An excess dopaminergic activity may be implicated in the etiology of schizophrenia. Our objective was to identify nucleotide variants in the 5' region of the dopamine D2 receptor gene (DRD2) and to clarify their effects on schizophrenia. We identified two polymorphisms, the A-241G and -141CIns/Del,by examination of 259 bp in the 5'-flanking region and 249 bp of exon 1 ofDRD2. Reporter constructs containing the -141CDel allele cloned into a luciferase reporter plasmid drove 21% (Y-79 cells) and 43% (293 cells) expression compared with the -141CIns allele. In a case-control study, the -141CDel allele frequency was significantly lower in 260 schizophrenic patients than in 312 controls (OR = 0.60, 95%CI 0.44-0.81,P< 0.001). No significant association was found between the A-241G polymorphism andin vitro luciferase activity, or in allele frequency between the patients versus controls. These findings show that the -141CIns/Del may be a functional polymorphism in the 5'-promoter region ofDRD2 and may affect the susceptibility to schizophrenia.
The concept that an excess dopaminergic activity leads to the psychotic symptoms of schizophrenia is based on pharmacological observations (1 -3 ). Dopamine agonists can cause or exacerbate psychotic symptoms (4 ). The antipsychotic potency of a wide range of neuroleptic drugs is correlated with the ability to block dopamine D2 receptors (2 ,3 ). Also, the density of dopamine D2 and/or other D2-like receptors (D3 and D4) is elevated in the brain of schizophrenics obtained at post-mortem who had been neuroleptic-free for many years before death (5 ). However, the issue of whether the schizophrenic process itself is associated with an increase in dopamine receptor is controversial (6 ) and it is not yet clear which D2-like receptors are raised in post-mortem schizophrenic brains (7 ,8 ).
An increase in the density of D2 receptors in schizophrenia could result from abnormal processing of genetically abnormal receptors. However, an intensive search for nucleotide variants causing alterations in the amino acid sequence has identified only a few molecular variants. The identified variants, Ser311Cys (9 ,10 ), Val96Ala (10 ), and Pro310Ser (10 ), are relatively rare. An association has been reported between the Cys311 variant and type I schizophrenia (11 ,12 ) or with alcoholism (13 ). However, other investigators failed to find an association with schizophrenia (10 ,14 -19 ) or alcoholism (20 ). No other variant has been reported to be associated with schizophrenia or alcoholism (10 ).
Variation in the genomic sequence of the promoter region of the D2 receptor gene (DRD2) could affect the expression or regulation of the gene. The first exon ofDRD2 is ~250 kb apart from the second exon where the translation start codon exists (21 ). Since the possibility that promoter variants are associated with schizophrenia cannot be excluded, we investigated the sequences of the first exon and of the 5'-flanking region to find out functional variants associated with the expression ofDRD2.
Direct sequencing of selected individual samples in which sequence variations were suggested by SSCP analysis carried out in randomly selected 20 schizophrenic and 20 control subjects revealed variants at two positions. A-241 was substituted with G and an insertion/deletion (Ins/Del) variant was found at position -141 where one cytosine was deleted from a run of two cytosines when compared with the published sequence (22 ; Fig.1 ). In addition, base differences at two positions from the published sequence (not polymorphic) were found by PCR direct sequencing in five individuals (Fig.1 ).
Table 1.
To test the effect of a polymorphic sequence on gene expression, fragments containing the three haplotypes (the -141CIns and A-241 alleles, the -141CIns and G-241 alleles, the -141Del and A-241 alleles) were fused to luciferase reporter constructs and transiently transfected into D2-expressing human retinoblastoma Y79 cells (24 ) and D2-non-expressing human kidney 293 cells. Stable cultured tumor cells of human CNS origin constitutively expressing D2-receptors have rarely been reported (24 ,25 ). Y-79 cells express both neural- and glial-specific cellular protein markers (26 ) and D2 receptors expressed in Y-79 cells are functional (24 ). It has been confirmed that 293 cells do not express D2 receptors (27 ). The haplotype containing the A-241 and -141CIns alleles is the most common and may be the wild haplotype. The wild-type fragment with the A-241/-141C Ins alleles directed luciferase synthesis to a level of 180% and 48% of the SV40 promoter-reporter plasmid, or of 38- and 16-fold greater than the promoter-less basal plasmid in Y79 and 293 cells, respectively (Fig.2 ). By contrast, the fragment bearing the A-241/-141CDel alleles directed synthesis with significantly less promoter strength than the fragment with the wild haplotype (39 and 21% of the SV-40 promoter-reporter plasmid, or 8- and 7-fold greater than the promoter-less basal plasmid in Y79 cells and 293 cells, respectively). Reporter constructs containing the A-241/-141CDel allele cloned into a luciferase reporter plasmid drove 21% (Y-79 cells) and 43% (293 cells) expression compared with the wild A-241/-141C Ins haplotype. The promoter strength of the fragment with the G-241/-141CIns alleles did not significantly differ from that of the fragment with the wild haplotype in Y79 and 293 cells.
