Role of metabotropic glutamate receptor 7 in autism spectrum disorders: A pilot study

Life Sciences 92 (2013) 149–153

Contents lists available at SciVerse ScienceDirect

Life Sciences journal homepage: www.elsevier.com/locate/lifescie

Role of metabotropic glutamate receptor 7 in autism spectrum disorders: A pilot study You Yang a,⁎, Chunhua Pan b a b

Shanghai Children's Medical Center of Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Children's Environmental Health, Shanghai, China Shanghai Institute of Hematology of Shanghai Jiao Tong University School of Medicine, China

a r t i c l e

i n f o

Article history: Received 11 May 2012 Accepted 13 November 2012 Keywords: Autism spectrum disorders (ASDs) Metabotropic glutamate receptor 7 (GRM7) Single nucleotide polymorphisms (SNPs) Haplotype Linkage disequilibrium

a b s t r a c t Aims: The presence of genetic variants for autism spectrum disorders (ASDs) was investigated for the metabotropic glutamate receptor 7 (GRM7) gene in a case–control study. Main methods: Employing Affymetrix SNP microarrays, 297 single nucleotide polymorphisms (SNPs) covering the GRM7 gene were selected and analyzed in ASD patients (n = 22), non-ASD patients [n = 14, including seven patients with development delay (DD)/mental retardation (MR), four patients with language delay (LD), and three patients with attention deficit hyperactivity disorder (ADHD)] and normal control subjects (n = 18). Key findings: Twenty-one statistically significant SNPs with different inheritance models (recessive, dominant and allele) were demonstrated in three groups (ASDs vs. combined controls, ASDs vs. normal controls, ASDs vs. non-ASD patients). Associations of rs779867 and rs6782011 with ASDs were significant in all three groups and independent associations of rs779867 and rs6782011 with ASDs were found in the ASD vs. combined controls group, which are in modest linkage disequilibrium (D′ > 0.5). Further haplotype analysis showed that rs6782011/rs779867 (T-C) was statistically significantly related to ASDs in both the ASD vs. combined controls and ASD vs. normal controls groups (bootstrap P value = 0.013, permutation P value = 0.013 for the former group and bootstrap P value = 0.002, permutation P value = 0.020 for the latter). Significance: These findings support a role for genetic variants within the GRM7 gene in 3p26.1 in ASDs. © 2012 Elsevier Inc. All rights reserved.

Introduction Recent work has indicated that synaptic genes may be involved in susceptibility to autism spectrum disorders (ASDs) (Peca et al., 2011). In addition, metabotropic glutamate receptors (GRMs), which are found both pre- and postsynaptically, have been implicated in a diverse variety of neuronal functions and exaggerated signaling through GRM5 receptors can account for multiple cognitive and syndromic features of fragile X syndrome, the most common inherited form of mental retardation and autism (Dolen and Bear, 2008). Synaptic synthesis of fragile X mental retardation protein (FMRP) has itself been shown to be induced by GRM activation (Qiu et al., 2007). The disease phenotype is a result of excessive GRM receptor signaling arising from the absence of FMRP. Accordingly, the GRM5 receptor has been proposed as a possible drug target for symptoms of fragile X syndrome (Bear et al., 2004). Treatment with GRM antagonists, as proposed by Bear et al. (Bear, 2005), may reduce the phenotypic effects

⁎ Corresponding author at: Shanghai Children's Medical Center of Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Children's Environmental Health, 1678 Dongfang Road, Shanghai, 200127, China. Tel.: +86 21 38626161 6020; fax: +86 21 50904612. E-mail address: [email protected] (Y. Yang). 0024-3205/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.lfs.2012.11.010

