RESEARCH ARTICLE Neuropsychiatric Genetics

Association of the Manganese Superoxide Dismutase Gene Ala–9Val Polymorphism With Age of Smoking Initiation in Male Schizophrenia Smokers Xiang Yang Zhang,1,2* Wen-Wang Rao,3 Qiong Yu,3 Yaqin Yu,3 Changgui Kou,3 Yun-Long Tan,1 Da-Chun Chen,1 Lingjun Zuo,4 Xiangguang Luo,4 and Jair C. Soares2 1

Psychiatry Research Center, Beijing HuiLongGuan Hospital, Peking University, Beijing, China Department of Psychiatry and Behavioral Sciences, the University of Texas Health Science Center at Houston, Houston, Texas

2 3

School of Public Health, Jilin University, Changchun, China

4

Department of Psychiatry, Yale University School of Medicines, New Haven, Connecticut

Manuscript Received: 11 July 2015; Manuscript Accepted: 23 October 2015

Schizophrenia patients exhibit higher smoking rates than the general population. A growing body of evidence suggests that cigarette smoke impairs the antioxidant defense mechanisms, leading to oxidative damage. Manganese superoxide dismutase (MnSOD) is the major antioxidant in the mitochondria, catalyzing the metabolism of superoxide radicals to form hydrogen peroxide. Since the identification of a well-characterized functional polymorphism, Ala–9Val of MnSOD, a number of studies have evaluated the association between Val–9Ala and schizophrenia or cancer. In this study, we hypothesized that the functional polymorphism of MnSOD Ala–9Val was associated with smoking in patients with schizophrenia. This polymorphism was genotyped in 666 chronic male schizophrenia patients (smoker/neversmoker ¼ 507/159) and 660 male controls (smoker/never-smoker ¼ 360/300) using a case–control design. The cigarettes smoked per day (CPD) and smoking behaviors were evaluated by clinicianadministered questionnaires and the Fagerstrom Test for Nicotine Dependence (FTND). The results showed no significant differences in MnSOD Ala–9Val genotype and allele distributions between the patients and healthy controls or between smokers and never-smokers in either patients or healthy controls alone. The smokers with the Ala allele started smoking significantly earlier (19.9  5.8 vs. 21.7  6.5 years, P ¼ 0.005) only in patients. These results suggest that the MnSOD Ala–9Val polymorphism may not influence smoking status in a Chinese male schizophrenia population, but may influence the age at which smoking is started among schizophrenia smokers. Ó 2015 Wiley Periodicals, Inc.

Key words: schizophrenia; manganese superoxide dismutase; smoking; nicotine; genotype; association

INTRODUCTION Schizophrenia patients have a higher prevalence of smoking than the general population or other severely mentally ill patients [de

Ó 2015 Wiley Periodicals, Inc.

How to Cite this Article: Zhang XY, Rao W-W, Yu Q, Yu Y, Kou C, Tan Y-L, Chen D-C, Zuo L, Luo X, Soares JC. 2016. Association of the Manganese Superoxide Dismutase Gene Ala–9Val Polymorphism With Age of Smoking Initiation in Male Schizophrenia Smokers. Am J Med Genet Part B 171B:243–249.

Leon et al., 2002; Winterer, 2010; Dickerson et al., 2013; Hou et al., 2011]. The association between schizophrenia and smoking is consistently observed in samples from several different countries and has a relatively constant strength of association across different cultures, supporting a biological explanation for the association [Xu et al., 2014]. Furthermore, smoking is associated with vulnerability to schizophrenia in as much as most schizophrenia patients start smoking before the onset of their illness [de Leon and Diaz, 2005; Diaz et al., 2008; Zhang et al., 2010a]. High rates of smoking among those with psychotic disorders may contribute to the 20% Xiang Yang Zhang and Wen-Wang Rao have made the same contribution to this study. They should be considered as joint First author. Conflict of interest: No conflict of interest was disclosed for each author. Grant sponsor: National Natural Science Foundation of China; Grant number: 81371477; Grant sponsor: Beijing Municipal Natural Science Foundation; Grant numbers: 7132063, 7072035; Grant sponsor: NARSAD Independent Investigator; Grant number: 20314.  Correspondence to: Xiang Yang Zhang, M.D., Ph.D, UT Center of Excellence on Mood Disorders (UTCEMD), Biomedical and Behavioral Sciences Building (BBSB), 1941 East Road, TX 77054. E-mail: [email protected] Article first published online in Wiley Online Library (wileyonlinelibrary.com): 6 November 2015 DOI 10.1002/ajmg.b.32398

