells, the agonist-induced increase in [Ca2+]i mediated by hOXTR-376G-EGFP or hOXTR-376C-EGFP is smaller than the increase associated with hOXTR-376R-EGFP. These results provide new insights into the genetic architecture and therapeutic aspects of ASDs.
The present study is unique in associating non-synonymous allelic variations of the OXTR gene with ASDs. These variations at rs35062132 have not been reported in other disorders. To date, several studies have found ASD-associated SNPs in the OXTR gene, which reside in introns (rs4564970 , rs237897 , rs53576 [23, 24], rs2254298 [23–25], rs2268493 , rs7632287 , rs11720238 ), 3'-untranslated region (rs1042778) , and intergenic regions (rs7632287 and rs11720238) . The functional consequences of these SNPs remain unclear. Importantly, all minor alleles of these SNPs have a frequency more than 5%, and thus can be classified as common variations. In contrast, the frequencies of the G and T alleles of rs35062132 are 0.4% and 0.6%, respectively, in controls; these minor alleles can be categorized as rare [43–45]. Therefore, most previous studies favor the common disease–common variant model, in which most of the risk is caused by common genetic variations (>5% allele frequency), each allele conferring a slight risk [43–47]. By contrast, our results support the common disease–rare variant model, in which the risk is mostly attributable to rare variations (<5% allele frequency), each variant conferring a moderate but readily detectable increase in the relative risk [43–47]. In agreement with this model, we observed that the minor alleles clearly affected OXT-induced cellular responses. Future studies will be conducted to test whether behavioral changes caused by these variations are moderate and reasonably deleterious. Also it will be interesting to examine whether the rs35062132 variations cosegregate with the risk alleles in the non-coding region [22–24, 41, 42].
The limitation of this study is that sample size is not sufficient to analyze the rare variant alleles at rs35062132. Future studies with larger sample size or family-based association testing are necessary to conclude that the G allele of the rs35062132 is a genetic risk factor for ASDs. The result reported here should be replicated in independent populations with various ethnic backgrounds.
The amino acid variation R376G/C is located in the intracellular carboxy (C)-terminal tail of the receptor protein, which is critical for desensitization, internalization, and recycling of the OXTR, as in many other GPCRs [9–13]. Arg376 is associated with two structural components involved in these processes: (i) Arg376 is a part of a PKC consensus motif (Ser374-His375-Arg376; Figure 1A), which is thought to interact with PKC after OXT stimulation ; and (ii) Arg376 is in conjunction with one of the two Ser triplets in the C-terminus (Figure 1A) that are primary sites of agonist-induced phosphorylation and β-arrestin-2 binding . When either one of the two Ser triplets was mutated to alanine residues, the stability of the β-arrestin-2-OXTR complex was altered, and the ability of β-arrestin-2 to internalize with the receptor was eliminated . In the vasopressin receptor V2, which is highly homologous to OXTR in structure, the equivalent Ser triplets function as a retention motif. GRK phosphorylation of these Ser residues promotes the formation of stable receptor-β-arrestin complexes, followed by cotrafficking and colocalization of the complexes to endocytotic vesicles and slow recycling to the cell surface . Therefore, it is suggested that the R376G substitution in the OXTR might suppress the phosphorylation of Ser residues in the Ser triplets or reduce the stability of β-arrestin binding to the triplets, resulting in faster receptor recycling.
We demonstrated that, both in HEK-293 cells and in NG108-15 neuronal cells, agonist-induced [Ca2+]i elevation mediated by variant receptors, hOXTR-376G-EGFP and hOXTR-376C-EGFP, is smaller than that by the common receptor hOXTR-376R-EGFP. Despite the marked reduction in the peak [Ca2+]i, the decrease in IP3 levels was slight. Provided that Arg376 is not located in the known site for Gq binding (Figure 1A) , the Gq/11/PLCβ/IP3-independent, unidentified signaling pathway for [Ca2+]i elevation might be affected by the R376G/C substitution.
Ca2+ signaling pathways in neurons have been implicated in the pathogenesis of ASDs . However, the involvement of OXTR-mediated, PLC/IP3-dependent Ca2+ signaling in ASDs is still largely unknown. Recently, Ninan  has demonstrated that U73122, a PLC inhibitor, reduced OXT-induced suppression of glutamatergic synaptic transmission in pyramidal neurons of the medial prefrontal cortex, possibly by inhibiting PLC-dependent increase in postsynaptic calcium. Given that this process is critical for the OXT effects on social cognition, it is conceivable that the OXTR variations may serve as a risk factor for ASDs.
We have previously demonstrated that cADPR, which may act as an intracellular second messenger downstream of the OXTR, activates Ca2+ release from intracellular stores through the ryanodine receptor Ca2+ release channel. cADPR also initiates Ca2+ influx through melastatin-related transient receptor potential 2 (TRPM2) channels . Our previous SNP analysis has suggested that R140W substitution of CD38 protein, a regulator of ryanodine receptor-mediated Ca2+-induced Ca2+ release for OXT secretion , could be a potential risk factor for a subset of Japanese ASD patients . Although this study shares a part of the ASD patients with our previous analysis , the 140W allele of CD38 has not been found in the patients carrying the OXTR-376G or OXTR-376C until now (unpublished data). It is therefore conceivable that the underlying signaling process affected by the R376G/C substitution is independent of the CD38-mediated OXT release from hypothalamic neurons .
Besides their relevance to the etiology of ASDs, the results presented here might contribute to the development of new pharmacological treatments. Genetic polymorphisms of many drug targets predict responsiveness to drugs [52, 53]. It is likely that the alteration in the cellular functions mediated by the variant OXTRs could cause some individual differences in both behavioral and non-behavioral responses to OXT. Although no side effects specific to intranasal OXT administration have been reported , new clinical information regarding functional OXTR variants will be helpful in the determination of individual administration protocols to maximize therapeutic benefit with least adverse effects.