of the soluble amyloid precursor protein (sAPP), produced in excess when FMRP is absent, as occurs in fragile X syndrome. Given that many fragile X syndrome patients exhibit autistic features, the finding suggests that GRMs may play a role in susceptibility to autism (Bear, 2005). Furthermore, linkage and association of the inotropic GRM6 gene on chromosome 6 with autism has been reported (Jamain et al., 2002). The 7q31 region contains the GRM8 gene, which is also a good positional and functional candidate for susceptibility to autism (Carlsson, 1998). The data suggest the presence of a susceptibility mutation in linkage disequilibrium with variants of the GRM8 gene (Serajee et al., 2003). For GRM7, five isoforms have been characterized with most variants found to be expressed in the brain and retina at varying levels (Schulz et al., 2002). Although the precise physiological functions of the GRM7 subtypes are still unclear, targeted disruption of the orthologous murine GRM7 gene locus has been shown to cause a deficit in fear response and an impairment of taste aversion. This suggests a critical role for GRM7 in amygdala function, which is essential in mediating these behavioral traits (Masugi et al., 1999). Upon drug induction, mice lacking GRM7 are susceptible to epileptic seizures, indicating that GRM7 may be particularly important in the regulation of neuronal excitability (Enz, 2007; Okamoto et al., 1994). In particular, the GRM7_v1 isoform has been localized exclusively to the inner plexiform layer. Electron microscopy further demonstrated

150

Y. Yang, C. Pan / Life Sciences 92 (2013) 149–153

that GRM7_v1 is present asymmetrically at pre- and postsynaptic sites at certain cone bipolar cell ribbon synapses, possibly reflecting functional activity in the differential activation of postsynaptic neurons (Brandstatter et al., 1996; Martin et al., 2010). Additionally, a paternally inherited duplication (3p26.1), which encompasses GRM7, was reported in children with autism and additional developmental abnormalities (Davis et al., 2009). In the current study, we have completed an association analysis of GRM7 single nucleotide polymorphisms (SNPs) using an Affymetrix SNP Array 6.0 in a cohort of 22 patients with ASDs, 14 non-ASD patients [4 patients with language delay (LD), and 3 patients with attention deficit hyperactivity disorder (ADHD)] and 18 normal controls.

took the gender as a covariate and the analysis of association was adjusted by sex. The significance (P) level of all these tests was 0.05. The statistics were performed in three groups: 1) ASDs vs. combined controls, using 14 non-ASD patients and 18 normal subjects as the control; 2) ASDs vs. normal controls, using 18 normal subjects as the control; and 3) ASDs vs. non-ASDs controls, using 14 non-ASD patients as the control. Statistical analyses were performed by the SNPAlyze software (Version 8.0) and SPSS (Version 11.0). Results Statistically significant SNPs of the GRM7 gene in three groups

Materials and methods Subject This study was approved by the Ethics Committee of Shanghai Children's Medical Center of Shanghai Jiaotong University School of Medicine and all subjects gave informed consent for the genetic analyses. Blood samples were obtained from 22 unrelated ASD cases (mean age: 3.5 years; 17 males and 5 females) and 14 non-ASDs patients (7 cases with DD/MR, 4 cases with LD and 3 cases with ADHD; mean age: 4.0 years; 10 males and 4 females) recruited as outpatients of our hospital. All patients were diagnosed using the Diagnostic and Statistical Manual of Mental Disorders (DSM)-IV criteria (American Psychiatric and American Psychiatric Association. Task Force on, 2000). Physical examinations and molecular tests were carried out in order to exclude patients who had fragile X syndrome and tuberous sclerosis as well as any other neurological or medical condition suspected to be associated with autism. Eighteen unrelated controls (mean age: 3.6 years; thirteen males and seven females) who were not diagnosed with any psychiatric disorders were recruited as outpatients of our hospital.

For all the 297 SNPs, the genotypic distribution conformed to the HWE in controls. Table 1 lists the 21 SNPs with statistical significance in any of the inheritance models (recessive, dominant or allele) in three groups when a chi-square test was used to carry out the association analysis (P b 0.05). Two statistically significant SNPs of the GRM7 gene Table 2 summarizes the two SNPs of different inheritance models shared in three groups according to Table 1. Pairwise linkage disequilibrium between the two SNPs of the GRM7 gene was calculated for the cases and combined controls. We found modest linkage disequilibrium (D′ = 0.67, r 2 = 0.09) between the two SNPs in the combined controls and weak linkage disequilibrium (D′ = − 0.29, r 2 = 0.07) in the cases. In order to determine whether the two effects observed were independent or not, logistic regression analysis was undertaken and a significant result was found in the ASD vs. combined controls group (Table 3). The association analysis of haplotypes with ASDs using logistic regression

