Significant outcomes
• rs520688 in NOTCH4 is associated with the susceptibility of schizophrenia.
• rs2030324 in brain-derived neurotrophic factor (BDNF) is associated with the susceptibility of schizophrenia.
• rs520688 and rs203032 have combined effects on the susceptibility to schizophrenia but this is not caused by allele–allele interaction.
Limitations
• To further validate these results, more case and control samples are needed.
• Further investigations into the functional consequence of NOTCH4 and BDNF on schizophrenia would be very interesting.
• The single-nucleotide polymorphisms (SNPs) may act in haplotypes, and analyses of more SNPs are needed.
Introduction
Schizophrenia is a serious mental disorder with a lifetime prevalence rate of 1% in the general population worldwide (Reference Eaton1,Reference Sartorius, Jablensky and Korten2). As the illness places heavy economic and social burdens on families and society, it is important to establish ways to treat and prevent schizophrenia. However, the pathogenesis of schizophrenia is unclear. Although there are clearly environmental contributors to the disease, genetic predisposition is the major determinant of who develops schizophrenia, with heritability estimates as high as 80% (Reference Cardno and Gottesman3,Reference Sullivan, Kendler and Neale4), placing schizophrenia among the most heritable of common diseases. Genome-wide association studies (GWAS) and candidate gene approaches to schizophrenia have produced many positive results (Reference Ripke, Sanders and Kendler5), but most of these have poor reproducibility. Several studies have shown that NOTCH4 and BDNF are associated with the pathophysiology of schizophrenia (Reference Wei and Hemmings6–Reference Watanabe, Nunokawa, Kaneko and Someya11). In the first association study (Reference Wei and Hemmings6), we found that NOTCH4 polymorphisms were associated with the risk of schizophrenia in British patients. Associations between NOTCH4 and schizophrenia were replicated in other studies (Reference Glatt, Wang, Yeh, Tsuang and Faraone7–Reference Shibata, Ohnuma and Hiqashi9); however, the results of these studies are inconsistent across geographic regions. Furthermore, GWAS and meta-analyses revealed that NOTCH4 SNPs are associated with schizophrenia (Reference Ikeda, Aleksic and Kinoshita12).
BDNF is another well-defined schizophrenia-related gene. Decreased BDNF concentrations have been found in the cortical areas and the hippocampus of schizophrenics (Reference Durany, Michel and Zöchling13), and the concentration is also associated with the treatment of schizophrenia (Reference Pedrini, Chendo and Grande14). SNPs in BDNF have been shown to play an important role in structural and functional plasticity in schizophrenia and are potential markers for schizophrenia prevention and target therapy (Reference Favalli, Li, Belmonte-de-Abreu, Wong and Daskalakis15). Both BDNF and NOTCH4 are involved in neural development; however, there is no report of a direct relationship between the two proteins.
To investigate the relationship between polymorphisms in NOTCH4 and BDNF and schizophrenia, four SNPs (rs520688, rs415929, rs2030324, and rs12273539) in NOTCH4 and BDNF were examined in a Han Chinese population from southern China, using the MassARRAY iPLEX platform. Gene–gene interactions were explored and a genotype recombination analysis was performed.
Materials and methods
Subjects
The subjects who participated in our study were recruited between July 2008 and September 2011 from Xiangya Hospital, Xiangya, Hunan Province, China. All participants were permanent residents of Hunan from the Han Chinese population. Clinical information on each subject was collected from medical records.
The study recruited 470 patients with schizophrenia diagnosed by psychiatrists using the International Statistical Classification of Diseases and Related Health Problems, 10th Revision (ICD-10), and the Chinese Classification and Diagnostic Criteria of Mental Disorders, 2nd Revision (CCMD-II-R). Blood samples were collected from the subjects. As genotyping failed for six samples, 464 case subjects were included in the final analysis. Their ages ranged from 16 to 62 years. In addition, blood samples were collected from 464 healthy controls selected randomly from outpatients ranging in age from 18 to 68 years, during the same period with no history of schizophrenia or other mental disease.
