Resequencing of the vesicular glutamate transporter 2 gene (VGLUT2) reveals some rare genetic variants that may increase the genetic burden in schizophrenia

https://doi.org/10.1016/j.schres.2010.05.015Get rights and content

Abstract

Objectives

Vesicular glutamate transporters (VGLUT1-3) package glutamate into vesicles in the presynaptic terminal and regulate the release of glutamate. In mesencephalic dopamine neuron culture, the majority of isolated dopamine neurons express VGLUT2, but not VGLUT1 or 3, have been demonstrated. As related to the dysregulated glutamatergic hypothesis of schizophrenia, the gene encoding VGLUT2 is the most plausible candidate involved in the pathogenesis of this illness.

Methods

We searched for genetic variants in the promoter region and 12 exons (including UTR ends) of the VGLUT2 gene using direct sequencing in a sample of Han Chinese schizophrenic patients (n = 375) and non-psychotic controls (n = 366) from Taiwan, and conducted a case-control association study.

Results

We identified 8 common SNPs in the VGLUT2 gene. SNP and haplotype-based analyses showed no association with schizophrenia. Besides, we identified 9 rare variants in 13 out of 375 patients, including 3 variants located at the promoter region, 2 synonymous variants located at protein coding regions, and 4 variants located at UTR ends. No rare variants were found in the control subjects. Collectively, these rare variants were significantly overrepresented in the patient group (3.5% versus 0, p value of Fisher's exact test = 2.3 × 105), suggesting they may contribute to the pathogenesis of schizophrenia.

Conclusion

Although the functional significance of these rare variants remains to be characterized, our study may lend support to the multiple rare mutations hypothesis of schizophrenia, and may provide genetic clues to indicate the involvement of the glutamate transmission pathway in the pathogenesis of schizophrenia.

Introduction

The putative role for dysregulated glutamate neurotransmission in schizophrenia was first proposed based on the observation that N-methyl-d-aspartate receptor (NMDAR) antagonists such as phencyclidine, ketamine and MK-801 can induce schizophrenia-like symptoms in normal individuals, and exacerbate these symptoms in schizophrenic patients (Javitt and Zukin, 1991). Following this observation, accumulating evidence of NMDAR hypofunction in schizophrenia has been deduced from postmortem studies finding altered expression of NMDAR subunits and their intracellular interacting proteins in selective brain regions, from in vivo studies finding altered glutamate and glutamate metabolite levels, or from clinical studies finding that treatment with NMDAR modulators, such as glycine, d-serine, and glycine transporter inhibitors, can improve negative and cognitive symptoms (Javitt, 2007). Due to the predominantly postsynaptic localization of NMDARs, abnormalities of postsynaptic glutamate neurotransmission and signaling were included in the pathogenesis of schizophrenia (Stephan et al., 2006).

Then, another refinement of the hypothesis was introduced with the discovery that not only postsynaptic but also presynaptic factors caused aberrant glutamatergic transmission in schizophrenia (Honer and Young, 2004). For example, abnormal expression of several presynaptic proteins, such as synaptophysin, syntaxin, complexions, synapsins, Rab3a, synaptotagmin, synaptosomal-associated protein 25 (SNAP-25), vesicle-associated integral membrane protein (VAMP), vesicular glutamate transporters (VGLUTs), and n-ethylmaleimide-sensitive factor (NSF), has been found in postmortem brain studies of schizophrenic patients (Eastwood & Harrison, 2005, Harrison et al., 2003, Honer & Young, 2004, Nudmamud-Thanoi et al., 2007, Oni-Orisan et al., 2008, Smith et al., 2001, Uezato et al., 2009). These findings indicate that presynaptic components of glutamatergic synapses may be abnormal, and suggest that dysfunction in the molecular machinery that modulates the glutamate release into the synaptic cleft may be associated with this illness.

As one of these important presynaptic proteins, the VGLUTs are known to package glutamate into vesicles for subsequent release into the synaptic cleft (Wilson et al., 2005). Four types of vesicular glutamate transporters are known, VGLUTs 1–3 and the novel glutamate/aspartate cotransporter sialin (Aihara et al., 2000, Fremeau et al., 2002, Herzog et al., 2001, Miyaji et al., 2008). VGLUTs 1–3 are structurally and functionally conserved, but display highly contrasted and almost mutually exclusive expression profiles (Fremeau et al., 2004, Hisano, 2003, Shigeri et al., 2004). VGLUT1-expressing neurons mostly reside in cortical glutamatergic regions, as well as in the hippocampus and thalamus. VGLUT2-expressing neurons cover most subcortical regions from the thalamus to the spinal cord (Aihara et al., 2000, Fremeau et al., 2004, Herzog et al., 2001). VGLUT1 and 2 are expressed in the terminals of all well-characterized glutamatergic synapses. VGLUT3, on the other hand, appears to be expressed in neuronal populations that release other neurotransmitters, such as GABAergic interneurons of the hippocampus and cortex, and cholinergic interneurons of the striatum (Fremeau et al., 2002, Fremeau et al., 2004).

