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Association study between glutathione S-transferase P1 polymorphism and schizophrenia in the Korean population
Progress in Neuro-Psychopharmacology & Biological Psychiatry 27 (2003) 519 – 523 www.elsevier.com/locate/pnpbp
Association study between glutathione S-transferase P1 polymorphism and schizophrenia in the Korean population Chi-Un Paea, Jung-Jin Kima,b,*, Soo-Jung Leea, Chang-Uk Leea, Chul Leea, In-Ho Paika, Ho-Ran Parkc, Soo Yangc, Alessandro Serrettid a
Department of Psychiatry, Kangnam St. Mary’s Hospital, The Catholic University of Korea College of Medicine, Banpo-Dong, Seocho-Gu, Seoul, South Korea b Department of Psychiatry, Thomas Detre Hall of the Western Psychiatric Institute and Clinic, University of Pittsburgh, Pittsburgh, OH, USA c Department of Nursing, The Catholic University of Korea College of Nursing, Banpo-Dong, Seocho-Gu, Seoul, South Korea d Department of Psychiatry, Vita-Salute University, San Raffaele Institute, Milan, Italy Accepted 14 February 2003
Abstract This study is aimed to test the association between the coding sequence functional polymorphism (Ile105Val) of glutathione S-transferase P gene (GSTP1) and schizophrenia in the Korean population. Two hundred fourteen patients with schizophrenia according to the Diagnostic and Statistical Manual of Mental Disorders-IV (DSM-IV) criteria and 110 healthy controls were enrolled in this study. Patients and controls were biologically unrelated age and sex-matched native Koreans. Genotyping for GSTP1 polymorphism was performed by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). Genotype and allele distributions of GSTP1 polymorphism in patients with schizophrenia were not significantly different from those of the controls. Comparisons of clinical variables including Positive and Negative Syndrome Scale (PANSS), change of Brief Psychiatric Rating Scale (BPRS), number of admission, and onset age also were not different according to genotype distribution. The present study suggests that GSTP1 polymorphism may not confer susceptibility to development of schizophrenia in the Korean population. D 2003 Elsevier Science Inc. All rights reserved. Keywords: Association study; Glutathione S-transferase P gene (GSTP1) polymorphism; Korea; Schizophrenia
1. Introduction Higher production of cytotoxic reactive oxygen species (ROS), including hydrogen peroxide (H2O2) and nitric oxide (NO), than resources of antioxidant defenses, such as glutathione and ascorbate, can make oxidative stress. Growth and pruning of the neuron in the brain is partly regulated by a
Abbreviations: BPRS, Brief Psychiatric Rating Scale; JNK, c-Jun Nterminal kinase; ERK, extracellular signal-related receptor kinase; GSTP1, glutathione S-transferase P gene; MAP, mitogen-activated protein; PCP, phencyclidine; PCR, polymerase chain reaction; PANSS, Positive and Negative Syndrome Scale; ROS, reactive oxygen species; RFLP, restriction fragment length polymorphism; SAPK, stress-activated protein kinase; TAS, total antioxidant status. * Corresponding author. Department of Psychiatry, Kangnam St. Mary’s Hospital, College of Medicine, Catholic University of Korea, 505 Banpo-Dong, Seocho-Gu, Seoul 137-701, South Korea. Tel.: +82-2-5901532x1533; fax: +82-2-594-3870. E-mail address: [email protected] (J.-J. Kim).