No significant difference in genotype or allele frequency of the A-241G polymorphism was observed between the schizophrenic and control groups (Table1 ). The frequency of the -141CDel allele was significantly decreased in the schizophrenic subjects compared with the controls. The odds ratio for schizophrenia associated with the -141CIns allele was 0.60 (P< 0.001).
TheDRD2 5'-promoter fragments drove the transcription of heterologous luciferase constructs in Y79 cell line expressingDRD2 as well as inDRD2 non-expressing 293 cells. Although the three D2 5'-fragment constructs exhibited from 1.9- to 3.7-fold higher expression in Y79 cells than those in 293 cells, the promoter-less basic plasmid also showed 1.6-fold higher expression in Y79 cells than that in 293 cells. It is likely that the 304 bp sequence ofDRD2 analyzed in the present study did not contain the elements required to confer a tissue-specific expression of the gene. It has been reported that the 1 kb rat D2 promoter and the 450 bp human D5 promoter do not show cell-specific expression for D2 or D5 expressing cells (28 ,29 ). Also, there may be repressor elements between position -1140 and -352 of the rat m4 cholinergic muscarinic receptor gene that repress transcription in non-expressing cells (30 ).
The fragment that contained the -141CDel allele showed a decrease in promoter strength as compared with the fragment that contained the -141CIns allele in Y-79 and 293 cells. Although the transcription factors involved in this allelic difference have not been identified, the position of the polymorphism is part of a putative binding site for Sp-1, 5'-CCAGGCCGGGGATCGCC.
Whether the -141CIns/Del polymorphism is actually related toDRD2 gene expression in human brains is not yet clear. Our preliminary data showed that the number of spiperone binding sites (Bmax) in the putamen of the post-mortem brains tended to be decreased in four non-schizophrenics who carried the -141CDel allele compared with six non-schizophrenics who did not: the mean ± SD (fmol/mg protein) of the former and latter groups were 216.8 ± 56.8 and 144.1 ± 58.7, respectively,P< 0.09, Student'st-test, two-tailed. This trend was in the expected direction from the results of thein vitroluciferase assay experiments. However, the binding of spiperone to D4 receptor could interfere with measurements of Bmax.
A negative association betweeen the -141C allele associated with lower luciferase activity and schizophrenia was suggested by the results of the present study. Previous post-mortem and neuroimaging studies indicate that striatal D2 receptor density is slightly increased in drug-free schizophrenics as compared with controls (5 ). A decreased frequency of the -141CDel allele in schizophrenics may contribute to elevation of D2 receptor density in schizophrenics. If this association is confirmed by family samples, it will be evidence supporting the dopamine hypothesis for schizophrenia.
While it is generally accepted that genetic heterogeneity is a less likely bias in Japan, various biases including undetected population stratification may affect case-control comparisons. Furthermore, data that suggest a polymorphism inDRD2 associated with schizophrenia appear to be incompatible with linkage studies, since no study has reported positive linkage between schizophrenia andDRD2(31 ,32 ). However, if the association suggested by the present study is true, the effect is small. Linkage tests using large numbers of sib pairs and/or transmission disequilibrium test are more suitable for confirming or excluding the allelic susceptiblity to schizophrenia.
In conclusion, a possible functional polymorphism in the 5'-promoter region ofDRD2was identified and the polymorphism may be associated with schizophrenia. However, both case-control comparisons and promoter studies are not without potential pitfalls; our conclusions must be considered tentative until they have been subjected to the test of independent replication.
To evaluate the association between the identified polymorphisms and schizophrenia, we examined 260 unrelated Japanese patients [151 men and 119 women, aged 19-81 years (mean 44.4); age at disease onset 12-39 years (mean 22.4)] who met the criteria for schizophrenia of the American Psychiatric Association's Diagnostic and Statistical Manual of Mental Disorders (DSM-III-R) (1987). The patients were receiving treatment at eight hospitals within 200 km of Tokyo. The control subjects were 312 unrelated Japanese [173 males and 139 females, aged 29-75 years (mean 48.7)]. Among the controls, 135 were hospital staff members documented to be free of psychosis. The remainder were corporate employees who had requested annual physical examinations but had not been evaluated for psychiatric disorders by a psychiatrist. The cases and controls were resident in the same area of Japan. No minorities in Japan were included in this study. Samples of venous blood were collected after written informed consent had been obtained. The present study was approved by the ethics committees of Tokyo Medical and Dental University and University of Tsukuba.
