Introduction

Bipolar disorder (BP) is a severe, complex psychiatric condition characterized by the recurrence of episodes of depression and mania.¹ The lifetime prevalence in the general population is 1%, but the approximate morbid risk in first-degree relatives of BP probands is estimated to be up to 10%.² From the observation of this familial pattern of the disease, as well as from data arising from twin studies and molecular genetic studies, a strong genetic component has been suggested for BP.^2,3,4 On the other hand, the genetic mechanisms involved in this disease are complex and are most likely to be explained by the contribution of multiple genes of small effect.²

Candidate gene studies have focused mainly on genes coding for the serotonin and dopamine receptors. These candidates have been chosen because data from pharmacological studies suggested the involvement of these two neurotransmitters in the pathogenesis of BP.^5,6,7,8 Results from these studies are conflicting and none of them is suggestive of the presence of genes of major effect in conferring susceptibility to BP.^{9,10,11,12,13,14,15,16}

The amino acid glutamate has been hypothesized to be implicated in the pathogenesis of major psychoses, and a model involving glutamatergic deficiency has been proposed for schizophrenia.¹⁷ The glutamate receptors are currently classified according to their activation by different pharmacological agonists: the N-Methyl-D-aspartic acid (NMDA) receptor (NMDAR) is the one activated by the NMDA. The NMDAR is a major site for excitatory neurotransmission, and its antagonists (eg phencyclidine and ketamine) have been found to induce cognitive and behavioral symptoms, including psychotic symptoms in humans.^18,19

Particularly interesting with respect to BP are the data on the mechanism of action of the most commonly used compounds for the long-term treatment of the disease, namely lithium and valproate. There is good evidence that lithium acts via the NMDAR, either by stimulating glutamate release and inositol 1,4,5-triphosphatase accumulation.²⁰ or by inhibiting the NMDAR-mediated calcium influx.²¹ There is also evidence that valproate increases gamma-amino-butyric-acid (GABA) synthesis and release and attenuates the neuronal excitation induced by NMDA-type glutamate receptors.²² These findings suggest a role for the genes coding for the NMDAR as candidate genes for BP.

The NMDAR is composed of different combinations of subunits (NMDA-receptor 1 and NMDA-receptors 2A–D). A silent polymorphism in the gene encoding the NMDA-2B receptor (GRIN2B) has recently been investigated in a case–control association study including 164 schizophrenia patients with negative results.²³

The NMDAR1 subunit is encoded by a gene located on chromosome 9q34.3²⁴ (GRIN1). Two silent polymorphisms in the coding region and five in the noncoding regions have been recently identified and genotyped in a sample of schizophrenic patients and matched controls with negative results.²⁵

We previously reported the results of a family-based association study showing linkage disequilibrium between the 1001-G/C polymorphism of the GRIN1 and BP.²⁶

The aim of this study was to determine whether there is linkage disequilibrium between three polymorphisms of GRIN1 and BP.

Methods

Sample and assessment

A total of 288 probands (112 men, 176 women) with a diagnosis of Bipolar I (N=199), Bipolar II (N=77), or schizoaffective disorder, manic type (N=12) with their living parents were recruited from hospital clinics and newspaper advertisements in Toronto and across Central Canada. Diagnoses were done according to a 'best estimate' procedure, and were assessed by a structured interview for DSM-IV (SCID-I) administered by trained interviewers blind with respect to the genotypes of the probands.

In all, 279 probands (96.8%) were of European Caucasian origin, six (2.1%) were Asian, and three (1.1%) were Native American (aboriginal).

Written informed consent to participate in this study was obtained from all patients and their parents.

Genotyping

Genomic DNA was extracted from whole blood kept frozen at -80°C for an average of 2 weeks from the time they were drawn. The extraction procedure utilized a nonenzymatic, high salt method as described by Lahiri and Nurnberger.²⁷

We genotyped the 1001-G/C (located in the putative promoter of the gene), the 1970-A/G (located in exon 6), and the 6608-A/G (located in intron 11) polymorphisms of the NMDAR1 gene.