The study protocol was approved by the Clinical Research Ethics Committee of Xiangya Hospital. Written informed consent was obtained from the guardians of the participants.
DNA extraction
Peripheral blood samples were drawn from the participants at Xiangya Hospital, Xiangya, Hunan Province, China. The samples were delivered frozen by express mail to the School of Biotechnology, Southern Medical University, Guangzhou, Guangdong Province, China, and stored at −70°C. Genomic DNA was extracted from 200 μl of peripheral blood using a Genomic DNA Purification kit (Tiangen Biotech, Beijing, China) according to the manufacturer's instructions and stored at −70°C until use.
Genotyping
All SNPs were genotyped using the Sequenom MassARRAY matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry platform (Sequenom, San Diego, CA, USA). Primers were designed using a semi-automated method (Assay Design 3.1, Sequenom). The primer sequences are listed in Table 1.
Table 1 PCR primers designed using Sequenom MassARRAY Assay Design 3.1
PCR, polymerase chain reaction.
Statistical analyses
Hardy–Weinberg equilibrium (HWE) for the four SNPs in the control group was assessed using Haploview 4.2 (Daly Lab, Cambridge, MA, USA). Differences in genotype distributions between the cases and controls were evaluated by χ2 analysis. Associations between the polymorphisms and risk of schizophrenia were estimated using odds ratios (ORs) and 95% confidence intervals (95% CIs) with binary logistic regression analyses, controlling for age and sex as covariates. Their associations with schizophrenia were analysed using the web-based tool SNPStats (http://bioinfo.iconcologia.net/SNPstats). p < 0.05 was considered statistically significant. Allele–allele interactions were tested by logic regression cross-validation and combination analyses between rs520688 and rs2030324 with an overall χ2 test in a logistic regression.
Results
After adjusting for age and gender, all SNPs were in HWE, except rs520688, which was out of HWE for both the patients and controls. The distributions of the rs415929, rs520688, rs2030324, and rs12273539 polymorphisms in the schizophrenia and control groups are shown in Table 2. For BDNF rs2030324, the case group was 33.3% TT and 66.7% CT-CC, which differed significantly from the controls (39.7% TT and 60.3% CT-CC). The CC/CT genotype in rs2030324 increased the risk of schizophrenia with an OR of 1.34 (95% CI, 1.01–1.78, p = 0.044). For NOTCH4 rs520688, the case group was 79.9% AA-GG and 20.1% GA, which differed significantly from the controls (72.8% AA-GG and 27.2% GA). The GA genotype of rs520688 (p = 0.035) decreased the risk of schizophrenia with an OR of 0.71 (95% CI, 0.51–0.98, p = 0.035). The genotype distributions of rs415929 in NOTCH4 and rs12273539 in BDNF did not differ significantly between the case and control groups.
Table 2 Distribution of the rs415929, rs520688, rs2030324, and rs12273539 polymorphisms in schizophrenia and control groups
OR, odds ratio.
Bold values indicate p < 0.05.
Gene–gene combinations can be tested using an overall χ2-test in a logistic regression (Reference Pierri, Volk, Auh, Sampson and Lewis16). Recombination of rs520688 and rs2030324 was analysed using this method. There was a significant difference (χ2 = 12.461, p = 0.006); AA/GG-CT/CC increased the risk of schizophrenia with an OR of 1.424 (p = 0.022, 95% CI = 1.051–1.398; Table 3).
Table 3 Combination analysis of rs520688 and rs2030324 between the case and control groups
OR, odds ratio.
aOR (95% CI) was adjusted for age.
Bold value indicate p < 0.05.
If two SNPs have a combined effect, it might arise because they have a biological interaction, which can be tested using logic regression cross-validation (Reference Pierri, Volk, Auh, Sampson and Lewis16). As both BDNF and NOTCH4 are involved in neural development, there may be an interaction between NOTCH4 rs520688 and BDNF rs2030324. To confirm this hypothesis, the risk of rs520688 and rs2030324 and the interaction between rs520688 and rs2030324 were analysed using logic regression cross-validation; however, no significant result was obtained (χ2 = 0.933, p = 0.334; Table 4).
