Non-synonymous single-nucleotide variations of the human oxytocin receptor gene and autism spectrum disorders: a case–control study in a Japanese population and functional analysis

We recruited 132 ASD subjects (102 males, 30 females; 15.9 ± 0.7 years) from the outpatient psychiatry department of the Kanazawa University Hospital. All subjects fulfilled the DSM-IV criteria for pervasive developmental disorder. The diagnoses were made by two experienced child psychiatrists through interviews and clinical record reviews as described previously [31], and the subjects had no apparent physical anomalies. The two experienced child psychiatrists independently confirmed the diagnosis of ASD for all patients by semi-structured behavior observation and interviews with the subjects and their parents. During each of these interviews with parents, which were helpful in the evaluation of autism-specific behaviors and symptoms, the examiners used one of the following methods: the Asperger Syndrome Diagnostic Interview [32], the Autism Diagnostic Interview-Revised (ADI-R)[33], the Pervasive Developmental Disorders Autism Society Japan Rating Scale [34], the Diagnostic Interview for Social and Communication Disorders [35], or the Tokyo Autistic Behavior Scale [36]. Based on these evaluation methods, 97 patients were classified as autistic disorder (autism), 10 as Asperger’s disorder, and 25 as pervasive developmental disorder not otherwise specified (PDD-NOS). The 248 controls (143 males, 105 females; 31.3 ± 0.6 years) were unrelated healthy Japanese volunteers. All patients and controls were Japanese with no non-Japanese parents or grandparents. This study was approved by the ethics committees of Kanazawa University School of Medicine. All examinations were performed after informed consent according to the Declaration of Helsinki.

Genomic DNA was extracted from venous blood samples using the Wizard Genomic DNA Purification kit (Promega, Madison, WI, USA), or from nails using the ISOHAIR DNA extraction kit (Nippon Gene, Tokyo, Japan). DNA fragments containing exons 3 and 4 of the hOXTR gene were amplified by PCR in a 20-μl reaction mixture containing 2.5 ng genomic DNA, 200 μM dNTPs, 200 nM of each primer, 4 μl Ampdirect G/C (Shimadzu, Kyoto, Japan), 4 μl Ampdirect-4 (Shimadzu), and 1 unit Ex Taq DNA polymerase (Takara Shuzo, Otsu, Japan). The amplification procedure consisted of denaturation at 96°C for 1 min, followed by 35 cycles of 96°C for 30 s, 65°C for 1 min, and 72°C for 1 min. The primers used were FWD1 or FWD4 and REV1 for exon 3, and FWD5 and REV3 for exon 4 (sequences are given in Table 1). After amplification, PCR products were purified with the High Pure PCR Cleanup Micro kit (Roche, Mannheim, Germany), subjected to cycle sequencing reaction (BigDye version 1.1; Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocol, and analyzed on an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). The primers used were FWD1–FWD5 and REV1–REV6 (Table 1).

Two primers, FWD8 and REV7 (Table 1), were designed to amplify a 138-bp product encompassing rs35602132 at the mRNA position 1748 (g.8734707; c.1126). Three custom-designed TaqMan minor groove binder (MGB) probes were obtained from Applied Biosystems: 376G-FAM and 376C-FAM targeted to the variant alleles, and 376R-VIC to the common allele (Table 1). PCR was performed in 20-μl mixtures containing 10 μl TaqMan universal master mix (Applied Biosystems), 200 nM of each primer, 100 nM 376R-FAM, 100 nM 376G-VIC or 376C-VIC, and 10 ng of sample DNA. Thermocycling was performed on the ViiA 7 Real-Time PCR System (Applied Biosystems). The amplification was conducted at 60°C for 30 s, 95°C for 5 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min. Data were analyzed with SDS2.3 software (Applied Biosystems).

cDNAs encoding hOXTR variants at amino acid residue 376 were generated by site-directed mutagenesis as described previously [37]. pCHOXTR [38] harboring hOXTR-376R, a common-type receptor, was used as a template for PCR; the primers used were R376G-FWD and R376G-REV (Table 1) for the R376G substitution, and R376C-FWD and R376C-REV (Table 1) for R376C. The amplified products were treated with EX Taq DNA polymerase (Takara Shuzo) at 72°C for 10 min and cloned into a pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA, USA) to yield pCHOXTR-376G and pCHOXTR-376C. The cDNA inserts were verified by nucleotide sequencing.

