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Keywords:

  • gene–environment interaction;
  • GRIN2B;
  • herpes simplex virus-2;
  • NMDA receptor;
  • schizophrenia

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. SUBJECTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

N-methyl-D-aspartate (NMDA) receptors are very important for proper brain development and several lines of evidence support that hypofunction of the NMDA receptors are involved in the pathophysiology of schizophrenia. Gene variation and gene–environmental interactions involving the genes encoding the NMDA receptors are therefore likely to influence the risk of schizophrenia. The aim of this study was to determine (1) whether SNP variation in the genes (GRIN1, GRIN2A, GRIN2B, GRIN2C, and GRIN2D) encoding the NMDA receptor were associated with schizophrenia; (2) whether GRIN gene variation in the offspring interacted with maternal herpes simplex virus-2 (HSV-2) seropositivity during pregnancy influencing the risk of schizophrenia later in life. Individuals from three independently collected Danish case control samples were genotyped for 81 tagSNPs (in total 984 individuals diagnosed with schizophrenia and 1,500 control persons) and antibodies against maternal HSV-2 infection were measured in one of the samples (365 cases and 365 controls). Nine SNPs out of 30 in GRIN2B were significantly associated with schizophrenia. One SNP remained significant after Bonferroni correction (rs1806194, Pnominal = 0.0008). Significant interaction between maternal HSV-2 seropositivity and GRIN2B genetic variation in the offspring were observed for seven SNPs and two remained significant after Bonferroni correction (rs1805539, Pnominal = 0.0001 and rs1806205, Pnominal = 0.0008). The significant associations and interactions were located at the 3′ region of GRIN2B suggesting that genetic variation in this part of the gene may be involved in the pathophysiology of schizophrenia. © 2011 Wiley Periodicals, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. SUBJECTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Schizophrenia is a severe and often life-long mental disorder, affecting approximately one percent of the population worldwide. It is a complex disorder, believed to involve many contributing factors, genetic and environmental, as well as the interaction between the two [van Os et al., 2008].

Much of the neurobiological understanding of schizophrenia has evolved around the development of drugs, targeting the dopaminergic system, but a parallel and increasing interest has focused on the role of the glutaminergic system in schizophrenia [Carlsson, 2006; Coyle, 2006; Javitt, 2006]. Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system and serves as the neurotransmitter of the pyramidal cells. These are the source of efferent and interconnecting pathways of the cerebral cortex and limbic system, which are the brain regions implicated in the pathophysiology of schizophrenia [Tsai and Coyle, 2002]. A key factor in glutamatergic neurotransmission is the N-methyl-D-aspartate (NMDA) receptors, which are involved in brain development, excitatory neurotransmission, synaptic plasticity, and memory formation [Bliss and Collingridge, 1993; Yang et al., 1999; Akashi et al., 2009; Zhuo, 2009]. The NMDA receptors are composed of multiple subunits including at least one NR1 subunit (encoded by the GRIN1 gene) and one or more NR2 subunits (encoded by the GRIN2A-D genes)[McBain and Mayer, 1994], and less commonly, a NR3 subunit (encoded by the GRIN3A-B genes) [Das et al., 1998; Chatterton et al., 2002]. Several lines of evidence support that hypofunction of the NMDA receptors may be involved in the pathophysiology of schizophrenia. Noncompetitive antagonists of the NMDA receptors such as phenocyclidine, ketamine, and MK-801 may cause psychotic conditions similar to schizophrenia in healthy humans. Conversely NMDA receptor agonists (glycine and D-cycloserine) may improve schizophrenia symptoms [Javitt, 2006, 2008; Lisman et al., 2008; Labrie and Roder, 2010]. The involvement of NMDA receptors in schizophrenia is also supported by the observation of changes in glutamate receptor binding and subunit expression in postmortem brains of patients with schizophrenia [Meador-Woodruff et al., 2003; Woo et al., 2008]. Genetic investigations involving mutation screening and association analysis have mainly focused on the NR1 subunit, which is the binding site for the co-agonists glycine and D-serine on the NR2 subunits where the agonist binding site for glutamate is located [Lang et al., 2007]. Less attention has been given to the NR3 subunit, which regulates the calcium permeability and magnesium sensitivity of the receptor [Galliant et al., 2007]. Several studies have found association of schizophrenia with GRIN1 gene polymorphisms [Begni et al., 2003; Georgi et al., 2007; Galehdari et al., 2009], GRIN2A variations [Itokawa et al., 2003; Iwayama-Shigeno et al., 2005; Tang et al., 2006], and with GRIN2B variations [Ohtsuki et al., 2001; Miyatake et al., 2002; Di Maria et al., 2004; Qin et al., 2005; Martucci et al., 2006]. However, a number of other studies have not been able to replicate these findings [Nishiguchi et al., 2000; Williams et al., 2002; Shen et al., 2009].