Genotyping and SNP selection The genotyping platform used was the Affymetrix Genome-Wide Human SNP Array 6.0 (Affymetrix Inc., Santa Clara, California, USA) (McCarroll et al., 2008), performed using 1 μg of genomic DNA (diluted in 1 × 6 TE buffer and at 50 ng/μl). Genotypes were called using Birdseed version 2. Genotype quality control measures included sex check, Hardy–Weinberg Equilibrium [HWE; P b 0.01, minor allele frequency (MAF) >0.01, and a call rate of more than 0.985, as reported elsewhere (Smith et al., 2009)]. We selected 297 SNPs with a call rate of 100% in the GRM7 gene for analysis in our SNP dataset (see the list of the 297 SNPs in supplementary material online). Statistical analysis Deviation from the HWE was examined in controls at P b 0.01. Based on the logistic regression method, the case-control association of genotypes in three inheritance models (recessive, dominant and allele) was tested and the odds ratios (OR) and 95% confidence intervals (95% CI) were given. D′ and r2 were calculated to evaluate the magnitude of linkage disequilibrium. Logistic regression was used to determine whether independent effects existed. Haplotype frequencies were estimated and both bootstrap and permutation tests were applied. The association analysis of haplotypes was similar to that of genotypes with logistic regression and results were shown as OR and 95% CI. The most frequent haplotype was automatically selected as the reference category and rare haplotypes were pooled together in a group. To estimate the significance of the best results, we used the permutation procedure (n= 1000). A parametric bootstrap was used in order to obtain empirical significance for the test (n= 1000, twister = 2,000,000). Because of the skew ratios of the prevalence of male and female patients of ASDs, we

The haplotypes were then constructed and the results are shown in Table 4. The P values of the bootstrap test by SPSS software and the permutation test by SNPAlyze software were consistent for the rs6782011/rs779867 T–C haplotype in the three groups. Table 1 Statistically significant SNPs in the three comparison models. SNPs (21)

ASDs vs. combined controls (11)

ASDs vs. normal controls (8)

ASDs vs. non-ASDs controls (14)

rs779867 rs6782011 rs712786 rs11915789 rs983534 rs924806 rs342045 rs329044 rs329042 rs2875257 rs163420 rs163325 rs162805 rs162722 rs6810141 rs1499209 rs3828472 rs712768 rs7622749 rs951557 rs6791111 rs17216349 rs17046472 rs12630300

1 1

1 1 1

1 1 1 1 1 1 1 1 1 1 1 1 1 1

1 1

1 1

1 1 1 1 1

1 1 1 1 1

1.00 4.71 (1.23–18.14)⁎ 1.00 3.08 (1.14–8.38)⁎ ⁎ P value b 0.05, OR and 95% CI were further calculated using logistic regression and were adjusted by sex.

OR(95%CI) Control (%)

25 (89.29) 3 (10.71) 19 (67.86) 9 (32.14) 28 (63.64) 16 (36.36) 18 (40.91) 26 (59.09) 1.00 4.30 (1.27–14.52)⁎ 1.00 1.84 (0.74–4.58)

Case (%)

151

Discussion

31 (86.11) 5 (13.89) 21 (58.33) 15 (41.67) 28 (63.64) 16 (36.36) 18 (40.91) 26 (59.09) 1.00 4.41 (1.62–12.03)⁎ 1.00 2.35 (1.07–5.19)⁎ (63.64) (36.36) (40.91) (59.09) 28 16 18 26 rs6782011 C T rs779867 C T

56 (87.50) 8 (12.50) 40 (62.50) 24 (37.50)

ASDs vs. Non-ASD controls

OR (95%CI) Control (%) Case (%)

ASDs vs. Normal controls

Case (%) Allele model

Control (%)

ASDs vs. Combined controls Genotype

9 (40.91) 13 (59.09) 2 (9.09) 20 (90.91) rs6782011 C/C T/T + C/T rs779867 C/C T/T + C/T

OR (95%CI)

5.87 (1.15–29.85)⁎

OR (95%CI)

1.00 5.50 (1.13–26.85)⁎ 1.00 6.10 (0.95–38.99) 11 (78.57) 3 (21.43) 5 (35.71) 9 (64.29)

Control (%) Case (%)

9 (40.91) 13 (59.09) 2 (9.09) 20 (90.91) 1.00 4.67 (1.08–20.23)⁎ 1.00 5.69 (0.96–33.91) 13 (72.22) 5 (27.78) 7 (38.89) 11 (61.11) 9 (40.91) 13 (59.09) 2 (9.09) 20 (90.91) 1.00 5.15 (1.45–18.33)⁎