The 1.4-kb Eco RI fragments from pCHOXTR [38], pCHOXTR-376G, and pCHOXTR-376C were cloned into the Eco RI site of the mammalian expression plasmid pcDNA3.1 (+) (Invitrogen) to yield pcDNAHOXTR-376R, pcDNAHOXTR-376G, and pcDNAHOXTR-376C, respectively.

In some experiments, hOXTRs of the common type and variants were fused to the enhanced green fluorescent protein (EGFP). Expression plasmids for these fusion proteins were constructed essentially as described previously [38]. In brief, the 1.4-kb Eco RI fragments obtained from pCHOXTR [38], pcDNAHOXTR-376G, and pcDNAHOXTR-376C were subjected to PCR using primers EGFP-FWD and EGFP-REV (Table 1). The amplified fragments were cleaved with Pst I and Bam HI. The resulting 0.46-kb Pst I (1334)/Bam HI (primer) fragments and the 0.78-kb Bam HI (556)/Pst I (1334) fragment from pCHOXTR were ligated to Bam HI/Bgl II-cleaved pEGFP-N3 (Clontech) to yield pHOXTR-376R-EGFP, pHOXTR-376G-EGFP, and pHOXT-R376C-EGFP. Restriction endonucleasesites are identified by numbers (in parentheses; in accordance with the data deposited in GenBank under accession number NM_000916) indicating the 5'-terminal nucleotide generated by cleavage.

Human embryonic kidney HEK-293 cells and COS-7 cells were maintained in DMEM supplemented with 10% FBS at 37°C in a humidified atmosphere of 95% air and 5% CO₂. NG108-15 neuroblastoma × glioma hybrid cells were cultured as described previously [39]. Cells were grown in culture dishes to 80 to 90% confluence and transfected with expression plasmids using FuGENE HD Transfection Reagent (Roche) following the manufacturer’s instruction.

COS-7 cells transfected with pcDNAHOXTR-376R, pcDNAHOXTR-376G, pcDNAHOXTR-376C, or pcDNA3.1 (+) were collected 48 h after transfection, and resuspended in homogenization buffer (25 mM Tris, pH 7.4, 1 mM EDTA, 250 mM sucrose). The cells were homogenized and centrifuged at 1,500 g at 4°C for 10 min, to pellet nuclei and intact cells. The resulting supernatants were centrifuged at 40,000 g at 4°C for 30 min, and crude membrane pellets were suspended in binding buffer containing 50 mM HEPES (pH 7.4), 5 mM MgCl₂, 1 mM CaCl₂, and 0.2% BSA. Protein concentration was determined by the bicinchoninic acid assay (Pierce, Rockford, IL, USA) using BSA as a standard. [Tyrosyl-³H]-oxytocin ([³H]-OXT; PerkinElmer Life Sciences, Waltham, MA, USA) was diluted in the binding buffer to concentrations of 0.05 to 4 nM. Specific binding assays were performed in 5-μg cell membrane preparations incubated at room temperature for 1 h with increasing concentrations of [³H]-OXT, in the absence or presence of 200-fold excess of unlabeled OXT. Binding reaction mixtures were filtered through a GF/C glass fiber filter (Whatman, Maidstone, Kent, UK) and washed three times with Wash Buffer (50 mM HEPES, pH 7.4, 500 mM NaCl, 0.1% BSA). Radioactivity associated with membranes retained by glass filters was quantified in a liquid scintillation counter (LSC-5100; Aloka, Tokyo, Japan). Specific binding was calculated by subtracting non-specific binding in the presence of 200-fold excess of OXT at each radioligand concentration from total binding. Affinity (K_d) and maximal binding capacity (B_max) of saturation binding were obtained from saturation isotherm specific binding data by nonlinear regression curve analysis using the standard equation for a rectangular hyperbola fitted to one site with the GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA, USA). Competition binding experiments were performed in duplicate by incubating 5 μg of cell membranes at room temperature for 1 h, in the same medium with 1 nM [³H]-OXT and increasing concentrations of unlabeled OXT (10⁻¹² to 10⁻⁵ M). Log IC₅₀ values were derived from nonlinear least-squares analysis using the GraphPad Prism 5 software (GraphPad Software Inc.).

