Association between mitochondrial DNA variations and schizophrenia in the northern Chinese Han population

To determine whether mitochondrial DNA (mtDNA) variations are associated with schizophrenia, 313 patients with schizophrenia and 326 unaffected participants of the northern Chinese Han population were included in a prospective study. Single-nucleotide polymorphisms (SNPs) including C5178A, A10398G, G13708A, and C13928G were analyzed by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP). Hypervariable regions I and II (HVSI and HVSII) were analyzed by sequencing. The results showed that the 4 SNPs and 11 haplotypes, composed of the 4 SNPs, did not differ significantly between patient and control groups. No significant association between haplogroups and the risk of schizophrenia was ascertained after Bonferroni correction. Drawing a conclusion, there was no evidence of an association between mtDNA (the 4 SNPs and the control region) and schizophrenia in the northern Chinese Han population.


Introduction
Schizophrenia is a chronic, severe metal dysfunction. Clinical manifestations of schizophrenia vary tremendously, and the pathogenesis of this disease is unclear. Results of studies in which twins or adopted children were associated with occurrence of schizophrenia indicated that genetics and environmental factors together can produce this disease [1]. It was reported that the rate of mental illness among offspring was higher for those with a maternal history of mental illness than for those with a paternal history [2]. Patients with mitochondrial disease also were more likely to exhibit symptoms of mental illness [3]. Therefore, the risk of schizophrenia might be related to mitochondrial dysfunction.
The coding region of mtDNA encodes 13 proteins, 22 tRNAs, and 2 rRNAs. The control region regulates replication and expression of the mitochondrial genes and harbors a replication initiation site and 2 major transcription initiation sites [4]. Mitochondria dysfunction can severely affect neuronal activity, including synaptic connection, axon formation, and neuronal plasticity [5]. Other investigators determined that variations in mtDNA altered the construction and expression levels of relevant proteins [6]. These variations can yield defects in respiration, enhance glycolysis, and induce overproduction of reactive oxygen species (ROS) [7]. ROS PLOS  Questionnaire (GHQ) [21], and individuals with potential psychiatric disease were excluded. Only unrelated participants with no personal history of psychiatric disease and a GHQ score <7 were considered for inclusion in the study [22]. The control group and patient group were matched for ethnicity, age, gender, and geographical region. Patients with schizophrenia (mean age ± SD, 41.1 ± 7.1 years; range, 21-65 years; 157 men, 156 women) had been hospitalized for <1 month and met DSM-IV criteria for the diagnosis of schizophrenia. Patients were evaluated by at least 2 psychiatrists who reached consensus on diagnosis. Participants were recruited from 2005 to 2010. All patients and participants provided written informed consent prior to inclusion in this study. Specimens were obtained and analyzed with approval from the Ethics Committee of China Medical University.

Methods
DNA extraction, amplification, and sequencing. DNA was extracted from sample blood using the Chelex-100 method. The mtDNA fragment (15869-740) was amplified using primers for polymerase chain reaction (PCR): L15869F and H719R [23] ( Table 2). The 20 μl PCR reactions contained 2.0 μl 5×buffer, 1.6 μl 2.5 mM dNTP mix, 0.8 μl each of reverse (R) and forward (F) PCR primers (8 pM each), 0.2 μl of KOD Enzyme (1.0 U/μl), and 20 ng of template DNA. PCR was performed under the following cycle conditions: initial denaturation of 94˚C for 5 minutes; followed by 35 cycles of 94˚C denaturation for 30 seconds, 55˚C annealing for 30 seconds, and 72˚C elongation for 40 seconds; followed by a final extension at 72˚C for 10 minutes. The production fragment was sequenced with the following primers: L15869F and 80R for HVSI, 16539F and H719R for HVSII. The purified PCR products were sequenced by ABI 377 DNA automatic sequencer. We deposited the laboratory protocols in protocols.io (dx. doi.org/10.17504/protocols.io.ipccdiw).
PCR amplification and restriction fragment length polymorphism analysis of the mtDNA coding region. The four SNPs (C5178A, A10398G, G13708A, and C13928G) in the mtDNA coding region were detected using PCR-RFLP analysis. The primers listed in able 2 were used to amplify target fragments. The mismatch method was applied to generate an HpyCH4Ⅲ artificial restriction endonuclease site in the amplified fragment that included the C13928G SNP. The 20 μl PCR reactions contained 2.0 μl 10×buffer, 2 μl 2.5 mM dNTP mix, Table 2. Primers used for the analysis of mtDNA polymorphisms in the hypervariable region and the coding region.

