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Plasma and cerebrospinal fluid G72 protein levels in schizophrenia and major depressive disorder
Psychiatry Research 254 (2017) 244–250
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Plasma and cerebrospinal fluid G72 protein levels in schizophrenia and major depressive disorder
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Sayuri Ishiwataa,b, Kotaro Hattoria,c, Daimei Sasayamaa, Toshiya Teraishia, Tomoko Miyakawac, ⁎ Yuuki Yokotac, Ryo Matsumurac, Fuyuko Yoshidaa, Toru Nishikawab, Hiroshi Kunugia, a b c
Department of Mental Disorder Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo 187-8502, Japan Department of Psychiatry and Behavioral Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo 113-8519, Japan Medical Genome Center, National Center of Neurology and Psychiatry, Tokyo 187-8551, Japan
A B S T R A C T G72 is a modulator of D-amino acid oxidase, the enzyme that degrades D-serine, an amino acid that plays a critical role in glutamate neurotransmission, and has been implicated in psychiatric disorders. The aim of this study was to examine whether plasma or cerebrospinal fluid (CSF) G72 protein levels were altered in either schizophrenia or major depressive disorder (MDD) and whether any correlation between G72 levels and disease severity existed. Initially, 27 schizophrenic patients, 26 MDD patients, and 27 healthy controls matched for age, sex, and ethnicity were enrolled. Compared to those of controls, plasma or CSF G72 levels were not significantly different in patients with schizophrenia or MDD. Although we found a significant positive correlation between plasma G72 levels and a positive symptoms score on the positive and negative syndrome scale (PANSS), this was not replicated in the second study (40 schizophrenic patients). CSF G72 levels showed no significant correlation with PANSS scores. In MDD, neither plasma nor CSF G72 levels correlated significantly with depression severity. Since severity of our patients were relatively mild, further investigations in a large number of subjects including drug-free patients, younger patients, and more severely affected patients are warranted.
1. Introduction As an endogenous co-agonist for the N-methyl-D-aspartate-type glutamate receptor (NMDAR), D-serine plays an important role in the regulation of glutamate neurotransmission. A body of evidence suggests that NMDAR dysfunction plays a role in the pathophysiology of schizophrenia and major depressive disorder (MDD) (Nishikawa, 2011). A number of studies have shown decreased D-serine levels in serum (Hashimoto et al., 2003) and cerebrospinal fluid (CSF) (Bendikov et al., 2007) in patients with schizophrenia. In contrast, other studies have reported increased D-serine levels in serum (Hashimoto et al., 2016) and CSF (Madeira et al., 2015) in patients with MDD. However, these reported changes are disputed (Brouwer et al., 2013). G72 protein was identified as an activator of D-amino acid oxidase (DAO), the enzyme that catalyzes the oxidative deamination of D-amino acids such as D-serine and D-3,4-dihydroxyphenylalanine (D-DOPA) (Kawazoe et al., 2007; Wu et al., 2006). Previous genetic studies and meta-analyses have identified loci on chromosome 13q32-q34 as genetically linked with schizophrenia and bipolar disorder (Bander et al., 2002; Blouin et al., 1998; Levinson et al., 2000; Liu et al., 2001).
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The chromosomal 13q33 region contains the G72 and G30 genes, which are transcribed from opposite strands, and G72 was found to be associated with schizophrenia in two independent disease cohorts (Chumakov et al., 2002). Subsequently, several studies have reported that genetic variations within the G72 gene are associated with multiple psychiatric conditions, including schizophrenia (Addington et al., 2004; Li et al., 2007; Ma et al., 2009; Bass et al., 2009) and schizophreniarelated characteristics, such as frontal lobe volume change (Hartz et al., 2010), bipolar disorder (Bass et al., 2009; Schulze et al., 2005), MDD (Rietschel et al., 2008), panic disorder (Schumacher et al., 2005), methamphetamine psychosis (Kotaka et al., 2009), and response to antipsychotic treatment (Pae et al., 2010). Gene homologs of human G72 have not been found in any nonprimate species, including mice (Chumakov et al., 2002). The longest G72 open-reading frames (ORFs) in humans were detected at low levels in the amygdala, caudate nucleus, spinal cord and testis (Chumakov et al., 2002; Hattori et al., 2003). On the other hand, pLG72 protein was reported to exist in primary astrocytes from human prefrontal cortex (Sacchi et al., 2008) and in the mitochondria enriched membrane fraction from human amygdala and caudate nucleus (Kvajo et al.,
Corresponding author. E-mail address: [email protected] (H. Kunugi).
http://dx.doi.org/10.1016/j.psychres.2017.04.060 Received 17 September 2016; Received in revised form 11 April 2017; Accepted 12 April 2017 Available online 27 April 2017 0165-1781/ © 2017 Elsevier B.V. All rights reserved.
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severe head injury, or substance abuse/dependence were excluded. Depression severity was assessed using the Japanese version of the 17item Hamilton Depression Rating Scale (HAMD-17) (Hamilton, 1960), and the cut-off score for remission was set at ≤7 (Zimmerman et al., 2013). Eight of the 26 MDD patients were remitted. Schizophrenia symptoms were assessed by the Positive and Negative Syndrome Scale (PANSS) (Kay et al., 1987). The majority of the patients were medicated (24 schizophrenia and 22 MDD patients in the (A) groups and 34 patients in the (B) groups). Daily doses of anti-depressants and antipsychotics were converted to equivalent doses of imipramine and chlorpromazine, respectively, using published guidelines (Inagaki et al., 2013). The dose was considered zero if neither class of drug was prescribed. All participant procedures were conducted in accordance with the Declaration of Helsinki. The study protocol was approved by the ethics committee at the NCNP (No. 305). After the exact nature and intent of the study had been explained in detail, written informed consent was obtained from all subjects.