NMDA receptors are important for normal brain development due to their involvement in the regulation of synapse maturation [Cohen and Greenberg, 2008]. NR1 and NR2B knockout mice die shortly after birth, which indicates a requirement for NMDA receptor function in nerve circuit development [Forrest et al., 1994; Kutsuwada et al., 1996]. It has also been shown that NMDA receptor blockade in rodents during postnatal early brain development leads to alterations in NMDA receptor binding still evident at adulthood [du Boris et al., 2009], and maternal stress in rodents had influenced brain development and NMDA function of the offspring for life [Fumagalli et al., 2009]. These investigations suggest that disturbance of NMDA receptor function early in life has an impact on brain development which in turn could be influenced by the underlying genetic variation in the GRIN genes.

Genetic factors account for around 80% of the variation in liability to schizophrenia and the findings that the concordance rate of the disorder is approximately 50% for monozygotic twins indicate that the environment plays an important role in the development of the disease. Especially early in life, the environment may be important for disease risk as maternal infection during pregnancy, such as influenza [Brown et al., 2004; Byrne et al., 2007], herpes simplex virus-2 (HSV-2)[Buka et al., 2008; Mortensen et al., 2010], and toxoplasmosis [Brown et al., 2005; Mortensen et al., 2007] have been shown to increase the risk of the child-developing schizophrenia in adulthood. Maternal infection and fever has also been associated with altered synaptic transmission in the brain of the offspring [Lowe et al., 2008]. Furthermore, genetic and environmental risk factors are likely to interact as seen in a recent study of mice where a common mutation in a disease susceptibility gene for Crohn's disease in combination with a specific viral infection had profound effect when combined [Cadwell et al., 2010], and several findings suggest the presence of widespread gene–environment interactions in the etiology of schizophrenia [van Os et al., 2008]. Such interactions may also occur early in life since maternal pregnancy complications have been found to interact with the genotype of the fetus influencing the risk of developing schizophrenia later in life [Nicodemus et al., 2008].

Here, we present an association study of genetic variation in the GRIN1 and GRIN2A-D genes with schizophrenia in three Danish case control samples. Since the NMDA receptors are very important for proper brain development GRIN gene variation could also interact with environmental factors occurring early in life influencing the risk of schizophrenia. Such an environmental factor could be maternal HSV-2 seropositivity during pregnangy. A previous study of one of the samples included in this investigation has found maternal HSV-2 seropositivity on its own to be associated with increased risk of schizophrenia in the offspring (P = 0.002) [Mortensen et al., 2010]. This motivated us to conduct an investigation of interaction between GRIN gene variation in the offspring and maternal HSV-2 seropositivity in relation to the risk of schizophrenia in adulthood. The interaction analysis is unique because it is based on neonatal blood samples stored in the Danish Newborn Screening Biobank [Nørgaard-Pedersen and Hougaard, 2007], enabling assessment of maternal HSV-2 IgG antibody levels around the time of birth.

SUBJECTS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. SUBJECTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Subjects

We investigated genetic association with schizophrenia using three Danish samples. Individuals who occurred in more than one of the samples were identified by performing Graphical Representation Analysis [Abecasis et al., 2001] and included in one sample only.

Denmark I

This sample included 385 schizophrenia patients and 780 controls matched by gender and date of birth. The patients were recruited to the Danish Psychiatric Biobank and diagnosed with schizophrenia according to the ICD-10 criteria. The control individuals were ethnically Danish blood donors.

