 1997 Oxford University Press
Human Molecular Genetics, 1997, Vol. 6, No. 4
577–582
A functional polymorphism in the promoter region of
the dopamine D2 receptor gene is associated with
schizophrenia
Tadao Arinami*, Ming Gao, Hideo Hamaguchi and Michio Toru1
Department of Medical Genetics, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba 305, Japan
and 1Department of Neuropsychiatry, Tokyo Medical and Dental University School of Medicine, Tokyo 113, Japan
Received November 25, 1996; Revised and Accepted January 22, 1997
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 –141C Ins/Del, by
examination of 259 bp in the 5′-flanking region and
249 bp of exon 1 of DRD2. Reporter constructs
containing the –141C Del allele cloned into a luciferase
reporter plasmid drove 21% (Y-79 cells) and 43% (293
cells) expression compared with the –141C Ins allele.
In a case-control study, the –141C Del 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 and in vitro
luciferase activity, or in allele frequency between the
patients versus controls. These findings show that the
–141C Ins/Del may be a functional polymorphism in the
5′-promoter region of DRD2 and may affect the
susceptibility to schizophrenia.
INTRODUCTION
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).
*To whom correspondence should be addressed
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 of DRD2 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 of DRD2.
RESULTS
Identification of polymorphisms in the 5′-region of DRD2
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).
No evidence for linkage disequilibrium between the A–241G
and –141C Ins/Del polymorphisms was obtained (delta
value = 0.012, χ2 = 1.66, df = 1, P = 0.20) (23). The –141C
Ins/Del polymorphism was not in linkage disequilibrium with the
S311C polymorphism in exon 7 of DRD2 (delta value = –0.001,
P = 0.79, n = 273), or with the TaqIA polymorphism located 3′
to DRD2 (delta value = –0.01, P = 0.31, n = 179).
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Human Molecular Genetics, 1997, Vol. 6, No. 4
Figure 1. Polymorphisms in the 5′-region of the human DRD2. (A) Nucleotide sequence of the 5′ flanking region and exon 1 of the human D2 receptor gene. Upper case
letters represent exon 1 (33) and lower case letters, the 5′-flanking sequence. The location and orientation of oligonucleotides used for PCR are shown by arrows. The
nucleotide sequence is numbered from the 5′ end of the D2A cDNA (33) as indicated at the right of each line. Bases different from the published sequence (22) are shown
by asterisks. The position of the –141C Ins/Del polymorphism and the A–241G polymorphism are shown (box). (B1) Direct sequencing from individuals homozygous for
the A–241G polymorphism; (B2) genotyping for the A–241G polymorphism. The fragments amplified by PCR with primers D2-676 and -677 were digested withMaeIII.
Lane M represents pGEM marker (Promega); lane AA is a homozygote for the A–241 allele; lane AG is a heterozygote for the A–241 and G–241 alleles; lane GG is a
homozygote for the G–241 allele. (C1) Direct sequencing from individuals homozygous for the –141C Ins/Del polymorphism; (C2) genotyping for the –141C Ins/Del
polymorphism. The fragments amplified by PCR with primers D2-676 and -677 were digested with BstNI. Lane M represents pGEM marker (Promega); lane II is a
homozygote for the –141C Ins allele; lane DI is a heterozygote for the –141C Ins and –141C Del alleles; lane DD is a homozygote for the –141C Del allele.
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Figure 2. Transient expression of luciferase enzymatic activity driven by the DRD2 5′-flanking 304 bp containing the A–241 and –141C Del alleles, the A–241 and
–141C Ins alleles, and the G–241 and –141C Ins alleles in Y-79 cells (A) and in 293 cells (B). All data are normalized to β-galactosidase expression driven by
co-transfected pSV-β-galactosidase control plasmid (Promega). Activities are expressed as percentages of the activity of the positive control plasmid, pGL3-promoter
(Promega). Each value is the mean ± SEM for three independent experiments each performed in duplicate. P values are from a t-test (two-tailed).
