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Introduction
Schizophrenia is a complex and devastating brain disorder that affects 1% of the population and is ranked as one of the
most costly disorders to afflict
humans[1]. Chlorpromazine is a typical antipsychotic drug used for the treatment of
schizophrenia since the 1950s, and became a milestone in the development of treatments for psychotic disorders. Although
chlorpromazine is no longer used in some countries, it is still widely used to treat schizophrenia in China and many other
developing countries. During the treatment of schizophrenics with antipsychotics, especially the typical antipsychotics, it
can cause a high rate of extrapyramidal syndrome (EPS), including akathisia, acute dystonia and pseudoparkinsonism, and
tardive dyskinesia, which is a serious drawback in neuroleptic treatment. However, the occurrence of EPS can be the
bottleneck of chlorpromazine treatment.
In recent years, investigators have been trying to find genetic factors contributing to drug-induced EPS by paying close
attention to dopamine receptor genes. Ser9Gly polymorphism in
DRD3 was studied extensively and inconsistent reports
were published[ 2_4]. In addition, several studies have aimed to identify the relationship between the
DRD2 gene polymorphisms and the drug response or adverse effects, but the results are also
controversial[5_7]. Two studies revealed that
TaqIA and -141Cins>del were
associated with drug
response[6,7], but one showed a negative
association[2]. Meanwhile, an in
vitro study demonstrated that the
-141Cins>del polymorphism had a functional role in affecting
DRD2 expression[8]. In addition,
Ser311Cys polymorphism partly affected the neuroleptics binding affinity to cause the blockade of functional
activity[9]. Hence, DRD2 is likely to be a promising candidate gene for the inducement of EPS in schizophrenic patients. In our previous
study, we found that -141Cins>del in the
DRD2 gene may be related to the therapeutic effects of chlorpromazine in
schizophrenic patients[10].
The dopamine D2 receptor (DRD2) is the primary binding target of all antipsychotics. It belongs to the family of receptors
coupled to heterotrimeric cyclic guanine nucleotide binding regulatory proteins (G-proteins). DRD2 activates intracellular
signaling by the inhibition of cAMP synthesis through interaction with
G1-like proteins[5]. The development of EPS has been
seen as a consequence of the action of typical neuroleptics on striatal DRD2. Dopamine receptor blockade
in the basal ganglia is considered as the mechanism of
EPS[11]. Farde et
al[12_15], in a series of studies, have shown that: (1) typical
neuroleptics from different chemical classes used at conventional doses occupy 65%_89% of the available DRD2; and (2)
individuals who experience EPS have significantly higher (82%±4%) levels of DRD2 blockade as compared to those patients
without EPS (74%±4%). Otherwise, clozapine, an atypical neuroleptic, has a significantly lower level of DRD2 occupancy and
produces virtually no EPS at conventional
doses[16]. Another study has also reported a consistent
result[17]. The degree of DRD2 occupancy can be an indicate of
EPS[18]. All of the above suggests that DRD2 is closely related to the onset of EPS.
In order to evaluate whether variations in the
DRD2 gene are related to drug-induced EPS, we identified more SNP
(-141Cins>del, TaqIB,
TaqID, Ser311Cys, rs6275, rs6277
and TaqIA) in the DRD2 gene of 146 Chinese schizophrenic inpatients
treated with chlorpromazine.
Materials and methods
Patients and drug treatment We recruited 146 patients, who were of Han Chinese origin, from Shanghai Mental Health
Center. Informed consent was obtained from all participating patients. All patients were acute inpatients with schizophrenia
(mean age of onset=27.3 years, SD=9.2, 38.7% female) diagnosed according to Diagnostic and Statistical Manual of Mental
Disorder, Third Edition, Revised
(DSM-III-R)[19]. None of the patients had any medication for at least 1 month before this
study. The dosage of chlorpromazine used in the comparison study was in a range of 300_600 mg/d. Patients treated with
any other antipsychotic drugs were not included. The diagnosis for each patient with EPS was made in terms of the
Simpson-Angus Scale (SAS) by at least two psychiatrists, independently, after the patients were treated with chlorpromazine for 8
weeks. As a result, 59 of 146 patients experienced EPS. The participating psychiatrists were blinded to the patientsĄŻ
genotypes.