Denmark II

In Denmark, nationwide screening of newborns for phenylketonuria (PKU) and other metabolic diseases has been carried out since May 1975 and since 1981 surplus of the samples have been stored as a routine procedure in the Danish Newborn Screening Biobank at −20°C in accordance with regulations from the Ministry of Health [Nørgaard-Pedersen and Hougaard, 2007]. Annually around 65,000 newborns (5–7 days of age) are screened [Nørgaard-Pedersen and Hougaard, 2007] using capillary blood from heel pricks collected on filter paper. The Denmark II sample was established using the unique personal identification number assigned to all live-born children (CPR-number) and stored in the Danish Civil Registration System (DCRS) [Pedersen et al., 2006]. Information from the DCRS was linked with the information stored in the nationwide Danish Psychiatric Central Register [Munk-Jorgensen and Mortensen, 1997]. In this way archived dry blood spot samples from 365 Danish born individuals with schizophrenia diagnosed according to ICD-10 (1994–2005) were obtained from the Danish Newborn Screening Biobank. For each case we randomly selected at least one individually time-matched control with same gender, date of birth and age, from the Biobank. In total 434 controls with no record of mental illness were included in the study.

Denmark III

This sample included 234 incident schizophrenia cases and 286 medical student volunteers as controls [Nyegaard et al., 2010]. The patients were all interviewed using the semi-structured interview Schedules for Clinical Assessment in Neuropsychiatry (SCAN) version 2.1 (World Health Organization) and fulfilled the ICD 10-DCR criteria for schizophrenia. Consensus best estimate of lifetime diagnoses was made independently by two senior psychiatrists. The cases and controls were of Danish parentage for three generations.

This study has been approved by the Danish Data Protection Agency and the ethics committees in Denmark.

Genotyping

Genotyping of individuals from the Denmark I and III sample was performed using genomic DNA isolated from blood samples following standard procedures. Individuals from the Denmark II sample were genotyped using DNA extracted from dried blood spots obtained from the Danish Newborn Screening Biobank using Extract-N-Amp Blood PCR kit (Sigma Aldrich, St. Louis, MO). To ensure a sufficient amount of DNA for genotyping, the extracted material was whole-genome-amplified using the AmpliQ Genomic Amplifier kit (Ampliqon, Skovlunde, Denmark) following Sørensen et al. [Sørensen et al., 2007]. The genotyped single nucleotide polymorphisms (SNPs) were identified from HapMap data (version 22 dbSNP 126 (NCBI build 36)) (http://www.hapmap.org/) using the tagger selection algorithm in Haploview (pair-wise tagging function, r2 threshold = 0.8) [Bakker et al., 2005]. Furthermore missense SNPs were included (four in GRIN1, one in GRIN2A and one in GRIN2B) as well as SNPs found to be associated with schizophrenia in other studies (14 SNPs). Due to the large size of GRIN2A and GRIN2B, these genes were tagged only in exons, intron–exon boundaries (300 bp up- and downstream for each exon) and putatively regulatory regions upstream (2,000 bp) and downstream (2,000 bp) of the genes. We genotyped 124 SNPs: 13 in GRIN1, 46 in GRIN2A, 44 in GRIN2B, 7 in GRIN2C, and 14 in GRIN2D. Genotyping was carried out using the Sequenom MassARRAY Genotyping platform (Sequenom, San Diego, CA) using the protocol described in Nyegaard et al. [2010]. Primer sequences and assay conditions are available from the authors upon request.

Serological Analysis

We were able to perform serological analysis of maternal HSV-2 antibodies in the Denmark II sample due the unique collection procedure of the blood samples. The blood spots obtained from the Danish Newborn Screening Biobank were measured for type specific antibodies to the gG2 glycoprotein of HSV-2 by enzyme immunoassay [Buka et al., 2008] at the Stanley Neurovirology Laboratory. Maternal IgG antibodies are transferred across the placenta to the fetus, and at the time when the blood sample is taken from the baby (day 5–7) it has not yet produced its own IgG antibodies. Consequently the IgG antibody level in the newborn blood sample is of maternal origin. An assay value greater than or equal to 0.11 optical density units was used to indicate exposure to HSV2 infection (See Mortensen and co-workers [Mortensen et al., 2010] for details). This cut-off value corresponded to a prevalence among control women around 17%, which is in accordance with Looker et al. [2008] who reported a HSV-2 prevalence of 18% among western European women.