ASDs vs. Non-ASD controls

OR (95%CI) Control (%) Case (%)

ASDs vs. Normal controls

OR (95%CI) Case (%) Dominate model

Control (%)

ASDs vs. Combined controls Genotype

24 (75.00) 8 (25.00) 12 (37.5) 20 (62.5)

1.00 – 1.00 – 14 (100.0) 0 (0.0) 14 (100.0) 0 (0.0) 19 (86.36) 3 (13.64) 16 (72.73) 6 (27.27) 1.00 – 1.00 1.19 (0.27–5.25) 18 (100.0) 0 (0.0) 14 (77.78) 4 (22.22) 19 (86.36) 3 (13.64) 16 (72.73) 6 (27.27) 1.00 – 1.00 2.50 (0.61–10.31) 19 (86.36) 3 (13.64) 16 (72.73) 6 (27.27) rs6782011 C/C + C/T T/T rs779867 C/C + C/T T/T

32 (100.0) 0 (0.0) 28 (87.50) 4 (12.50)

Control (%) Case (%) Control (%) Case (%) Case (%) Recessive model

Control (%)

ASDs vs. Combined controls Genotype

Table 2 Two statistically significant SNPs of the GRM7 gene.

OR (95%CI)

ASDs vs. Normal controls

OR (95%CI)

ASDs vs. Non-ASD controls

OR (95%CI)

Y. Yang, C. Pan / Life Sciences 92 (2013) 149–153

ASDs are neurodevelopmental disorders that share a set of complex behavioral phenotypes involving difficulties in communication and reciprocal social interaction, as well as stereotypical repetitive behavior and a restricted range of interest (Strock and National Institute of Mental H, 2008). The glutamate pathway has been considered to play important roles in neural plasticity, neural development, and neurodegeneration (Nakanishi et al., 1998; Shimizu et al., 2000) and, through animal models and neurochemical and neuropharmacological studies, autism has been proposed to be a disorder involving distorted glutamate concentration (Carlsson, 1998; McCoy et al., 2002; Moreno-Fuenmayor et al., 1996; Purcell et al., 2001). The above findings suggest that GRM7, previously not associated with ASDs, may play a role in susceptibility to autism. The GRM7 gene is mapped to 3p26.1, which encompasses approximately 859.7 kb of DNA and includes 11 exons. We tested the hypothesis that the GRM7 gene plays a role in the pathogenesis of ASDs. In our study, rs6782011, which is located in intron 6 of the GRM7 gene, showed significant association with ASDs in both dominant (OR: 4.67-5.50, 95% CI: 1.08–26.85) and allele (OR: 4.30-4.71, 95% CI: 1.23-18.14) inheritance models in three groups. On the other hand, rs779867, which is located in intron 5 of GRM7 gene, showed significant association with ASDs in dominant (OR: 5.87, 95% CI: 1.15-29.85) and allele (OR: 2.35-3.08, 95% CI: 1.07-8.38) inheritance models in one or two of the three comparison models. Independent associations of rs779867 and rs6782011 with ASDs were found in the group of ASDs vs. combined controls, which are in modest linkage disequilibrium (D′ > 0.5). As the r2 is greatly influenced by population size (Pritchard and Przeworski, 2001), the r2 results are, however, of less value here. Though the two SNPs are located in the intron region, there is steadily accumulating evidence to support a role for intronic variants in complex diseases (Wu et al., 2005). Alternatively, it is also possible that these two SNPs are not directly involved but are in linkage disequilibrium with other functional polymorphisms within the same gene or with another nearby locus that is the true susceptibility locus. To verify our hypothesis, we further tested the linkage disequilibrium of these two SNPs with the CNTN4 gene, which is located in 3p25-26, an autism susceptibility locus very close to the breakpoints of the 3p syndrome (Zeng et al., 2002). Fifteen SNPs out of 417 SNPs with a call rate of 100% in the CNTN4 gene were significantly related to ASDs in the ASDs vs. combined controls group (data not shown). A strong linkage disequilibrium between rs779867, rs6782011 and the 15 SNPs in the CNTN4 gene was found in the cases and combined controls (D′ b −0.7 between rs6782011, rs779867 in GRM7 gene and rs12494352 in the CNTN4 gene for the combined controls; D′ =−1 between rs6782011, rs779867 in GRM7 gene and rs7643747 in CNTN4 gene for the cases). However, further study of the relationship of the GRM7 gene with the CNTN4 gene in autism is still required. Furthermore, the result of association analysis for the rs6782011/rs779867 haplotype including introns 5 and 6 was consistent with individual SNP analysis in this study (P ≤ 0.02), suggesting that in Chinese children at least one susceptibility locus for ASDs lies within or very close to the region spanning rs6782011 and rs779867. GRM7 is found in the presynaptic membrane, has voltage-gated calcium channel activity and has been shown to be involved in synaptic transmission (Perroy et al., 2001). A function for GRM7 in neural pathways has been supported by experimental evidence in mouse and it is involved in memory, learning, behavioral fear response and conditioned taste aversion (Callaerts-Vegh et al., 2006; Cryan et al., 2003; Holscher et al., 2004, 2005; Masugi et al., 1999; Perroy et al., 2001, 2002; Sansig et al., 2001). Clinically, it has been reported that better fear conditioning is associated with reduced symptom severity in patients with ASDs (South et al., 2011). Memory and learning are usually impaired in ASDs and excitatory neurotransmitter signaling