redox mechanism that controls a balance between neurodestructive oxidants and neuroprotective antioxidants (Smythies, 1999). Dopamine (Yoshioka et al., 2002) and estradiol (Behl and Moosmann, 2002) have antioxidant effects, involving redox mechanism (Smythies, 1999). Interestingly, dopamine is also easily turned into dopamine – quinones, which is highly neurotoxic free radical (Smythies, 1996). This redox mechanism has been involved in impairment of glutamate synapse (Smythies, 1999) and it is altered in patients with schizophrenia (Reddy and Yao, 1996; Yao et al., 1999). Plasma total antioxidant status (TAS) (Yao et al., 1998) and individual antioxidant proteins, such as albumin and bilirubin (Yao et al., 2000), were reduced in patients with schizophrenia, suggesting that an impaired antioxidant defense system may be a liability factor in the development of schizophrenia. The ROS reduce glutathione level, increases intracellular calcium (Hoyt et al., 1997), mediates b-amyloid protein (Zhang et al., 1997), and glutamate neurotoxicity (Smythies, 1999). These findings suggest that oxid-
0278-5846/03/$ – see front matter D 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0278-5846(03)00043-5
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C.-U. Pae et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 27 (2003) 519–523
ative stress may have a role in etiopathogenesis in schizophrenia. Moreover, Sagara (1998) found that some of the side effects of antipsychotic drug are related to oxidative stress and to the level of ROS that is controlled by antioxidant such as glutathione. Moreover, the phenomenon of apoptosis, or programmed cell death, could be related to etiopathogenesis of schizophrenia with mechanisms connected to the neurodevelopment (Catts and Catts, 2000). Cytoarchitectural disturbances of the cerebral cortex and synaptic connectivity (Kawasak et al., 2000; Harrison and Eastwood, 1998; Arnold and Trojanowski, 1996) have been supposed in patients with schizophrenia as well as progressive volumetric changes of the brain (DeLisi et al., 1995, 1997, 1998). These findings could be related to the increased rate of apoptosis in schizophrenia during neurodevelopment (Catts and Catts, 2000). This critical phenomenon in normal development could result in an abnormal number of neurons and pathological neural development (Margolis et al., 1994). In addition, apoptosis is linked to process of oxidative stress through modulation of stress protein kinases such as mitogen-activated protein (MAP) kinases (e.g., c-Jun N-terminal kinase [JNK] and extracellular signal-related receptor kinase [ERK]) (Yoshizumi et al., 2002). Glutathione S-transferase P (GSTP) is an interesting enzyme that binds to reduced glutathione and is involved in the detoxification of ROS, thus, keeping the balance of cellular redox state (Sato, 1989). Alterations in GSTP could be involved in oxidative stress and apoptosis, contributing to the development of schizophrenia through alterations in neurodevelopment. Moreover, it has a pivotal role in the regulation of stress kinases such as MAP, including p38, ERK, and JNK/SAPK (stress-activated protein kinase), which are involved in the intracellular signal transduction pathways that control brain function and neural plasticity against stress (Duman et al., 1994; Yin et al., 2000; Kyosseva et al., 2001). Recently, a study demonstrated that chronic phencyclidine (PCP) administration caused alteration of MAP kinases under regulation of GSTP, suggesting the role of the signal pathway and the enzyme in PCP-induced psychosis (Kyosseva et al., 2001). Moreover, GSTP has been found to suppress in vitro the dopamine-induced apoptosis in neurons through the modulation of MAP kinase activity (Ishisaki et al., 2001) implying possible role of this enzyme in the development of schizophrenia considering that a characteristic of this disease has been found to be the degeneration of dopaminergic neurons of the nigrostriatal and mesolimbic systems (Ishisaki et al., 2001; Smythies, 1996). It is also noteworthy that GSTP1 is located on chromosome 11q13 (Jeronimo et al., 2002), a locus possibly associated to genetic susceptibility to schizophrenia and located close to dopamine receptor D2 gene (Gelernter et al., 1992; Mulcrone et al., 1995). A functional polymorphism at codon 105 in exon 5 of GSTP1 has been found (Hu et al., 1998), which differs by a single base pair substitution (adenosine to guanosine) at nucleotide 313 resulting in an
amino acid substitution (isoleucine to valine) (Harries et al., 1997). Further, the biophysical characteristics of this polymorphism have been reported that the valine allele could make the enzymatic activity low and detoxification capacity less (Hu et al., 1998). These findings sufficiently provide us with a theoretical rationale to investigate the association between GSTP1 polymorphism and schizophrenia in a Korean sample. The authors therefore genotyped the polymorphism in patients with schizophrenia and controls and further analyzed the relationship among clinical variables such as onset age, scale score for psychotic symptom, duration of illness and numbers of admission, and GSTP1 polymorphism in patients with schizophrenia.
2. Methods 2.1. Subjects Two hundred fourteen (214) in-patients with schizophrenia admitted to Kangnam St. Mary’s Hospital, Catholic University of Korea, Seoul, and 110 normal controls participated in this study. Most of patients were chronic case and hospitalized in acute phase of the clinical course of their disease. The diagnosis was made based on the consensus between two board-certified psychiatrists (J.J.K. and C.U.L.) according to the Diagnostic and Statistical Manual of Mental Disorders-IV (DSM-IV) (American Psychiatric Association, 1994). The Structured Clinical Interview for DSM-IV Axis I Disorders—Clinician Version (SCID-CV) (First et al., 1997) was administered to all patients. Other available resources, such as clinical course, family information, and medical records, were also used. Those having a history of, or suffering from, other mental or neurological disorders other than schizophrenia were excluded. The control group consisted of volunteers from the paramedical and medical staffs or students at the hospital who had no personal or family history of major mental disorders and neurological diseases. Board-certified psychiatrists (J.J.K. and C.U.L.) also evaluated through personal interview the control group whether they had either current or past history of psychiatric problems. A written informed-consent form was obtained from all subjects after full description of the study. The Ethics Committee of the Kangnam St. Mary’s Hospital approved this study. Ninety-nine (46.3%) patients with schizophrenia and 48 (43.6%) normal controls were male. No difference was present in the gender distribution between the two groups. The average age was older in the patient group (31.2 ± 9.9 years) than in the control group (26.6 ± 10.2 years). 2.1.1. Assessment of clinical variables The clinical variables according to genotypes in patients group were evaluated by examining the symptom severity
C.-U. Pae et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 27 (2003) 519–523 Table 1 Genotype and allele distribution of GSTP1 polymorphism in patients with schizophrenia and the controls in the Korean population
GSTP1 * A/A GSTP1 * A/B GSTP1 * B/B GSTP1 * A GSTP1 * B
Schizophrenia (n = 214)
Controls (n = 110)
139 68 7 346 82
74 33 3 181 39
(65.0) (31.8) (3.3) (80.8) (19.2)
(67.3) (30.0) (2.7) (82.3) (17.7)
Values represent number (%).