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244 reduction in life expectancy reported in this population [Kelly et al., 2011, 2012]. The adverse health consequences of smoking have been largely attributed to the abundance of reactive oxygen species (ROS) and reactive nitrogen species (RNS) that readily react with various biomolecules [Bar-Shai et al., 2006]. It has long been known that the tar and gas phases of tobacco smoke contain many oxidizing species [Valavanidis et al., 2009], and smokers may incur a high and sustained free radical load [Purkis et al., 2012], as they inhale large amounts of potentially injurious free radicals derived from tobacco [Aravamudan et al., 2014]. One of the principal reactive oxygen species produced in aerobic organisms is O2, which is highly cytotoxic. With the cytotoxicity of this oxidant, exposure to cigarette smoke results in increased levels of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), peroxidase (POx), and glutathione peroxidase (GSH-Px). SOD is the key antioxidant defense enzyme that detoxifies the superoxide radical (O2) and generates hydrogen peroxide (H2O2), which in turn is detoxified [Finkel, 2005]. The three isoforms of SOD contain different prosthetic groups: the manganese (Mn) isoform (SOD2) is found in mitochondria, and the copper and zinc (CuZn) isoforms are cytoplasmic (SOD1) and extracellular (SOD3). Mitochondrial manganese SOD (MnSOD) is the only SOD essential for life, and the major antioxidant in the mitochondria involved in the defense against ROS-induced oxidative damage [Macmillan-Crow and Cruthirds, 2001] because mitochondria produce 95% of ROS and superoxide anions from oxygen consumption [Robinson, 1998]. The Ala–9Val polymorphism (rs4880) in exon 2 of the MnSOD gene is the most extensively studied SNP, with the Ala-to-Val substitution possibly resulting in a conformational change in the target sequence from b-sheet to ahelix, which can induce a 30–40% increase in mitochondrial MnSOD activity [Carillon et al., 2013]. As MnSOD plays a vital role in the antioxidant defense system as well as neurodevelopment, a change in enzyme concentration through the action of Ala–9Val, may result in ROS accumulation and hence cell injury. This functional Ala–9Val polymorphism of MnSOD has been extensively investigated for association with schizophrenia, but with conflicting results in different ethnic groups [Hori et al., 2000; Zhang et al., 2002, 2014]. There is accumulating evidence linking the mitochondria in general, and MnSOD in particular, to the development of different kinds of cancer [Wang et al., 2009]. A number of molecular epidemiological studies have been conducted to examine the association between MnSOD Val–9Ala and cancer susceptibility [Dhar and St. Clair, 2012], suggesting that the MnSOD Val–9Ala polymorphism may contribute to cancer development through a disturbed antioxidant balance [Wang et al., 2009]. It is well known that smoking is one of the main and definite risk factors for cancer development. However, to our surprise, there is no study reporting the relationship of MnSOD gene polymorphisms and smoking. The purpose of the present study, therefore, was to investigate the possible relationship between the MnSOD Val–9Ala polymorphism and smoking behavior in schizophrenia versus healthy controls in a Chinese population. Because smoking is substantially more common among Chinese men than in women with

AMERICAN JOURNAL OF MEDICAL GENETICS PART B schizophrenia [Zhang et al., 2010b], as well as gender differences in smoking behaviors [de Leon and Diaz, 2005], we included only male subjects.

