Schizophrenia is a chronic, severe, and disabling brain disorder that affects ∼1% of the world’s population. Mitochondrial dysfunction has been reported frequently in patients with schizophrenia and other psychiatric disorders (Shao et al., 2008). As the largest complex of the respiratory chain, mitochondrial complex I plays a major role in controlling oxidative phosphorylation and its abnormality can lead to a variety of diseases associated with mitochondrial dysfunction (Distelmaier et al., 2009). Hitherto, many studies have reported that both the nuclear-encoded and the mtDNA-encoded genes for mitochondrial complex I may be involved in schizophrenia and bipolar disorder (Karry et al., 2004; Rollins et al., 2009), although some of the previous conclusions were controversial (Bandelt et al., 2007; Rollins et al., 2009). In our recent study, we found no association between the NDUFV2 gene (encoding a subunit of mitochondrial complex I) and schizophrenia (Zhang et al., 2010); thus, it failed to verify the previous report for a positive association between this gene and schizophrenia (Washizuka et al., 2006). However, whether other counterparts of the mitochondrial complex I would be involved in schizophrenia remains to be determined.
The NADH dehydrogenase Fe-S protein 7 (NDUFS7) gene, encoding a 20 kD subunit of complex I, has been reported to be associated with Leigh syndrome (Triepels et al., 1999; Lebon et al., 2007). A decreased level of NDUFS7, which might cause a decreased activity of complex I and a resultant resultant increase in protein oxidation and nitration, has been found in bipolar patients compared with healthy controls (Andreazza et al., 2010). These lines of evidence indicated that NDUFS7 might be a susceptible gene for psychiatric diseases. In this study, we aimed to investigate the association of NDUFS7 gene polymorphisms with schizophrenia in a cohort of Han Chinese patients with schizophrenia and healthy controls from Hunan Province (China).
Materials and methods
A total of 330 unrelated patients with schizophrenia [mean age of onset 24.4±8.3 years old (range: 10–57); 70% men] and 330 matched healthy controls [mean age 42.9±15.3 years old (range: 17–74); 66% men], all of Han Chinese origin, were recruited from Hunan Province in South Central China. These patients were analyzed for the NDUFV2 gene in our recent study (Zhang et al., 2010). Schizophrenia patients were diagnosed independently by two psychiatrists according to the DSM-IV criteria for schizophrenia and had at least a 2-year history of schizophrenia. The controls were clinically diagnosed as having no psychiatric disorders or other diseases and were well matched in geographic origin and ethnicity with the schizophrenia patients. All participants or supervisors of patients signed informed consent. This study was approved by the institutional review boards of the Kunming Institute of Zoology and the 2nd Xiangya Hospital of the Central South University.
Selection of tagging single-nucleotide polymorphisms and genotyping
Genomic DNA was isolated from the peripheral blood of patients and controls according to the standard phenol–chloroform procedure. To set inclusion criteria for tagging single-nucleotide polymorphisms (SNPs), we retrieved the CHB data from the HapMap database (http://hapmap.ncbi.nlm.nih.gov/; HapMap III, release 2) and defined linkage disequilibrium (LD) blocks using the Haploview Program (version 4) (Barrett et al., 2005). All SNPs listed in the entire NDUFS7 gene region were included in the LD analysis (Fig. 1). Haplotype-tagging SNPs (htSNPs) were selected at the cutoff level of r2 of at least 0.80 and a minor allele frequency (MAF) of at least 0.2 (Fig. 1). All selected htSNPs were amplified and genotyped by direct sequencing. Nested PCRs were carried out using three primers: for the first step PCR, the primer pair F1 (5′-ACGACATGGACCGCTTTG-3′) and R1 (5′-AAACTAATGGCAAGTGAGAAGG-3′) was used; for the second step PCR, the primer pair F2 (5′-GGCGTCGCACTTGGTATGT-3′) and R1 was used. Amplification was carried out in a total volume of 25 µl. Purified PCR products were sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit on an ABI PRISM 3730 Genetic Analyzer (Applied Biosystems, Foster City, California, USA).
Deviation from the Hardy–Weinberg equilibrium was calculated using the HWsim program (http://krunch.med.yale.edu/hwsim/). PHASE2.1.1 program (Stephens et al., 2001) was used to reconstruct the haplotype of the three htSNPs. We compared the allele, genotype, and haplotype frequencies of the three htSNPs between the case and the control samples using the χ2-test. A P-value less than 0.05 was considered as statistically significant.
