Genetic association and identification of a functional SNP at GSK3β for schizophrenia susceptibility

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Abstract

Objective

GSK3β is a key gene in neurodevelopment, and also an important target of antipsychotics. Several lines of evidence including association and gene expression studies have suggested GSK3β as a susceptibility gene for schizophrenia, but the underlying genetic mechanism is still unknown. In this study, we test whether the genetic variants in GSK3β contribute to the risk of schizophrenia in Chinese population.

Methods

We first conducted an association analysis of 9 representative SNPs spanning the entire genomic region of GSK3β in two independent Han Chinese case–control samples from southwestern China (the Kunming sample and the Yuxi sample, a total of 2550 subjects).Then using EMSA and reporter gene assays, we tested the functional impact of the identified risk SNP on transcriptional factor binding affinity and promoter activity.

Results

We observed weak allelic associations of three GSK3β SNPs (rs3755557, rs7431209 and rs13320980) with schizophrenia in the combined Han Chinese samples. Further analysis using genotypes (under recessive genetic model) supported the association of rs3755557 (p = 0.01, corrected), which is located in the GSK3β promoter region. The functional assays demonstrated that the risk SNP (rs3755557) could influence the transcription factor binding affinities, resulting in a higher promoter activity of the risk allele.

Conclusion

Our findings suggest that GSK3β is likely a risk gene for schizophrenia, and its expression alteration caused by the risk SNP in the promoter region may contribute to the etiology of schizophrenia.

Introduction

Schizophrenia (MIM181500) is one of the most severe mental disorders with worldwide lifetime prevalence approaching 1%, and characterized by psychotic features (delusions and hallucinations), disorganization, dysfunction in normal affective responses, and altered cognitive function (Mueser and McGurk, 2004). It has been demonstrated that genetic component plays an important role in the etiology of schizophrenia, but the underlying genetic risk factors are yet to be identified.

The currently prevailing hypothesis has proposed that schizophrenia is a neurodevelopmental disorder, and disturbances in the early stage of brain development may cause abnormal alterations during embryonic neurogenesis, including regulating neuronal progenitor proliferation (Mao et al., 2009), changing synaptic morphology and physiology, altering differentiation, migration and survival of neurons, etc., which may result in impaired neuronal network in the mature brain and eventually lead to schizophrenia (Fatemi and Folsom, 2009).

The GSK3β gene, spanning about 286 kb on human chromosome 3q13.33 with 12 exons, is highly expressed in the brain and plays numerous roles in development, including synaptic plasticity modulation, intracellular trafficking, apoptosis and regulation of gene transcription etc. (Hur and Zhou, 2010). GSK3β is a key player in the Wnt and PI3K/AKT signaling pathways, which are crucially involved in neurodevelopment and also show abnormalities in schizophrenia (Cotter et al., 1998, Emamian et al., 2004, Lie et al., 2005). It has been suggested that GSK-3β is a master regulator of neural progenitor homeostasis, supporting the pivotal role of GSK-3β in brain development and function (Kim et al., 2009). Additionally, there are abundant evidences that GSK3β interacts with many known schizophrenia risk genes during neurodevelopment, implying potential involvement of GSK3β in schizophrenia susceptibility (Figure S1).

The involvement of GSK3β in schizophrenia are also supported by previous association studies, in which several markers have been identified, though the results were inconsistent among different studies (Scassellati et al., 2004, Ikeda et al., 2005, Lee et al., 2006, Szczepankiewicz et al., 2006, Meng et al., 2008, Souza et al., 2008). In addition, the postmortem studies indicated that GSK3β may play an important role in the pathogenesis of schizophrenia, and a reduction of approximately 40% in GSK3β protein, mRNA and kinase activity was reported in the postmortem frontal cortex of schizophrenia patients (Kozlovsky et al., 2000, Kozlovsky et al., 2001, Kozlovsky et al., 2004, Kozlovsky et al., 2005). However, there have been inconsistent reports (Nadri et al., 2004). Collectively, whether GSK3β contributes to schizophrenia susceptibility awaits further studies.

To search for the susceptible genetic variants of GSK3β for schizophrenia, we first conducted a two stage case–control association analysis using samples independently collected from two cities (Kunming and Yuxi) of southwestern China (a total of 2550 subjects), and we identified a risk SNP (rs3755557) which is located in the GSK3β promoter region. With the use of EMSA and reporter gene assays, we proved that rs3755557 is a functional polymorphism.

Section snippets

Subjects

We recruited two case–control samples independently from Kunming (432 cases, mean age 35.5 ± 9.8 years and 568 controls, mean age 36.1 ± 6.8 years) and Yuxi (520 cases, mean age 38.6 ± 10.4 years and 1030 controls, mean age 36.7 ± 6.9 years) of southwestern China. The patients were diagnosed with schizophrenia according to The Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) criteria (Kunming) and The International Classification of Diseases 10 (ICD-10) (Yuxi) respectively.

Case–control association analysis

The genotypic distributions of the 9 SNPs are in Hardy–Weinberg equilibrium in all samples, except for rs7431209 in the Yuxi case sample (p = 0.01). We double checked the eletromorphs of the original genotype data and no calling errors were found (Figure S2).

Table 1 presents the results of allelic association. For the Kunming sample, among the 9 tested SNPs, one SNP (rs13320980) showed nominally significant association with schizophrenia (p = 0.002, uncorrected). There are two SNPs with marginal

Discussion

Previous studies have suggested GSK3β gene as a potential susceptibility gene for schizophrenia due to its critical role in neurodevelopment, and this was supported by the reported aberrant expression of GSK3β in schizophrenia patients. But the efforts in identifying risk genetic variants in GSK3β gene for schizophrenia have harvested little. Potential susceptibility loci for schizophrenia on chromosome 3q was detected by early genome-wide analysis using microsatellite markers, which is

Role of funding source

This work was supported by grants from the National 973 project of China (grant numbers, 2011CBA00401 and 2007CB815705), the National Natural Science Foundation of China (grant numbers, 30871343 and 31071101), and the Natural Science Foundation of Yunnan Province of China (grant number, Y003721111).

Contributors

Authors ML, XJL and BS designed the experiments. Authors ML, YM, XX, XJL, LS and YMP performed the experiments. Authors ML and XX analyzed the data. Authors ML, BS, XX and XJL wrote the paper. Authors YM, XYL, LDY and HBD conducted the sample collection. All authors contributed to and have approved the final manuscript.

Conflict of interest

The authors have no conflicts of interest to declare.

Acknowledgements

We are grateful to the volunteers who participated in the genetic studies summarized in this article. We thank the participating psychiatrists in the Second People's Hospital of Yuxi City for helping with the ascertainment of the study subjects (Drs. Shun-ying Yang, Xing-fu Pu and Jin Yu). We also thank Hui Zhang from Kunming Institute of Zoology for her technical assistance.

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