Association between Ala–9Val polymorphism of Mn-SOD gene and schizophrenia

www.elsevier.com/locate/pnpbp

Progress in Neuro-Psychopharmacology & B

Association between Ala–9Val polymorphism of Mn-SOD

gene and schizophrenia

Omer Akyola,*, Medaim Yanikb, Halit Elyasa, Mustafa Namlic, Halit Canatana,d, Haluk Akina,

Huseyin Yucea, H. Ramazan Yilmaze, Hamdi Tutkunf, Sadik Sogutg, Hasan Herkenf,

Hqseyin Ozyurth, Haluk Asuman Savasf, Suleyman Salih Zorogluf

aDepartment of Medical Biology and Genetics, Firat University Medical School, Elazig, TurkeybDepartment of Psychiatry, Harran University Medical School, Urfa, Turkey

cElazig Psychiatry and Neurology State Hospital, Elazig, TurkeydDepartment of Pharmacology and Toxicology, Faculty of Medicine, Health Sciences Centre, Kuwait University, Safat, Kuwait

eDepartment of Medical Biology and Genetics, Suleyman Demirel University Medical School, Isparta, TurkeyfDepartment of Psychiatry, Gaziantep University Medical School, Gaziantep, Turkey

gDepartment of Biochemistry, Mustafa Kemal University Medical School, Hatay, TurkeyhDepartment of Biochemistry, Gaziosmanpasa University Medical School, Tokat, Turkey

Accepted 15 October 2004

Abstract

Reactive oxygen species (ROS) have been suggested to play an important role in physiopathology of schizophrenia. The major

intracellular antioxidant enzymes, copper–zinc superoxide dismutase in the cytoplasm and manganese superoxide dismutase (Mn-SOD) in

the mitochondria, rapidly and specifically reduce superoxide radicals to hydrogen peroxide. Polymorphisms in the genes encoding

antioxidant enzymes should therefore result in predisposition to schizophrenia. The present study was performed to assess whether there is a

genetic association between a functional polymorphism (Ala–9Val) in the human Mn-SOD gene in schizophrenic patients (n=153) and

healthy controls (n=196) using a PCR/RFLP method. Significant differences in the genotypic distribution between schizophrenics and

controls were observed. Genotypic distribution with 14 (9.2%) Ala/Ala, 106 (69.3%) Ala/Val and 33 (21.6%) Val/Val subjects in

schizophrenia was different from those of controls with 46 (23.5%), 83 (42.3%) and 67 (34.2%), respectively ( pb0.0001). When the patients

with schizophrenia were divided into the subgroups as disorganized, paranoid and residual, there was a significant difference in genotypic

distribution among the subgroups (v2=11.35, df=4, p=0.023). This association between –9Ala Mn-SOD allele and schizophrenia suggests

that –9Ala variant may have a contribution in the physiopathogenesis of schizophrenia. Further investigations are warranted in larger

populations with other susceptible genes that might be associated with schizophrenia.

D 2004 Published by Elsevier Inc.

Keywords: Gene polymorphism; Manganese superoxide dismutase; Schizophrenia

0278-5846/$ - s

doi:10.1016/j.pn

Abbreviation

CAT, catalase; C

deoxy adenosin

dithiotreitol; EC

manganese supe

radicals; !OH, hdismutase; TBE

* Correspon

1652x113; fax:

E-mail addr

iological Psychiatry 29 (2005) 123–131

ee front matter D 2004 Published by Elsevier Inc.

pbp.2004.10.014

s: AIMS, Abnormal Involuntary Movement Scale; Ala, alanine; ALS, amyotrophic lateral sclerosis; BPRS, Brief Psychiatric Rating Scale;

NS, central nervous system; Cu,Zn-SOD, copper- and zinc-containing superoxide dismutase; dNTP, deoxy nucleotide triphosphate; dATP,

e triphosphate; dCTP, deoxy cytidine triphosphate; dGTP, deoxy guanosine triphosphate; dTTP, deoxy tymidine triphosphate; DTT,

-SOD, extracellular SOD; EDTA, ethylene diamine tetraacetic acid; GSH-Px, glutathione peroxidase; H2O2, hydrogen peroxide; Mn-SOD,

roxide dismutase; MTS, mitochondrial targeting sequence; NADPH, reduced nicotinamide adenine dinucleotide phosphate; O2!�, superoxide

ydroxyl radical; PCR, polymerase chain reaction; PUFA, polyunsaturated fatty acids; ROS, reactive oxygen species; SOD, superoxide

, Tris–borate–EDTA; TD, tardive dyskinesia; Val, valine.

ding author. Hacettepe University, Faculty of Medicine, Department of Biochemistry, TR-06100 Sihhiye, Ankara, Turkey. Tel.: +90 312 305

+90 312 310 0580.

ess: [email protected] (O. Akyol).

O. Akyol et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 29 (2005) 123–131124

1. Introduction

The hypothesis that reactive oxygen species (ROS) play

an important role in schizophrenia as well as neurodegener-

ative disorders remains speculative. Some of the neuro-

pathological changes in neuropsychiatric disorders may be

the result of increased free radical-mediated or ROS-

mediated neuronal injury (Mahadic and Mukherjee, 1996).