As related to the current dysregulated glutamatergic hypothesis and the classical subcortical dopaminergic hypothesis of schizophrenia (Howes and Kapur, 2009), VGLUT2 is the most plausible candidate involved in the pathogenesis of this illness. In the literature review, we found that a mesencephalic dopamine neuron culture established from rats demonstrated that the majority of isolated dopamine neurons express VGLUT2, but not VGLUT1 or 3 (Dal Bo et al., 2004). When VGLUT2 expression was selectively deleted in the cortex, hippocampus, and amygdala in preadolescent mice, they presented schizophrenia-like behaviors such as increased locomotor activity, altered social dominance and risk assessment, decreased sensorimotor gating, and impaired long-term spatial memory (Wallen-Mackenzie et al., 2009). Abnormal VGLUT2 expression has also been linked to schizophrenia in postmortem brain studies (Eastwood & Harrison, 2005, Smith et al., 2001, Uezato et al., 2009). Increased VGLUT2 mRNA expression has been found in the thalamus and inferior temporal gyrus. However, the potential effect of altered VGLUT2 expression in these regions was unclear.

The VGLUT2 gene (official name: SLC17A6; GeneID: 57084) was mapped to chromosome 11p14. This region has been reported to be linked to schizophrenia (Suarez et al., 2006). The function of a transporter is to a large extent affected by its genetic variations. To our knowledge, there is no report of a genetic association study of the VGLUT2 gene and schizophrenia in the literature. As part of our series of molecular genetic studies of schizophrenia, we were interested in understanding whether the VGLUT2 gene plays a role in conferring genetic liability to schizophrenia. To test this possibility, we systematically searched for variants of the VGLUT2 gene in a sample of Han Chinese schizophrenic patients and non-psychotic controls from Taiwan, and conducted a case-control association study.

Section snippets

Subjects

All subjects recruited into this study were Han Chinese from Taiwan. The structured clinical interview for the DSM-IV was used for the diagnosis. Patients meeting the diagnostic criteria of schizophrenia were recruited into this study. Exclusion criteria included psychosis due to general medical conditions, substance-related psychosis, and mood disorder with psychotic features. Non-psychotic controls were recruited from the Department of Family Medicine of a medical center located in eastern

SNP identification and association study with schizophrenia

After sequencing all the amplicons of the 375 patients and 366 control subjects, we detected 8 common SNPs, including g.-269C > A (rs11026523) located at 5′UTR, g.2887T > A (rs3816363) located at the putative core promoter region, and 6 SNPs (c.2577_2578insAA (rs3993089), c.2587C > A (rs1979072), c.2734A > G (rs1979073), c.2945T > G (rs12420269), c.3167A > G, c.3278C > T (rs11026539)) located at 3′UTR. The locations of these SNPs are illustrated in Fig. 1. The nomenclature of the identified genetic variation

Discussion

In spite of the strong rationale for VGLUT2 being a plausible candidate gene for schizophrenia (Dal Bo et al., 2004, Eastwood & Harrison, 2005, Smith et al., 2001, Uezato et al., 2009, Wallen-Mackenzie et al., 2009), no significant differences were observed between the patients and controls in allelic, genotypic, and haplotype distributions of the identified common SNPs in this study. Although the genetic bases of schizophrenia have been attributed to the joint effects of multiple common SNPs

Role of funding source

The funding sources had no further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication

Contributors

Author Chia-Hsiang Chen designed the study and wrote the protocol. Author Yu-Chih Shen managed the literature searches and did the genetic analyses. Authors Ding-Lieh Liao, Chao-Lin Lu, Jen-Yeu Chen, Ying-Jay Liou, Tzu-Ting Chen undertook patient evaluation and recruitment. Author Yu-Chih Shen wrote the first draft of the manuscript. All authors contributed to and have approved the final manuscript.

Conflict of interest

The authors declared no conflict of interest.

Acknowledgement

Funding for this study was provided by the National Health Research Institutes, the National Science Council, and the Hualien Armed Forces General Hospital in Taiwan.

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