2008). However, further investigations about G72 expression, mechanisms of regulation and secretion into CSF and plasma are needed. To investigate the behavioral consequences of the presence of the G72 gene, Otte et al. generated transgenic mice carrying the entire human G72/G30 genomic region and observed behavioral symptoms related to psychiatric diseases, such as impaired pre-pulse inhibition, higher sensitivity to phencyclidine (PCP), increased compulsive behaviors, motor coordination deficits, and impaired odor discrimination (Otte et al., 2009). The mechanisms underlying the link between G72 and psychiatric disorders remain unclear. pLG72, the main protein product of the G72 gene, was reported to contribute to NMDAR function through activation of DAO, the enzyme responsible for reducing Dserine levels (Sacchi et al., 2016). On the other hand, it has been suggested that G72 is a negative regulator of human DAO (Sacchi et al., 2008). In either case, LG72 regulation of DAO activity should influence D-serine levels and thus modulate NMDAR function. An alternative hypothesis is that LG72 is a mitochondrial protein that can promote mitochondrial fragmentation and dendritic arborization (Kvajo et al., 2008). An association between schizophrenia and mitochondria has been reported; platelet mitochondrial complex I activity was measured in schizophrenic patients and was found to be positively correlated with schizophrenic symptoms as well as with increased brain region-specific glucose metabolism, measured using FDG-PET (Ben-Shachar et al., 2007). A positive correlation between mitochondrial complex I and III gene mRNA levels and psychotic symptomatology, especially positive symptoms, has also been reported (Akarsu et al., 2014). Recently, Lin et al. reported that plasma G72 protein levels were approximately 2-fold higher in schizophrenia patients (both medicated and drug-free patients) compared with healthy control subjects (Lin et al., 2014). More recently, Akyol et al. also reported that serum G72 levels in schizophrenia patients were significantly increased (Akyol et al., 2016). To our knowledge, however, no study has examined CSF G72 levels in patients with schizophrenia. Furthermore, there has been no report examining plasma or CSF G72 protein levels in patients with MDD. In the present study, we sought to determine whether CSF and/or plasma G72 levels are altered in patients with schizophrenia and MDD, compared to healthy controls. We further examined the correlation between G72 levels and disease severity.
2.2. Sample collection CSF samples were obtained by lumbar puncture between 10:00 h and 16:00 h from the L4–5 or L3–4 interspace of participants in the left decubitus position. CSF samples were immediately placed on ice, and then centrifuged at 4000× g. Supernatants were aliquoted and stored at −80 °C until assays were performed. Plasma samples were obtained immediately before the lumbar puncture in tubes containing ethylenediamineteraacetic acid. The centrifuged samples were stored at −80 °C until assay.
2.3. ELISA Human G72 (D-amino acid oxidase activator: DAOA) ELISA kits were purchased from Cusabio (Cusabio Biotech Co. Ltd, China; catalog no. CSB-EL006495HU). The specificity of the human G72 antibody in this ELISA kit was further confirmed using immunoprecipitation and western blotting (Supplementary Methods). Samples were diluted to 1:2 for the CSF and to 1:4 for the plasma using the diluent supplied with the kit. Kit strips were first blocked using Super Block™ (TBS) Blocking Buffer (Thermo Scientific, Japan) and the sample was then added. Samples were incubated with the strips at 37 °C for 2 h and then at 4 °C overnight. The next day, samples were aspirated and a biotinylated-G72 antibody, diluted in Can Get Signal (R) Immunoreaction Enhancer Solution 1 (Toyobo Co., Osaka, Japan), was added to the kit strips, and incubated for 2 h at 37 °C. Strips were then washed three times, for 2 min each time, with wash buffer. An HRP-conjugated antibody diluted in Can Get Signal (R) Immunoreaction Enhancer Solution 2 (Toyobo Co., Osaka, Japan) was then added to the kit strips, incubated for 2 h 37 °C, and washed again with the wash buffer as mentioned above. Thereafter, TMB was added to the kit strips and incubated for 25 min at 37 °C. Subsequently, stop solution added and the absorbance at 450 nm was measured within 5 min.
2. Methods 2.1. Subjects The initial groups (A) consisted of 27 patients with schizophrenia (14 males, 13 females, mean ± standard deviation (SD) age: 41.0 ± 7.4 years), 26 patients with MDD (13 males, 13 females, 41.4 ± 7.3 years), and 27 healthy controls (14 males, 13 females, 41.6 ± 9.1 years), matched for age, sex, and ethnicity (Japanese). The replication groups (B) consisted of 40 patients with schizophrenia (20 males, 20 females, 41.2 ± 10.8 years) and 40 healthy controls (20 males, 20 females, 41.2 ± 9.5 years). CSF was collected only from (A) groups whereas plasma was collected from both (A) and (B) groups. All patients were biologically unrelated and of Japanese ethnicity, who were recruited at the National Center of Neurology and Psychiatry (NCNP) hospital (Tokyo, Japan), or through advertisements in free local magazines and our website. Healthy control subjects were also of Japanese ethnicity and were from the same geographical area (i.e., western Tokyo metropolitan area), recruited through advertisements. A Japanese version of the structured Mini-International Neuropsychiatric Interview (M.I.N.I.) (Sheehan et al., 1998) was given to all participants. A consensus diagnosis by at least two psychiatrists was made according to the Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM-IV) criteria (American Psychiatric Association, 1994) based on the M.I.N.I., an additional unstructured interview, and information from medical records, if available. Individuals with a prior medical history of central nervous system disease,
2.4. Statistical analysis Data are reported as mean ± standard deviation (SD). Analysis of covariance (ANCOVA), controlling for age and sex, was performed to compare CSF or plasma G72 protein levels between patients and controls. The relationships between protein levels and clinical variables (PANSS, HAMD-17, age, or drug equivalent) were analyzed using partial correlation analysis, controlling for age and sex. Statistical significance for a two-tailed P-value was < 0.05 that was corrected for multiple comparisons by Bonferroni method. All analyses were performed using the SPSS Statistics 22.0 Japanese version (IBM Japan, Tokyo, Japan). 245
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Fig. 1. Plasma and cerebrospinal fluid (CSF) G72 protein levels in the initial (A) groups. Comparison of plasma (a) or CSF (b) G72 protein levels among patients with schizophrenia, those with major depressive disorder (MDD), and healthy controls. Correlation between plasma and CSF G72 levels in healthy controls (c).
3. Results
We observed no statistically significant correlation of the total HAMD17 score with plasma or CSF G72 levels in the MDD patients (plasma Fig. 2e; CSF Fig. 3e). Plasma or CSF G72 protein levels also showed no significant correlation with typical or atypical chlorpromazine equivalent or benzodiazepine equivalent (Supplementary Fig. 2). The possible association between plasma G72 protein levels and schizophrenia symptoms was further examined in the replication (B) groups. As seen in the initial sample (A), plasma G72 protein levels were not significantly different between schizophrenic patients and controls (Fig. 4a, F (78, 1)=1.705, p=0.196 corrected). There was no significant correlation between plasma G72 levels and PANSS positive (Fig. 4b) score, negative score, general psychopathology, or total scores (data not shown), in the schizophrenic patients in the replication (B) group.