152

Y. Yang, C. Pan / Life Sciences 92 (2013) 149–153

Acknowledgments

Table 3 Logistic regression of two SNP models. SNPs

OR (95% CI)

P value

rs6782011 rs779867

4.57 (1.21–25.86) 5.11 (1.51–1.6E + 09)

0.023 0.009

OR and 95% CI were calculated using logistic regression with enter-method and bootstrap resampling and were adjusted by sex.

This work was funded by the National Natural Science Fund (30801345), Shanghai Municipal Education Commission (08YZ41) and Shanghai University Scientific Selection and Cultivation for Outstanding Young Teachers in Special Fund (jdy09144), China. References

via glutamate receptors modulates these cognitive functions (Choudhury et al., 2012). Associative learning capabilities are considered a relative strength for children with ASDs. GRM7 is also involved in associative learning (Callaerts-Vegh et al., 2006). Children with ASDs show cognitive difficulties with regard to learning picture-word and picture-object relations via an associative mechanism and have difficulty understanding the symbolic nature of pictures (Preissler, 2008). In this study, we selected two SNPs shared in three comparison models and, due to the small number of samples, performed detailed association analysis. We did find some significant association of other SNPs in the GRM7 gene, as indicated in Table 1, which need further validation with more samples. The association with the GRM7 gene was based on SNPs in an intron region and how this specific intron relates to ASDs is difficult to comprehend. It would be worthwhile, therefore, to continue the association research of GRM7 located in this region with a greater number of samples. Conclusions Our results, together with the findings of the previous studies, suggest that the GRM7 gene may be implicated in the predisposition to ASDs. It is noteworthy that this is the first reported association analysis in autism for the GRM7 gene, and therefore it is necessary to replicate the finding in other samples and to perform further functional studies to determine a possible role for GRM7 in predisposition to ASDs. In addition, since the changes in brain development and GRM7 levels could also be due to other genes in this pathway, their possible associations with ASDs also need to be investigated. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.lfs.2012.11.010. Conflict of interest statement We declare no involvement of study sponsors in the study design; collection, analysis and interpretation of data; the writing of the manuscript; the decision to submit the manuscript for publication.