ROS-mediated neuronal injury occurs when oxidative stress

exists. Oxidative stress is a state in which there is an

imbalance between the oxidant and antioxidant defense

system, and generally occurs as a consequence of increased

production of ROS, or when the antioxidant defense system

is inefficient. The controversial data reported in the

literature show a variation for the activities of antioxidant

enzymes in schizophrenia. Erythrocyte superoxide dismu-

tase (SOD) activity was found to be increased (Vaiva et al.,

1994), unchanged (Herken et al., 2001) or decreased

(Mukerjee et al., 1996); and catalase (CAT) and glutathione

peroxidase activities (GSH-Px) were found to be increased

(Herken et al., 2001) or unchanged (Yao et al., 1998) in

schizophrenic patients. Lately, we found decreased plasma

SOD activity and unchanged plasma GSH-Px activity in

schizophrenic patients compared to the healthy control

Fig. 1. The schematic representation of oxidant and antioxidant system in hum

ion, proton, H2O: water, SOD: superoxide dismutase, CAT: catalase, H2O2

glutathione, GSSG: oxidized glutathione, GSH-R: glutathione reductase, NAD

oxidized nicotinamide adenine dinucleotide phosphate, Fe2+: ferrous iron, OH

radical), tNOS: total nitric oxide synthases, NO.: nitric oxide radical, ONO

peroxidation of membrane phospholipids), NO2�: nitrite, PUFA: polyunsaturated

Neuropsychopharmacol. Biol. Psychiatry 2002;26:995–1005).

subjects (Akyol et al., 2002). According to this study, the

dose and the duration of treatment with drugs had no

influence on the results, so it can be interpreted that the

changes in enzyme activities are more likely to be related

mainly to the underlying disease rather than the drugs used

in the treatment.

SOD is an enzyme catalyzing the dismutation reaction

of superoxide radicals (O2�) to hydrogen peroxide (H2O2)

(Fig. 1). Manganese-containing SOD (Mn-SOD), even

though not coded in the mitochondrial DNA, is involved

in controlling dioxygen toxicity in mitochondria, an

organelle of extreme oxidative load (Fridovich, 1995).

Using enzymatic analysis of mouse/human hybrids, the

Mn-SOD gene was initially localized to chromosome 6

(Creagan et al., 1973). Later, the Mn-SOD gene was

sublocalized to region 6q25 by fluorescence in situ

hybridization and somatic cell hybrid mapping (Church

et al., 1992). As is the case with most mitochondrial

proteins, the enzyme translocates to the mitochondria after

translation of the protein containing an N-terminal leader

signal which is 24 amino acids length in the cytosol and

removed during the transport of the molecule to the

mitochondria. Shimoda-Matsubayashi et al. (1996) found

a structural mutation of T to C substitution in the coding

ans. O2!�: superoxide anion radical, O2: molecular oxygen, H+: hydrogen

: hydrogen peroxide, GSH-Px: glutathione peroxidase, GSH: reduced

PH+H+: reduced nicotinamide adenine dinucleotide phosphate, NADP+:�: hydroxyl ion, .OH: hydroxyl radical (the most potent free oxygen

O�: peroxynitrite, MDA: malondialdehyde (the last product of lipid

fatty acid, XO: xanthine oxidase (adapted from Akyol, O., et al. Prog.

Table 1

Demographic data of the patients and control subjects (results were

expressed as meanFstandard deviation)

O. Akyol et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 29 (2005) 123–131 125

sequence which changes the amino acid codon at �9

position in the signal peptide from valine (GTT) to

alanine (GCT). This signal peptide is removed during the

processing to a mature enzyme and plays a key role in

targeting the enzyme to the mitochondria (Shimoda-

Matsubayashi et al., 1996). The valine-to-alanine sub-

stitution induces a conformational change of the mito-

chondrial targeting sequence (MTS) from a h-sheet to an

a-helix. Single amino acid substitutions in the leader

signal were previously reported for ornithine transcarba-

mylase to change the mitochondrial processing efficiency

depending on its conformational change (Horwich et al.,

1986).

Because the mitochondria are protected from O2.� by Mn-

SOD enzyme, neurons may become susceptible to O2.�-

related damages when the activity of Mn-SOD in the

mitochondria is reduced. Previous studies revealed that the

variant –9Ala allele associates with sporadic motor neuron

disease (Van Landeghem et al., 1999a), exudative age-

related macular degeneration (Kimura et al., 2000), Parkin-

son’s disease (Shimoda-Matsubayashi et al., 1996), diabetic

nephropathy (Nomiyama et al., 2003) and ankylosing

sypondylitis (Yen et al., 2003), which are all related to

oxidative stress and abnormal free radical defence mecha-

nisms. Recently, a significant increase in the wild-type Val

allele in Japanese schizophrenic patients with tardive

dyskinesia (TD) was found as compared to the patients

without TD, suggesting the –9Ala allele may act protec-

tively against free radical damage in TD (Hori et al., 2000).

Contrary to the previous finding, Zhang et al. (2002) found

neither a significant difference in the frequency of the Ala

allele in the TD group compared to the non-TD or normal

control subjects, nor a relationship between Mn-SOD

activity and the Ala variant.

Oxidative/antioxidative balance in the brain constitutes

the primary defense against oxidant stress of the brain.

Although the expression of SODs has been characterized in

human brain, the specific role of the antioxidant enzymes in

the pathogenesis of most of the brain diseases remains

unclear. For instance, individual variability of these

enzymes (polymorphisms) leading to lower antioxidant

activity in brain cells may be hypothesized to play a role

in schizophrenia. Several polymorphisms have in fact been

characterized, but their role in the pathogenesis of various

brain disorders has not yet been systematically investigated.

The present study was aimed to assess whether there is a

genetic association between a functional polymorphism

(Ala–9Val) in the human Mn-SOD gene and schizophrenia

including subtypes of the disease and TD as a side effect of

the treatment.

Schizophrenia Control

n 153 196

Age 37.6F10.8 35.6F14.8

Gender (male/female) 94/59 102/94

Neuroleptic doses

(chlorp. equivalent, g/day)

394F175 –

2. Methods

The study was conducted at the Department of Psychia-

try, Gaziantep University Medical Faculty and Elazig

Mental and Neurology State Hospital between 2001 and

2002. The study was performed in accordance with the

Declaration of Helsinki (1964), revised in Tokyo (1975) and

the subsequent Venice (1983) and Hong Kong (1989)

amendments. The project was carried out with the approval

of the Medical Human Ethical Committee at Gaziantep

University, Faculty of Medicine.