HEK-293 cells were plated on poly-D-ornithine-coated glass coverslips and cultured overnight. After transfection with pcDNAHOXTR-376R, pcDNAHOXTR-376G, or pcDNAHOXTR-376C, the cells were incubated at 37°C in serum-free DMEM for different times, in the absence or presence of OXT (100 nM). Reactions were stopped by removing the medium and fixing the cells with 4% paraformaldehyde at room temperature for 10 min. The cells were then blocked with PBS containing 1% BSA and 1.5% normal horse serum for 30 min, and incubated with goat anti-OXTR antibody (N-19; 1:200; sc-8103, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight. After washing in PBS, the cells were incubated with donkey anti-goat IgG (H + L) antibody conjugated with Alexa Fluor 488 (1:500; Invitrogen) combined with 4',6-diamidino-2-phenylindole (DAPI; 1 μg/ml; Dojindo, Kumamoto, Japan). In some experiments, pSNAP_f-ADRβ2 (New England Biolabs, Ipswich, MA, USA), an expression plasmid for β2 adrenergic receptor fused to SNAP_f, was cotransfected into HEK-293 cells. The cells were labeled with SNAP-Surface Alexa Fluor 488 (2 μM; New England Biolabs), fixed with 4% paraformaldehyde, and reacted with goat anti-OXTR antibody (N-19; 1:200; Santa Cruz) at 4°C overnight, followed by incubation with donkey anti-goat IgG (H + L) antibody conjugated with Alexa Fluor 594 (1:500; Invitrogen); none of the solutions contained any cell-permeabilizing reagents. Fluorescent images were obtained using confocal laser scanning microscopes (LSM5 Pascal; Carl Zeiss, Jena, Germany; FluoView FV10i, Olympus). Data were analyzed using the FV10-ASW software (Olympus).

We measured [Ca²⁺]_i using the fluorescent Ca²⁺ indicator fura-2-acetoxymethyl ester (fura-2/AM). HEK-293 cells or NG108-15 cells transfected with pHOXTR-376R-EGFP, pHOXTR-376G-EGFP, or pHOXTR-376C-EGFP were loaded with fura-2/AM to a final concentration of 3 μM in complete medium and incubated at 37°C. After 30-min loading, the cells were washed three times with HEPES-buffered saline (HBS) solution (145 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 20 mM HEPES-NaOH, 2 mM CaCl₂, 20 mM glucose, pH 7.4). Cells expressing EGFP-tagged OXTRs were visualized at a wavelength of 488 nm before OXT application. The fluorescence of the cells loaded with fura-2/AM was then measured at 37°C, at the determined sites, through a pinhole (10 to 20 μm in diameter). We used alternating excitation wavelengths of 340 and 380 nm in a Ca²⁺ microspectrofluorometric system (OSP-3 Model; Olympus, Tokyo, Japan), as described previously [39]. The Ca²⁺ emission was detected every 10 s for 5 min after OXT application. The ratio of fluorescence at 340 nm and 380 nm (F₃₄₀/F₃₈₀) was used to determine [Ca²⁺]_i. All data were normalized to the baseline fluorescence (F₀) recorded 10 s before OXT addition, and given as F/F_0.