American Psychiatric AAmerican Psychiatric Association. Task Force on D–I. Diagnostic and statistical manual of mental disorders: DSM-IV-TR. Washington, DC: American Psychiatric Association; 2000. Bear MF. Therapeutic implications of the mGluR theory of fragile X mental retardation. Genes Brain Behav 2005;4:393–8. Bear MF, Huber KM, Warren ST. The mGluR theory of fragile X mental retardation. Trends Neurosci 2004;27:370–7. Brandstatter JH, Koulen P, Kuhn R, van der Putten H, Wassle H. Compartmental localization of a metabotropic glutamate receptor (mGluR7): two different active sites at a retinal synapse. J Neurosci 1996;16:4749–56. Callaerts-Vegh Z, Beckers T, Ball SM, Baeyens F, Callaerts PF, Cryan JF, et al. Concomitant deficits in working memory and fear extinction are functionally dissociated from reduced anxiety in metabotropic glutamate receptor 7-deficient mice. J Neurosci 2006;26:6573–82. Carlsson ML. Hypothesis: is infantile autism a hypoglutamatergic disorder? Relevance of glutamate–serotonin interactions for pharmacotherapy. J Neural Transm 1998;105: 525–35. Choudhury PR, Lahiri S, Rajamma U. Glutamate mediated signaling in the pathophysiology of autism spectrum disorders. Pharmacol Biochem Behav 2012;100:841–9. Cryan JF, Kelly PH, Neijt HC, Sansig G, Flor PJ, van Der Putten H. Antidepressant and anxiolytic-like effects in mice lacking the group III metabotropic glutamate receptor mGluR7. Eur J Neurosci 2003;17:2409–17. Davis LK, Meyer KJ, Rudd DS, Librant AL, Epping EA, Sheffield VC, et al. Novel copy number variants in children with autism and additional developmental anomalies. J Neurodev Disord 2009;1:292–301. Dolen G, Bear MF. Role for metabotropic glutamate receptor 5 (mGluR5) in the pathogenesis of fragile X syndrome. J Physiol 2008;586:1503–8. Enz R. The trick of the tail: protein–protein interactions of metabotropic glutamate receptors. Bioessays 2007;29:60–73. Holscher C, Schmid S, Pilz PK, Sansig G, van der Putten H, Plappert CF. Lack of the metabotropic glutamate receptor subtype 7 selectively impairs short-term working memory but not long-term memory. Behav Brain Res 2004;154:473–81. Holscher C, Schmid S, Pilz PK, Sansig G, van der Putten H, Plappert CF. Lack of the metabotropic glutamate receptor subtype 7 selectively modulates Theta rhythm and working memory. Learn Mem 2005;12:450–5. Jamain S, Betancur C, Quach H, Philippe A, Fellous M, Giros B, et al. Linkage and association of the glutamate receptor 6 gene with autism. Mol Psychiatry 2002;7:302–10. Martin R, Durroux T, Ciruela F, Torres M, Pin JP, Sanchez-Prieto J. The metabotropic glutamate receptor mGlu7 activates phospholipase C, translocates munc-13-1 protein, and potentiates glutamate release at cerebrocortical nerve terminals. J Biol Chem 2010;285:17907–17. Masugi M, Yokoi M, Shigemoto R, Muguruma K, Watanabe Y, Sansig G, et al. Metabotropic glutamate receptor subtype 7 ablation causes deficit in fear response and conditioned taste aversion. J Neurosci 1999;19:955–63. McCarroll SA, Kuruvilla FG, Korn JM, Cawley S, Nemesh J, Wysoker A, et al. Integrated detection and population-genetic analysis of SNPs and copy number variation. Nat Genet 2008;40:1166–74.

Table 4 Haplotypes of two statistically significant SNPs. Haplotype

Case (%)

Control (%)

OR

95% CI

rs6782011/rs779867 (ASDs vs. combined controls) C–C 14 (31.82) C–T 14 (31.82) T–T 12 (27.27) T–C 4 (9.09)

39 (60.94) 18 (28.13) 6 (9.38) 1 (1.56)

1.00 2.29 4.94 13.92

Referent 0.83-6.70 1.64-24.09 1.38-1.6E+10

rs6782011/rs779867 (ASDs vs. normal controls) C–C 14 (31.82) C–T 14 (31.82) T–T 12 (27.27) T–C 4 (9.09)

21 (58.33) 11 (30.56) 4 (11.11) 0 (0.00)

1.00 1.93 3.61 3.7E+09

Referent 0.63-6.89 0.91-22.76 1.5E+9-1.3E+10

rs6782011/rs779867 (ASDs vs. non-ASD controls) C–C 14 (31.82) C–T 14 (31.82) T–T 12 (27.27) T–C 4 (9.09)

18 (0.64) 7 (0.39) 2 (0.29) 1 (0.50)

1.00 2.85 7.37 6.36

Referent 0.84-13.48 1.86-3.1E+9 0.46-1.1E+10

P value*

P value**

0.101 0.004 0.013

0.001 0.000 0.161 0.389 0.013

0.231 0.046 0.002

0.012 0.006 0.330 0.659 0.020

0.085 0.012 0.088

0.038 0.001 0.235 0.367 0.314

P value* indicates the bootstrap test and P value** indicates the permutation test. OR and 95% CI were calculated using logistic regression and bootstrap resampling and were adjusted by sex.