METHODS Subjects We recruited 666 male schizophrenia inpatients from Beijing Hui-Long-Guan hospital, a Beijing-city-owned psychiatric hospital, and HeBei Province Veterans Psychiatric Hospital in BaoDing city, 50 miles from Beijing. All patients met the DSM-IV diagnosis of schizophrenia, which two psychiatrists confirmed based on the Structured Clinical Interview for DSM-IV (SCID). All schizophrenia patients were of the chronic type, with at least 5 years of illness, were Han Chinese, and between 25 and 75 years old. The mean duration of illness was 24.6  4.6years. All patients had been receiving stable doses of oral antipsychotic drugs for at least 12 months before entry into the study. Antipsychotic drug treatment consisted mainly of drug monotherapy including: clozapine (n ¼ 301), risperidone (n ¼ 147), chlorpromazine (n ¼ 49), sulpiride (n ¼ 36), perphenazine (n ¼ 34), quetiapine (n ¼ 29), haloperidol (n ¼ 24), aripiprazole (n ¼ 21), and others (n ¼ 25). Mean antipsychotic dose (in chlorpromazine equivalents) was 453  387 mg/day [Woods, 2003]. We also recruited 660 male normal controls from the local community. None of them had any personal or family history nor demonstrated any clinical psychiatric disorders. All were Han Chinese from the Beijing area. All subjects were in good physical health, and any subjects with major medical illnesses or drug and alcohol abuse/dependence were excluded. All subjects gave written informed consent, which was approved by the Institutional Review Board of Beijing HuiLong-Guan hospital.

Clinical Measures Each subject filled out a detailed questionnaire that recorded general information, sociodemographic characteristics, smoking behavior, and medical and psychological conditions. In addition, we administered a cigarette smoking questionnaire to record smoking history and family history of smoking from each subject, including the pack years. The Chinese translation of the standardized Fagerstrom Test for Nicotine Dependence (FTND) was employed to measure the degree of nicotine dependence [Fagerstrom, 1978]. Additional information was collected from available medical records and collateral data (from family and/or treating clinician). Additional visits were requested for subjects with missing or ambiguous data. In order to maintain consistency in the use of various terms while gathering data on smoking behavior, the US Centers for Disease Control and Prevention have developed and updated the following definitions (US Centers for Disease Control and Prevention, 2010): Never smokers—Adults who have never smoked a cigarette or who smoked fewer than 100 cigarettes in their entire lifetime. Former Smokers—Adults who have smoked at least 100 cigarettes in their lifetime, but they currently do not smoke. Nonsmokers—Adults who currently do not smoke cigarettes, including both former smokers and never smokers.

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Current Smokers—Adults who have smoked 100 cigarettes in their lifetime and currently smoke cigarettes every day (daily) or some days (nondaily). In our present study, former smokers were excluded, and only never smokers were included in the following analysis. In addition, the age of smoking initiation was defined as age of onset of daily smoking.

Psychopathological Assessment Four psychiatrists who had previously completed training in scoring the Positive and Negative Syndrome Scale (PANSS) assessed patient psychopathology using it. After training, repeated assessment showed that they maintained an inter observer correlation coefficient greater than 0.8 for the PANSS total score.

Genotyping DNA was extracted using standard protocols. The genotypes of the MnSOD Ala–9Val polymorphism were identified as reported in a previous study [Zhang et al., 2014]. Genotyping was duplicated and carried out blind to the clinical status.

Statistical Analysis Deviations from Hardy–Weinberg equilibrium (HWE) were assessed using the HWSIM program [Cubells et al., 1997]. Differences of the MnSOD Ala–9Val allele and genotype frequencies between groups were evaluated using x2 tests. Furthermore, a logistic regression (schizophrenia and controls, or smokers and never-smokers), or a linear regression (smoking-related parameters, such as age of smoking, cigarettes smoked per day [CPD], or nicotine dependence severity) was employed to investigate the association with the MnSOD Ala–9Val genotype. Group differences were compared using Student’s two-sample ttest or one way analysis of variance (ANOVA) for continuous variables and chi squared for categorical variables. Bonferroni corrections were applied to each test to adjust for multiple testing. A univariate analysis was performed to examine the relationship between demographic variables (independent variable) and smoking as the dependent variable. Only those variables that were statistically significantly associated with smoking were included

in the following logistic regression. Then a logistic regression analysis was performed to adjust for potential confounding factors using smoking as a dependent variable and those variables showing significant difference between smoker and never-smoker groups as independent variables. The power of the sample was calculated with Quanto Software [Gauderman, 2002], with known risk allele frequencies, a schizophrenia population prevalence of 0.01, or the prevalence of smoking in schizophrenia and examining dominant and recessive models.