using Positive and Negative Syndrome Scale (PANSS) (Kay et al., 1988), age of onset, change of Brief Psychiatric Rating Scale (BPRS) (Overall and Gorham, 1962) at the time of admission and discharge, and number of admission.
521
tions. Statistical significance was determined at P values less than .05. The power of our sample to detect differences between variants was calculated using a two-tailed alpha value of .05. Power analysis was performed with the GPOWER program (Erdfelder et al., 1996). With these parameters, considering the allele frequencies in our sample, the power analysis showed that our sample size had a high power (.80) to detect a small effect size (w=.11), which corresponds to a difference of 8% between the two alleles (odds ratio [OR] = 1.83).
3. Results 3.1. Genotype and allele frequencies of GSTP1 polymorphism in patients with schizophrenia and normal controls
2.2. Genotyping for GSTP polymorphism 2.2.1. DNA preparation DNA was extracted from whole blood using by proteinase K, as described elsewhere (Miller et al., 1988). DNA samples were stored at 4 C until used as template DNAs for polymerase chain reaction (PCR). 2.2.2. PCR-restriction fragment length polymorphism (RFLP) Amplification of the target site in GSTP1 was carried out by PCR using forward and reverse primers (50-ACC CCA GGG CTC TAT GGG AA-30 and 50-TGA GGG CAC AAG AAG CCC CT-30, respectively), as previously described (Jeronimo et al., 2002). Briefly, PCR was performed using 30 ml of reaction mixture (1 buffer [50 mM KCl, 10 mM Tris –HCl pH 8.3, 1.5 mM MgCl2], 200 ng of each primer, 200 mM of each dNTP, 50 ng of the genomic DNA, 1 unit of Taq DNA polymerase; Boehringer Mannheim, Mannheim, Germany) using the following conditions in a Perkin Elmer 9600 thermocycler (Foster City, CA, USA). After the initial denaturation step at 94 C for 5 min, the sample were processed through 35 cycles of 94 C for 45 s, 55 C for 45 s, and 72 C for 1 min. Finally, the final extension at 72 C for 7 min was done. The 176-bp PCR products were digested for 2 h at 37 C with 2 units of BsmAI (New England Biolabs, Hertfordshire, UK). The digested samples were separated on ethidium bromide 4% agarose gel electrophoresis. 2.3. Data analysis SPSS version 10.0 (SPSS, Chicago, IL, USA) was used for statistical analysis. Chi-square test was used to compare allele and genotype frequencies in patients and controls. Analysis of variance and Student’s t test were used to analyze PANSS score, onset age, BPRS change, and number of admission according to genotype and allele distribu-
Genotype distributions in patients with schizophrenia (c2 = 3.2, P=.201) and controls (c2 = 0.0004, P > .1) were in Hardy– Weinberg equilibrium. The genotype and allele distributions in patients with schizophrenia were not significantly different from those of the controls as shown in Table 1 (c2 = 0.203, df = 2, P=.904; c2 = 0.196, df = 1, P=.658, respectively). Even if we classified the genotype according to sex, it showed no difference between patients and controls (data not shown, male: c2 = 0.791, P=.673; female: c2 = 0.466, P=.792, respectively). 3.2. Relationship between GSTP1 polymorphism and clinical variables in patients group There was no significant difference in scores of PANSS (positive, negative, and total score), age of onset, BPRS change, and number of admission according to GSTP1 genotype (Table 2) and allele (data not shown, P=.670,
Table 2 Comparison of clinical variables with GSTP1 genotypes in patients with schizophrenia Genotype
PANSS total PANSS positive PANSS negative Age of onset (year) Number of admission BPRS change
P value
GSTP1 * A/A (n = 139)
GSTP1 * A/B (n = 68)
GSTP1 * B/B (n = 7)
92.82 ± 12.70
92.83 ± 14.08
88.42 ± 15.95
.689
24.73 ± 5.15
24.75 ± 4.16
24.00 ± 7.32
.927
21.07 ± 4.76
21.25 ± 5.08
19.85 ± 5.42
.771
25.6 ± 7.87
26.6 ± 7.70
23.3 ± 5.90
.485
2.9 ± 2.51
2.5 ± 2.01
2.1 ± 1.21
.357
7.9 ± 6.4
.349
11.7 ± 7.8
Values represent means ± S.D.