2.1. Subjects

The patients with schizophrenia were all chronic inpa-

tients at the above-mentioned hospitals. The characteristics of

the patient and control groups were summarized in Table 1.

All patients had complete medical reports from the first

admission. Clinical as well as demographic data was obtained

from the medical records and interview with the patients. All

the patients with schizophrenia were diagnosed using DSM-

IV (American Psychiatric Association, 1994) criteria by four

psychiatrists with consensus based on cross-sectional inter-

views and case records, which included the Brief Psychiatric

Rating Scale (BPRS) (Overal and Gorhan, 1962). A total of

153 patients with schizophrenia were recruited who were 94

man and 59 women with age ranging from 16 to 71 years

(Table 1). Patients were divided into three subgroups

according to DSM-IV criteria as disorganized (n=32), para-

noid (n=81) and residual (n=35) type schizophrenic patients

(Table 2). Familial cases of schizophrenia were excluded

from the study. None of the subjects had significant neuro-

logical comorbidity, epilepsy, mental retardation or history of

drug abuse. Patients with chronic systemic diseases such as

diabetes mellitus, hypertension, etc., severe head injury were

also excluded from the study. All patients were being treated

with stable doses of neuroleptics (haloperidol, chlorproma-

zine, thioridazine, fluphenazine, olanzapine, sulpride, risper-

idone or zuclopenthixol). The patients were assessed for TD

by the Abnormal Involuntary Movement Scale (AIMS)

employing a standard evaluation method (Guy, 1976) during

the study. So the patients with schizophrenia were also

divided into two subgroups as having TD (n=23, mean age

39.8 years) or not (n=130, mean age 37.3 years). In the

AIMS, dyskinesia was observed in different regions of the

body including the facial and oral regions, the extremities and

the trunk. Subjects with two or more two-point ratings, or one

or more three-point ratings, on the first seven items of the

AIMS were diagnosed as having TD according to the

Table 2

Clinical characteristics of the patients

Subtypes of schizophrenia Tardive dyskinesia

Paranoid Disorganized Residual (+) (�)

n 81 32 35 23 130

Age 35.5F10.4 37.9F10.1 42.1F11.3 39.8F11.5 37.3F10.7

Gender (M/F) 53/28 20/12 16/19 12/11 82/48

Neuroleptic doses

(chlorp. equivalent, mg/day)

422F190 377F194 396F176 363F114 399F184


Schooler-Kane criteria (Schooler and Kane, 1982). Severity

ratings were made along a 5-point ordinal scale from 0 to 4

(none, minimal, mild, moderate and severe).

Control individuals were selected from the healthy

individuals who had no psychiatric disorders. Also, none

of the control subjects had a history of severe head injury,

seizure or chronic medical illness. Thus, a total of 196

individuals were recruited as controls, who were 102 man

and 94 women with age ranging from 17 to 73 years (mean

35.6 years). Informed consents were obtained from all

subjects, and if cooperation with patients was not possible

(mostly), from the patients’ relatives.

2.2. The instruments and chemicals used

Microcentrifuge (Ole Dich Instrumentmakers APS, type

157.MP, Germany, and Eppendorf microcentrifuge type

5415C, Germany), electronical balance (Shimadzu Corpo-

ration Libror AEG-320, Japan), pH meter (Hanna Instru-

ments H18521 pHmeter, Italy), UV/visible spectro

photometer (LKB biochrom Ultraspec Plus 4054, Cam-

bridge, England), vortex (Labinco L46, The Netherlands),

electrophoresis apparatus (1200 V-500 mA E815, Belgium),

electrophoresis box (Consort N.V. Parklaan 36 B-2300

Turnhout, Belgium), Eppendorf Mastercycler gradient (Neth-

eler Mlnz, 22331 Hamburg, Germany) and reading-imaging-

saving unit of electrophoresis gels (TCP-20-M, Vilber

Lourmat, Cedex, France) were used. Isopropanol, ethanol,

boric acid, ethylenediamine tetraacetic acid (EDTA), brom-

phenol blue, xylene cyanole, ficoll, agarose, high resolution

agarose, ethidium bromide, TRIS-base, potassium acetate,

magnesium acetate, TRIS-acetate, dithiotreitol (DTT) and

bovine serum albumin (BSA-acetylated) were purchased

from Sigma (Steinheim, Germany). Genomic DNA purifica-

tion kit, deoksinqkleotid trifosfat (dNTP) sets (dATP, dGTP,

dCTP, dTTP), Taq DNA polymerase, restriction endonu-

clease NgoM IV (formerly NgoM I) and B(phi)-X174DNA/HaeIII and B(phi)-X174DNA/HinfI DNA markers were

from Promega (Madison, WI, USA). Polymerase chain

reaction (PCR) primers were from IDT Integrated DNA

Technologies (Coralville, IA, USA).