RESULTS Allele and Genotype Frequencies of MnSOD Ala–9Val of Samples Subgrouped by Smoking The patients included 507 (76.1%) smokers, and healthy controls included 360 (54.5%) smokers. Allele and genotype frequencies of samples for the MnSOD Ala–9Val polymorphism are given in Table I. Distributions of the MnSOD Ala–9Val genotypes were consistent with Hardy–Weinberg equilibrium in both patients with schizophrenia and healthy controls (both P > 0.05). No significant differences were found in MnSOD Ala–9Val genotype and allele distributions between the patients and healthy controls (X2 ¼ 0.001, df ¼ 1, P ¼ 0.97; X2 ¼ 0.001, df ¼ 1, P ¼ 0.97, respectively). The distribution of the allele and genotype frequencies did not differ between smokers and never-smokers in patients (X2 ¼ 1.49, df ¼ 1, P ¼ 0.22; X2 ¼ 1.26, df ¼ 1, P ¼ 0.26, respectively), or between smokers and never-smokers in healthy controls (X2 ¼ 0.67, df ¼ 1, P ¼ 0.42; X2 ¼ 0.56, df ¼ 1, P ¼ 0.45, respectively; Table I). Furthermore, no significant differences in the frequencies of genotype and alleles were observed between the smokers and never-smokers in the combined subjects or when the smokers and never-smokers were analyzed separately (in all P > 0.05).

Relationship Between the MnSOD Ala–9Val Genotype and Clinical Variables To further explore the relationship between the MnSOD gene and clinical variables, we used the quantitative trait test to determine

TABLE I. MnSOD Val–9Ala Allele and Genotype Frequencies in Smokers and Never-smokers of Schizophrenia and Healthy Controls Genotype distribution Schizophrenia (n ¼ 666) Smokers (n ¼ 507) Never-smokers (n ¼ 159) Controls (n ¼ 660) Smokers (n ¼ 360) Never-smokers (n ¼ 300)

Ala/Ala + Ala/Va (%) 180 (27.0) 143 (28.2) 37 (23.3) 179 (27.1) 93 (25.8) 86 (28.7)

Allele frequency Val/Val (%) 486 (73.0) 364 (71.8) 122 (76.7) 481 (72.9) 267 (74.3) 214 (71.3)

Ala (%) 180 (13.5) 143 (14.1) 37 (11.6) 179 (13.6) 93 (12.9) 86 (14.3)

Val (%) 1152 (86.5) 871 (85.9) 281 (88.4) 1141 (86.4) 627 (87.1) 514 (85.7)

Note: No significant differences were found in MnSOD Val–9Ala genotype and allele distributions between the patients and healthy controls (x2 ¼ 0.001, df ¼ 1, P ¼ 0.97; x2 ¼ 0.001, df ¼ 1, P ¼ 0.97, respectively), or between the smokers and never-smokers in both patients (X2 ¼ 1.49, df ¼ 1, P ¼ 0.22; X2 ¼ 1.26, df ¼ 1, P ¼ 0.26, respectively) and healthy controls (X2 ¼ 0.67, df ¼ 1, P ¼ 0.42; X2 ¼ 0.56, df ¼ 1, P ¼ 0.45, respectively).

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the association of individual phenotypes with the MnSOD Ala– 9Val genotype. We found no association between MnSOD Ala– 9Val and any clinical phenotypes, include age, education, body mass index (BMI), onset of illness, duration of illness, hospitalization, antipsychotic treatment (type, dose and duration of treatment) as well as clinical symptoms assessed on the PANSS (all P > 0.05; Table II).

regression analysis for associations with smoking, the negative symptom subscore of PANSS (odds ratio ¼ 0.97, 95%CI ¼ 0.93– 1.0, Wald X2 ¼ 4.56, df ¼ 1, P ¼ 0.03) and the number of hospitalizations (odds ratio ¼ 1.09, 95%CI ¼ 1.01–1.19, Wald x2 ¼ 4.14, df ¼ 1, P ¼ 0.04) remained significant, suggesting that smoking was associated with lower PANSS negative symptom subscore and larger number of hospitalizations.