Quality Control

The open source software PLINK (http://pngu.mgh.harvard.edu/∼purcell/plink/summary.shtml) [Purcell et al., 2007] was used to perform quality control, and calculate the association analysis below. After quality control 81 SNPs with nice cluster plots, minor allele frequency above 0.01, no deviation from Hardy–Weinberg equilibrium (HWE) (nominal P > 0.001 in controls) and a call rate higher than 95% were left for analysis (5 SNPs in GRIN1, 34 SNPs in GRIN2A gene, 30 SNPs in GRIN2B, 4 SNPs in GRIN2C, and 8 SNPs in GRIN2D). Of the failed SNPs 15 failed genotyping on the Sequenom in all three samples and 26 SNPs were excluded due to low call rate. The low call rate was caused by failed genotyping of the SNPs in the Denmark II sample, which has a lower DNA quality than the other samples due to the origin of the DNA from dried blood spots. Finally two SNPs were excluded due to deviation from HWE. Twenty-five individuals (13 cases and 12 controls) were excluded from the analysis due to low genotyping rate. The remaining individuals had a genotyping rate greater than 98%.

Genetic Homogeneity of Cases and Controls

In order to check for homogeneity we measured the pair-wise fixation index, FST, values between cases and controls for each sample independently. The FST values were estimated with the FST calculation included in the Tracy–Widom statistics in the software Eigensoft [Patterson et al., 2006] using genotype data from the 81 SNPs in the present study together with 144 SNPs genotyped in relation to other projects. We found no FST deviation from zero between cases and controls in any of the three samples. FST is an estimate of genetic divergence based on drift and markers used for estimating the true FST should therefore be neutral. However, this is not the case for the markers we have used, since they are located within candidate genes potentially associated with schizophrenia or other psychiatric disorders. Due to the markers' possible association with the outcome, we might expect the estimated FST measures to overestimate the true genetic divergence between cases and controls. The finding of no significant FST values between the cases and controls therefore reinforces that we are analyzing homogenous samples.

Association Analysis

A stratified association analysis was performed using the Cochran–Mantel–Haenszel (CMH) test in PLINK [Purcell et al., 2007]. For the Denmark I and Denmark II samples, each case and its individually matched control(s) during sample collection were treated as one cluster. The unmatched cases and controls from the Denmark III sample were analyzed as a single cluster. Correction for multiple testing was performed using Bonferroni correction and following Miller's [1981] suggestion we applied a separate probability statement for each gene.

Evidence for association at the level of the whole gene was evaluated based on the method described by Curtis et al. [2008]. First the association P-values obtained from the CMH analysis for all SNPs in the gene were combined according to the method of Fisher [1925] into a summative measure for each gene. Then the statistical significance of the summative measure was assessed using permutation testing where the case-control labels were permuted while maintaining the genotypes (10,000 permutations were used). In this way an empirical distribution of the summative statistic could be calculated taking into account the interdependencies of the markers due to linkage disequilibrium (LD) between them. The calculations were done using Stata 9.1 (Stata Corp., College Station, TX).

The homogeneity of odds ratios was analyzed using the “Partitioning of the total association chi-square test” in PLINK which includes a between-strata heterogeneity test of the odds ratios. In this analysis each of the three samples (Denmark I, Denmark II, and Denmark III) was treated as one cluster. A SNP haplotype association test was performed using the haplotype association test in PLINK. Each gene was analyzed separately using a sliding window approach of two- and three-marker haplotypes.

GRIN Gene Variation and Maternal HSV-2 Infection

In the Danish II sample 167 individuals (95 cases and 72 controls) were born to HSV-2 seropositive mothers and 619 individuals were born to seronegative mothers (265 cases and 354 controls) (Table I). The numbers refer to the number of individuals after genotypic quality control. Analysis of interaction between GRIN gene variations and maternal HSV-2 seropositivity was conducted using conditional logistic regression incorporating an interaction term. For each SNP, the number of minor alleles was included into the model as a continuous variable, corresponding to an additive genetic model. P-values were two-sided and based on likelihood ratio tests. Bonferroni correction was performed as described above. As a measure of gene–environment interaction at the level of the whole gene Fischer's summative statistic was calculated from the interaction P-value for each SNP within each of the five genes. In order to correct for LD between markers the summative statistic was multiplied by the estimated number of independent tests number (calculated using matSpD [Nyholt, 2004] http://gump.qimr.edu.au/general/daleN/matSpD/) divided by the total number of SNPs and two times the number of estimated independent tests was used as degree of freedom value in Fisher's method.

Table I. Number (#) of Cases (SZ) and Controls With HSV-2 Seropositive or Seronegative Mothers
 SZ # (%)Controls # (%)
  1. Only individuals passing genotypic quality control are included.