2.3. Blood collection and genetic analyses

Venous blood samples (5–10 ml), taken in ethylenedi-

amine-tetraacetic acid as anticoagulant, were obtained

during the routine blood sampling for biochemical and

hematological analyses from the patients and controls after

they gave their informed consent. Genomic DNA was

isolated from 300 Al aliquots of venous blood using

Promega DNA isolation kit according to the manufactur-

er’s recommendations. An alanine/valine polymorphism in

the signal peptide of Mn-SOD gene was evaluated using a

primer pair (forward 5VACCAGCAGGCAGCTGG-

CGCCGG3V and reverse 5VGCGTTGATGTG-

AGGTTCCAG3V as defined by Mitrunen et al., 2001) to

amplify a 107-bp fragment. Polymerase chain reaction

(PCR) amplification of the genomic DNA was performed

as described by Akyol et al. (2004) in a total volume of 50

Al, containing 50 ng of genomic DNA, 20 pmol/Al of eachprimer, 1.25 U Taq polymerase (in 50 mM Tris–HCl [pH

8.0], 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 50%

glycerol and 1% Triton X-100), 2 mM dNTP, 2 mM

MgCl2, 1� PCR buffer containing 50 mM KCl, 10 mM

Tris–HCl (pH 8.3 at 25 8C). The PCR conditions involved

an initial denaturation of DNA at 95 8C for 5 min,

followed by 35 cycles of amplification at 95 8C for 1 min

(melting), 61 8C for 1 min (annealing) and 72 8C for 2

min, and a final extension at 72 8C for 7 min. The

resulting 107-bp PCR product was digested with the

restriction endonuclease NgoM IV at 37 8C for 16 h

according to the manufacturer’s recommendations and

digestion products were visualized with electrophoresis in

3% agarose gel stained with ethidium bromide (0.5 Ag/ml).

Restriction enzyme digestion results in a 107-bp product

(allele 1 Val-9) or 89 and 18 bp products (allele 2 Ala-9).

2.4. Statistical tests

All statistical analyses were performed using the Stat-

istical Package for the Social Sciences (SPSS) version 9.0

for Windows (SPSS, Chicago, IL). Differences in the

demographic characteristics of patients, duration of neuro-

leptic treatment, current neuroleptic dose and total AIMS

scores were assessed among the three Mn-SOD genotypes

using one-way analyses of variance (ANOVA). The Chi-

square–goodness-of-fit test was used to test the distribution

of genotypes and allele frequencies for deviations from

Hardy-Weinberg equilibrium. Chi-square (or Fisher’s exact

test) was used to compare the allele and genotype

frequencies between the patient groups and controls.

Differences of allelic distribution between patients with

Table 5

Genotypes and allele frequencies of the Ala–9Val polymorphism in patients

with and without TD

Genotypesa Allele frequencies

Ala/Ala

n (%)

Ala/Val

n (%)

Val/Val

n (%)

–9Ala –9Val

TD+ (n=23) 2 (8.7) 12 (52.2) 9 (39.1) 0.35 0.65

TD� (n=130) 12 (9.2) 94 (72.3) 24 (18.5) 0.45 0.55

Control (196) 46 (23.5) 83 (42.3) 67 (34.2) 0.43 0.57

a Significant difference in genotype frequencies between control group

and patients with and without TD (v2=30.91, df=4, p=0.0001). No

significant difference in genotype frequencies between patients with TD

and patients without TD (v2=5.02, df=2, p=0.081).

Table 3

The genotypes of Ala–9Val polymorphism and allele frequencies in

controls and schizophrenic patients

Genotypesa Allele

frequenciesb

Ala/Ala

n (%)

Ala/Val

n (%)

Val/Val

n (%)

–9Ala –9Val

Patients (153) 14 (9.2) 106 (69.3) 33 (21.6) 0.46 0.54

Control (196) 46 (23.5) 83 (42.3) 67 (34.2) 0.44 0.56

a Significant difference in genotype frequencies between patients and

controls (v2=26.53, df=2, p=0.0001).b No significant difference in allele frequencies between patients and

controls.


TD and without TD, gender, schizophrenia subgroups and

cholorpromazine equivalent of neuroleptics were analyzed

using Fisher’s exact test.

Table 6

Frequencies of genotypes in schizophrenia patients and controls and

3. Results

Clinical demographic characteristics of schizophrenic

patients and control subjects are shown in Tables 1 and 2.

The allele and genotype frequencies of the subjects for the

Ala–9Val Mn-SOD polymorphism are shown in Tables 3, 4

and 5. The frequencies of genotypes calculated from Hardy-

Weinberg equilibrium were within the range of 95%

confidence interval (CI) of genotype frequencies in the

control subjects but not in the patients (Table 6). If the �9

codon was GCT (Ala), then digestion with NgoM IV

produces two DNA fragments, 89 and 18 bp in length. If

codon was GTT (Val), the amplified product was not

digested with NgoM IV and remained as a whole 107-bp

DNA fragment (Fig. 2).

3.1. Mn-SOD genotypes and allele frequencies in patients

with schizophrenia and disease-free controls

Genotype data for Mn-SOD were available from 196

patients with schizophrenia and 153 disease-free controls. In

the schizophrenic patients, the frequency of Ala/Ala

genotype was approximately 3-fold lower and that of Ala/

Val was 1.3-fold higher compared to the control group.

Significant difference in genotype frequencies was found

between patients and controls (v2=26.53, df=2, p=0.0001).

Table 4

The relationship between schizophrenia subgroups and the genotypes of

Ala–9Val polymorphism

Genotypesa Allele frequencies

Ala/Ala

n (%)

Ala/Val

n (%)

Val/Val

n (%)

–9Ala –9Val

Paranoid 4 (4.9) 64 (79) 13 (16) 0.44 0.56

Disorganized 1 (3.1) 21 (65.6) 10 (31.3) 0.36 0.64

Residual 6 (17.1) 19 (54.3) 10 (28.6) 0.44 0.56

a Significant difference in genotype frequencies between schizophrenia

subgroups (v2=11.35, df=4, p=0.023).

The frequency of Val-9 was more common allele both in

healthy subjects and patients (Table 3).