HEK-293 cells were plated at a density of approximately 5 × 10⁴ per well in a 24-well plate, and transiently transfected with pcDNAHOXTR-376R, pcDNAHOXTR-376G or pcDNAHOXTR-376C, or pcDNA3.1(+). Before treatment with OXT, the transfected cells were washed and incubated in HBS containing 2 mM CaCl₂, 10 mM glucose, and 10 mM LiCl at 37°C for 10 min. After OXT treatment, the reaction was terminated at designated time points by the addition of 50 μl of 10% (w/v) perchloric acid. The acid-soluble extract was neutralized with 150 μl of 1.53 M KOH-75 nM HEPES, and perchloric acid was precipitated on ice and removed by a brief centrifugation. The IP₃ aqueous extract was then examined with an Inositol-1,4,5-triphosphate [³H]-Radio Receptor Assay Kit (PerkinElmer Life Sciences).

Data obtained from genetic studies were analyzed using a contingency table and the Fisher’s exact test (GraphPad Prism 5; GraphPad Software Inc.). All data from the in vitro studies are shown as mean ± standard error of the mean. Statistical significance was determined by the Student’s t test or two-way analysis of variance (ANOVA) using an interactive fitting program (GraphPad Prism 5; GraphPad Software Inc.); P values smaller than 0.05 were considered to be statistically significant.

As the entire amino acid sequence of the hOXTR is encoded by exons 3 and 4, as illustrated in Figure 1A, we initially re-sequenced both exons for genetic variations. Of 27 single-nucleotide variations listed in the NCI dbSNP database, we detected minor alleles of rs4686302, rs151257822, and rs35062132 in 59 ASD patients and 30 unrelated healthy Japanese volunteers (Figure 1A, Table 2). As c.1126 G>C (rs35062132; R376G) was detected in two ASD patients but not in controls (Table 2, Figure 1B), we selected rs35062132 for further analysis.

We genotyped an additional 73 ASD patients and 218 healthy volunteers for rs35062132 variation using the TaqMan-based real-time PCR method. In total, we obtained data from 132 ASD patients and 248 controls. Six individualsin the ASD group (4.5%) and two in the control group (0.8%) were identified as heterozygotes carrying the R376G variation (rs35062132; c.1126C>G). One person in the ASD group (0.8%) and three in the control group (1.2%) carried R376C (c.1126C>T; Table 3). No homozygous carriers for the minor alleles were observed. The χ² goodness-of-fit test showed that the genotype frequency distribution of rs35062132 did not deviate from Hardy-Weinberg equilibrium in the patient group (χ² = 0.098, P = 0.99) or the control group (χ² = 0.026, P = 1.00). The genotype and allele frequencies of rs35062132 obtained from all participants are summarized in Table 3. The C/G genotype was significantly correlated with an increased risk of ASDs (odds ratio (OR) = 5.83; 95% confidence interval (CI) = 1.16 to 29.3; P = 0.024, Fisher’s exact test; Table 3). Consistently, the G allele also showed a correlation with an increased likelihood of ASDs (OR = 5.73, 95% CI = 1.15 to 28.6; P = 0.024, Fisher’s exact test; Table 3). The frequencies of the C/T genotype and the T allele did not significantly differ between the ASD and control groups (Table 3).

To find out whether the amino acid changes at the residue 376 affect the receptor functions, we compared ligand-binding properties of the common and variant hOXTRs. In saturation binding and Scatchard analysis of ³[H]-OXT binding to membranes prepared from COS-7 cells transfected with hOXTR-expression plasmids, the variants hOXTR-376G and hOXTR-R376C, as well as the common-type hOXTR-376R, exhibited single high-affinity binding sites. Values of K_d were 0.90 ± 0.28, 1.1 ± 0.12, and 1.2 ± 0.19 nM (n = 15 in each group), and values of B_max were 3153 ± 531, 3202 ± 234, and 3120 ± 339 fmol/mg of protein (n = 15 in each group) for hOXTR-376R, hOXTR-376G, and hOXTR-376C, respectively. Competition binding experiments confirmed that the variant and common receptors did not differ significantly: values of IC₅₀ were 1.05 ± 0.47, 0.49 ± 0.23, and 0.71 ± 0.30 nM (n = 15 in each group) for hOXTR-376R, hOXTR-376G, and hOXTR-376C, respectively. These data indicate that both variations have no or little effect on OXT binding.