12.1 ± 6.3
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P=.879, P=.670, P=.837, P=.156, and P=.655, respectively).
4. Discussion 4.1. Speculation on the role of GSTP1 polymorphism in schizophrenia As stated in the Introduction, GSTP has been supposed to have a regulatory function in the process of antioxidant defenses and apoptosis through modulation of various kinases, which was suggested to be related to the development of schizophrenia (Arnold and Trojanowski, 1996; Catts and Catts, 2000; Harrison and Eastwood, 1998; Kawasak et al., 2000; Smythies, 1996, 1999). Further, the enzyme activity of GSTP shows age-related changes in brain (Gopal et al., 2000). GSTP1 gene variants have different influence on enzymatic activity: GSTP1 * B has lower enzyme activity and less capacity for detoxification (Hu et al., 1998). Our hypothesis therefore was that patients with schizophrenia would display different distribution of genotype and/or allele compared with the controls, since schizophrenic patients were supposed to have alteration in process of antioxidant defense and apoptosis, which is under the regulation of GSTP. To the best of our knowledge, this is the first study to examine the association between GSTP1 polymorphism at codon 105 in exon 5 and schizophrenia. Unfortunately, we found no significant differences in distribution of genotype and allele frequencies between patients with schizophrenia and the controls. Moreover, any association between distribution of genotypes or alleles and clinical variables, such as scores of PANSS, were not found. The present findings suggest that GSTP1 polymorphism is not associated with susceptibility to the development of schizophrenia itself, as well as clinical heterogeneity of the disorder, at least in our Korean sample. 4.2. Deliberation on the negative finding These negative findings for the association between GSTP1 polymorphism and schizophrenia should lead to some considerations. This polymorphism may not affect the development of schizophrenia or exert a very weak influence on the etiopathogenesis of the disorder. The presence of other unknown modulating factors and/or unidentified surrounding genes for GSTP1 polymorphism that may confer susceptibility to schizophrenia could be also conceivable. Given that GSTP has a regulatory function for signaling pathways through modulation of variable kinases (Duman et al., 1994; Kyosseva et al., 2001; Yin et al., 2000) and antioxidant defense mechanism are interactive (Smythies, 1999), it might be a feasible speculation that the role of GSTP in the development of schizophrenia would be an intricate modulatory function rather than a direct influence. In addition, the
negative result of Ile105Val GSTP1 polymorphism in this study does not completely exclude the association of GSTP1 with schizophrenia, because there might be other mutations in this gene that might be associated with schizophrenia. An ethnic difference in GSTP1 polymorphism has been reported. Asians have more GSTP1 * A/A and less GSTP1 * B/B and GSTP1 * A/B than Caucasians (Nerurkar et al., 2000). In addition, the frequencies of GSTP1 * A/A, GSTP1 * A/B, and GSTP1 * B/B was 0.67, 0.30, and 0.03, respectively, in our controls, compared to 0.43, 0.48, and 0.09, respectively, in Caucasian controls (Jeronimo et al., 2002). The power of the study should be also considered when a negative result is presented. The power analysis showed that our sample size had a high power (.80) to detect a small allele association (w=.11, OR = 1.83). Thus, further studies with larger samples and coming from different ethnicities should be performed to confirm the present findings that GSTP1 polymorphism is not associated with schizophrenia. 4.3. Limitation of the study This study has several limitations. Our controls consisted of hospital staffs and students so that they could not adequately represent general Korean population. A further limitation of the present study is a possible population stratification that is a liability to association study (Spielman et al., 1993); although methods controlling for stratification were not applied (Pritchard and Rosenberg, 1999), our subjects are known to be genetically homogeneous (Benkmann et al., 1989).
5. Conclusion Evidence for a contribution of GSTP1 polymorphism to susceptibility to the development of schizophrenia in the Korean population was not found in this study. Further investigations should be launched to provide us more useful information on the role of GSTP1 polymorphism in the pathogenesis of schizophrenia.