3.2. Mn-SOD polymorphism in patients with schizophrenia:

association with subgroups of schizophrenia

When the patients with schizophrenia were divided into

three subgroups as disorganized, paranoid and residual,

there was a significant difference in genotypic distribution

between the subgroups (v2=11.35, df=4, p=0.023). Among

the subgroups of schizophrenia, the Ala/Ala genotype in

residual subgroup was approximately five-fold higher

compared to disorganized subgroups and three-fold higher

compared to paranoid subgroup (Table 4). However,

relatively small sample size for Ala/Ala genotype may lead

to questionable of the results in schizophrenia subgroups.

3.3. Mn-SOD polymorphism in patients with schizophrenia:

association with tardive dyskinesia

Twenty-three of the 153 patients were suffered from TD

during the treatment. There was a significant difference in

genotypic distribution (v2=30.91, df=4, p=0.0001) betweenthe schizophrenic subjects with and without TD and

control group (Table 5). The difference was not significant

(v2=5.02, df=2, p=0.081) between patients with and

without TD.

expected frequencies according to the Hardy-Weinberg equilibrium

% Genotypes

(95% CI)

Ala/Ala Ala/Val Val/Val

Patients

Expected

according to

H-W equilibrium

9.2

(5.1–14.8)

69.3

(61.3–76.5)

21.6

(15.3–28.9)

21.1 49.7 29.1

Control

Expected

according

to H-W equilibrium

23.5

(17.7–30.4)

42.3

(35.5–49.6)

34.2

(27.6–41.3)

19.4 49.3 31.4

Fig. 2. Restriction analysis of Ala–9Val polymorphism in the Mn-SOD gene. Lanes 1–12: 12 samples after digestion with NgoM IV; lanes 6 and 7: Ala/Ala

genotype; lanes 1, 3, 4, 5, 10, 11 and 12: Ala/Val genotype; lanes 2, 8 and 9: Val/Val genotype; line A: molecular weight DNA marker B(phi)-X174DNA/HaeIII DNA and line B: molecular DNA marker B(phi)-X174DNA/HinfI DNA fragments are shown. Bands represent 107 bp (up) and 89 bp (down) DNA

fragments in lanes 1–12.


4. Discussion

4.1. Role of Mn-SOD polymorphism in the disease

Free radicals involve in the pathophysiology of

numerous neuropsychiatric disorders including schizophre-

nia. The aim of this study was to explore whether the

genetic polymorphism of antioxidant enzymes is associ-

ated with the development of schizophrenia. As a first

step, we dealt with Mn-SOD the gene of which are

known to be polymorphic (Rosenblum et al., 1996).

Moreover, abnormal antioxidant defense mechanism has

been shown in patients with schizophrenia (Herken et al.,

2001; Akyol et al., 2002; Sarsilmaz et al., 2003; Reddy

and Yao, 1996). The results suggest that the gene for Mn-

SOD is associated with schizophrenia. The findings that

Ala/Val genotype is lower and Ala/Ala genotype is higher

in control group than those of schizophrenia group may

suggest the importance of Ala/Val genotype in the

predisposition to schizophrenia and Ala/Ala genotype in

protecting against schizophrenia. Three previous studies

found no significant relationship of the Ala genotype with

schizophrenia in Japanese and Chinese population, but

found significant relationship of the Ala genotype with

TD (Hori et al., 2000; Zhang et al., 2002; Zhang et al.,

2003). Zhang et al. (2002) investigated if and how the

functional polymorphism site may influence Mn-SOD

activity in plasma, and whether variations in this activity

may relate to the presence of TD. They found that

genotype did not significantly affect the activity of Mn-

SOD in plasma in the patient groups; however, there was

a significant increase in the activity of Mn-SOD in

plasma in the patients with TD as compared with the

patients without TD and normal controls. No significant

difference in the activity was found between two

genotypic groups within all patients as well as within

the patients with TD group, although the mean Mn-SOD

activity was higher in the Val/Val genotype groups as

compared to the Ala/Val genotype group in the patients

with TD. Although they did not find any significant

difference in Mn-SOD polymorphism, the above-men-

tioned study may enlighten our study. We could not

estimate the enzyme activities together with the genotyp-

ing of Mn-SOD gene because of some technical

difficulties. In our study, Val/Val and Ala/Ala genotypes

were found to be higher in controls than those of

schizophrenics; nevertheless, Ala/Val genotype was found

to be higher in schizophrenia than that of controls. These

findings are parallel with those of the enzyme activities.

When two studies are thought together, Mn-SOD activity

should be increased in our control subjects that that of

schizophrenics, because the researchers in the above-

mentioned study was found higher enzyme activity in

Val/Val genotype. In the previous studies, SOD activities

were found to be decreased in the biological materials of

schizophrenia patients when compared with the healthy

subjects (Mukerjee et al., 1996; Akyol et al., 2002).

Significantly higher Val/Val and Ala/Ala genotypes in

control group than those of the patients may support the

decreased Mn-SOD activity in schizophrenics. Ala/Ala

genotype has been suggested to be related with increased

enzyme activity and characterized as a protecting factor

against TD (Hori et al., 2000). From this point of view,

schizophrenic patients in our study has lower Ala/Ala

genotype frequency than controls, and so they have

possibly decreased Mn-SOD activity. Researchers found

the –9Val allele more frequent in patients with schizo-

phrenia having TD than in those without and suggested

that this genotype may be a risk factor of TD (Hori et al.,

2000). The nonsignificant difference between the patients

with TD and without TD in our study may be just because

of unequally distributed subjects in the mentioned groups.

The interpretation of the results might be that another

unknown polymorphism, which is in linkage disequili-

brium with the Val–9Ala polymorphism and contributes

susceptibility to TD, exists in the Mn-SOD gene. Another

interpretation is that the –9Ala allele may play a role in

protecting against schizophrenia, based on the relatively

lower frequency of this allele in subjects with schizophre-

nia than in those of controls. The findings, in conjunction

with the relatively lower frequency of the –9Ala (high

activity) allele in patients with schizophrenia in our study,


suggest that controls may show higher Mn-SOD activity

and may be more capable of detoxifying O2.� than those

with schizophrenia.