HSV-2 seropositive95(26.4)72 (16.9)
HSV-2 seronegative265 (73.6)354 (83.1)

Imputation

To increase the information about the GRIN2B region the genotyping coverage was increased by imputing additional genotypes, using the software MACH 1.0 [Li et al., 2010]. The 1000Genomes August 2009 (CEU population) was used as reference haplotypes and imputation was done by a two-stage process with a model estimation based on 200 randomly selected subjects and 50 Monte Carlo iterations, followed by application of the resulting model to the rest of the data. Imputation was done for the region chr12:13.595.677–14.034.289 (NCBI36/hg 18) using quality controlled genotypes. Only imputed SNPs with Rsq values (squared correlation between imputed and true genotypes) above 0.3 and a quality above 0.9 were kept for further analysis. Association analysis and test for interaction based on the imputed dataset were performed as described above.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. SUBJECTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Association Analysis

In the GRIN2B gene nine SNPs were significantly associated with schizophrenia (Table II). One SNP (rs1806194, Pnominal = 0.0008) remained significantly associated after Bonferroni correction (correction for 30 tests). We observed no significant association of SNPs in the other four analyzed GRIN genes with schizophrenia, except rs17203234 in GRIN2A (Pnominal = 0.0473) (Table II).

Table II. SNPs Showing Association With Schizophrenia (Pnominal < 0.05). Genotypes, Number (#) of Genotypes and Minor Allele Frequencies (MAF) in Schizophrenia (SZ) and Control Individuals
SNPGeneLocationSNP typeGenotypes# of genotypesMAFP -valuesOR
SZ (n = 971)Controls (n = 1489)SZControls
  • Nominal P-values (P-values) and odds ratio (OR) from the CMH test.

  • a

    Significant after Bonferroni correction.

rs890GRIN2B13606575untranslated-3TT/TG/GG250/488/229320/766/3910.5110.4760.01271.161
rs1806191GRIN2B13607905synonym exon13GG/GA/AA260/455/230336/727/4020.5160.4780.00491.185
rs10772692GRIN2B13612415intronAA/AC/CC206/476/274266/748/4580.4640.4350.04161.131
rs2270359GRIN2B13613580intronTT/TG/GG254/476/226349/737/3870.5150.4870.04421.127
rs1806194GRIN2B13614394intronCC/CT/TT141/458/346283/728/4620.3920.4390.0008a0.816
rs1806195GRIN2B13614659intronGG/GT/TT245/456/240314/702/4090.5030.4670.00641.179
rs1805482GRIN2B13656041synonym exon7AA/AG/GG95/404/463182/663/6300.3090.3480.00550.836
rs1805199GRIN2B13660931intronAA/AG/GG41/297/62943/422/10120.1960.1720.01621.202
rs1805554GRIN2B13798708intronAA/AG/GG0/55/9150/62/14170.0280.0210.04741.464
rs17203234GRIN2A9778018intronCC/CT/TT0/14/9371/35/14400.0070.0130.04730.539

There was significant gene-wise association of GRIN2B with schizophrenia (P = 0.0047) and no gene-wise association was observed for the other genes. Gene–region P-values for GRIN2B were calculated in order to localize the region of GRIN2B which demonstrated association (using the method that was used to calculate the gene-wise P-values). This was done for SNPs placed in the 5′ region of the gene (chr12:13.884.751–14.024.289), the middle of the gene (chr12:13.745.214–13.884.751) and the 3′ region of the gene (chr12:13.605.677–13.745.214). Significant gene–region association was only found for the 3′ region of the gene (P = 0.0046).

After Bonferroni correction no tests for homogeneity of odds ratios were significant (two SNPs demonstrated nominal significant differences between samples, rs10772692 Pnominal = 0.045 and rs1806205 Pnominal = 0.026). When performing haplotype analysis we found no increased association when compared to the CMH single marker analyses. The two and three marker haplotypes most strongly associated with schizophrenia were the G–C haplotype of rs2270359–rs1806194 and the C–T–T haplotype of rs1806194–rs1806195–rs1806213 in GRIN2B (Pnominal= 0.001 for both haplotypes).