Acknowledgements This study was supported by a grant number R01-2000000-00091-0 and M6-0225-00-0001 from the Basic Research Program of the Korean Science and Engineering Foundation.
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Association study between glutathione S-transferase P1 polymorphism and schizophrenia in the Korean population Chi-Un Paea, Jung-Jin Kima,b,*, Soo-Jung Leea, Chang-Uk Leea, Chul Leea, In-Ho Paika, Ho-Ran Parkc, Soo Yangc, Alessandro Serrettid a
Department of Psychiatry, Kangnam St. Mary’s Hospital, The Catholic University of Korea College of Medicine, Banpo-Dong, Seocho-Gu, Seoul, South Korea b Department of Psychiatry, Thomas Detre Hall of the Western Psychiatric Institute and Clinic, University of Pittsburgh, Pittsburgh, OH, USA c Department of Nursing, The Catholic University of Korea College of Nursing, Banpo-Dong, Seocho-Gu, Seoul, South Korea d Department of Psychiatry, Vita-Salute University, San Raffaele Institute, Milan, Italy Accepted 14 February 2003
Abstract This study is aimed to test the association between the coding sequence functional polymorphism (Ile105Val) of glutathione S-transferase P gene (GSTP1) and schizophrenia in the Korean population. Two hundred fourteen patients with schizophrenia according to the Diagnostic and Statistical Manual of Mental Disorders-IV (DSM-IV) criteria and 110 healthy controls were enrolled in this study. Patients and controls were biologically unrelated age and sex-matched native Koreans. Genotyping for GSTP1 polymorphism was performed by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). Genotype and allele distributions of GSTP1 polymorphism in patients with schizophrenia were not significantly different from those of the controls. Comparisons of clinical variables including Positive and Negative Syndrome Scale (PANSS), change of Brief Psychiatric Rating Scale (BPRS), number of admission, and onset age also were not different according to genotype distribution. The present study suggests that GSTP1 polymorphism may not confer susceptibility to development of schizophrenia in the Korean population. D 2003 Elsevier Science Inc. All rights reserved. Keywords: Association study; Glutathione S-transferase P gene (GSTP1) polymorphism; Korea; Schizophrenia
1. Introduction Higher production of cytotoxic reactive oxygen species (ROS), including hydrogen peroxide (H2O2) and nitric oxide (NO), than resources of antioxidant defenses, such as glutathione and ascorbate, can make oxidative stress. Growth and pruning of the neuron in the brain is partly regulated by a
Abbreviations: BPRS, Brief Psychiatric Rating Scale; JNK, c-Jun Nterminal kinase; ERK, extracellular signal-related receptor kinase; GSTP1, glutathione S-transferase P gene; MAP, mitogen-activated protein; PCP, phencyclidine; PCR, polymerase chain reaction; PANSS, Positive and Negative Syndrome Scale; ROS, reactive oxygen species; RFLP, restriction fragment length polymorphism; SAPK, stress-activated protein kinase; TAS, total antioxidant status. * Corresponding author. Department of Psychiatry, Kangnam St. Mary’s Hospital, College of Medicine, Catholic University of Korea, 505 Banpo-Dong, Seocho-Gu, Seoul 137-701, South Korea. Tel.: +82-2-5901532x1533; fax: +82-2-594-3870. E-mail address: [email protected] (J.-J. Kim).