Results and discussion
The role of mitochondria in psychiatric disorders has received increasingly more attention in recent years (Shao et al., 2008; Rezin et al., 2009). Genetic variants and abnormal gene expression of nuclear-encoded and mtDNA-encoded genes for the respiratory chain have been reported to be associated with a disturbance in energy metabolism and excessive oxidative stress, which were actively involved in schizophrenia, bipolar disorder, and other psychiatric disorders (Karry et al., 2004; Rollins et al., 2009; Clay et al., 2011; Verge et al., 2011). We hypothesize that genetic variants of the structural subunits of the respiratory chain complexes would confer susceptibility to schizophrenia. We have started a systematic analysis for the main subunits of the electron transport chain in Han Chinese with schizophrenia, with the aim of understanding energy impairment as a mechanism underlying the pathophysiology of schizophrenia.
In this study, we genotyped three htSNPs (rs2074896, rs2074897, and rs2074898) of the NDUFS7 gene, which encodes a subunit of mitochondrial complex I, and was reported recently to be associated with bipolar disorders (Andreazza et al., 2010), in 330 Han Chinese patients with schizophrenia and 330 matched normal individuals. These three htSNPs were selected for genotyping because they could capture all nine common variants of the NDUFS7 gene according to the analysis of the CHB data in HapMap III dataset (release 2) (Fig. 1). No deviation from the Hardy–Weinberg equilibrium was observed for each of the three htSNPs in our control individuals or schizophrenia patients. There was no statistical difference for the three htSNPs in the allele and genotype frequencies between our case and control populations (Table 1). However, we observed a marked difference for allele frequencies and LD pattern of the nine SNPs in the NDUFS7 gene (including the three htSNPs) between East Asian and European populations according to the HapMap data (Table S1 and Fig. S1, http://links.lww.com/PG/A63), which indicated a huge ethnic difference. A total of six haplotypes were reconstructed on the basis of the three htSNPs (rs2074896–rs2074897–rs2074898). Four main haplotypes (GGA, TGG, GAG, and GGG) were prevalent and accounted for ∼99% of all observations. However, we found no statistical difference in the haplotype distribution in the case and the control populations (Table 2).
The lack of association between the three htSNPs of the NDUFS7 gene and schizophrenia in the current study is not completely surprising when considering the complex pathogenesis of this psychiatric disorder. The current observation, together with our negative result of the NDUFV2 gene (Zhang et al., 2010), could not completely reject a possibility of mitochondrial dysfunction as a cause for schizophrenia, because the mitochondrion was composed of 1000–1500 proteins (Pagliarini et al., 2008), and each impaired counterpart might lead to dysfunction of the mitochondrion. Another limitation of our current study is the incompleteness of the clinic information available, which did not allow us to draw a conclusion on the association between the NDUFS7 gene SNPs and schizophrenia subtypes. Furthermore, schizophrenia would more likely fit for the ‘common disease-rare variants’ hypothesis (McClellan et al., 2007; International Schizophrenia Consortium, 2008; Sebat et al., 2009); we could not rule out the possibility that rare variants in the NDUFS7 gene would contribute to schizophrenia. We compared the matrilineal genetic component of our case and control samples and found that both populations were very similar (W. Zhang, J. Tang, A.-M. Zhang, M.-S. Peng, H.-B. Xie, L. Xu, Y.-P. Zhang, X. Chen, Y.-G. Yao, unpublished data), which indicated that the lack of association was unlikely caused by population stratification.
We genotyped three htSNPs of the NDUFS7 gene in our patients with schizophrenia, but found no positive association. Replication studies in other independent populations with a large sample size would help to further define the potential role of the NDUFS7 gene in schizophrenia. Although our analysis for multiple structural subunits of complex I encoded by nuclear genes, such as the NDUFS7 gene and the NDUFV2 gene (Zhang et al., 2010), showed no association with schizophrenia, we would expect that other subunits of this complex may confer genetic susceptibility to schizophrenia. Our ongoing project to screen all mitochondrial complex I genes may provide information to answer this question.
We thank the participants in this study and the two anonymous reviewers for helpful comments on the early version of the text. This work was supported by the National Natural Science Foundation of China (30900486, 31000557) and Yunnan Province (2009CI119).
Conflicts of interest
There are no conflicts of interest.
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Han Chinese; NDUFS7; schizophrenia; tagging single-nucleotide polymorphism
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