Locus
Annealing Temperature (˚C) Primer Sequences (5´! 3´) 2 μl each of R and F PCR primers (10 pM each), 0.2 μl of rTaq Enzyme (5 U/μl), and 20 ng of template DNA. PCR was performed under the following cycle conditions: initial denaturation of 94˚C for 5 minutes; followed by 30 cycles of 94˚C denaturation for 30 seconds, annealing at 61-65˚C (Table 2) for 30 seconds, and elongation at 72˚C for 30 seconds; followed by a final extension at 72˚C for 5 minutes. PCR products were digested with restriction enzymes (S1 Table), and fragments were detected on 6% polyacrylamide gel. Data analysis. The purified PCR products were sequenced by ABI 377 DNA automatic sequencer. Sample sequences were validated twice with Sequencher 4.1.4 software (Gene Codes Corp, Ann Arbor, MI, USA) to ensure accuracy of the data set. The sequences then were aligned and compared with rCRS [24]. The mtDNA haplogroups were classified according to the mtDNA Build16 (19 February 2014) using MitoTool [25]. Differences between the control and patient groups were ascertained using SPSS PASW Statistics v. 18.0 (IBM, Chicago, IL). The significance threshold for the haplogroup tests was 0.0020 (0.05/25); 0.0022 (0.05/22) for females and 0.0021 (0.05/24) for males [26]. Power analysis was conducted by PS program [27] statistical software.

No association between the 4 SNPs in the coding region and schizophrenia
Based on sequences of the hypervariable regions and PCR-RFLP fragments in the coding region, the 326 samples in the control group were divided into 312 haplotypes; the 313 samples in the patient group were divided into 301 haplotypes (S1 and S2 Tables). Allelic distributions of the 4 SNPs in the patient and control groups were summarized in Table 3. In the northern Chinese Han population, the minor frequencies of C5178A, A10398G, G13708A, and C13928G were 0.255, 0.472, 0.058, and 0.123, respectively. These substitutions have been reported in approximately 2700 mtDNA sequences at rates of 0.111, 0.541, 0.064, and 0.033, respectively [28]. There were no significant between-group differences in allele frequencies of the 4 SNPs (Table 4). Genotype frequencies of the 4 SNPs also were not significantly different between the control and patient groups (Table 5).

Discussion
Herein, no associations were detected between 4 SNPs and schizophrenia in the northern Chinese Han population. Investigators have shown previously that the C5178A polymorphism was associated with bipolar disorder in the Japanese population [29], and that the 5178C-10389A haplotype was a risk factor for bipolar disorder [15]. It was reported that patients with schizophrenia or bipolar disorder had the same risk-associated genetic variations [30]. Our findings of a lack of an association might be attributable to the different genetic background of population and the small sample size. In the current study, no association was found between the A10398G polymorphism and schizophrenia, which was in agreement with the findings of Zhang et al. [31]. In a study involving a small sample size, it was determined that the G13708A polymorphism was related to schizophrenia; but this relationship was not detected in large sample replicates [32]. In addition, the substitutions of the 3 SNPs (A10398G, G13708A, and C13928G) were predicted to be benign using PolyPhen-2 [33], and these predictions were consistent with our findings. In the northern Han population, the frequencies of 11 haplotypes comprising the 4 SNPs did not significantly differ between the control and patient groups. It was reported that the total frequency of the 3 haplogroups (M, B, and D) was 0.630 in 11,240 Asian mtDNA sequences [34]; we observed a total frequency of 0.606 in the northern Han Chinese population. In this study, there was no evidence of an association between haplogroups and schizophrenia in the northern Chinese Han population. In Europe, similar results were found [35,36]. Ueno et al. [37] did not observe an association between haplogroups and schizophrenia among the Japanese population. In a study of the Israeli population, Amar et al. [38] found that the frequency of haplogroup HV (including H and HV Ã ) in a group of patients with schizophrenia was significantly higher than in the control group. However, these authors did not conduct multiple testings or include a suitable control group. Rollins et al. [39] reported that haplogroup HV might be a risk factor for schizophrenia and bipolar disorder, but these authors evaluated a small sample. Discordant results have been obtained regarding an association of haplogroups with schizophrenia in the Han population. It was found that haplogroup N9a was probably a protective factor for schizophrenia in the Han population [40]. In the Hunan Han population, haplogroup B5a was reported to be a risk factor for schizophrenia [31]. In the study, no proof was found for an association of haplogroups with schizophrenia. In the Han population, discrepancies in observed associations among haplogroups and schizophrenia may be attributable to geographical differences.
Associations between mtDNA polymorphisms and schizophrenia were inconsistent among studies. Several factors may explain these discrepancies. First, the sample sizes of many of these studies were relatively small, so results may be not representative of true relationships between mtDNA and schizophrenia. Second, immigration, environment, intermarriage, and genetic factors affected polymorphisms of mtDNA. Specifically, mtDNA polymorphisms were known to differ among ethnic groups [41][42][43] and geographic regions [44]. Third, many of these studies lacked multiple testing [35].
In summary, we found on evidence of between-group differences in 4 SNPs (C5178A, A10398G, G13708A, and C13928G) thoughts to be associated with schizophrenia. Moreover, there were no significant differences between control and patient groups in the 11 haplotypes comprising of the 4 SNPs. No evidence was observed for an association of any haplogroup with schizophrenia. The control and coding regions of mtDNA should be both analyzed to detect the relationship between mitochondrial DNA and schizophrenia. Except for the polymorphisms of 4 SNPs (C5178A, A10398G, G13708A, and C13928G) and the control region, the Characteristics of other mtDNA regions and rare variants were not examined. Associations