Initially, we identified the specificity of the G72 antibody used in the ELISA employed here. A band with the expected size of G72 was detected in western blots of CSF as well as in anti-G72 immunoprecipitates of CSF (Cusabio or Santa Cruz) (Supplementary Fig. 1a). Furthermore, we confirmed the precision of G72 ELISA kit by calculating relative standard deviation (RSD) of G72 standard protein measured values and lower concentration in CSF than detection range of this kit showed less than 30% RSD (Supplementary Fig. 1b). In current samples, there were no correlations between G72 protein levels and age in participants (Supplementary Fig. 3). There were strikingly large inter-individual differences in both plasma and CSF G72 protein levels (Fig. 1a, b). In the initial (A) groups, there was no significant difference in G72 levels in plasma (Fig. 1a) or CSF (Fig. 1b) between schizophrenic or MDD patients, compared with the control group. Using ANCOVA and controlling for age and sex, the following statistics were obtained: Plasma; F (77, 2)=0.676, schizophrenia vs. control p=1.000 corrected, MDD vs. control, p=1.000 corrected, CSF; F (77, 2)=0.967, schizophrenia vs. control p=1.000 corrected, MDD vs. control, p=1.000 corrected. G72 protein levels were approximately 10-fold higher in plasma than those observed in CSF and there was no significant correlation between them (Fig. 1c), suggesting independent regulation of G72 expression in the central nervous and peripheral systems. When the possible correlation between G72 levels and disease severity was examined in the initial (A) group, for the schizophrenic patients, we found a significant positive correlation between plasma G72 levels and PANSS positive score (r=0.42, p=0.042, Fig. 2a), but not with PANSS negative, general psychopathology, or total scores (Fig. 2b, c and d). None of the 4 PANSS subscale scores showed any significant correlation with CSF G72 protein levels (Fig. 3a, b, c and d).
4. Discussion To our knowledge, this is the first study that examined both plasma and CSF G72 levels in schizophrenic and MDD patients and evaluated the correlation between G72 levels and disease symptoms. We measured CSF and plasma G72 protein levels in patients with schizophrenia and MDD, and compared them with the levels of healthy controls. There were no significant differences in CSF or plasma G72 levels in schizophrenic or MDD patients relative to controls. Although we found that plasma G72 levels positively correlated with PANSS positive scores (r=0.42) in schizophrenic patients in the initial (A) group, this result was not reproduced in the subsequent replication (B) group. Lin et al. reported that plasma G72 levels were significantly increased in Han Chinese patients with schizophrenia, compared to those in the controls (Lin et al., 2014). Furthermore, Akyol et al. also reported that serum G72 levels of schizophrenia were significantly increased (Akyol et al., 2016). However, we failed to replicate their 246
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Fig. 2. Correlation of plasma G72 protein levels with Positive and Negative Syndrome Scale (PANSS) scores and the 17-item Hamilton Depression Rating Scale (HAMD-17) score in schizophrenia and major depressive disorder (MDD) patients. Correlation of plasma G72 levels with PANSS positive (a), negative (b), general psychopathology (c), and total (d) scores in schizophrenic patients. Correlation of plasma G72 protein levels with the HAMD-17 score in MDD patients (e).
between plasma or CSF G72 levels and age were observed in controls, schizophrenia and MDD patients (Supplementary Fig. 3). Furthermore, to ensure that ELISA measured the appropriate molecule in our study, we initially confirmed, using western blotting, that the G72 antibody in our ELISA binds to a molecule with the correct molecular weight (Supplementary Fig. 1). However, the possibility remains that the epitopes recognized are different between the different G72 antibodies, particularly since the G72 gene has complex splice variants (Sacchi et al., 2016) yielding different isoforms (Otte et al., 2009). On the other hand, Akyol et al. (2016) used the same G72 antibody as the one used in this study. The discrepancy between the study of Akyol et al. and this study might have arisen from the use of different samples, i.e., serum vs. plasma samples, or different background variables such as ethnicity and antipsychotic treatment (such as the use of clozapine for patients in the study of Akyol et al.). Because chlorpromazine (Iwana et al., 2008)
results with two independent data sets. The discrepancy between the study of Lin et al. and the present study may be attributed to the differences in ethnicity, patient disease severity, age and methods of measuring G72 protein, i.e., western blotting and ELISA. Actually, Lin et al. showed the mean of PANSS total scores in medicated schizophrenia was 87.6 and 88.1 in two cohorts, respectively. On the other hand, our PANSS total scores in medicated schizophrenia showed 62.1 and 61.9 in initial (A) and replication (B) sample, respectively. The mean age of subjects was also different from that of Lin. The age-matched subjects in Lin's study (controls; 33.0 ± 9.1, medicated schizophrenia; 35.9 ± 7.8) were younger than those in our study (initial sample (A) controls; 41.3 ± 9.2 (Plasma), 41.6 ± 9.1 (CSF), schizophrenia; 41.0 ± 7.4, replication sample (B) controls; 41.2 ± 9.5, schizophrenia; 41.6 ± 11.7). Therefore, we examined the relation between G72 levels and age. No correlations 247
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Fig. 3. Correlation of cerebrospinal fluid (CSF) G72 protein levels with the Positive and Negative Syndrome Scale (PANSS) scores and the 17-item Hamilton Depression Rating Scale (HAMD-17) score in schizophrenia and major depressive disorder (MDD) patients. Correlation of CSF G72 levels with PANSS positive (a), negative (b), general psychopathology (c), and total (d) scores in schizophrenia patients; Correlation of plasma G72 protein levels with the HAMD-17 score in MDD patients (e).