4.2. The pathophysiological mechanisms of ROS-induced

brain damage and its possible relation with psychiatric

diseases

ROS are generated in vivo during many of the normal

biochemical reactions involving oxygen, including the

mitochondrial electron transfer chain, microsomal electron

transport system, NADPH-dependent oxidases and oxidation

of polyunsaturated fatty acids (PUFA) and catecholamines

(Fig. 1). During the past decade, reduction–oxidation

reactions that generate ROS including H2O2, O2� and OH

have been identified as important chemical mediators in the

regulation of signal transduction processes involved in cell

growth and differentiation (Sauer et al., 2001). Production

and release of O2� occurs towards the cytosolic side of the

inner mitochondrial membrane. However, due to the high

concentrations of mitochondrial SOD, the mitochondrial O2�

levels are kept at low steady state levels and only H2O2 which

is able to permeate the mitochondrial membrane may escape

to the cytoplasm (Boveris and Chance, 1973; Han et al., 2001;

Chance et al., 1979). Central nervous system (CNS) cells are

more vulnerable to the toxic effects of free radicals when

compared with other organs of the body because they have a

high rate of oxidative metabolic activity (e.g., catecholamines

degradation, etc.) and high oxygen uptake, a low level of

protective antioxidant enzymes namely CAT and GSH-Px, a

high ratio of membrane surface area to cytoplasmic volume, a

neuronal anatomical network vulnerable to disruption and

high concentrations of readily oxidisable membrane PUFA

(Evans, 1993). The PUFA located in cellular membranes of

CNS can readily react with free radicals and undergo

peroxidation. Lipid peroxidation on membrane lipids can

markedly alter membrane function such as transport mech-

anisms, receptor interactions, ion channels functions. Since

lipid peroxidation as a result of antioxidant enzyme lack and/

or excessive ROS production can alter the quality and

quantity of membrane phospholipids, these changes will

contribute to the physiopathology of schizophrenia by

altering distribution of phospholipids in membranes, chang-

ing the physicochemical properties of membranes, influenc-

ing the activities of membrane proteins and reducing levels of

neurotransmitter receptor-mediated generation of second

messengers such as diacylglycerol and inositol phosphate

(Akyol, 2002).

Mn-SOD is polymorphic and at least two functional

variants exist in human populations (Shimoda-Matsubaya-

shi et al., 1996; Borgstahl et al., 1996). One of these

(Ile58Thr) involves a C to T substitution at nucleotide

residue 339 leading to a substitution of isoleucine by

threonine at amino acid residue 58. A number of methods

have been described for detection of the Ala–9Val Mn-SOD

gene dimorphism. PCR amplification of the polymorphic

region, followed by NgoM IV treatment (i.e. PCR/restric-

tion fragment length polymorphism) is one of the commonly

used methods as validated by Akyol et al. (2004). The Ala–

9Val polymorphism in the MTS causes premature aging or

progeria (Rosenblum et al., 1996) and have been associated

with an increased risk of sporadic motor neuron disease

(Van Landeghem et al., 1999b) and with nonfamilial

idiopathic cardiomyopathy (Hiroi et al., 1999) but has no

effect on the occurrence of Parkinson disease (Farin et al.,

2001) or amyotrophic lateral sclerosis (ALS) (Parboosingh

et al., 1995).

Accordingly, a valine-to-alanine substitution may

increase the targeting efficiency by a conformational change

of the targeting sequence, consequently leading to an

increase in mitochondrial ROS scavenging. The presence

of more than one signal sequence for this vital enzyme

suggests a combinatorial mechanism determining rates of

targeting, membrane translocation and/or signal sequence

cleavage with concomitant folding of Mn-SOD (Rosenblum

et al., 1996). An in vitro study has suggested that the

enzyme content in the mitochondrial fraction is higher when

the signal peptide has alanine as the position 16 amino acid

than when it has valine (Hiroi et al., 1999).

The transport of Mn-SOD into mitochondria is mediated

through the interaction of the MTS with receptors on the

mitochondrial membrane. The Ala–9Val (C1183T) poly-

morphisms in the MTS may influence the efficiency of Mn-

SOD transport into mitochondria. The –9Ala polymorphism

results in the formation of a-helix and the –9Val takes a h-sheet structure (Shimoda-Matsubayashi et al., 1996). On the

other hand, the a-helix structure is known to be important

for the effective transport of precursor proteins into

mitochondria (Lemire et al., 1989). The amino acid

substitution (Ala/Val) at position �9 of the MTS may lead

to misdirected trafficking, followed by the alteration of Mn-

SOD activity in human mitochondria leading ineffective

struggle with ROS produced by mitochondrial electron

transport chain.

Van Landeghem et al. (1999b) investigated the poly-

morphism (Ala–9Val) in the MTS of Mn-SOD gene in

various ethnic groups by means of the oligonucleotide

ligation assay. There were significant variations in this

polymorphism between three different language groups:

Baltic, Finnish and Germanic. The Ala frequency in an

Asiatic population (Chinese and Japanese) has been found

to be significantly lower than in most European populations

(Kimura et al., 2000; Farin et al., 2001). We found that 44%

of control subjects had the allele alanine and the genotype

distribution was in the Hardy-Weinberg equilibrium.