redox mechanism that controls a balance between neurodestructive oxidants and neuroprotective antioxidants (Smythies, 1999). Dopamine (Yoshioka et al., 2002) and estradiol (Behl and Moosmann, 2002) have antioxidant effects, involving redox mechanism (Smythies, 1999). Interestingly, dopamine is also easily turned into dopamine – quinones, which is highly neurotoxic free radical (Smythies, 1996). This redox mechanism has been involved in impairment of glutamate synapse (Smythies, 1999) and it is altered in patients with schizophrenia (Reddy and Yao, 1996; Yao et al., 1999). Plasma total antioxidant status (TAS) (Yao et al., 1998) and individual antioxidant proteins, such as albumin and bilirubin (Yao et al., 2000), were reduced in patients with schizophrenia, suggesting that an impaired antioxidant defense system may be a liability factor in the development of schizophrenia. The ROS reduce glutathione level, increases intracellular calcium (Hoyt et al., 1997), mediates b-amyloid protein (Zhang et al., 1997), and glutamate neurotoxicity (Smythies, 1999). These findings suggest that oxid-
0278-5846/03/$ – see front matter D 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0278-5846(03)00043-5
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ative stress may have a role in etiopathogenesis in schizophrenia. Moreover, Sagara (1998) found that some of the side effects of antipsychotic drug are related to oxidative stress and to the level of ROS that is controlled by antioxidant such as glutathione. Moreover, the phenomenon of apoptosis, or programmed cell death, could be related to etiopathogenesis of schizophrenia with mechanisms connected to the neurodevelopment (Catts and Catts, 2000). Cytoarchitectural disturbances of the cerebral cortex and synaptic connectivity (Kawasak et al., 2000; Harrison and Eastwood, 1998; Arnold and Trojanowski, 1996) have been supposed in patients with schizophrenia as well as progressive volumetric changes of the brain (DeLisi et al., 1995, 1997, 1998). These findings could be related to the increased rate of apoptosis in schizophrenia during neurodevelopment (Catts and Catts, 2000). This critical phenomenon in normal development could result in an abnormal number of neurons and pathological neural development (Margolis et al., 1994). In addition, apoptosis is linked to process of oxidative stress through modulation of stress protein kinases such as mitogen-activated protein (MAP) kinases (e.g., c-Jun N-terminal kinase [JNK] and extracellular signal-related receptor kinase [ERK]) (Yoshizumi et al., 2002). Glutathione S-transferase P (GSTP) is an interesting enzyme that binds to reduced glutathione and is involved in the detoxification of ROS, thus, keeping the balance of cellular redox state (Sato, 1989). Alterations in GSTP could be involved in oxidative stress and apoptosis, contributing to the development of schizophrenia through alterations in neurodevelopment. Moreover, it has a pivotal role in the regulation of stress kinases such as MAP, including p38, ERK, and JNK/SAPK (stress-activated protein kinase), which are involved in the intracellular signal transduction pathways that control brain function and neural plasticity against stress (Duman et al., 1994; Yin et al., 2000; Kyosseva et al., 2001). Recently, a study demonstrated that chronic phencyclidine (PCP) administration caused alteration of MAP kinases under regulation of GSTP, suggesting the role of the signal pathway and the enzyme in PCP-induced psychosis (Kyosseva et al., 2001). Moreover, GSTP has been found to suppress in vitro the dopamine-induced apoptosis in neurons through the modulation of MAP kinase activity (Ishisaki et al., 2001) implying possible role of this enzyme in the development of schizophrenia considering that a characteristic of this disease has been found to be the degeneration of dopaminergic neurons of the nigrostriatal and mesolimbic systems (Ishisaki et al., 2001; Smythies, 1996). It is also noteworthy that GSTP1 is located on chromosome 11q13 (Jeronimo et al., 2002), a locus possibly associated to genetic susceptibility to schizophrenia and located close to dopamine receptor D2 gene (Gelernter et al., 1992; Mulcrone et al., 1995). A functional polymorphism at codon 105 in exon 5 of GSTP1 has been found (Hu et al., 1998), which differs by a single base pair substitution (adenosine to guanosine) at nucleotide 313 resulting in an
amino acid substitution (isoleucine to valine) (Harries et al., 1997). Further, the biophysical characteristics of this polymorphism have been reported that the valine allele could make the enzymatic activity low and detoxification capacity less (Hu et al., 1998). These findings sufficiently provide us with a theoretical rationale to investigate the association between GSTP1 polymorphism and schizophrenia in a Korean sample. The authors therefore genotyped the polymorphism in patients with schizophrenia and controls and further analyzed the relationship among clinical variables such as onset age, scale score for psychotic symptom, duration of illness and numbers of admission, and GSTP1 polymorphism in patients with schizophrenia.
2. Methods 2.1. Subjects Two hundred fourteen (214) in-patients with schizophrenia admitted to Kangnam St. Mary’s Hospital, Catholic University of Korea, Seoul, and 110 normal controls participated in this study. Most of patients were chronic case and hospitalized in acute phase of the clinical course of their disease. The diagnosis was made based on the consensus between two board-certified psychiatrists (J.J.K. and C.U.L.) according to the Diagnostic and Statistical Manual of Mental Disorders-IV (DSM-IV) (American Psychiatric Association, 1994). The Structured Clinical Interview for DSM-IV Axis I Disorders—Clinician Version (SCID-CV) (First et al., 1997) was administered to all patients. Other available resources, such as clinical course, family information, and medical records, were also used. Those having a history of, or suffering from, other mental or neurological disorders other than schizophrenia were excluded. The control group consisted of volunteers from the paramedical and medical staffs or students at the hospital who had no personal or family history of major mental disorders and neurological diseases. Board-certified psychiatrists (J.J.K. and C.U.L.) also evaluated through personal interview the control group whether they had either current or past history of psychiatric problems. A written informed-consent form was obtained from all subjects after full description of the study. The Ethics Committee of the Kangnam St. Mary’s Hospital approved this study. Ninety-nine (46.3%) patients with schizophrenia and 48 (43.6%) normal controls were male. No difference was present in the gender distribution between the two groups. The average age was older in the patient group (31.2 ± 9.9 years) than in the control group (26.6 ± 10.2 years). 2.1.1. Assessment of clinical variables The clinical variables according to genotypes in patients group were evaluated by examining the symptom severity
C.-U. Pae et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 27 (2003) 519–523 Table 1 Genotype and allele distribution of GSTP1 polymorphism in patients with schizophrenia and the controls in the Korean population
GSTP1 * A/A GSTP1 * A/B GSTP1 * B/B GSTP1 * A GSTP1 * B
Schizophrenia (n = 214)
Controls (n = 110)
139 68 7 346 82
74 33 3 181 39
(65.0) (31.8) (3.3) (80.8) (19.2)
(67.3) (30.0) (2.7) (82.3) (17.7)
Values represent number (%).