GRIN Gene Variation and Maternal HSV-2 Infection

Evidence for significant interaction between GRIN gene variation and maternal HSV-2 seropositivity affecting the risk of schizophrenia was found for three SNPs in GRIN2A and seven SNPs in GRIN2B (Table III). After Bonferroni correction (correction for 30 tests) two SNPs remained significant in the GRIN2B gene (rs1805539, rs1806205) (Table III). The two GRIN2B SNPs both demonstrated an odds ratio below 1 when having a seropositive mother, and the opposite when having a seronegative mother (Table III). The whole–gene interaction P-value was highly significant for GRIN2B (P = 3.44 × 10−05). None of the other genes demonstrated significant whole–gene interaction P-values. The gene–region interaction P-value (calculated for the regions defined above) was only significant for the 3′ region of GRIN2B (P = 2.84 × 10−6).

Table III. Minor Allele Frequency (MAF) for Individuals With Schizophrenia (SZ) and Controls Having HSV-2 Seropositive (HSV-2+) and Negative Mothers (HSV-2)
SNPGeneAlleles minor/majorHSV-2 minor+Alleles minor/majorHSV-2 minorP (ADD × HSV-2)ORposORneg
Freq. casesFreq. controlsFreq. casesFreq. controls
  • Significant nominal P-values (PADDxHSV-2) for the interaction between SNP variation in the offspring and maternal HSV-2 infection. Odds ratios depending on maternal HSV-2 infection are calculated separately for individuals with a seropositive (ORpos) or a seronegative (ORneg) mother using logistic regression with an additive model.

  • a

    Significant after Bonferroni correction.

rs10492827GRIN2AG/A0.2500.169G/A0.1810.2040.02461.6360.861
rs11866328GRIN2AT/G0.3680.465T/G0.4410.4040.01360.6351.156
rs2267772GRIN2AC/G0.4030.296C/G0.2920.3550.00881.6370.757
rs1805199GRIN2BA/G0.2390.100A/G0.1830.1620.01932.7381.145
rs1805482GRIN2BA/G0.3120.290A/G0.2900.3830.02550.4590.661
rs1805539GRIN2BC/G0.3530.528C/G0.4310.3750.0001a0.4811.241
rs1806191GRIN2BG/A0.5050.443G/A0.5350.4400.00951.3161.463
rs1806195GRIN2BT/G0.5220.465G/T0.5150.4340.03961.2911.392
rs1806201GRIN2BA/G0.2340.352A/G0.2960.2350.00170.5531.372
rs1806205GRIN2BC/G0.2700.381C/G0.3530.2700.0008a0.5801.446

Imputation

Imputation allowed us to get genotypic information from additionally 219 SNPs (having an Rsq value > 0.3, quality > 0.9, MAF > 0.01, call rate > 0.9) in GRIN2B. No SNPs demonstrated strong deviation from HWE in either controls or cases (nominal P < 0.001). The CMH association test found 39 SNPs significantly associated with schizophrenia (nominal P < 0.05) in the imputed dataset, however none of them with a smaller P-value than observed for our genotyped marker rs1806194. The imputed SNP with the lowest P-value was rs1805475 (nominal P = 0.002), which is in high LD with rs1806194 (r2 = 0.95). After imputing 33 SNPs demonstrated nominal significant interaction P-values. The imputed SNP with the lowest interaction P-value was rs1805476 (Pnominal = 0.0003), which is in moderate LD with three genotyped SNPs demonstrating significant interaction P-values (rs1806191, r2 = 0.64; rs1806201, r2 = 0.59; rs1806195, r2 = 0.62).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. SUBJECTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

In this study one SNP (rs1806194) was associated with schizophrenia in the GRIN2B gene after Bonferroni correction and additional eight SNPs showed nominal significance (Table II). Taking the evidence of association of these nine SNPs into account the gene-wise P-value for association was low (P = 0.0047), providing strong evidence that GRIN2B is associated with schizophrenia in the Danish population. Moreover, all of the nine SNPs except one (rs180554) are located at the 3′ region of the gene showing limited LD with SNPs placed in other regions of the gene. Since the probability of this occurring by chance is low, we suggest that genetic variation within this part of GRIN2B in particular, could influence the susceptibility to schizophrenia. This conclusion is supported by the imputation results since the imputed SNP demonstrating the strongest association with schizophrenia is in high LD with rs1806194. The level of LD between the genotyped SNPs at the 3′ region of the gene is low (Fig. 1), indicating the presence of multiple risk variants. The results of this study confirm the findings of other studies demonstrating association between schizophrenia and GRIN2B gene variation at the 3′ region of the gene [Ohtsuki et al., 2001; Di Maria et al., 2004]. However, this study was not able to replicate the results of the meta-analysis performed by Li and He [2007]. They found replicated evidence for association of four SNPs in GRIN2B with schizophrenia. Three of these four SNPs were also included in this study (rs1806201, rs7301328, rs1805502) but they were not significantly associated with schizophrenia. In this study, six SNPs previously found to be associated with schizophrenia in the literature [Forrest et al., 1994; Ohtsuki et al., 2001; Begni et al., 2003; Di Maria et al., 2004; Qin et al., 2005; Martucci et al., 2006; Zhao et al., 2006] were successfully genotyped. Only one of them was nominal significantly associated with schizophrenia (rs890 in GRIN2B). All the studies from which the literature SNPs had been identified were performed using a smaller sample size than the one used in this study, and therefore the lack of replication is unlikely the result of low power.