Table 1. Distribution of the A–241G and the –141C Ins/Del polymorphisms of DRD2
Number (frequency) of genotype
Subjects
Number (frequency) of allele
A–241G
Schizophrenics
Controls
(n = 229)
(n = 170)
Schizophrenics
Controls
(n = 260)
(n = 312)
aχ2=10.8,
Odds ratio
(95% confidence interval)
A–241G
AA
186 (0.81)
140 (0.82)
–141C Ins/Del
AG
40 (0.17)
29 (0.17)
GG
3 (0.01)
1 (0.01)
A
412 (0.90)
309 (0.91)
–141C Ins/Del
G
46 (0.10)
31 (0.09)
1.11 (0.69–1.78)
Ins/Ins
190 (0.73)
193 (0.62)
Del/Ins
66 (0.25)
102 (0.33)
Del/Del
4 (0.02)
17 (0.05)
Ins
446 (0.86)
488 (0.78)
Del
74 (0.14)
136 (0.22)
0.60 (0.44–0.81)a
P < 0.001, two-tailed.
Transient transfection and luciferase assay
To test the effect of a polymorphic sequence on gene expression,
fragments containing the three haplotypes (the –141C Ins and
A–241 alleles, the –141C Ins and G–241 alleles, the –141 Del 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 –141C Ins 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/–141C Del 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/–141C Del 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/–141C Ins alleles did not
significantly differ from that of the fragment with the wild
haplotype in Y79 and 293 cells.
Association of DRD2 5′-promoter polymorphism with
schizophrenia
No significant difference in genotype or allele frequency of the
A–241G polymorphism was observed between the schizophrenic
and control groups (Table 1). The frequency of the –141C Del
allele was significantly decreased in the schizophrenic subjects
compared with the controls. The odds ratio for schizophrenia
associated with the –141C Ins allele was 0.60 (P < 0.001).
DISCUSSION
The DRD2 5′-promoter fragments drove the transcription of
heterologous luciferase constructs in Y79 cell line expressing
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DRD2 as well as in DRD2 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 of DRD2 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 –141C Del allele showed a
decrease in promoter strength as compared with the fragment that
contained the –141C Ins 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 –141C Ins/Del polymorphism is actually related
to DRD2 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 –141C
Del 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’s
t-test, two-tailed. This trend was in the expected direction from
the results of the in vitro luciferase 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 –141C Del 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 in DRD2
associated with schizophrenia appear to be incompatible with
linkage studies, since no study has reported positive linkage
between schizophrenia and DRD2 (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 of DRD2 was 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.
MATERIALS AND METHODS
Population
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.
Amplification of exon 1 and the 5′-flanking region of DRD2
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 of DRD2 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 Gandelman et al. (22). Native Pfu polymerase
(Stratagene) was used to amplify the fragments by incubation at
98C for 1 min followed by 35 cycles of 98C for 20 s and 74C
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 77C. PCR reaction buffers were supplemented
with formamide at 4% final concentration.
Single strand conformation polymorphism (SSCP) and
direct sequencing
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 using Taq polymerase and dye terminators on an Applied
Biosystems automated sequencer (Perkin-Elmer).
Genotyping
PCR-restriction fragment length polymorphism (RFLP) analysis
was performed on amplified fragments digested with BstN1 (for
the –141C Ins/Del polymorphism) or with MaeIII (for the
A–241G polymorphism). The digested fragments were electro-
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phoresed in 2% agarose gel and were visualized by ethidium
bromide staining.
Construction of D2-promoter-luciferase plasmids
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 the MluI linker added D2-677 and the BglII linker added
D2-676. The fragments were cloned into MluI/BglII-cut
pGL3-basic plasmid (Promega). The clones of the promoter
segments used were sequenced in full to rule out any sequence
alterations.
Cell culture, plasmid transfections and luciferase assays
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-β-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
β-galactosidase enzymatic activity according to the manufacturer’s recommendations (Promega).
Statistical procedures
Luciferase activity was normalized to β-galactosidase activity and
compared by Student’s t-test. Group difference in allele frequency
was evaluated by the χ2 test in a case-control study. A two-tailed
α criterion of <0.05 was considered statistically significant.
ACKNOWLEDGEMENTS
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.
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