using Positive and Negative Syndrome Scale (PANSS) (Kay et al., 1988), age of onset, change of Brief Psychiatric Rating Scale (BPRS) (Overall and Gorham, 1962) at the time of admission and discharge, and number of admission.
521
tions. Statistical significance was determined at P values less than .05. The power of our sample to detect differences between variants was calculated using a two-tailed alpha value of .05. Power analysis was performed with the GPOWER program (Erdfelder et al., 1996). With these parameters, considering the allele frequencies in our sample, the power analysis showed that our sample size had a high power (.80) to detect a small effect size (w=.11), which corresponds to a difference of 8% between the two alleles (odds ratio [OR] = 1.83).
3. Results 3.1. Genotype and allele frequencies of GSTP1 polymorphism in patients with schizophrenia and normal controls
2.2. Genotyping for GSTP polymorphism 2.2.1. DNA preparation DNA was extracted from whole blood using by proteinase K, as described elsewhere (Miller et al., 1988). DNA samples were stored at 4 C until used as template DNAs for polymerase chain reaction (PCR). 2.2.2. PCR-restriction fragment length polymorphism (RFLP) Amplification of the target site in GSTP1 was carried out by PCR using forward and reverse primers (50-ACC CCA GGG CTC TAT GGG AA-30 and 50-TGA GGG CAC AAG AAG CCC CT-30, respectively), as previously described (Jeronimo et al., 2002). Briefly, PCR was performed using 30 ml of reaction mixture (1 buffer [50 mM KCl, 10 mM Tris –HCl pH 8.3, 1.5 mM MgCl2], 200 ng of each primer, 200 mM of each dNTP, 50 ng of the genomic DNA, 1 unit of Taq DNA polymerase; Boehringer Mannheim, Mannheim, Germany) using the following conditions in a Perkin Elmer 9600 thermocycler (Foster City, CA, USA). After the initial denaturation step at 94 C for 5 min, the sample were processed through 35 cycles of 94 C for 45 s, 55 C for 45 s, and 72 C for 1 min. Finally, the final extension at 72 C for 7 min was done. The 176-bp PCR products were digested for 2 h at 37 C with 2 units of BsmAI (New England Biolabs, Hertfordshire, UK). The digested samples were separated on ethidium bromide 4% agarose gel electrophoresis. 2.3. Data analysis SPSS version 10.0 (SPSS, Chicago, IL, USA) was used for statistical analysis. Chi-square test was used to compare allele and genotype frequencies in patients and controls. Analysis of variance and Student’s t test were used to analyze PANSS score, onset age, BPRS change, and number of admission according to genotype and allele distribu-
Genotype distributions in patients with schizophrenia (c2 = 3.2, P=.201) and controls (c2 = 0.0004, P > .1) were in Hardy– Weinberg equilibrium. The genotype and allele distributions in patients with schizophrenia were not significantly different from those of the controls as shown in Table 1 (c2 = 0.203, df = 2, P=.904; c2 = 0.196, df = 1, P=.658, respectively). Even if we classified the genotype according to sex, it showed no difference between patients and controls (data not shown, male: c2 = 0.791, P=.673; female: c2 = 0.466, P=.792, respectively). 3.2. Relationship between GSTP1 polymorphism and clinical variables in patients group There was no significant difference in scores of PANSS (positive, negative, and total score), age of onset, BPRS change, and number of admission according to GSTP1 genotype (Table 2) and allele (data not shown, P=.670,
Table 2 Comparison of clinical variables with GSTP1 genotypes in patients with schizophrenia Genotype
PANSS total PANSS positive PANSS negative Age of onset (year) Number of admission BPRS change
P value
GSTP1 * A/A (n = 139)
GSTP1 * A/B (n = 68)
GSTP1 * B/B (n = 7)
92.82 ± 12.70
92.83 ± 14.08
88.42 ± 15.95
.689
24.73 ± 5.15
24.75 ± 4.16
24.00 ± 7.32
.927
21.07 ± 4.76
21.25 ± 5.08
19.85 ± 5.42
.771
25.6 ± 7.87
26.6 ± 7.70
23.3 ± 5.90
.485
2.9 ± 2.51
2.5 ± 2.01
2.1 ± 1.21
.357
7.9 ± 6.4
.349
11.7 ± 7.8
Values represent means ± S.D.
