Recent studies have implicated the excitatory amino acid transporter (EAAT) in schizophrenia.This study aimed to investigate the association of EAAT1 and EAAT2 gene polymorphisms with schizophrenia in Chinese Han population.
A total of 233 patients with schizophrenia and 342 healthy controls were enrolled.Two SNPs (rs2269272, rs2731880) in the EAAT1 gene and four SNPs (rs12360706, rs3088168, rs12294045, rs10836387) in the EAAT2 gene were genotyped by SNaPshot.Clinical characteristics were collected using a self-made questionnaire.Psychotic symptoms were measured using the Positive and Negative Symptoms Scale (PANSS), and cognitive function was assessed using the Matrics Consensus Cognitive Battery (MCCB).
Significant differences in allelic distribution between cases and controls were demonstrated at the locus rs12294045 (P = 0.004) of the EAAT2 gene.Different genotypes of rs12294045 were associated with family history (P=0.046), and patients with CT genotype had a higher proportion of family history of mental illness.The polymorphism of rs12294045 was associated with working operational memory (LNS: P = 0.016) and language learning function (HVLT-R: P = 0.042) in patients with lower CT genotype scores.However, these differences were no longer significant after Bonferroni correction.
Our study suggests that the polymorphism of rs12294045 in the EAAT2 gene may be associated with schizophrenia in the Chinese Han population.CT genotype may be one of the risk factors for patient family history and cognitive deficits.
Schizophrenia is a common psychiatric disorder with unknown etiology and clinical manifestations of positive symptoms, negative symptoms, and cognitive deficits [1].Cognitive deficits are now generally recognized as a core symptom of schizophrenia, have their own pathological mechanisms, and are associated with neurodevelopmental abnormalities [2].
The glutamate hypothesis for schizophrenia suggests that abnormal transmission of glutamate in the brain contributes to the cognitive and behavioral abnormalities associated with schizophrenia [3, 4].Changes in glutamate concentrations in the central nervous system may disrupt the structural connectivity and integrity of neurons, leading to programmed cell death and cell proliferation.Such changes may affect the ability to adapt to environmental changes and resist various physiological insults.About 40% of synapses in the central nervous system are glutamatergic [5, 6].When glutamate is at a normal level, it plays an important role in synaptic plasticity, learning and memory, neurodevelopment, etc. [6].Elevated levels of glutamate in the glutamatergic synaptic cleft lead to overactivation of the corresponding glutamate receptors, resulting in an excessive increase in intracellular calcium concentration in neurons, leading to excitatory neuron damage and even neuronal cell death [7,8] .Relevant studies have shown that this excitotoxicity may be involved in the occurrence and development of a variety of neuropsychiatric diseases, including schizophrenia, bipolar disorder, attention deficit hyperactivity disorder, epilepsy, stroke, amyotrophic lateral sclerosis and special tremor [9,10,11,12].On the other hand, the N-methyl-D-aspartate (NMDA) hypofunction hypothesis in schizophrenia suggests that inhibition of synaptic NMDA receptors that lead to reduced glutamate release is associated with schizophrenia [13, 14] ].Glutamate levels in the synaptic space are mainly regulated by the excitatory amino acid transporter (EAAT) [15,16,17].
Previous studies have found that EAAT1 and EAAT2 genes are associated with schizophrenia.Region-specific increases in cortical EAAT1 and EAAT2 mRNA have been implicated in the pathophysiology of schizophrenia [18, 19].The expression level of EAAT2 protein in the brain of patients is lower than that of normal people [20,21,22], and the impaired cognitive functions such as speech fluency and language learning function of patients are related to the decreased expression level of this gene [20,21,22] .15, 23].In addition, the EAAT2 gene polymorphism was found to be associated with schizophrenia in the Japanese population [24], and the G allele of SNP rs4354668 was associated with poor clinical manifestations of abstract thinking and working memory [25, 26].According to the glutamate hypothesis of schizophrenia [27,28,29,30], researchers suggest that reduced glutamate levels in schizophrenia may be associated with impaired cognitive and social functioning [31,32].
Therefore, we conducted a case-control study to investigate the association between EAAT1 and EAAT2 gene polymorphisms and clinical features, symptom severity, and cognitive deficits in Chinese Han Chinese patients with schizophrenia.