We then examined the agonist-induced internalization and recycling of hOXTRs in HEK-293 cells, which have been most extensively used for investigation of GPCR internalization and trafficking. HEK-293 cells transfected with an expression plasmid for hOXTR-376R, hOXTR-376G, or hOXTR-376C were stimulated with 100 nM OXT for 5, 10, 15, 30, 45, 60, 90, and 120 min. At each time point, the cells were fixed and stained with antibody pecific for the amino (N)-terminus, located extracellularly, of the OXTR under impermeable conditions. The specificity of the staining was confirmed by the comparison with the staining of mock-transfected and non-transfected HEK-293 cells (Additional file 1: Figure S1).

Immunoreactivity for hOXTRs was visualized by confocal laser scanning microscopy (Figure 2A). Immunofluorescence was measured and expressed as a percentage of the intensity for receptors remaining at the cell surface. Cell-surface intensity for hOXTR-376R reached the lowest value of 43.9 ± 2.3% (n = 30) after 60 min, and then gradually increased to 77.1 ± 8.7% (n = 29) of the prestimulatory level at 120 min. OXT-induced internalization in the cells expressing hOXTR-376C were very similar to that for hOXTR-376R: the surface intensity decreased to 45.1 ± 3.3% (n = 30) at 60 min, and then elevated to 79.4 ± 18.1% (n = 8) at 120 min. In contrast, the surface intensity of hOXTR-376G showed a significantly faster decrease than that for hOXTR-376R at 5 min after OXT application (62.5 ± 4.7% (n = 25) for hOXTR-376G, 78.7 ± 6.7% (n = 15) for hOXTR-376R; P <0.05; Student’s t test). In addition, hOXTR-376G showed significantly higher levels of surface intensities from 30 min to 120 min after OXT application (Figure 2B). The values at 30, 45, 60, and 120 min were 59.6 ± 2.7 (n = 30, P <0.05; Student’s t test), 61.4 ± 3.0 (n = 30, P <0.05; Student’s t test), 67.7 ± 3.7 (n = 30, P <0.01; Student’s t test), and 96.1 ± 6.6% (n = 23, P <0.05; Student’s t test) for hOXTR-376G, compared with 52.0 ± 2.5 (n = 30), 51.1 ± 3.1 (n = 30), 43.9 ± 2.3 (n = 30), and 77.1 ± 8.7% (n = 29) for hOXTR-376R, respectively (Figure 2B). The majority of the hOXTR-immunoreactivity was colocalized with fluorescence for SNAP_f-tagged β2-adrenergic receptor as a plasma membrane marker (>84%; Additional file 2: Figure S2). These data demonstrate that, upon agonist stimulation, hOXTR-376G is internalized into the cytoplasm and recycled to the plasma membrane more rapidly than hOXTR-376R and hOXTR-376C.

To examine whether the amino acid substitutions altered the coupling to intracellular signaling system, we measured OXT-induced Ca²⁺ mobilization. Both HEK-293 cells and NG108-15 neuroblastoma × glioma hybrid cells were transfected with an expression plasmid for EGFP-tagged hOXTR-376R, hOXTR-R376G, or hOXTR-376C. Before OXT stimulation, EGFP fluorescence was homogenously distributed in both HEK-293 cells and NG108-15 neuronal cells expressing hOXTR-376R-EGFP, hOXTR-376G-EGFP, and hOXTR-376C-EGFP, as previously reported [13] (Figure 3A,B). After loading these cells with Ca²⁺-sensitive fluorescent dye fura-2/AM, the effect of the amino acid variations on OXT-induced increase in [Ca²⁺]_i was analyzed using a dual-wavelength (340 and 380 nm) fluorescence spectrophotometer.