Y. Yang, C. Pan / Life Sciences 92 (2013) 149–153 McCoy PA, Shao Y, Wolpert CM, Donnelly SL, Ashley-Koch A, Abel HL, et al. No association between the WNT2 gene and autistic disorder. Am J Med Genet 2002;114: 106–9. Moreno-Fuenmayor H, Borjas L, Arrieta A, Valera V, Socorro-Candanoza L. Plasma excitatory amino acids in autism. Invest Clin 1996;37:113–28. Nakanishi S, Nakajima Y, Masu M, Ueda Y, Nakahara K, Watanabe D, et al. Glutamate receptors: brain function and signal transduction. Brain Res Brain Res Rev 1998;26: 230–5. Okamoto N, Hori S, Akazawa C, Hayashi Y, Shigemoto R, Mizuno N, et al. Molecular characterization of a new metabotropic glutamate receptor mGluR7 coupled to inhibitory cyclic AMP signal transduction. J Biol Chem 1994;269:1231–6. Peca J, Ting J, Feng G. SnapShot: autism and the synapse. Cell 2011;147:706:e1. Perroy J, Gutierrez GJ, Coulon V, Bockaert J, Pin JP, Fagni L. The C terminus of the metabotropic glutamate receptor subtypes 2 and 7 specifies the receptor signaling pathways. J Biol Chem 2001;276:45800–5. Perroy J, El Far O, Bertaso F, Pin JP, Betz H, Bockaert J, et al. PICK1 is required for the control of synaptic transmission by the metabotropic glutamate receptor 7. EMBO J 2002;21:2990–9. Preissler MA. Associative learning of pictures and words by low-functioning children with autism. Autism 2008;12:231–48. Pritchard JK, Przeworski M. Linkage disequilibrium in humans: models and data. Am J Hum Genet 2001;69:1-14. Purcell AE, Jeon OH, Zimmerman AW, Blue ME, Pevsner J. Postmortem brain abnormalities of the glutamate neurotransmitter system in autism. Neurology 2001;57:1618–28. Qiu C, De Ronchi D, Fratiglioni L. The epidemiology of the dementias: an update. Curr Opin Psychiatry 2007;20:380–5.

153

Sansig G, Bushell TJ, Clarke VR, Rozov A, Burnashev N, Portet C, et al. Increased seizure susceptibility in mice lacking metabotropic glutamate receptor 7. J Neurosci 2001;21:8734–45. Schulz HL, Stohr H, Weber BH. Characterization of three novel isoforms of the metabotrobic glutamate receptor 7 (GRM7). Neurosci Lett 2002;326:37–40. Serajee FJ, Zhong H, Nabi R, Huq AH. The metabotropic glutamate receptor 8 gene at 7q31: partial duplication and possible association with autism. J Med Genet 2003;40:e42. Shimizu E, Tang YP, Rampon C, Tsien JZ. NMDA receptor-dependent synaptic reinforcement as a crucial process for memory consolidation. Science 2000;290: 1170–4. Smith EN, Bloss CS, Badner JA, Barrett T, Belmonte PL, Berrettini W, et al. Genome-wide association study of bipolar disorder in European American and African American individuals. Mol Psychiatry 2009;14:755–63. South M, Larson MJ, White SE, Dana J, Crowley MJ. Better fear conditioning is associated with reduced symptom severity in autism spectrum disorders. Autism Res 2011;4: 412–21. Strock M, National Institute of Mental H. Autism spectrum disorders pervasive developmental disorders. Bethesda, Md: Dept. of Health and Human Services, National Institutes of Health, National Institute of Mental Health; 2008. Wu S, Jia M, Ruan Y, Liu J, Guo Y, Shuang M, et al. Positive association of the oxytocin receptor gene (OXTR) with autism in the Chinese Han population. Biol Psychiatry 2005;58:74–7. Zeng L, Zhang C, Xu J, Ye X, Wu Q, Dai J, et al. A novel splice variant of the cell adhesion molecule contactin 4 (CNTN4) is mainly expressed in human brain. J Hum Genet 2002;47:497–9.