5. Conclusions

Finally, our results suggest that Mn-SOD is associated

with the physiopathology of schizophrenia. A greater

understanding of the function of the Ala–9Val poly-


morphism and analyses in conjunction with other ROS-

related polymorphisms will help to fully elucidate the

contribution of this gene to schizophrenia. Therefore, the

results of this study are encouraging for further studies of

genetic polymorphisms of other antioxidant enzymes

including copper- and zinc-containing superoxide dismu-

tase (Cu,Zn-SOD), extracellular SOD, GSH-Px and

glutathione-S-transferases as well as oxidant enzymes

including xanthine oxidase and myeloperoxidase, as the

underlying cause of schizophrenia. Genetic variation in

other ROS defense genes and ROS producing genes

mentioned above could be important effect modifiers in

the relationship of Mn-SOD with schizophrenia. A basic

understanding about the expression and regulation of

antioxidant enzymes in normal brain and the changes that

occur in neuropsychiatric diseases is necessary to develop

therapeutic interventions to control oxidative stress in the

brain. So, more efforts should be directed not only toward

understanding the significance of the variability of these

enzymes in individuals and various diseases but also their

potential role in therapeutic approaches. Further inves-

tigations are warranted in larger populations with other

susceptible genes that might be associated with schizo-

phrenia.

Acknowledgement

This work was supported by the Fund of FUBAP

(Scientific Research Management Unit of Firat University)

with Grand number 654.

References

Akyol, O., 2002. Increased lipid peroxidation in schizophrenia: a marker of

membrane breakdown. Eur. Psychiatr. 17 (Suppl. 1), 75S.

Akyol, O., Herken, H., Uz, E., Fadillioglu, E., Unal, S., Sogut, S., Ozyurt,

H., Savas, H.A., 2002. The indices of endogenous oxidative and

antioxidative processes in plasma from schizophrenic patients: the

possible role of oxidant/antioxidant imbalance. Prog. Neuropsycho-

pharmacol. Biol. Psychiatry 26, 995–1005.

Akyol, O., Canatan, H., Yilmaz, H.R., Yuce, H., Ozyurt, H., Sogut, S.,

Gulec, M., Elyas, H., 2004. PCR/RFLP based cost-effective identi-

fication of SOD2 signal (leader) sequence polymorphism (Ala–9Val)

using NgoM IV: a detailed methodological approach. Clin. Chim. Acta

345, 151–159.

American Psychiatric Association, 1994. Diagnostic and Statistical Manual

of Mental Disorders, 4th ed. American Psychiatric Association,

Washington, DC.

Borgstahl, G.E., Parge, H.E., Hickey, M.J., Johnson, M.J., Boissinot, M.,

Hallewell, R.A., Lepock, J.R., Cabelli, D.E., Tainer, J.A., 1996. Human

mitochondrial manganese superoxide dismutase polymorphic variant

Ile58Thr reduces activity by destabilizing the tetrameric interface.

Biochemistry 35, 4287–4297.

Boveris, A., Chance, B., 1973. The mitochondrial generation of hydrogen

peroxide. General properties and effect of hyperbaric oxygen. Biochem.

J. 134, 707–716.

Chance, B., Sies, H., Boveris, A., 1979. Hydroperoxide metabolism in

mammalian organs. Physiol. Rev. 59, 527–605.

Church, S.L., Grant, J.W., Meese, E.U., Trent, J.M., 1992. Sublocalization

of the gene encoding manganese superoxide dismutase (MnSOD/

SOD2) to 6q25 by fluorescence in situ hybridization and somatic cell

hybrid mapping. Genomics 14, 823–825.

Creagan, R., Tischfield, J., Ricciuti, F., Ruddle, F.H., 1973. Chromosome

assignments of genes in man using mouse–human somatic cell hybrids:

mitochondrial superoxide dismutase (indophenol oxidase-B, tetrameric)

to chromosome 6. Humangenetik 20, 203–209.

Evans, P.H., 1993. Free radicals in brain metabolism and pathology. Br.

Med. Bull. 49, 577–587.

Farin, F.M., Hitosis, Y., Hallagan, S.E., Kushleika, J., Woods, J.S., Janssen,

P.S., Smith-Weller, T., Franklin, G.M., Swanson, P.D., Checkoway, H.,

2001. Genetic polymorphisms of superoxide dismutase in Parkinson’s

disease. Mov. Disord. 16, 705–707.

Fridovich, I., 1995. Superoxide radical and superoxide dismutases. Annu.

Rev. Biochem. 64, 97–112.

Guy, W., 1976. ECDEU assessment for psychopharmacology. HEW Public

Health Service, Washington, DC.

Han, D., Williams, E., Cadenas, E., 2001. Mitochondrial respiratory chain-

dependent generation of superoxide anion and its release into the

intermembrane space. Biochem. J. 353, 411–416.

Herken, H., Uz, E., Ozyurt, H., Sogut, S., Virit, O., Akyol, O., 2001.

Evidence that the activities of erythrocyte free radical scavenging

enzymes and the products of lipid peroxidation are increased in

different forms of schizophrenia. Mol. Psychiatry 6, 66–73.

Hiroi, S., Harada, H., Nishi, H., Satoh, M., Nagai, R., Kimura, A., 1999.

Polymorphisms in the SOD2 and HLA-DRB1 genes are associated with

nonfamilial idiopathic dilated cardiomyopathy in Japanese. Biochem.

Biophys. Res. Commun. 261, 332–339.

Hori, H., Ohmori, O., Shinkai, T., Kojima, H., Okano, C., Suzuki, T.,

Nakamura, J., 2000. Manganese superoxide dismutase gene poly-

morphism and schizophrenia: relation to tardive dyskinesia. Neuro-

psychopharmacology 23, 170–177.

Horwich, A.L., Kalousek, F., Fenton, W.A., Pollock, R.A., Rosenberg, L.E.,

1986. Targeting of pre-ornithine transcarbamylase to mitochondria:

definition of critical regions and residues in the leader peptide. Cell 44,

451–459.