The addition of 100 nM OXT to HEK-293 cells expressing hOXTR-376R-EGFP induced a rapid initial increase of [Ca²⁺]_i; the levels reached 223 ± 19.6% (n = 24) of the prestimulatory level and then returned to the basal level within 3 min (Figure 3C,E). In contrast, both hOXTR-376G-EGFP- and hOXTR-376C-EGFP-expressing HEK-293 cells exhibited significantly smaller initial peaks: the maximum [Ca²⁺]_i was 142 ± 13.2% (n = 24) and 159 ± 3.8% (n = 31) for hOXTR-376G-EGFP and hOXTR-376C-EGFP, respectively (Figure 3C,E). Mock-transfected control cells did not show Ca²⁺ mobilization at all (Figure 3C,E).

The same tendency was observed in NG108-15 neuronal cells. [Ca²⁺]_i initially increased to relatively low levels compared with those in HEK-293 cells. The maximum [Ca²⁺]_i levels at the initial peak in hOXTR-376G-EGFP- and hOXTR-376C-EGFP-expressing NG108-15 cells were 137 ± 5.1% (n = 28) and 128 ± 2.0% (n = 18), respectively, significantly lower than those in hOXTR-376R-EGFP-expressing cells, which reached 161 ± 5.1% (n = 24; Figure 3D,F).

We further examined the OXT dose-dependence during the increase in [Ca²⁺]_i in HEK-293 cells expressing the common and variant hOXTRs. OXT concentrations ranging from 10⁻¹¹ to 10⁻⁶ M were tested. When the OXT concentration was lower than 10⁻¹⁰ M, no differences in the initial [Ca²⁺]_i rise were observed in the cells expressing hOXTR-376R-EGFP, hOXTR-376G-EGFP, and hOXTR-376C-EGFP (Figure 4A,B). In the cells expressing hOXTR-376R-EGFP, the maximum initial increase in [Ca²⁺]_i was at 10⁻⁷ M OXT (Figure 4A,B).

In HEK293 cells expressing hOXTR-376G-EGFP, the maximal [Ca²⁺]_i increase was obtained at 10⁻⁸ M OXT (158 ± 5.2% (n = 24); Figure 4A,B). OXT at 10⁻⁷ and 10⁻⁶ M resulted in significantly smaller [Ca²⁺]_i increases compared with those for hOXTR-376R-EGFP, with initial peak values being 142 ± 13.2% (n = 24) and 129 ± 7.2% (n = 16), respectively.

In HEK-293 cells expressing hOXTR-376C-EGFP, the maximal [Ca²⁺]_i increase was obtained at 10⁻⁷ M (159 ± 3.8% (n = 31); Figure 4A,B). At concentrations higher than 10⁻⁹ M, the OXT-induced increase in [Ca²⁺]_i was significantly reduced (Figure 4A,B). These data suggested that the rs35062132 variations (R376G/C) might alter the OXTR-mediated changes in the intracellular calcium concentrations.

IP₃ is known to mediate OXT-induced increase in [Ca²⁺]_i levels. Therefore, we investigated whether the amino acid substitutions could affect IP₃ production. As shown in Figure 5, in HEK-293 cells expressing hOXTR-376R-EGFP, 100 nM OXT elicited an increase in IP₃ level within initial 10 s; this increase was maintained for 30 s and the level returned to its basal value after 3 min. Mock-transfected cells did not show any changes. The cells expressing hOXTR-376G-EGFP and hOXTR-376C-EGFP also stimulated IP₃ production. During the whole period of 3 min after OXT application, IP₃ production in cells expressing hOXTR-376G-EGFP was not statistically different compared with hOXTR-376R-EGFP (P = 0.1705, F(1, 217) = 1.89; two-way ANOVA), whereas cells expressing hOXTR-376C-EGFP showed significantly smaller IP₃ production (P = 0.003, F(1, 210) = 9.01; two-way ANOVA). During the initial 5 to 30 s after OXT application, a slight but significant drop from the maximum IP₃ levels was observed in these cells, compared with the values for hOXTR-376R-EGFP (P = 0.0210, F(1, 107) = 5.49 for hOXTR-376G-EGFP; P = 0.0209, F(1, 103) = 5.5 for hOXTR-376C-EGFP; two-way ANOVA).