Table 4 Allele–allele interaction analysis between rs520688 and rs2030324 (χ2 = 12.461, p = 0.006)
OR, odds ratio.
aOR (95% CI) was adjusted for age.
The bold values indicate p < 0.05.
Discussion
NOTCH4, which is located on chromosome 6p21.3, is associated with neuronal development. The genetic knockout of NOTCH4 and NOTCH1 leads to abnormal vascular morphogenesis in mouse embryos (Reference Krebs, Xue and Norton17). BDNF, which is located on chromosome 11p13, is expressed in neurons and endothelial cells during development (Reference Lee, Duan and Mattson18). During the development of the cerebral cortex and hippocampus, BDNF induces the differentiation of neural stem cells into neurons and promotes the survival of newly generated neurons (Reference Barnabé-Heider and Miller19,Reference Cheng, Wang, Cai, Rao and Mattson20). BDNF expression in endothelial cells is involved in neurogenesis in the canary song system (Reference Louissaint, Rao, Leventhal and Goldman21). BDNF also plays an important role in preventing the death of neurons during development, and it promotes cell survival under stressful conditions (Reference Larsson, Nanobashvili, Kokaia and Lindvall22).
Studies show that schizophrenia is related to a reduction in the soma of prefrontal cortex neurons (Reference Benes23–Reference Fatemi and Folsom25). In addition, the distribution of neurons in the prefrontal cortex is altered in schizophrenia, with fewer surface white matter and grey matter neurons and more deep white matter neurons (Reference Benes, Todtenkopf and Taylor26,Reference Schmidt-Kastner, Van Os, Wm Steinbusch and Schmitz27). Numerous studies have shown that schizophrenia involves apoptosis via an abnormal pathway, changing the apoptotic activity of neurons and glial cells (Reference Glantz, Gilmore, Lieberman and Jarskog28,Reference Honig and Rosenberg29). BDNF and NOTCH4 are expressed in neurons and endothelial cells during development and are closely related to neuron development. BDNF and NOTCH4 SNPs are associated with the susceptibility of individuals to schizophrenia (Reference Wei and Hemmings6–Reference Ikeda, Aleksic and Kinoshita12). However, schizophrenia is caused by the interaction of many genes and environmental factors. To study the association between susceptibility genes and schizophrenia, it is insufficient to analyse just one SNP in a single gene, as this cannot describe the exact relationship between the disease and the gene. Analyses of SNP–SNP, gene–gene, and gene–environment interactions are also necessary. This study examined NOTCH4 and BDNF as candidate genes and SNPs as genetic markers in a case–control study using correlation analysis to analyse the relationship between polymorphisms in NOTCH4 and BDNF and schizophrenia. Four SNPs (rs520688 and rs415929 in NOTCH4 and rs2030324 and rs12273539 in BDNF) were genotyped in 464 schizophrenics and 464 healthy controls from Southern China using the Sequenom MassARRAY® iPLEX platform. Of these four SNPs, rs520688 in NOTCH4 and rs2030324 in BDNF were significantly associated, whereas rs415929 and rs12273539 had no associations with schizophrenia in our study population. Furthermore, there were significant differences in the combinations of genotypes and model testing. The AA/GG-CC/CT genotype was more frequent in the cases, indicating that it is a risk marker for schizophrenia. If two SNPs interact, this may be caused by a biological interaction; however, no significant allele–allele interaction was found between rs520688 and rs2030324. Consequently, the combined effect of rs520688 and rs2030324 may not be caused by a functional interaction between NOTCH4 and BDNF.
The combined effects of rs520688 and rs2030324 on the susceptibility to schizophrenia imply that NOTCH4 and BDNF are closely linked to schizophrenia. These results may not only help to reveal the mechanism of action of NOTCH4 and BDNF in schizophrenia, but also provide data for developing individualised therapy for schizophrenia.
Acknowledgements
This work was supported by Key Programmes for Science and Technology Development of Guangzhou (Grant no. 2008A1-E4151). The authors express their deepest gratitude to all the patients and healthy controls who agreed to participate in this study.