To explore why we could not reproduce the correlation of plasma G72 protein levels with positive symptoms in the replication (B) group, we compared clinical variables between the initial (A) group and the replication (B) group. These two groups were similar in age distribution, sex ratio, and symptom severity (PANSS scores). However, the daily dose (chlorpromazine equivalent dose) of the schizophrenic patients in the initial (A) group was found to be significantly higher than that in the replication (B) group (Supplementary Table 1, P=0.013), which may indicate a possible effect of antipsychotic medication on G72 expression. We showed the list of antipsychotic and anti-anxiety drugs in supplementary Table 2. In contrast, PANSS negative, general psychopathology, and total scores were not significantly related to plasma G72 levels. CSF G72 levels were not correlated with any PANSS score (Fig. 3a, b, c, and d),
and risperidone (Abou El-Magd et al., 2010) inhibit DAO, plasma G72 levels may be affected by the use of these antipsychotics. In order to investigate the possible effect of drug use on the measured levels of G72, we examined the correlation of G72 levels with antipsychotic (chlorpromazine equivalent) dose. There was no correlation between the medication dose and plasma or CSF G72 protein levels. Further investigations will be required to examine the possible association between G72 expression and use of antipsychotic drugs. In patients with schizophrenia in the initial (A) group, plasma G72 protein levels positively correlated with a PANSS positive score (r=0.42, p=0.042, Fig. 3a). However, there was no such a correlation in the replication (B) group (Fig. 4b). Akyol et al. (2016) also reported that there was no correlation between the G72 levels in serum and the severity of schizophrenia. 248
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Fig. 4. Comparison of plasma G72 protein levels and correlation with the Positive and Negative Syndrome Scale (PANSS) positive score in the replication (B) group. (a) Comparison of plasma G72 protein levels between patients with schizophrenia and healthy controls in the replication (B) group. (b) Correlation between plasma G72 protein levels and PANSS positive score in schizophrenia patients in the replication (B) group.
that G72 levels may be higher in younger and more severe patients but the trend may diminish in older and less severe individuals. Further investigations in a large number of subjects including drug-free patients, younger patients and more severely affected patients with schizophrenia and MDD are warranted to clarify the relationship between the body fluid levels of G72 and the psychiatric disorders.
indicating that plasma or CSF G72 levels are unlikely to be a marker for negative symptoms or general psychopathology in schizophrenia. Previous studies have reported that D-serine levels in serum (Hashimoto et al., 2015), and total serine levels in plasma (Maes et al., 1995; Sumiyoshi et al., 2004), were significantly elevated in MDD patients compared to those in controls. Another study reported that Dserine plasma levels in a (R, S)-ketamine responder were significantly lower than those in a (R, S)-ketamine non-responder (Moaddel et al., 2015). These previous reports raised the possibility that D-serine plays a role in the pathophysiology of MDD. Indeed, a genetic study has suggested a possible role of G72 in the etiology of MDD (Rietschel et al., 2008). However, other studies in MDD patients have reported that D-serine levels in plasma (Mitani et al., 2006), or in postmortem brain (Hashimoto et al., 2007), showed no significant difference compared to those from controls. Therefore, we hypothesized that G72, an activator of a D-serine degrading enzyme, might be altered in MDD. Contrary to our expectation, however, we found no significant difference in plasma or CSF G72 levels in MDD patients, compared with those in controls (Fig. 1a and b). Previously, Mitani et al. (2006) reported that there was a significant negative correlation between plasma L-serine levels and HAMD-21 score in MDD and that D-serine levels showed a similar trend, which may raise the possibility that G72 levels are related to depression severity. In our study, however, neither plasma nor CSF G72 levels were shown to be significantly correlated with HAMD-17 score in MDD patients (Fig. 3e). Therefore, our results suggest that plasma or CSF G72 protein levels are unlikely to be a biomarker for MDD. To our knowledge, there is no direct evidence that G72 protein level in the serum or CSF is related to D-serine levels in the central nervous system. Further, it is largely unknown as to how G72 proteins are secreted to blood or CSF. In conclusion, we did not obtain data supporting an alteration in G72 protein expression in plasma or CSF in schizophrenia. Since (1) the plasma and CSF contents of G72 did not correlate each other in the schizophrenia and MDD patients and controls and (2) neither of the contents associated with the symptom severities in the respective two patient groups, it is suggested that G72 contents might not serve as biomarkers for these disorders and therapeutic monitoring. There are several limitations in this study. First, the sample size was relatively small; therefore, minor effects may have been missed. Second, the majority of the patients were medicated and thus the possible influences of medication on the G72 levels cannot be excluded. Third, the severity of medicated schizophrenia patients was milder than that of previous reports (Akyol et al., 2016; Lin et al., 2014). Fourth, the subjects in this study were older than other population of previous reports. Taking into account the findings across studies, it is possible
Conflict of interest The authors declare no conflict of interest. Acknowledgements This work was supported by grants from an Intramural Research Grant (24-11; 27-1; 27-6) for Neurological and Psychiatric Disorders of NCNP (H.K. and K.H.), and the Health and Labour Sciences Research Grants for Comprehensive Research on Persons with Disabilities from Japan Agency for Medical Research and development, AMED (15dk0310061h0001 and 27260301) (H.K. and K.H.), and Grant-inAid for Scientific Research (A) from the Japan Society for the Promotion of Science (25253075) (H.K. and K.H.), and SENSHIN Medical Research Foundation (K.H.), and KAKENHI (26461731) (K.H.), and Grant-in-Aid for Young Scientists (B) from the Japan Society for the Promotion of Science (15K19715) (S.I). Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.psychres.2017.04.060. References Abou El-Magd, R.M., Park, H.K., Kawazoe, T., Iwana, S., Ono, K., Chung, S.P., Miyano, M., Yorita, K., Sakai, T., Fukui, K., 2010. The effect of risperidone on D-amino acid oxidase activity as a hypothesis for a novel mechanism of action in the treatment of schizophrenia. J. Psychopharmacol. 24, 1055–1067. Addington, A.M., Gornick, M., Sporn, A.L., Gogtay, N., Greenstein, D., Lenane, M., Gochman, P., Baker, N., Balkissoon, R., Vakkalanka, R.K., Weinberger, D.R., Straub, R.E., Rapoport, J.L., 2004. Polymorphisms in the 13q33.2 gene G72/G30 are associated with childhood-onset schizophrenia and psychosis not otherwise specified. Biol. Psychiatry 55 (10), 976–980. Akarsu, S., Torun, D., Bolu, A., Erdem, M., Kozan, S., Ak, M., Akar, H., Uzun, Ö., 2014. Mitochondrial complex I and III gene mRNA levels in schizophrenia, and their relationship with clinical features. J. Mol. Psychiatry 2 (1), 6. Akyol, E.S., Albayrak, Y., Aksoy, N., Şahin, B., Beyazyuz, M., Kuloğlu, M., Hashimoto, K., 2016. Increased serum G72 protein levels in patients with schizophrenia: a potential candidate biomarker. Acta Neuropsychiatr. 1–7. American Psychiatric Association, 1994. Diagnostic and statistical manual of mentaldisorders DSM-IV. American Psychiatric Press, Washington,D.C.