A total of 233 patients with schizophrenia diagnosed in the Diagnostic and Statistical Manual of Mental Disorders Fourth Edition (DSM-IV) who were hospitalized in Shandong Province Mental Health Center from November 2015 to March 2018 were included in this study.Patients also met the following criteria: they were between the ages of 18 and 60; their biological parents were Han Chinese; they were able to co-complete the Matrics Consensus Cognitive Battery (MCCB) cognitive test; they had not taken antipsychotics for at least one month or six Months did not receive modified electroconvulsive therapy; they had no history of drug, smoking, or alcohol abuse.At the same time, we included 342 healthy controls between the ages of 18 and 60.They also met criteria including that their biological parents were Han Chinese, that they had no family history of mental illness, and that they had no substance abuse and no recent plans to become pregnant.
A self-made general clinical data questionnaire was used to collect the participants’ age, gender, educational level, occupation, age of onset, course of disease, family history, interpersonal relationship, marital status and other disease-related data.The Positive and Negative Symptoms Scale (PANSS) was used to assess the severity of symptoms in patients.Cognitive function of patients was assessed using the Matrix Consensus Cognitive Battery (MCCB), which has been shown to be an effective tool for testing cognitive deficits in Chinese patients with schizophrenia [33].
The SNPs information of EAAT1 and EAAT2 genes in Chinese Han population were downloaded from International Genome Database and Genome Reference Consortium Human Genome Build 38 (GRch38) version, and then analyzed by Haploview software (version 4.2).Tag SNPs were screened for MAF > 0.05 and Υ2 ≥ 0.8.Two loci for EAAT1 (rs2269272 and rs2731880) and four loci for EAAT2 (rs12360706, rs3088168, rs12294045 and rs10836387) were selected.
A total of 5 ml of peripheral venous blood from patients and healthy controls was collected and placed in an anticoagulant tube containing 0.5 mol/L EDTA.The tubes were then centrifuged at 3000 rpm/min for 10 minutes to remove leukocytes and serum.Deoxyribonucleic acid (DNA) from blood samples was extracted using a modified potassium iodide method.
Primers were designed using an online primer design tool and synthesized by the Beijing Institute of Genomics (Beijing, China) (Table 1).PCR was performed using a 29 μl volume including 2 μl genomic DNA, 3 μl 10 Ex Taq buffer (Takara), 1 μl MIX Primer, 2 μl dNTPs (2.5 mM each), 20.8 μl H2O, and 0.2 μl Ex Taq [5 U/ μl] (Takara).The reaction conditions for PCR were as follows: denaturation at 96°C for 20 s after an initial step of 2 min at 96°C; annealing at 54°C for 10 s; extension at 72°C for 30 s; a total of 35 cycles.The amplified product was then digested and purified.SNaPshot extension reactions were performed using the SNaPshot Multiplex PCR Kit (Applied Biosystems) using a 5 μl volume, including 3 μl PCR product, 1 μl MIX Primer (5 PM), 0.5 μl SNaPshot MIX (ABI), and 0.5 μl ddH2O.The reaction procedure was as follows: after an initial step of 2 min at 95 °C, denaturation at 95 °C for 10 s; annealing at 50 °C for 5 s; extension at 60 °C for 30 s; a total of 35 cycles.Following the above reaction, 5ul SNaPshot PCR production and 0.5 U SAP were performed at 37°C for 60 minutes and 75°C for 15 minutes.Finally, 8 μl volumes (including 1 μl SNaPshot product, 6.5 μl Hi-Di Formamide, and 0.5 μl GS-120 LIZ (ABI)) were genetically analyzed using a PRISM 3730 XI Genetic Analyzer (Applied Biosystems) and the results were analyzed with GeneMapper v4. 1 Software (Applied Biosystems) analysis.
Statistical analysis was performed using the SPSS software package (version 21.0).Goodness-of-fit chi-square test was used to verify whether the allele and genotype frequencies of the members of the case and control groups were in Hardy-Weinberg equilibrium (HWE).The chi-square test was used to analyze the differences in various qualitative data of the subjects, including allele frequency and genotype frequency, gender, onset form, family history of mental illness, interpersonal relationship, pre-morbid characteristics, current marital status, occupational status, use of single Factorial analysis of variance (ANOVA) was used to compare differences in age of onset, total disease duration, PANSS scores (except PANSS positive scores) and cognitive function among patients with schizophrenia.PANSS positive scores were tested nonparametrically using the Kruskal-Wallis test.The least significant difference (LSD) method was used to perform multiple analyses.Linkage disequilibrium analysis and haplotype analysis were performed by Haploview software (version 4.2).