1001-G/C

The PCR conditions for this polymor-phism were as follows: the total volume of the PCR reaction was 25 l containing 100 ng of DNA, reaction buffer containing 100 mM Tris-HCl and 500 mM KCl, 1 mM MgCl₂, 0.6 M of both primers (5'-AAACGGCTGGCCCCGAACACA-3' and 5'CTCTTG CAGACTGGAGCTCCAGA-3', respectively), 0.2 mM. dNTP, and 1 U of Taq polymerase. The DNA was denatured for 5 min at 95°C, then it was subject to five cycles (95°C for 25 s, 72°C for 1 min), another set of 30 cycles (95°C for 25 s, 67°C for 45 s) and a final extension period of 10 min at 72°C. An aliquot of 5 l PCR product in a total reaction volume of 10 l was digested overnight at 37°C with 0.125 l of BseRI whose endonuclease activity is at

The resulting DNA fragments were resolved by gel electrophoresis in a 3.0% agarose gel containing ethidium bromide, and visualized by ultraviolet light. The fragment sizes were compared to molecular length standards. The following restriction fragments were produced from a PCR product of 304 bp: 168 bp (G allele) and 136 bp (C allele).

1970-A/G

The PCR reaction volume was 20 l in which 50 g of genomic DNA was amplified using PCR buffer containing 100 mM Tris-HCl and 500 mM KCl, 1.95 M of MgCl_2, 0.4 M. of each primer (5'AGTGCTGGAGTCCTGGCCCGT-3' and 5'- CGTCGC TGATGTGGGCCGACT-3'), 0.16 mM dNTP, 4% DMSO and 1 U of Taq polymerase. The PCR conditions were the same as that described for the amplification of 1001-G/C polymorphism. An aliquot of 5.0 l of DNA was digested with 0.5 l of MspI, whose endonuclease activity is at

at 37°C overnight and was resolved in a 3.0% agarose gel electrophoresis. The resulting fragments were produced from a PCR product of 292 bp: 136 bp (A allele) and 97 bp and 59 bp (G allele).

6608-A/G

The total reaction volume was 25 l containing 150 ng of DNA, reaction buffer containing 100 mM Tris-HCl and 500 mM KCl, 0.8 mM MgCl₂, 0.6 M. each of each primer (5'-AGCAGTTACC GCCCGCACCTA-3' and 5'-TGCAAGCCCCGCCTACT CCAT-3'), 0.048 mM dNTP, and 1 U of Taq polymerase. The DNA was denatured at 95°C for 5 min, then subject to 30 cycles (95°C for 30 s, 67°C for 30 s, 72°C for 30 s), and a final extension at 72°C for 10 min. An aliquot of 3.0 l of amplified DNA was digested with 1.5 l of BfaI enzyme with endonuclease activity at

The resulting DNA fragments were resolved by gel electrophoresis in a 3.5% agarose gel containing ethidium bromide, and visualized by ultraviolet light. The following restriction fragments were produced from a PCR size of 244 bp: 159 bp (A allele), and 85 bp (G-allele).

Statistical analyses

To describe clinically the sample in more detail we tabulated mean age, age at onset of BP or schizoaffective disorder, and, for BP patients only, presence/absence of seasonal pattern, and presence/absence of rapid-cycling course. Data were computed using the entire sample of probands and in the two subsamples defined by gender. Quantitative variables were compared using Student's t-tests, while categorical ones were compared using ² tests.

We tested for the presence of linkage or linkage disequilibrium between individual polymorphisms of the NMDAR1 gene and BP with the transmission disequilibrium test (TDT).²⁸ In addition, the program TRANSMIT for multiple marker haplotype transmission^29,30 was used. This program can analyze the transmission of markers from parents to offspring even if the phase is unknown. In our analysis, the minimum haplotype frequencies were fixed at 0.03; thus, haplotypes with a frequency lower than 3% were excluded, and the ambiguity of haplotypes was set at 3.

Finally, we determined whether the three polymorphisms we genotyped were in linkage disequilibrium with each other in the entire sample, in the sample of affected individuals, and in the sample of unrelated parents (STATA for Windows, version 7.1).

Results

The demographic and clinical data of all probands included in the study are shown in Table 1. No significant differences were found between genders.

Table 1 - Demographic and clinical characteristics of the sample of probands.