DNA was prepared from blood using standard techniques. The genomic sequence of 284 bp of the 5'-flanking region and 274 bp of exon 1 ofDRD2 was amplified by PCR with the primer pair D2-677 (5'-ACTGGCGAGCAGACGGTGAGGACCC; nn -284 ~ -260) and D2-676 (5'-TGCGCGCGTGAGGCTGCCGGTTCGG; nn -5 ~ +20), and the pair of D2-1073 (5'-CGCCGAGGAGGTACAGCTCCTTTGGTG; nn -354~ -325) or D2-977 (5'-GCCGAACCGGCAGCCTCACGCGCGCA; nn -6 ~ +21) and D2-976 (5'-GGGGCAGAGACGGCGCCGGCTGCTT; nn +250 ~ +274). Nucleotides are numbered from the 5' end of the human D2A cDNA (33 ), modified as described in the paper by Gandelmanet al. (22 ). NativePfu polymerase (Stratagene) was used to amplify the fragments by incubation at 98oC for 1 min followed by 35 cycles of 98oC for 20 s and 74oC for 5 min for primers of D2-676 and -677. When using primers D2-1073 or D2-977 and D2-976, the annealing/extension temperature was 77oC. PCR reaction buffers were supplemented with formamide at 4% final concentration.
The SSCP method was used to screen polymorphisms using PhastSystem (Pharmacia). Direct sequencing was carried out using a Sequenace kit (US Biochemical Corp.) or cycle sequencing usingTaq polymerase and dye terminators on an Applied Biosystems automated sequencer (Perkin-Elmer).
PCR-restriction fragment length polymorphism (RFLP) analysis was performed on amplified fragments digested withBstN1 (for the -141CIns/Del polymorphism) or withMaeIII (for the A-241G polymorphism). The digested fragments were electrophoresed in 2% agarose gel and were visualized by ethidium bromide staining.
Fragments consisting of 284 bp of the 5'-flanking sequence and 20 bp of the first exon were obtained from individuals with identified haplotypes. The fragments were amplified by PCR with theMluI linker added D2-677 and theBglII linker added D2-676. The fragments were cloned intoMluI/BglII-cut pGL3-basic plasmid (Promega). The clones of the promoter segments used were sequenced in full to rule out any sequence alterations.
Human retinoblastoma Y-79 cells purchased from The American Type Culture Collection (ATCC) were grown in suspension in RPMI 1640 supplemented with 15% fetal bovine serum. Human embryonal kidney 293 cells purchased from ATCC were grown in Eagle's minimal essential medium (MEM) supplemented with 10% horse serum.
The DNA purified by a Qiagen column was transiently transfected into cells using Tfx-50 (Promega). An aliquot of 3 µg of promoter-luciferase fusion plasmid, pGL3-basic and pGL3-promoter plasmid DNAs was co-transfected with pSV-[beta]-galactosidase control plasmid (Promega). After 5 h, cells were washed in phosphate-buffered saline and maintained in the appropriate media supplemented with sera. After an additional 24 h, cells were harvested, lysed, and assayed for luciferase and [beta]-galactosidase enzymatic activity according to the manufacturer's recommendations (Promega).
Luciferase activity was normalized to [beta]-galactosidase activity and compared by Student'st-test. Group difference in allele frequency was evaluated by the [chi]2 test in a case-control study. A two-tailed [alpha] criterion of <0.05 was considered statistically significant.
This study was supported by a scientific research grant (No. 08672594) from the Ministry of Education, Science and Culture of Japan, and a scientific research grant from the National Center of Neurology and Psychiatry of the Ministry of Health and Welfare of Japan.
Human Molecular Genetics
Pages
Introduction
Results
Identification of polymorphisms in the 5'-region ofDRD2
Transient transfection and luciferase assay
Association ofDRD2 5'-promoter polymorphism with schizophrenia
Discussion
Materials And Methods
Population
Amplification of exon 1 and the 5'-flanking region ofDRD2
Single strand conformation polymorphism (SSCP) and direct sequencing
Genotyping
Construction of D2-promoter-luciferase plasmids
Cell culture, plasmid transfections and luciferase assays
Statistical procedures
Acknowledgements
References
Subjects
Number (frequency) of genotype
Number (frequency) of allele
Odds ratio
(95% confidence interval)
A-241G
A-241G
AA
AG
GG
A
G
Schizophrenics (n= 229)
186 (0.81)
40 (0.17)
3 (0.01)
412 (0.90)
46 (0.10)
1.11 (0.69-1.78)
Controls (n= 170)
140 (0.82)
29 (0.17)
1 (0.01)
309 (0.91)
31 (0.09)
-141CIns/Del
-141C Ins/Del
Ins/Ins
Del/Ins
Del/Del
Ins
Del
Schizophrenics (n= 260)
190 (0.73)
66 (0.25)
4 (0.02)
446 (0.86)
74 (0.14)
0.60 (0.44-0.81)a
Controls (n= 312)
193 (0.62)
102 (0.33)
17 (0.05)
488 (0.78)
136 (0.22)
REFERENCES
This page is maintained by OUP admin. Last updated Thu Mar 13 17:29:21 GMT 1997. Part of the OUP Journals World Wide Web service.Copyright Oxford University Press, 1996