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Levinson, D.F., Holmans, P., Straub, R.E., Owen, M.J., Wildenauer, D.B., Gejman, P.V., Pulver, A.E., Laurent, C., Kendler, K.S., Walsh, D., Norton, N., Williams, N.M.,
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Plasma and cerebrospinal fluid G72 protein levels in schizophrenia and major depressive disorder
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Sayuri Ishiwataa,b, Kotaro Hattoria,c, Daimei Sasayamaa, Toshiya Teraishia, Tomoko Miyakawac, ⁎ Yuuki Yokotac, Ryo Matsumurac, Fuyuko Yoshidaa, Toru Nishikawab, Hiroshi Kunugia, a b c
Department of Mental Disorder Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo 187-8502, Japan Department of Psychiatry and Behavioral Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo 113-8519, Japan Medical Genome Center, National Center of Neurology and Psychiatry, Tokyo 187-8551, Japan
A B S T R A C T G72 is a modulator of D-amino acid oxidase, the enzyme that degrades D-serine, an amino acid that plays a critical role in glutamate neurotransmission, and has been implicated in psychiatric disorders. The aim of this study was to examine whether plasma or cerebrospinal fluid (CSF) G72 protein levels were altered in either schizophrenia or major depressive disorder (MDD) and whether any correlation between G72 levels and disease severity existed. Initially, 27 schizophrenic patients, 26 MDD patients, and 27 healthy controls matched for age, sex, and ethnicity were enrolled. Compared to those of controls, plasma or CSF G72 levels were not significantly different in patients with schizophrenia or MDD. Although we found a significant positive correlation between plasma G72 levels and a positive symptoms score on the positive and negative syndrome scale (PANSS), this was not replicated in the second study (40 schizophrenic patients). CSF G72 levels showed no significant correlation with PANSS scores. In MDD, neither plasma nor CSF G72 levels correlated significantly with depression severity. Since severity of our patients were relatively mild, further investigations in a large number of subjects including drug-free patients, younger patients, and more severely affected patients are warranted.
1. Introduction As an endogenous co-agonist for the N-methyl-D-aspartate-type glutamate receptor (NMDAR), D-serine plays an important role in the regulation of glutamate neurotransmission. A body of evidence suggests that NMDAR dysfunction plays a role in the pathophysiology of schizophrenia and major depressive disorder (MDD) (Nishikawa, 2011). A number of studies have shown decreased D-serine levels in serum (Hashimoto et al., 2003) and cerebrospinal fluid (CSF) (Bendikov et al., 2007) in patients with schizophrenia. In contrast, other studies have reported increased D-serine levels in serum (Hashimoto et al., 2016) and CSF (Madeira et al., 2015) in patients with MDD. However, these reported changes are disputed (Brouwer et al., 2013). G72 protein was identified as an activator of D-amino acid oxidase (DAO), the enzyme that catalyzes the oxidative deamination of D-amino acids such as D-serine and D-3,4-dihydroxyphenylalanine (D-DOPA) (Kawazoe et al., 2007; Wu et al., 2006). Previous genetic studies and meta-analyses have identified loci on chromosome 13q32-q34 as genetically linked with schizophrenia and bipolar disorder (Bander et al., 2002; Blouin et al., 1998; Levinson et al., 2000; Liu et al., 2001).
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The chromosomal 13q33 region contains the G72 and G30 genes, which are transcribed from opposite strands, and G72 was found to be associated with schizophrenia in two independent disease cohorts (Chumakov et al., 2002). Subsequently, several studies have reported that genetic variations within the G72 gene are associated with multiple psychiatric conditions, including schizophrenia (Addington et al., 2004; Li et al., 2007; Ma et al., 2009; Bass et al., 2009) and schizophreniarelated characteristics, such as frontal lobe volume change (Hartz et al., 2010), bipolar disorder (Bass et al., 2009; Schulze et al., 2005), MDD (Rietschel et al., 2008), panic disorder (Schumacher et al., 2005), methamphetamine psychosis (Kotaka et al., 2009), and response to antipsychotic treatment (Pae et al., 2010). Gene homologs of human G72 have not been found in any nonprimate species, including mice (Chumakov et al., 2002). The longest G72 open-reading frames (ORFs) in humans were detected at low levels in the amygdala, caudate nucleus, spinal cord and testis (Chumakov et al., 2002; Hattori et al., 2003). On the other hand, pLG72 protein was reported to exist in primary astrocytes from human prefrontal cortex (Sacchi et al., 2008) and in the mitochondria enriched membrane fraction from human amygdala and caudate nucleus (Kvajo et al.,
Corresponding author. E-mail address: [email protected] (H. Kunugi).
http://dx.doi.org/10.1016/j.psychres.2017.04.060 Received 17 September 2016; Received in revised form 11 April 2017; Accepted 12 April 2017 Available online 27 April 2017 0165-1781/ © 2017 Elsevier B.V. All rights reserved.
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severe head injury, or substance abuse/dependence were excluded. Depression severity was assessed using the Japanese version of the 17item Hamilton Depression Rating Scale (HAMD-17) (Hamilton, 1960), and the cut-off score for remission was set at ≤7 (Zimmerman et al., 2013). Eight of the 26 MDD patients were remitted. Schizophrenia symptoms were assessed by the Positive and Negative Syndrome Scale (PANSS) (Kay et al., 1987). The majority of the patients were medicated (24 schizophrenia and 22 MDD patients in the (A) groups and 34 patients in the (B) groups). Daily doses of anti-depressants and antipsychotics were converted to equivalent doses of imipramine and chlorpromazine, respectively, using published guidelines (Inagaki et al., 2013). The dose was considered zero if neither class of drug was prescribed. All participant procedures were conducted in accordance with the Declaration of Helsinki. The study protocol was approved by the ethics committee at the NCNP (No. 305). After the exact nature and intent of the study had been explained in detail, written informed consent was obtained from all subjects.