Full table

For the 1001-G/C polymorphism, the genotype frequencies were GG=0.7%, GC=12.4%, and CC=86.9%, in Hardy–Weinberg equilibrium. Of the 288 triads comprising the total sample, 73 were informative for the TDT, while the other 205 were triads with homozygous parents. With respect to the 1970-A/G polymorphism, the genotype frequencies were AA=48.8%, AG=43.8%, and GG=7.4%, in Hardy–Weinberg equilibrium. A total of 174 triads were suitable for the TDT. For the 6608-G/C polymorphism, the genotype frequencies were GG=0%, GC=10%, and CC=90%, in Hardy–Weinberg equilibrium. In all, 48 triads were informative for the TDT.

Allele frequencies and results of the TDT on individual markers investigated are summarized in Table 2a. For both the 1001-G/C and the 6608-G/C polymorphisms, we found a preferential transmission of the G allele to the affected probands (²=4.765, df=1, P=0.030 and ²=8.395, df=1, P=0.004, respectively). In Table 2b, the results of the haplotype analysis are reported.

Table 2 - (a) Results of the TDT on NMDAR1 polymorphisms in Bipolar triads. (b) results of the haplotype-TDT.

Full table

The three polymorphisms resulted in strong linkage disequilibrium with each other in the entire sample (1001-G/C vs 1970-A/G, P<0.04; 1001-G/C vs 6608-G/C, P=9.929e-15; 1970-A/G vs 6608-G/C, P<0.001) and in the sample of parents (1001-G/C vs 1970-A/G, P<0.04; 1001-G/C vs 6608-G/C, P=4.282e-15; 1970-A/G vs 6608-G/C, P=0.001), while were not in linkage disequilibrium in the sample of affected individuals only (1001-G/C vs 1970-A/G, P>0.7; 1001-G/C vs 6608-G/C, P>0.4; 1970-A/G vs 6608-G/C, P>0.2).

Discussion

To our knowledge, this is the first study investigating the role of GRIN1 in conferring susceptibility to BP. We have done a family-based association study using one of the largest samples of parent–offspring triads ever investigated, and its size guarantees reasonable power for the statistical analyses performed.³¹ This large cohort of families also allowed us to study the transmission of low-frequency alleles. Furthermore, the study design we used has been considered one of the best strategies to detect disease susceptibility in complex diseases where specific genes are thought to be of small effect.³²

Our results indicate the presence of significant linkage disequilibrium between both the 1001-G/C and the 6608-G/C polymorphisms of the GRIN1 and BP, with a preferential transmission of the G alleles to the affected individuals. These findings suggest a role of the 1001G and of the 6608G variants of the GRIN1 in conferring susceptibility to BP. Nonetheless, the possibility that the 1001C and the 6608C variants play a protective role should also be considered.

As mentioned, the hypothesis of the involvement of NMDAR in the pathogenesis of major psychoses is intriguing and consistent with the data derived from challenge and pharmacological studies. Antagonists of the receptor induce psychotic symptoms resembling symptoms observed during schizophrenia and manic episodes, such as conceptual disorganization, hallucinations, and delusions that have been suggested to be related mainly to the activation of the prefrontal cortex.¹⁹ Data on the putative mechanism of action of lithium and valproate, strengthen the significance of our findings. Both are successfully used for the long-term treatment of BP and both are considered to act via glutamatergic transmission.^20,21,22 In the light of these data, it is reasonable to hypothesize that alterations of the glutamatergic transmission and/or of the NMDAR are implicated in the pathogenesis of BP.

The two polymorphisms of the GRIN1 that we have found associated with BP consist of silent substitutions; thus, they are unlikely to affect directly the function of the receptor. On the other hand, there may be other explanations for the role of these polymorphisms in the pathogenesis of BP. First, as it has been hypothesized for serotonin receptor genes (eg 5HT2A),³³ one of the variants could induce different mRNA secondary structure, which affects the efficiency of translation. Second, other polymorphisms may exist in the gene that could be in linkage disequilibrium with these variants and be functionally relevant.

Given the possible important implication of our findings, further replications on independent samples are warranted. Furthermore, other polymorphisms of the gene should be investigated as well as genes coding for the different subunits of the NMDAR. Finally, given the involvement of the NMDAR in the mechanism of action of mood stabilizers, the variants of GRIN1 and of the other gene regions encoding for the different subunits of the receptor protein should be investigated with respect to alternative phenotype related to BP. It will be particularly interesting to test the role of these genes in predicting the response to mood stabilizers in BP.

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