SNP genotyping We chose 7 SNP
(-141Cins>del, TaqIB,
TaqID, Ser311Cys, rs6275, rs6277 from
http://www.ncbi.nlm.nih.gov/SNP/ and
TaqIA) from DRD2, which spans about 270 kb where
-141Cins>del is in the promoter region,
TaqIB and TaqID are in intron 1 and intron 2,
rs6275 and rs6277 are in exon 7.
TaqIA is in the 3ĄŻ-untranslated region, which is in fact located in
a novel gene, untitled X-kinase
gene[20]. Genomic DNA was extracted from peripheral blood leukocytes by a standard phenol
extraction procedure.
Genotyping of TaqIB, TaqID and
TaqIA was modified on the basis of Kaiser
et al[5], while analysis of
-141Cins>del was modified according to
Jönsson et al[21].
Rs6275 and rs6277 were analyzed by direct sequencing. All amplification reactions
were performed in a total volume of 25 µL, containing 10 ng DNA, 1×buffer, 200 µmol/L dNTP,
4×10-6 µmol/L of each primer,
1×Q solution, and 1 unit Taq DNA polymerase.
The PCR program of all these reactions consisted of 36 cycles, including an initial denaturation at 94 ºC for 5 min, and a
terminal extension period at 72 ºC using a Gene Amp 9700 thermcycler (Applied Biosystems, Foster City, CA). The condition
of the cyclic PCR was as follows. For the
-141Cins>del polymorphism: 94 ºC for 45 s, 55 ºC for 30 s, and 72 ºC for 1 min. For
TaqIB and TaqID: 94 ºC for 45 s, 53 ºC for 30 s, and 72 ºC for 1.5 min. For
rs6275 and rs6277: 94 ºC for 45 s, 55 ºC for 30 s, and
72 ºC for 45 s. For TaqIA: 94 ºC for 45 s, 56 ºC for 30 s, 72 ºC for 1 min; and for Ser311Cys, 94 ºC for 45 s, 60 ºC for 1 min, 72
ºC for 1.5 min.
All but rs6275 and rs6277 PCR products were digested with restriction enzymes according to the manufacturerĄŻs protocol,
separated by 2.0% agarose gel electrophoresis and stained with ethidium bromide for UV visualization. For
rs6275 and rs6277, we first amplified a fragment including the two SNP, and then purified the PCR product with shrimp alkaline phosphatase.
The purified PCR product was used to carry out sequencing reaction by using sense primer and a BigDye Terminator Cycle
Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA) as a total volume of 5 µL. The sequence analysis was
performed in an ABI PRISM model 3100 DNA sequencer (Applied Biosystems, Foster City, CA).
Statistical methods The difference of allele distribution between patients with EPS and without EPS was investigated
using CLUMP version 1.6[22] based on 1000 stimula-tions. The
P-values reported were two tailored and the limit of
significance was set to 0.05. The pair-wise linkage disequilibrium (LD), as measured by
DĄŻ[23], was estimated with 2LD
software from haplotype frequencies based on alleles at all possible pairs of SNP
loci[24]. EHPLUS was used to estimate the haplotype
frequency by performing model-free analysis and permutation tests of allelic association based on
EH[25]. It uses marker genotypes from a group of unrelated individuals or a group of cases and a group of controls and employs gene-counting
algorithm to estimate haplotype frequencies and output asymptotic and permutation test statistics. We used an online
calculator to test the departure from Hardy-Weinberg equilibrium in both groups (Online Hardy-Weinberg equilibrium
calculator http://www.kursus.kvl.dk/shares/vetgen/ Popgen/genetik/applets/kitest.htm).