A total of 575 subjects were recruited for this study, including 233 patients with schizophrenia and 342 healthy controls.There were no significant differences in gender (χ2 = 0.041, P = 0.084) and age (t = 1.858, P = 0.064) between the two groups (Table 2).All six SNPs were in Hardy-Weinberg equilibrium in both case and control groups.
Differences in the distribution of alleles at rs2731880 of the EAAT1 gene were observed (χ2 = 4.205, P = 0.040), while no significant differences were observed after Bonferroni correction (P < 0.008 corrected for 6 tests).The genotype and allele distribution of rs12294045 in the EAAT2 gene were significantly different between cases and controls (χ2 = 8.054, P = 0.018; χ2 = 8.144, P = 0.004, respectively).When correcting for multiple testing, differences in allele distribution remained significant, and the effect of differences in genotype distribution was only at a significant trend level.No association was found between schizophrenia and the other four tested SNPs (EAAT1: rs2269272; EAAT2: rs3088168, rs10836387, rs12360706).
A comparison of the allelic and genotype distributions of the six SNPs in the EAAT1 and EAAT2 genes between schizophrenia patients and controls is presented in Tables 3 and 4, respectively.
In patients and controls, pairwise linkage disequilibrium (LD) between the studied EAAT1 rs2269272 and rs2731880 and EAAT2 rs3088168, rs10836387, rs12294045 and rs12360706 SNPs was determined.The LD blocks of EAAT1 and EAAT2 gene SNPs and the values of the correlation coefficient R2 for the two groups are shown in Figures 1 and 3.1 and 2.
Linkage disequilibrium analysis of selected SNPs in patients.a represents the EAAT1 gene and b represents the EAAT2 gene.(Note: The number in the LD block is the value of R2, expressed as a percentage, for example, 11 means R2 = 0.11)
Linkage disequilibrium analysis of selected SNPs in controls.Figure 1a is the EAAT1 gene and b is the EAAT2 gene.(Note: The number in the LD block is the value of R2, expressed as a percentage, for example, 13 means R2 = 0.13)
No haplotypes were found by haplotype analysis using the loci of the EAAT1 or EAAT2 genes of the current study.
Based on the above results, we found that the polymorphism of rs12294045 in the EAAT2 gene may be associated with schizophrenia.Therefore, we further examined the three genotypes of rs12294045 in clinical data (sex, age of onset, onset form, overall disease course, family history of psychosis, interpersonal relationships, premorbid characteristics, current marital status, occupational status) and schizophrenia patients. PANSS score (PANSS Positive Scale Score, PANSS Negative Scale Score, PANSS Psychopathology Scale Score, PANSS Total Score).Results yielded a nominally significant P value (P = 0.046) in family history.Compared with patients without family history, patients with a family history of psychosis had a lower proportion of CC homozygous genotype and a higher proportion of CT heterozygous genotype.However, the P value did not stand up to Bonferroni’s correction for multiple testing (P < 0.0038 corrected for 13 tests).There were no significant differences in other clinical data and PANSS scores between the three genotypes of rs12294045 (Table 5).
One way to perform ANOVA is to determine whether different genotypes of rs12294045 are associated with cognitive function in patients.Differences were observed between the Alphanumeric Span Test (LNS) and the Hopkins Language Learning Test Revised (HVLT-R) (LNS: P = 0.016; HVLT-R: P = 0.042).LSD analysis showed that patients with the CT genotype had significantly lower scores than those with the CC and TT genotypes in the LNS representing working operational memory (P1 = 0.042, P2 = 0.011).Furthermore, patients with CT genotype had significantly lower HVLT-R scores than those with TT genotype (P2 = 0.020), and lower than those with CC genotype (P1 = 0.098).These results suggest that heterozygosity for rs12294045 (CT) may be considered a risk factor for cognitive function in schizophrenia.However, after Bonferroni correction (P < 0.005 for 10 tests), these differences were no longer significant.No significant differences were found in other subtests of MCCB (Table 6).