thumbnail image

Figure 1. Linkage disequilibrium (r2 values) between all genotyped SNPs in the GRIN2B 3′ region (Chr12: 13,603,492–13,661,207). This is the location of the SNPs showing the strongest association with schizophrenia and interaction with maternal HSV-2 infection. SNPs marked with “*” were significantly associated with schizophrenia, SNPs marked with “+” showed significant interaction with maternal HSV-2 infection.

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Recent genome-wide association (GWA) studies [O'Donovan et al., 2008; Stefansson et al., 2009] have not reported SNPs within GRIN2B to be associated with schizophrenia on a genome-wide scale. However the risk associated with rs11055621 (OR = 0.79, P = 0.0027) located in GRIN2B, reported in the GWA study of individuals with European ancestry by Shi and co-workers [Shi et al., 2009], was at the same level as observed in this study.

In the present study it was possible to investigate interactions between GRIN gene variation and maternal HSV-2 seropositivity in the Denmark II sample. This sample has an advantage of being population based, including study individuals from nation-wide registers, eliminating the risk of selection bias in the case ascertainment. A previous investigation of the Denmark II sample has shown maternal HSV-2 infection on its own to be significantly associated with increased risk of schizophrenia in the offspring [Mortensen et al., 2010] confirmed by Buka et al. [2008]. Very interestingly this study demonstrated strong evidence for interaction between GRIN2B gene variation and maternal HSV-2 seropositivity influencing the risk of schizophrenia in the offspring. Three SNPs were both significantly associated with schizophrenia and demonstrated a significant interaction P-value (rs1806191, rs1806195, rs1805199 (Tables II and III); however, none of these SNPs remained significant after Bonferroni correction. In the interaction analysis two SNPs (rs1805539, rs1806205) remained significant after Bonferroni correction (Table III). These two SNPs showed no association with schizophrenia on their own, which can be explained by the major allele increasing the risk of schizophrenia among individuals with HSV-2 seropositive mothers while having the opposite effect in the seronegative group, thus cancelling the SNP effect in the basic analysis. When calculating the odds ratios within the group of HSV-2 positive mothers and within the group of HSV-2 negative mothers for the 33 SNPs demonstrating significant interaction P-values in the imputed dataset, the picture was not so clear cut. Nine SNPs demonstrated an odds ratio below one in the seropositive group and above one in the seronegative group, 19 SNPs demonstrated the opposite pattern and five SNPs demonstrated an odds ratio above one in the seronegative group and an increased odds ratio in the seropositive group. The findings of SNPs demonstrating opposite odds ratios depending on HSV-2 status stress the importance of including environmental factors when evaluating the risk of schizophrenia.

The SNPs associated with schizophrenia and which demonstrate significant interaction P-values are all located in the 3′ region of the gene (except rs180554) (Fig. 2) and are not in LD with SNPs more upstream in the gene. This indicates that genetic variation particularly affecting the carboxyl terminal and posterior part of the NR2B subunit is involved in the pathophysiology of schizophrenia. The carboxyl domain is believed to extend into the cytoplasm where it binds various proteins. The carboxyl tail is also the location of a phosphorylation site for the Ca2+calmodulin-dependent protein kinase II [Pradeep et al., 2009] (The SNPs closest to this site genotyped in this study are rs1806191 and rs1805247 up- and downstream, respectively). Interestingly, maternal stress has been found to affect the brain of offspring in rats by reducing Ca2+calmodulin-dependent protein kinase II levels while increasing NR2B phosphorylation affecting the glutamate synapses for life [Fumagalli et al., 2009].