2008). However, further investigations about G72 expression, mechanisms of regulation and secretion into CSF and plasma are needed. To investigate the behavioral consequences of the presence of the G72 gene, Otte et al. generated transgenic mice carrying the entire human G72/G30 genomic region and observed behavioral symptoms related to psychiatric diseases, such as impaired pre-pulse inhibition, higher sensitivity to phencyclidine (PCP), increased compulsive behaviors, motor coordination deficits, and impaired odor discrimination (Otte et al., 2009). The mechanisms underlying the link between G72 and psychiatric disorders remain unclear. pLG72, the main protein product of the G72 gene, was reported to contribute to NMDAR function through activation of DAO, the enzyme responsible for reducing Dserine levels (Sacchi et al., 2016). On the other hand, it has been suggested that G72 is a negative regulator of human DAO (Sacchi et al., 2008). In either case, LG72 regulation of DAO activity should influence D-serine levels and thus modulate NMDAR function. An alternative hypothesis is that LG72 is a mitochondrial protein that can promote mitochondrial fragmentation and dendritic arborization (Kvajo et al., 2008). An association between schizophrenia and mitochondria has been reported; platelet mitochondrial complex I activity was measured in schizophrenic patients and was found to be positively correlated with schizophrenic symptoms as well as with increased brain region-specific glucose metabolism, measured using FDG-PET (Ben-Shachar et al., 2007). A positive correlation between mitochondrial complex I and III gene mRNA levels and psychotic symptomatology, especially positive symptoms, has also been reported (Akarsu et al., 2014). Recently, Lin et al. reported that plasma G72 protein levels were approximately 2-fold higher in schizophrenia patients (both medicated and drug-free patients) compared with healthy control subjects (Lin et al., 2014). More recently, Akyol et al. also reported that serum G72 levels in schizophrenia patients were significantly increased (Akyol et al., 2016). To our knowledge, however, no study has examined CSF G72 levels in patients with schizophrenia. Furthermore, there has been no report examining plasma or CSF G72 protein levels in patients with MDD. In the present study, we sought to determine whether CSF and/or plasma G72 levels are altered in patients with schizophrenia and MDD, compared to healthy controls. We further examined the correlation between G72 levels and disease severity.
2.2. Sample collection CSF samples were obtained by lumbar puncture between 10:00 h and 16:00 h from the L4–5 or L3–4 interspace of participants in the left decubitus position. CSF samples were immediately placed on ice, and then centrifuged at 4000× g. Supernatants were aliquoted and stored at −80 °C until assays were performed. Plasma samples were obtained immediately before the lumbar puncture in tubes containing ethylenediamineteraacetic acid. The centrifuged samples were stored at −80 °C until assay.
2.3. ELISA Human G72 (D-amino acid oxidase activator: DAOA) ELISA kits were purchased from Cusabio (Cusabio Biotech Co. Ltd, China; catalog no. CSB-EL006495HU). The specificity of the human G72 antibody in this ELISA kit was further confirmed using immunoprecipitation and western blotting (Supplementary Methods). Samples were diluted to 1:2 for the CSF and to 1:4 for the plasma using the diluent supplied with the kit. Kit strips were first blocked using Super Block™ (TBS) Blocking Buffer (Thermo Scientific, Japan) and the sample was then added. Samples were incubated with the strips at 37 °C for 2 h and then at 4 °C overnight. The next day, samples were aspirated and a biotinylated-G72 antibody, diluted in Can Get Signal (R) Immunoreaction Enhancer Solution 1 (Toyobo Co., Osaka, Japan), was added to the kit strips, and incubated for 2 h at 37 °C. Strips were then washed three times, for 2 min each time, with wash buffer. An HRP-conjugated antibody diluted in Can Get Signal (R) Immunoreaction Enhancer Solution 2 (Toyobo Co., Osaka, Japan) was then added to the kit strips, incubated for 2 h 37 °C, and washed again with the wash buffer as mentioned above. Thereafter, TMB was added to the kit strips and incubated for 25 min at 37 °C. Subsequently, stop solution added and the absorbance at 450 nm was measured within 5 min.
2. Methods 2.1. Subjects The initial groups (A) consisted of 27 patients with schizophrenia (14 males, 13 females, mean ± standard deviation (SD) age: 41.0 ± 7.4 years), 26 patients with MDD (13 males, 13 females, 41.4 ± 7.3 years), and 27 healthy controls (14 males, 13 females, 41.6 ± 9.1 years), matched for age, sex, and ethnicity (Japanese). The replication groups (B) consisted of 40 patients with schizophrenia (20 males, 20 females, 41.2 ± 10.8 years) and 40 healthy controls (20 males, 20 females, 41.2 ± 9.5 years). CSF was collected only from (A) groups whereas plasma was collected from both (A) and (B) groups. All patients were biologically unrelated and of Japanese ethnicity, who were recruited at the National Center of Neurology and Psychiatry (NCNP) hospital (Tokyo, Japan), or through advertisements in free local magazines and our website. Healthy control subjects were also of Japanese ethnicity and were from the same geographical area (i.e., western Tokyo metropolitan area), recruited through advertisements. A Japanese version of the structured Mini-International Neuropsychiatric Interview (M.I.N.I.) (Sheehan et al., 1998) was given to all participants. A consensus diagnosis by at least two psychiatrists was made according to the Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM-IV) criteria (American Psychiatric Association, 1994) based on the M.I.N.I., an additional unstructured interview, and information from medical records, if available. Individuals with a prior medical history of central nervous system disease,
2.4. Statistical analysis Data are reported as mean ± standard deviation (SD). Analysis of covariance (ANCOVA), controlling for age and sex, was performed to compare CSF or plasma G72 protein levels between patients and controls. The relationships between protein levels and clinical variables (PANSS, HAMD-17, age, or drug equivalent) were analyzed using partial correlation analysis, controlling for age and sex. Statistical significance for a two-tailed P-value was < 0.05 that was corrected for multiple comparisons by Bonferroni method. All analyses were performed using the SPSS Statistics 22.0 Japanese version (IBM Japan, Tokyo, Japan). 245
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Fig. 1. Plasma and cerebrospinal fluid (CSF) G72 protein levels in the initial (A) groups. Comparison of plasma (a) or CSF (b) G72 protein levels among patients with schizophrenia, those with major depressive disorder (MDD), and healthy controls. Correlation between plasma and CSF G72 levels in healthy controls (c).
3. Results
We observed no statistically significant correlation of the total HAMD17 score with plasma or CSF G72 levels in the MDD patients (plasma Fig. 2e; CSF Fig. 3e). Plasma or CSF G72 protein levels also showed no significant correlation with typical or atypical chlorpromazine equivalent or benzodiazepine equivalent (Supplementary Fig. 2). The possible association between plasma G72 protein levels and schizophrenia symptoms was further examined in the replication (B) groups. As seen in the initial sample (A), plasma G72 protein levels were not significantly different between schizophrenic patients and controls (Fig. 4a, F (78, 1)=1.705, p=0.196 corrected). There was no significant correlation between plasma G72 levels and PANSS positive (Fig. 4b) score, negative score, general psychopathology, or total scores (data not shown), in the schizophrenic patients in the replication (B) group.