Characteristics of Schizophrenia Smokers Based The Related Factors for Age of Smoking on Genotype Grouping Initiation We also examined the characteristics of smokers in the patient group based on genotype grouping (Table II). Among smokers, individuals with Ala allele started smoking significantly earlier than those with the Val/Val genotype (19.9  5.8 years for Ala carriers vs. 21.7  6.5 years for Val/Val; F ¼ 8.10, df ¼ 1,492, P ¼ 0.005; Bonferroni corrected: P < 0.05). However, there was no significant difference in other variables, including the number of cigarettes smoked per day (CPD), and the FTND score (all P > 0.05). In addition, we found no significant association between MnSOD Ala–9Val genotype and smoking parameters, including age of smoking onset, CPD, or FTND in control smokers (all P > 0.05).

Characteristics of Smoking and Never-smoking Male Schizophrenia Patients The univariate analyses comparing smokers and never-smokers in patients showed that there were no significant differences in age, education and BMI (all P > 0.05). However, smokers had significant lower PANSS negative symptom score (P ¼ 0.003) and total score (P ¼ 0.029), and longer duration of illness (P ¼ 0.038). However, only the significant difference in PANSS negative symptom subscore passed the Bonferroni test (P < 0.05). In the logistic

Stepwise multiple regression analysis including MnSOD Ala–9Val genotype, age, duration of illness, age of onset, CPD, FTND, duration of illness, the number of hospitalizations, PANSS, and its subscale scores and antipsychotic drugs (type, dose equivalent to chlorpromazine, duration of treatment) and age at smoking initiation (as dependent variable) in patient groups identified CPD (b ¼ 0.32, t ¼ 6.23, P < 0.001), the age of schizophrenia onset (b ¼ 0.14, t ¼ 2.74, P ¼ 0.006), and MnSOD Ala–9Val genotype (b ¼ 0.11, t ¼ 2.14, P ¼ 0.033) to be associated with age at smoking initiation.

The Power of the Sample To demonstrate the power of our sample size, we utilized the sliding odds ratio ranging from 1.4 to 2.0 with at least 80% power and a type I error level of 0.05 (a < 0.05, two-sided). According to the previous studies on Chinese Han population, the minor Ala allele frequency of patients was set to be 13.8 percent [Zhang et al., 2014]. With the case–control ratio of approximately 1:1 in this study, for the range of odds ratio of 1.4, 1.6, 1.8, and 2.0, the number of cases required for the desired power of 0.8 is 673, 338, 213, and 152 at dominant model, or 6,170, 2,975, 1,803, and 1,237 at recessive

TABLE II. Characteristics of Schizophrenia According to MnSOD Ala–9Val Polymorphism Genotype

Age (years) Education (years) BMI (kg/m2) Age of onset (years) Hospitalization PANSS P subscore N subscore G subscore Total score Smokers (N ¼ 507) Cigarettes/day Age at smoking initiation FTND score

Ala/Ala + Ala/Val 47.0  8.9 8.5  2.7 24.5  3.5 22.9  4.4 4.2  2.7 11.3 23.7 24.9 59.9

   

4.6 8.4 5.1 14.6

25.3  11.9 19.9  5.8 4.0  2.3

Val/Val 47.8  9.3 8.5  2.4 24.3  3.9 23.0  4.8 4.4  2.9

F 0.96 0.11 0.24 0.12 0.38

df 1,664 1,662 1,507 1,664 1,664

P-value 0.33 0.75 0.63 0.74 0.54

   

0.01 0.06 0.01 0.01

1,664 1,664 1,664 1,664

0.98 0.80 0.97 0.97

1.05 8.10 1.11

1,492 1,492 1,467

0.14 0.005 0.11

11.3 23.5 24.9 59.7

4.5 7.9 5.8 14.3

23.1  8.8 21.7  6.5 3.6  1.9

P, positive symptom; N, negative symptom; G, general psychopathology; PANSS, Positive and Negative Syndrome Scale (PANSS); FTND, Fagerstrom Test for Nicotine Dependence (FTND).  P < 0.01

ZHANG ET AL. model, respectively. Therefore, our sample size (666 cases and 660 controls) was deemed insufficient to suggest that the polymorphism does not contribute to the susceptibility to schizophrenia.