Kimura, K., Isashiki, Y., Sonoda, S., Kakiuchi-Matsumoto, T., Ohba, N.,

2000. Genetic association of manganese superoxide dismutase with

exudative age-related macular degeneration. Am. J. Ophthalmol. 130,

769–773.

Lemire, B.D., Fankhauser, C., Baker, A., Schatz, G., 1989. The

mitochondrial targeting function of randomly generated peptide

sequences correlates with predicted helical amphiphilicity. J. Biol.

Chem. 264, 20206–20215.

Mahadic, S.P., Mukherjee, S., 1996. Free radical pathology and antioxidant

defense in schizoprenia: a review. Schizophr. Res. 19, 1–17.

Mitrunen, K., Sillanpaa, P., Kataja, V., Eskelinen, M., Kosma, V.M.,

Benhamou, S., Uusitupa, M., Hirvonen, A., 2001. Association between

manganese superoxide dismutase (MnSOD) gene polymorphism and

breast cancer risk. Carcinogenesis 22, 827–829.

Mukerjee, S., Mahadik, S.P., Scheffer, R., Correnti, E.E., Kelkar, H., 1996.

Impaired antioxidant defence at the onset of psychosis. Schizophr. Res.

19, 19–26.

Nomiyama, T., Tanaka, Y., Piao, L., Nagasaka, K., Sakaim, K., Ogihara,

T., Nakajima, K., Watada, H., Kawamori, R., 2003. The poly-

morphism of manganese superoxide dismutase is associated with

diabetic nephropathy in Japanese type 2 diabetic patients. J. Hum.

Genet. 48, 138–141.

Overal, J.E., Gorhan, D.R., 1962. The Brief Psychiatric Rating Scale.

Psychol. Rep. 10, 799–812.

Parboosingh, J.S., Rouleau, G.A., Meninger, V., McKenna-Yasek, D.,

Brown Jr., R.H., Figlewicz, D.A., 1995. Absence of mutations in the

Mn superoxide dismutase or catalase genes in familial amyotrophic

lateral sclerosis. Neuromuscul. Disord. 5, 7–10.

Reddy, R.D., Yao, J.K., 1996. Free radical pathology in schizophrenia: a

review. Prostaglandins Leukot. Essent. Fatty Acids 55, 33–43.


Rosenblum, J.S., Gilula, N.B., Lerner, R.A., 1996. On signal sequence

polymorphisms and diseases of distribution. Proc. Natl. Acad. Sci.

U. S. A. 93, 4471–4473.

Sarsilmaz, M., Songur, A., Ozyurt, H., Kus, I., Ozen, O.A., Ozyurt, B.,

Sogut, S., Akyol, O., 2003. Potential role of dietary omega-3 essential

fatty acids on some oxidant/antioxidant parameters in rats’ corpus

striatum. Prostaglandins Leukot. Essent. Fatty Acids 69, 253–259.

Sauer, H., Wartenberg, M., Hescheler, J., 2001. Reactive oxygen species as

intacellular messengers during cell growth and differentiation. Cell.

Physiol. Biochem. 11, 173–186.

Schooler, N.R., Kane, J.M., 1982. Research diagnoses for tardive

dyskinesia. Arch. Gen. Psychiatry 39, 486–487.

Shimoda-Matsubayashi, S., Matsumine, H., Kobayashi, T., Nakagawa-

Hattori, Y., Shimizu, Y., Mizuno, Y., 1996. Structural dimorphism in the

mitochondrial targeting sequence in the human manganese superoxide

dismutase gene. A predictive evidence for conformational change to

influence mitochondrial transport and a study of allelic association in

Parkinson’s disease. Biochem. Biophys. Res. Commun. 226, 561–565.

Vaiva, G., Thomas, P., Leroux, J.M., Thomas, P., Leroux, J.M., Cottencin,

O., Dutoit, D., Erb, F., Goudemand, M., 1994. Erythrocyte superoxide

dismutase (eSOD) determination in positive movements of psychosis.

Therapies 49, 343–348.

Van Landeghem, G.F., Tabatabaie, P., Beckman, G., Beckman, L.,

Andersen, P.M., 1999a. Manganese-containing superoxide dismutase

signal sequence polymorphism associated with sporadic motor neurone

disease. Eur. J. Neurol. 6, 639–644.

Van Landeghem, G.F., Tabatabaie, P., Kucinskas, V., Saha, N., Beckman,

G., 1999b. Ethnic variation in the mitochondrial targeting sequence

polymorphism of MnSOD. Hum. Hered. 49, 190–193.

Yao, J.K., Reddy, R., McElhinny, L.G., Van Kammen, D.P., 1998. Effects

of haloperidol on antioxidant defence system enzymes in schizophrenia.

J. Psychiatr. Res. 32, 385–391.

Yen, J.H., Chen, C.J., Tsai, W.C., Lin, C.H., Ou, T.T., Hu, C.J., Liu, HW.,

2003. Manganese superoxide dismutase and cytochrome P450 1A1

genes polymorphisms in rheumatoid arthritis in Taiwan Hum. Immu-

nology 64, 366–373.

Zhang, Z.J., Zhang, X.B., Hou, G., Sha, W.W., Reynolds, G.P., 2002.

The increased activity of plasma manganese superoxide dismutase in

tardive dyskinesia is unrelated to the Ala–9Val polymophism. J.

Psychiatr. Res. 26, 317–324.

Zhang, Z.J., Zhang, X.B., Hou, G., Yao, H., Reynolds, G.P., 2003.

Interaction between polymorphisms of the dopamine D3 receptor and

manganese superoxide dismutase genes in susceptibility to tardive

dyskinesia. Psychiatr. Genet. 13, 187–192.

Association between Ala–9Val polymorphism of Mn-SOD gene and schizophrenia

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