12.1 ± 6.3
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P=.879, P=.670, P=.837, P=.156, and P=.655, respectively).
4. Discussion 4.1. Speculation on the role of GSTP1 polymorphism in schizophrenia As stated in the Introduction, GSTP has been supposed to have a regulatory function in the process of antioxidant defenses and apoptosis through modulation of various kinases, which was suggested to be related to the development of schizophrenia (Arnold and Trojanowski, 1996; Catts and Catts, 2000; Harrison and Eastwood, 1998; Kawasak et al., 2000; Smythies, 1996, 1999). Further, the enzyme activity of GSTP shows age-related changes in brain (Gopal et al., 2000). GSTP1 gene variants have different influence on enzymatic activity: GSTP1 * B has lower enzyme activity and less capacity for detoxification (Hu et al., 1998). Our hypothesis therefore was that patients with schizophrenia would display different distribution of genotype and/or allele compared with the controls, since schizophrenic patients were supposed to have alteration in process of antioxidant defense and apoptosis, which is under the regulation of GSTP. To the best of our knowledge, this is the first study to examine the association between GSTP1 polymorphism at codon 105 in exon 5 and schizophrenia. Unfortunately, we found no significant differences in distribution of genotype and allele frequencies between patients with schizophrenia and the controls. Moreover, any association between distribution of genotypes or alleles and clinical variables, such as scores of PANSS, were not found. The present findings suggest that GSTP1 polymorphism is not associated with susceptibility to the development of schizophrenia itself, as well as clinical heterogeneity of the disorder, at least in our Korean sample. 4.2. Deliberation on the negative finding These negative findings for the association between GSTP1 polymorphism and schizophrenia should lead to some considerations. This polymorphism may not affect the development of schizophrenia or exert a very weak influence on the etiopathogenesis of the disorder. The presence of other unknown modulating factors and/or unidentified surrounding genes for GSTP1 polymorphism that may confer susceptibility to schizophrenia could be also conceivable. Given that GSTP has a regulatory function for signaling pathways through modulation of variable kinases (Duman et al., 1994; Kyosseva et al., 2001; Yin et al., 2000) and antioxidant defense mechanism are interactive (Smythies, 1999), it might be a feasible speculation that the role of GSTP in the development of schizophrenia would be an intricate modulatory function rather than a direct influence. In addition, the
negative result of Ile105Val GSTP1 polymorphism in this study does not completely exclude the association of GSTP1 with schizophrenia, because there might be other mutations in this gene that might be associated with schizophrenia. An ethnic difference in GSTP1 polymorphism has been reported. Asians have more GSTP1 * A/A and less GSTP1 * B/B and GSTP1 * A/B than Caucasians (Nerurkar et al., 2000). In addition, the frequencies of GSTP1 * A/A, GSTP1 * A/B, and GSTP1 * B/B was 0.67, 0.30, and 0.03, respectively, in our controls, compared to 0.43, 0.48, and 0.09, respectively, in Caucasian controls (Jeronimo et al., 2002). The power of the study should be also considered when a negative result is presented. The power analysis showed that our sample size had a high power (.80) to detect a small allele association (w=.11, OR = 1.83). Thus, further studies with larger samples and coming from different ethnicities should be performed to confirm the present findings that GSTP1 polymorphism is not associated with schizophrenia. 4.3. Limitation of the study This study has several limitations. Our controls consisted of hospital staffs and students so that they could not adequately represent general Korean population. A further limitation of the present study is a possible population stratification that is a liability to association study (Spielman et al., 1993); although methods controlling for stratification were not applied (Pritchard and Rosenberg, 1999), our subjects are known to be genetically homogeneous (Benkmann et al., 1989).
5. Conclusion Evidence for a contribution of GSTP1 polymorphism to susceptibility to the development of schizophrenia in the Korean population was not found in this study. Further investigations should be launched to provide us more useful information on the role of GSTP1 polymorphism in the pathogenesis of schizophrenia.
Acknowledgements This study was supported by a grant number R01-2000000-00091-0 and M6-0225-00-0001 from the Basic Research Program of the Korean Science and Engineering Foundation.
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