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Figure 2. Distribution of –log10(P-values) values in GRIN2B from CMH association test and for interaction between SNP variation and maternal HSV-2 infection. rs-numbers for SNPs with P-values below 0.01 are given.

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The expression pattern of the NR2B subunit changes during brain development. Expression starts early in the developing brain of the fetus and predominates in prenatal cortex, the thalamus, and spinal cord [Monyer et al., 1994]. During early postnatal brain development the NR2 subunit has the highest levels of expression compared to the other NMDA subunits [Ewald and Cline, 2009]. The expression declines to low levels during cortical development when the NR2B receptors are replaced by NR2A containing NMDA receptors [Cohen and Greenberg, 2008; Liu et al., 2004]. The results from this study, suggesting that maternal HSV-2 seropositivity interacts with GRIN2B variations in the offspring, are therefore biologically plausible due to the crucial role of NR2B in early brain formation.

The pathophysiological pathways through which maternal HSV-2 might affect the NR2B subunit in the offspring are several. It is possible that some infants are directly infected with HSV-2 during pregnancy or delivery. However, this is very unlikely, since the incidence of neonatal herpes is very low with an estimated rate of 3.2 out of 100,000 live births in the Netherlands [Poeran et al., 2008] and 1.6 out of 100,000 live births the United Kingdom [Tookey and Peckham, 1996]. A more likely scenario might be that the pathophysiological processes result from a generally enhanced maternal immune activation due to HSV-2 infection. In this study it is not known if the antibodies measured in the immunoassay reflect primary HSV-2 infection during pregnancy or already established HSV-2 infection before pregnancy. However, the major part of the HSV-2 seropositive women in the study are more likely to have an established infection since only around 1.5% of women acquire primary HSV-2 during pregnancy [Brown et al., 1997]. The impact of established HSV-2 infection on the NR2B subunit in the offspring may not be that different from the impact of primary infection due to frequent reactivation of HSV-2 in latently infected cells of the sensory ganglia. Reactivation is known to be frequent with several episodes a year and HSV can travel via the neuronal axons back to the genital mucosa without clinical symptoms, which may characterize up to 81% of the recurrences [Wald et al., 2003; Watts et al., 2003; Crespi et al., 2007; Mark et al., 2008]. Among pregnant women with established genital HSV infection, 75% can expect at least one recurrence during pregnancy [Sheffield et al., 2006; Aga and Hollier, 2009].This suggests that the immune system in the major part of the HSV-2 seropositive mothers in this study has been activated due to the disease during pregnancy. It could therefore be speculated that the interaction between HSV-2 seropositivity and GRIN2B influencing the risk of schizophrenia in the offspring is not specific to the herpes virus but rather may result from a general increased maternal immune activation. In rats general maternal immune activation has been shown to alter brain development in the offspring [Smith et al., 2007; Lowe et al., 2008] and to be associated with offspring NMDA dysregulation [Samuelsson et al., 2006].

It could also be speculated that maternal HSV-2 recurrence might lead to stress responses in the mother. Maternal stress has been shown to alter the NMDA receptor mediated synaptic plasticity leading to long lasting malfunctions of the hippocampus which could cause psychopathological symptoms in adulthood [Son et al., 2006; Fumagalli et al., 2009].

In conclusion the comprehensive SNP tagging approach has made it possible to evaluate precisely the genetic variation in the five genes analyzed and there was strong evidence for association of SNPs in GRIN2B with schizophrenia. The results suggest that especially genetic variation in the 3′ region of GRIN2B is associated with schizophrenia, and that genetic variation in this region interacts with maternal HSV-2 seropositivity, affecting the risk of schizophrenia in the offspring later in life. A possible explanation for the observed interaction is that GRIN2B variation affects the susceptibility of the NR2B subunit in the offspring to maternal inflammatory- and/or stress responses induced by HSV-2 infection.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. SUBJECTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

The study was supported financially by the Stanley Medical Research Institute, the Novo Nordisk Foundation, and The Danish Council for independent Research – Medical Sciences (jr.nr. 9601612 and 9900734). The authors would like to thank Mette Bertelsen for collecting blood samples and Steen Staarup for interviewing patients and in the Denmark III sample.

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  2. Abstract
  3. INTRODUCTION
  4. SUBJECTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. SUBJECTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

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