Initially, we identified the specificity of the G72 antibody used in the ELISA employed here. A band with the expected size of G72 was detected in western blots of CSF as well as in anti-G72 immunoprecipitates of CSF (Cusabio or Santa Cruz) (Supplementary Fig. 1a). Furthermore, we confirmed the precision of G72 ELISA kit by calculating relative standard deviation (RSD) of G72 standard protein measured values and lower concentration in CSF than detection range of this kit showed less than 30% RSD (Supplementary Fig. 1b). In current samples, there were no correlations between G72 protein levels and age in participants (Supplementary Fig. 3). There were strikingly large inter-individual differences in both plasma and CSF G72 protein levels (Fig. 1a, b). In the initial (A) groups, there was no significant difference in G72 levels in plasma (Fig. 1a) or CSF (Fig. 1b) between schizophrenic or MDD patients, compared with the control group. Using ANCOVA and controlling for age and sex, the following statistics were obtained: Plasma; F (77, 2)=0.676, schizophrenia vs. control p=1.000 corrected, MDD vs. control, p=1.000 corrected, CSF; F (77, 2)=0.967, schizophrenia vs. control p=1.000 corrected, MDD vs. control, p=1.000 corrected. G72 protein levels were approximately 10-fold higher in plasma than those observed in CSF and there was no significant correlation between them (Fig. 1c), suggesting independent regulation of G72 expression in the central nervous and peripheral systems. When the possible correlation between G72 levels and disease severity was examined in the initial (A) group, for the schizophrenic patients, we found a significant positive correlation between plasma G72 levels and PANSS positive score (r=0.42, p=0.042, Fig. 2a), but not with PANSS negative, general psychopathology, or total scores (Fig. 2b, c and d). None of the 4 PANSS subscale scores showed any significant correlation with CSF G72 protein levels (Fig. 3a, b, c and d).
4. Discussion To our knowledge, this is the first study that examined both plasma and CSF G72 levels in schizophrenic and MDD patients and evaluated the correlation between G72 levels and disease symptoms. We measured CSF and plasma G72 protein levels in patients with schizophrenia and MDD, and compared them with the levels of healthy controls. There were no significant differences in CSF or plasma G72 levels in schizophrenic or MDD patients relative to controls. Although we found that plasma G72 levels positively correlated with PANSS positive scores (r=0.42) in schizophrenic patients in the initial (A) group, this result was not reproduced in the subsequent replication (B) group. Lin et al. reported that plasma G72 levels were significantly increased in Han Chinese patients with schizophrenia, compared to those in the controls (Lin et al., 2014). Furthermore, Akyol et al. also reported that serum G72 levels of schizophrenia were significantly increased (Akyol et al., 2016). However, we failed to replicate their 246
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Fig. 2. Correlation of plasma G72 protein levels with Positive and Negative Syndrome Scale (PANSS) scores and the 17-item Hamilton Depression Rating Scale (HAMD-17) score in schizophrenia and major depressive disorder (MDD) patients. Correlation of plasma G72 levels with PANSS positive (a), negative (b), general psychopathology (c), and total (d) scores in schizophrenic patients. Correlation of plasma G72 protein levels with the HAMD-17 score in MDD patients (e).
between plasma or CSF G72 levels and age were observed in controls, schizophrenia and MDD patients (Supplementary Fig. 3). Furthermore, to ensure that ELISA measured the appropriate molecule in our study, we initially confirmed, using western blotting, that the G72 antibody in our ELISA binds to a molecule with the correct molecular weight (Supplementary Fig. 1). However, the possibility remains that the epitopes recognized are different between the different G72 antibodies, particularly since the G72 gene has complex splice variants (Sacchi et al., 2016) yielding different isoforms (Otte et al., 2009). On the other hand, Akyol et al. (2016) used the same G72 antibody as the one used in this study. The discrepancy between the study of Akyol et al. and this study might have arisen from the use of different samples, i.e., serum vs. plasma samples, or different background variables such as ethnicity and antipsychotic treatment (such as the use of clozapine for patients in the study of Akyol et al.). Because chlorpromazine (Iwana et al., 2008)
results with two independent data sets. The discrepancy between the study of Lin et al. and the present study may be attributed to the differences in ethnicity, patient disease severity, age and methods of measuring G72 protein, i.e., western blotting and ELISA. Actually, Lin et al. showed the mean of PANSS total scores in medicated schizophrenia was 87.6 and 88.1 in two cohorts, respectively. On the other hand, our PANSS total scores in medicated schizophrenia showed 62.1 and 61.9 in initial (A) and replication (B) sample, respectively. The mean age of subjects was also different from that of Lin. The age-matched subjects in Lin's study (controls; 33.0 ± 9.1, medicated schizophrenia; 35.9 ± 7.8) were younger than those in our study (initial sample (A) controls; 41.3 ± 9.2 (Plasma), 41.6 ± 9.1 (CSF), schizophrenia; 41.0 ± 7.4, replication sample (B) controls; 41.2 ± 9.5, schizophrenia; 41.6 ± 11.7). Therefore, we examined the relation between G72 levels and age. No correlations 247
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Fig. 3. Correlation of cerebrospinal fluid (CSF) G72 protein levels with the Positive and Negative Syndrome Scale (PANSS) scores and the 17-item Hamilton Depression Rating Scale (HAMD-17) score in schizophrenia and major depressive disorder (MDD) patients. Correlation of CSF G72 levels with PANSS positive (a), negative (b), general psychopathology (c), and total (d) scores in schizophrenia patients; Correlation of plasma G72 protein levels with the HAMD-17 score in MDD patients (e).