DISCUSSION To our knowledge, this is the first study to investigate any association between the MnSOD gene polymorphism and smoking status in a schizophrenia population. Results revealed no evidence of association between the MnSOD Ala–9Val genotype polymorphism and smoking among male schizophrenia patients in a Chinese population. However, we did find an association between MnSOD Ala–9Val gene polymorphism and age at cigarette smoking initiation among the patients with schizophrenia. This study indicated that the MnSOD-9Ala variant may have a specific role in age at smoking initiation only in schizophrenia smokers. Schizophrenia smokers who carry the Ala allele started smoking significantly earlier than their Val-homozygous counterparts. The –9Ala allele is thought to be a mutant allele, which causes a conformational change in the mitochondrial targeting sequence (MTS) that misdirects intracellular trafficking of the protein, which can induce a 30–40% increase in mitochondrial MnSOD activity [Zai et al., 2010; Carillon et al., 2013]. We speculate that this association may be related to smoking effects on free radical metabolism. The studies have shown that cigarette smoke contains oxidants such as oxygen-free radicals and volatile aldehydes, which are probably the major causes of damage to biomolecules exposed to cigarette smoke [Giuca et al., 2010]. Cigarette smoke contains two classes of free radicals: one in the “gas phase” and the other in “tar.” There are >1,018 organic free radicals per puff in the gas phase of cigarette smoke, whereas the tar phase has 1,019 free radicals per gram. The obligatory use of the body reserve of antioxidants to detoxify the excess of these free radicals in smokers therefore results in an alteration in the level of different antioxidants [Dwyer, 2003]. Oxidative stress emerges as a consequence of imbalance between reducing and oxidizing capacity of the cell in favor of the latter. Those excess amounts of free radicals start to attack cellular macromolecules such as lipids, proteins, and DNA [Cano et al., 2010; Colombo et al., 2014]. Several studies showed that cigarette smoke impairs the antioxidant defense mechanisms of the cell membrane, and causes membrane deformity and death [Cano et al., 2010; Colombo et al., 2014]. A number of prevalent and severe disorders, such as atherosclerosis, mutagenesis and cancer in the upper respiratory system, as well as lungs, have been related to free radicals and smoking [Padmavathi et al., 2010]. Thus, in order to prevent free radical damage and promote restoring mechanisms, the antioxidant enzyme activities may be increased in response to increased oxidative tone or serve as a compensatory mechanism [Lohr et al., 2003]. Since SOD, especially MnSOD may play a critical role in the detoxification of superoxide and the reduction of cellular oxidative stress, it is reasonable that MnSOD may be associated with smoking behavior. On the other hand, individuals who started smoking earlier indicate that they have been smoking longer and more cigarettes, and probably more addicted to smoking, leading to more oxidative damage. Thus, more MnSOD is needed to keep the balance between the production and removal of free radicals antagonizing

247 oxidative damage. Only the Ala-MnSOD patients make the additional MnSOD needed to address the increasing oxidative damage mediated by earlier age of smoking. Hence, it is likely that the earlier age at smoking initiation may be associated with MnSOD-9Ala variant in schizophrenia smokers by affecting MnSOD expression within mitochondria. However, this is only our speculation. The mechanism linking the MnSOD Ala–9Val gene polymorphism and age at smoking initiation is still unknown, which warrants further investigation. It is worthy of mentioning that based on the present data, we are unable to explain why the MnSOD Ala–9Val polymorphism was associated with the age of smoking onset but not other smoking behaviors such as the CPD or smoking severity (as measured by the FTND). Studies show that different genetic factors are involved in different aspects of smoking behavior, smoking initiation, and progression to nicotine dependence [Kendler et al., 1999]. Other polymorphisms that affect smoking behaviors may include acetylcholine [Greenbaum et al., 2006], serotonin [Lerer et al., 2006], tryptophan, and dopamine [Sullivan et al., 2001] polymorphisms. For example, among male Japanese, a polymorphism of MAO-A influenced nicotine dependence and smoking initiation, but not severity of smoking [Ito et al., 2003]. Another study showed that both DRD1 markers tested (rs4532 and rs686) and the DRD3 marker (rs1025398) showed an association with quantity of tobacco smoked, but not with smoking initiation [Novak et al., 2010]. Taken together, it is reasonable to conclude that multiple systems and a variety of polymorphisms play a role in different aspects of smoking behavior and nicotine dependence in schizophrenia. Further, it is noteworthy that the schizophrenia patients with MnSOD Ala allele were more vulnerable to begin smoking earlier than those with the Val/Val genotype, but this effect was not observed in healthy controls. There are some possible reasons to explain this discrepancy between patients and normal controls. First, the sample size of the smokers in healthy controls is relatively small. Thus, our results may have appeared by chance. A replication study would be needed with a larger smoker sample size. Second, our recent study has shown that the patients with schizophrenia start to smoke before the onset of illness and they have a higher prevalence than controls of ever smoking [Zhang et al., 2010a], which are consistent with previous studies in the USA and Spain [de Leon and Diaz, 2005; Diaz et al., 2008], suggesting that people who are going to develop schizophrenia have some risk factors that make them more vulnerable to start smoking [de Leon and Diaz, 2005]. Leonard et al. found that that the presence of an alpha 7 promoter polymorphism was more frequent in schizophrenia patients than in controls and may be a marker for the neurophysiological abnormalities that increase the risk of schizophrenia [Leonard et al., 2002]. Two epidemiological surveys of the British general population showed that new psychotic symptoms were 70% more likely in smokers than nonsmokers [Wiles et al., 2006], and that cigarette smoking was associated with greater schizotypy in first degree relatives of schizophrenia patients [Esterberg et al., 2007]. Taken together, these findings suggest that there may be heritable factors that make subjects with a vulnerability to schizophrenia more prone to become smokers. Therefore, based on this discrepancy between the association of MnSOD Ala–9Val genotype