This study has two goals.Firstly, the relationship between EAAT1 and EAAT2 genes and schizophrenia was discussed.We found no significant association between EAAT1 gene polymorphisms and schizophrenia.For the EAAT2 gene, the polymorphism of rs12294045 may be associated with schizophrenia in the Chinese Han population.Second, we investigated the association between the polymorphism of rs12294045 and clinical features, symptom severity, and cognitive function in patients with schizophrenia.The results indicated that the rs12294045 locus may be associated with family history, working operational memory and language learning, of which CT genotype was a risk factor.However, after many tests, nothing makes sense.
This study only showed that the rs2731880 allele distribution of EAAT1 gene may be related to schizophrenia.Previous studies have reported an association between EAAT1 and schizophrenia.Spangaro M et al.It was found that rs2731880 in the European population may be associated with schizophrenia [25].In addition, it was found that the expression level of EAAT1 mRNA in the thalamus of schizophrenia patients was significantly higher than that of healthy subjects [34].Animal model studies [35] have shown that EAAT1 knockout mice have negative social behaviors, such as lack of pleasure, social withdrawal, and self-neglect, compared with their nested wild-type mice.Our inconsistent results may be due to small sample sizes or genetic differences between different ethnic groups.
Our study found evidence of an association between rs12294045 in the EAAT2 gene and schizophrenia.There are few previous studies on rs12294045 and schizophrenia.Appenzeler S et al.[36] explored the polymorphism of rs12294045 in Parkinson’s disease and found no association.Recent studies have shown mixed results regarding the relationship between the EAAT2 gene and schizophrenia.A study on EAAT2 mRNA found that a region-specific increase in cortical EAAT2 mRNA was associated with the pathophysiology of schizophrenia [18].A case-control study in a Japanese population [24] reported an association of EAAT2 gene polymorphisms with schizophrenia and concluded that at least one susceptibility locus for schizophrenia may be located within or near EAAT2.In contrast, no association was observed between the polymorphism in SLC1A2 (EAAT2) – 181 A/C (rs4354668) and the onset of schizophrenia and its psychopathology in the Polish population [37].
Our findings regarding the relationship between the EAAT2 gene and cognitive function in patients with schizophrenia are rather weak.Previous studies have shown that working memory represents a core component of the changes in schizophrenia, and it has been shown that working memory can predict the severity of cognitive impairment and play a key role in the performance of other cognitive tasks in schizophrenia [38, 39] .Results of a prospective study showed that patients carrying the G allele of the EAAT2 SNP rs4354668 had significantly reduced gray matter volume and impaired working memory [26].Spangaro M et al.[40] found that genotypes associated with low expression of EAAT2 were significantly associated with cognitive impairments such as executive function and working memory function.Consistent with the above findings, recent studies have also suggested that genetic variants in EAAT2 may be associated with impaired working memory and executive function in patients with schizophrenia [41, 42].Further research on the function of the rs12294045 locus is needed to explore how its polymorphism affects the expression of EAAT2 mRNA, which will help us better understand the relationship between this locus and cognitive dysfunction in schizophrenia patients.
Our findings are subject to a number of limitations.First, the small sample size of this study resulted in relatively low heritability.Second, our patients were identified through the hospital inpatient department; we did not include patients with schizophrenia in the outpatient clinic.Our findings may not apply to patients who do not seek treatment or require hospitalization.Third, only one method (MCCB) is used to test the cognitive function of patients, so cognitive impairment in patients may not be fully exploited.Fourth, we cannot completely rule out other possible factors, such as previous medical treatment.Last but not least, in recent schizophrenia studies, the EAAT1 and EAAT2 loci have not been identified by genome-wide association analysis [43].Schizophrenia is a complex disorder of unknown etiology, and many factors may contribute to its pathogenesis.Therefore, further genome-wide association studies (GWAS) and interaction studies between genes and other genetic and environmental factors in schizophrenia based on larger sample sizes are needed.
In our study, the polymorphism of the rs12294045 locus of the EAAT2 gene may be associated with schizophrenia.CT genotype may be one of the risk factors for the patient’s family history, working memory and language learning dysfunction.
Researchers interested in this study may contact the corresponding authors for relevant data by email: liulf521@163.com.
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Post time: Mar-31-2022