To explore why we could not reproduce the correlation of plasma G72 protein levels with positive symptoms in the replication (B) group, we compared clinical variables between the initial (A) group and the replication (B) group. These two groups were similar in age distribution, sex ratio, and symptom severity (PANSS scores). However, the daily dose (chlorpromazine equivalent dose) of the schizophrenic patients in the initial (A) group was found to be significantly higher than that in the replication (B) group (Supplementary Table 1, P=0.013), which may indicate a possible effect of antipsychotic medication on G72 expression. We showed the list of antipsychotic and anti-anxiety drugs in supplementary Table 2. In contrast, PANSS negative, general psychopathology, and total scores were not significantly related to plasma G72 levels. CSF G72 levels were not correlated with any PANSS score (Fig. 3a, b, c, and d),
and risperidone (Abou El-Magd et al., 2010) inhibit DAO, plasma G72 levels may be affected by the use of these antipsychotics. In order to investigate the possible effect of drug use on the measured levels of G72, we examined the correlation of G72 levels with antipsychotic (chlorpromazine equivalent) dose. There was no correlation between the medication dose and plasma or CSF G72 protein levels. Further investigations will be required to examine the possible association between G72 expression and use of antipsychotic drugs. In patients with schizophrenia in the initial (A) group, plasma G72 protein levels positively correlated with a PANSS positive score (r=0.42, p=0.042, Fig. 3a). However, there was no such a correlation in the replication (B) group (Fig. 4b). Akyol et al. (2016) also reported that there was no correlation between the G72 levels in serum and the severity of schizophrenia. 248
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Fig. 4. Comparison of plasma G72 protein levels and correlation with the Positive and Negative Syndrome Scale (PANSS) positive score in the replication (B) group. (a) Comparison of plasma G72 protein levels between patients with schizophrenia and healthy controls in the replication (B) group. (b) Correlation between plasma G72 protein levels and PANSS positive score in schizophrenia patients in the replication (B) group.
that G72 levels may be higher in younger and more severe patients but the trend may diminish in older and less severe individuals. Further investigations in a large number of subjects including drug-free patients, younger patients and more severely affected patients with schizophrenia and MDD are warranted to clarify the relationship between the body fluid levels of G72 and the psychiatric disorders.
indicating that plasma or CSF G72 levels are unlikely to be a marker for negative symptoms or general psychopathology in schizophrenia. Previous studies have reported that D-serine levels in serum (Hashimoto et al., 2015), and total serine levels in plasma (Maes et al., 1995; Sumiyoshi et al., 2004), were significantly elevated in MDD patients compared to those in controls. Another study reported that Dserine plasma levels in a (R, S)-ketamine responder were significantly lower than those in a (R, S)-ketamine non-responder (Moaddel et al., 2015). These previous reports raised the possibility that D-serine plays a role in the pathophysiology of MDD. Indeed, a genetic study has suggested a possible role of G72 in the etiology of MDD (Rietschel et al., 2008). However, other studies in MDD patients have reported that D-serine levels in plasma (Mitani et al., 2006), or in postmortem brain (Hashimoto et al., 2007), showed no significant difference compared to those from controls. Therefore, we hypothesized that G72, an activator of a D-serine degrading enzyme, might be altered in MDD. Contrary to our expectation, however, we found no significant difference in plasma or CSF G72 levels in MDD patients, compared with those in controls (Fig. 1a and b). Previously, Mitani et al. (2006) reported that there was a significant negative correlation between plasma L-serine levels and HAMD-21 score in MDD and that D-serine levels showed a similar trend, which may raise the possibility that G72 levels are related to depression severity. In our study, however, neither plasma nor CSF G72 levels were shown to be significantly correlated with HAMD-17 score in MDD patients (Fig. 3e). Therefore, our results suggest that plasma or CSF G72 protein levels are unlikely to be a biomarker for MDD. To our knowledge, there is no direct evidence that G72 protein level in the serum or CSF is related to D-serine levels in the central nervous system. Further, it is largely unknown as to how G72 proteins are secreted to blood or CSF. In conclusion, we did not obtain data supporting an alteration in G72 protein expression in plasma or CSF in schizophrenia. Since (1) the plasma and CSF contents of G72 did not correlate each other in the schizophrenia and MDD patients and controls and (2) neither of the contents associated with the symptom severities in the respective two patient groups, it is suggested that G72 contents might not serve as biomarkers for these disorders and therapeutic monitoring. There are several limitations in this study. First, the sample size was relatively small; therefore, minor effects may have been missed. Second, the majority of the patients were medicated and thus the possible influences of medication on the G72 levels cannot be excluded. Third, the severity of medicated schizophrenia patients was milder than that of previous reports (Akyol et al., 2016; Lin et al., 2014). Fourth, the subjects in this study were older than other population of previous reports. Taking into account the findings across studies, it is possible
Conflict of interest The authors declare no conflict of interest. Acknowledgements This work was supported by grants from an Intramural Research Grant (24-11; 27-1; 27-6) for Neurological and Psychiatric Disorders of NCNP (H.K. and K.H.), and the Health and Labour Sciences Research Grants for Comprehensive Research on Persons with Disabilities from Japan Agency for Medical Research and development, AMED (15dk0310061h0001 and 27260301) (H.K. and K.H.), and Grant-inAid for Scientific Research (A) from the Japan Society for the Promotion of Science (25253075) (H.K. and K.H.), and SENSHIN Medical Research Foundation (K.H.), and KAKENHI (26461731) (K.H.), and Grant-in-Aid for Young Scientists (B) from the Japan Society for the Promotion of Science (15K19715) (S.I). Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.psychres.2017.04.060. References Abou El-Magd, R.M., Park, H.K., Kawazoe, T., Iwana, S., Ono, K., Chung, S.P., Miyano, M., Yorita, K., Sakai, T., Fukui, K., 2010. The effect of risperidone on D-amino acid oxidase activity as a hypothesis for a novel mechanism of action in the treatment of schizophrenia. J. Psychopharmacol. 24, 1055–1067. Addington, A.M., Gornick, M., Sporn, A.L., Gogtay, N., Greenstein, D., Lenane, M., Gochman, P., Baker, N., Balkissoon, R., Vakkalanka, R.K., Weinberger, D.R., Straub, R.E., Rapoport, J.L., 2004. Polymorphisms in the 13q33.2 gene G72/G30 are associated with childhood-onset schizophrenia and psychosis not otherwise specified. Biol. Psychiatry 55 (10), 976–980. Akarsu, S., Torun, D., Bolu, A., Erdem, M., Kozan, S., Ak, M., Akar, H., Uzun, Ö., 2014. Mitochondrial complex I and III gene mRNA levels in schizophrenia, and their relationship with clinical features. J. Mol. Psychiatry 2 (1), 6. Akyol, E.S., Albayrak, Y., Aksoy, N., Şahin, B., Beyazyuz, M., Kuloğlu, M., Hashimoto, K., 2016. Increased serum G72 protein levels in patients with schizophrenia: a potential candidate biomarker. Acta Neuropsychiatr. 1–7. American Psychiatric Association, 1994. Diagnostic and statistical manual of mentaldisorders DSM-IV. American Psychiatric Press, Washington,D.C.
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