248 and the age at smoking initiation in schizophrenia and control smokers in the present study, we speculate that MnSOD gene variant, especially Ala–9Val polymorphism, might play a role in such heritable factors, supporting the shared vulnerability hypothesis between schizophrenia and smoking. However, this explanation is quite speculative, and we could not provide firm mechanisms to explain this difference due to cross-sectional nature of the present study, which deserves the further investigation. It is well known that both environmental and genetic factors play a significant role in smoking [Li et al., 2003; Munafo et al., 2004]. In the present study, we found that negative symptoms, duration of illness, and the number of hospitalizations were associated with smoking in schizophrenia, suggesting important environmental factors in smoking among the patients with schizophrenia. Interestingly, we also found that MnSOD genotype, the number of cigarettes smoked each year, and the age of schizophrenia onset were associated with age at smoking initiation, suggesting the interaction between genetic and environmental factors in smoking behavior among the patients with schizophrenia. This study has several limitations. First, only one polymorphism was genotyped in the present study. A neighboring variant or gene, which may be in linkage disequilibrium with this MnSOD Ala–9Val polymorphism, could also be functionally associated with smoking in schizophrenia. Examining nearby functional sites in or near the MnSOD Ala–9Val polymorphism is warranted. Second, a main methodological limitation is the unbalanced number of smokers and never-smokers in patient group, due to much high smoking rate among schizophrenia, which might lead to bias in the statistical analysis in comparing the association of this SNP with smoking severity among the patients. Third, our sample was limited to males and can not be applied to females. Further investigations should also be performed to assess whether the MnSOD Ala–9Val polymorphism plays similarly important roles in nicotine addiction in women. Fourth, a misclassification of genotypes is possible in spite of our quality controls, but such misclassification would typically bias the results toward no effect. Fifth, limitation of the present study includes the relatively limited sample size. Hence, our results need to be replicated in a larger population of ancestries other than Chinese such as Caucasian subjects. In summary, the present study shows that a functional polymorphism of MnSOD was associated with the age at smoking initiation, but not with the degree of smoking and severity of nicotine dependence, or not becoming a smoker in male schizophrenia smokers. Moreover, several environmental factors possibly interact with the MnSOD gene variant and appear to be associated with age of smoking initiation in schizophrenia, suggesting that the gene  environment interactions may play a role in different aspects of smoking behavior in schizophrenia. However, caution should be noted in interpreting our results. As the sample size in the present study is modest, a significant association between the MnSOD Ala–9Val genotypes and the age of smoking initiation might have resulted from type I error due to modest sample size or multiple testing. Hence, the current association finding should be viewed as preliminary until confirmed in other independent larger samples in various ethnic populations.

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ACKNOWLEDGMENTS Funding for this study was provided by grants from the National Natural Science Foundation of China (81371477), the Beijing Municipal Natural Science Foundation (7132063 and 7072035), and the NARSAD Independent Investigator Grant (20314).

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