Novel Polymorphism in the Gene Region Encoding the Carboxyl-Terminal Intracellular Domain of the NMDA Receptor 2B Subunit: Analysis of Association With Schizophrenia

This study was approved by the ethics committee of Kobe University School of Medicine. Written informed consent was obtained from all subjects. The study group consisted of 164 unrelated Japanese patients who met DSM-IV criteria for schizophrenia (105 men and 59 women; mean age=51.5 years, SD=14.1). The comparison group consisted of 171 unrelated healthy volunteers (104 men and 67 women; mean age=47.8 years, SD=14.0).

The deduced amino acid sequence of the human NR2B subunit gene has an overall identity of 90% with the reported mouse sequence (10). Adopting the gene structure topology proposed for the region encoding the carboxyl-terminal domain of the mouse NMDA receptor e3 subunit (NR2C subunit in human) (11), which consists of a small-sized exon (26 amino acids) and a large-sized exon (378 amino acids), that region of the human NR2B subunit appears to consist mostly of a large-sized exon. Seven sets of polymerase chain reaction primers were prepared to produce approximately 300 base-pair overlapping fragments covering the complete deduced large-sized exon. Standard polymerase chain reaction was performed, and 22 patients with schizophrenia were screened by using single-strand conformation polymorphism analysis. The DNA fragments displaying a different pattern during single-strand conformation polymorphism analysis were subjected to direct sequencing. An endonuclease Aci I was used to confirm a mutation identified by polymerase chain reaction direct sequencing and to carry out an analysis of the presence of the mutation in the patients with schizophrenia and in the comparison group.

Differences in the allele and genotype frequencies between the patients with schizophrenia and the comparison group were tested for significance using the chi-square test.

The fragment size of each polymerase chain reaction product obtained by the procedure mentioned above coincided with the expected size. We examined the mutations in the large-sized exon encoding the carboxyl-terminal intracellular domain of the human NR2B subunit. One point mutation was identified by single-strand conformation polymorphism and sequence analysis. This mutation was located at cDNA nucleotide position 2664, and was silent, changing codon 888 from ACC to ACT. We failed to detect other mutations that changed the amino acid sequence in the examined region. Table 1 shows the distribution of genotype and allele frequencies of the 2664C/T polymorphism for the patients with schizophrenia and the comparison group. The genotype and allele frequencies in the patients with schizophrenia did not differ from those in the comparison group (genotype, χ²=0.93, df=2, p>0.10; allele, χ²=0.08, df=1, p>0.10).

We found one nucleotide sequence variant (2664C/T) in the region encoding the carboxyl-terminal intracellular domain of the NR2B subunit. This report is the first we are aware of to describe a polymorphism in the human NR2B subunit gene.

Based on our data indicating no association between schizophrenia and the 2664C/T polymorphism, it is unlikely that the human NR2B subunit gene contributes to the etiology of schizophrenia. However, NMDA receptors mediate neuronal signaling, affect neuronal gene expression, and are involved in neuronal plasticity, outgrowth, and survival. Especially, the human NR2B mRNA distribution is restricted to the frontoparietotemporal cortex and hippocampus (10), which are the sites that are probably affected in schizophrenia. Therefore, the NR2B gene is still a possible candidate for association with schizophrenia. There may be polymorphisms in the NR2B subunit gene that cause alternations in protein function, and such alternations in function could contribute to the etiology of schizophrenia. Further attempts are needed to find genetic markers and/or functional mutations at sites of the NR2B subunit gene other than the region encoding the carboxyl-terminal intracellular domain and to determine whether such polymorphisms, if they exist, are associated with schizophrenia.

Received Oct. 7, 1999; revision received Dec. 27, 1999; accepted Dec. 30, 1999. From the Department of Psychiatry and Neurology, Kobe University School of Medicine. Address reprint requests to Dr. Shirakawa, Department of Psychiatry and Neurology, Kobe University School of Medicine, 7-5-1 Kusunoki-cho Chuo-ku, Kobe 650-0017, Japan; [email protected] (e-mail).Supported by a Grant-in Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture; a Research Grant for Nervous and Mental Disorders from the Ministry of Heath and Welfare; and the Medical Research Fund of Hyogo Medical Association.