Results
Extensive genetic variations, including restriction sites
TaqIB, TaqIA, and rs6275, exist in the
DRD2 gene of schizophrenics. However,
TaqID,Ser311Cys with an amino acid substitution and
rs6277 showed low frequency of variation in the subjects.
Frequencies of all SNP genotypes revealed no significant deviation from Hardy_Weinberg equilibrium. The result of analysis
of the SNP by CLUMP is presented in Table 1.
No statistical significance between 59 patients with EPS and 87 patients
without EPS was observed in both genotype and allele distribution on each single marker.
The results showed that TaqIB, rs6275, and
TaqIA were in relative strong linkage disequilibrium, or in a LD block. The
frequency of any two haplotypes consisting of the three SNP in linkage disequilibrium had no statistical difference (data not
shown).
Discussion
It is generally recognized that genetic variants in
DRD2 are promising as predictors for adverse affects of antipsychotic
medication in schizophrenia patients, including EPS. Many studies have reported on the relationship between the
DRD2 gene and the occurrence of schizophrenia and drug response in schizophrenia, but few have reported on the correlation
between SNP in DRD2 and response to chlorpromazine, which is widely and routinely used in China and developing
countries. In this work, however, we genotyped 7 SNPs, including
-141Cins>Del, TaqIA,
TaqIB, Ser311Cys, rs6275, rs6277,
and TaqID, but only four of them were informative enough to carry out statistical analysis. The results showed no statistical
differences in allele and genotype frequency of
-141Cins>del, TaqIB,
rs6275, and TaqIA (P>0.05). A strong level of LD was
detected in TaqIB, rs6275, and
TaqIA (DĄŻ>0.5). No significant difference was detected in distribution of haplotype
constituted by the three SNP. This indicates that the four variations of
DRD2 do not play an important role in the development of
EPS. Compared with other similar studies, our results are inconsistent with those of Suzuki
et al[8] and Mihara et
al[26], who found a positive association between the polymorphisms in
DRD2 and EPS, but more agreeable with other more
comprehensive studies that show no association between the polymorphisms of
DRD2 and EPS in Caucasian
people[5,27,28]. In our study, as only one drug was used and patients had no other medication at least one month before this study, the detecting
power was much higher than using different neuroleptics. In addition, relatively large sample sizes and more polymorphisms
were analyzed in our study and that of Kaiser et
al[5], although both showed negative results. The mechanism of EPS is more
complex than its phenotype. Although the D2 receptor is shown to have a direct effect on the inducement of EPS, the
polymorphisms themselves in DRD2 gene do not play a major role. Instead, they may cause EPS during medication with
antipsychotics by interaction with other genes, which code drug metabolizing enzymes and other receptors, such as
CYP2D6 and DRD3. Moreover, the impact of polymorphisms in
DRD2 is not large enough for detection using the current method, but
the effect may become obvious in specific gene-gene and gene-environment interactions. So, we cannot exclude a role of
DRD2 in further pharmacogenetic and pharmacogenomic studies.
One negative factor of the present study is that the diagnosis of the patients with EPS recruited was made after 8 weeks
of treatment with chlorpromazine, which was relatively short. The variations in
DRD2 analyzed here affected late-onset EPS after long-term treatment but had little effect on early-onset EPS. Chen
et al[28] showed that TaqIA polymorphism was
associated with the occurrence of tardive dyskinesia after long-term treatment.
In conclusion, DRD2 is the rational candidate gene as a predictor of the neurological adverse effects from treatment with
antipsychotic drugs. However, the 4 genetic variants in the
DRD2 gene analyzed here have not been shown to play a major
role in the inducement of EPS in Chinese schizophrenic patients. In our further study, more relative genes, such as
DRD3, will be studied to clarify this and the interaction of the genes involved will also be investigated.
Acknowledgements
We are deeply grateful to all members of the families
participating in this study, as well as the psychiatrists and mental health workers who helped us in identifying the families.
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