We aimed to investigate possible associations between three norepinephrine transporter gene (SLC6A2) single nucleotide polymorphisms (T182C, A3081T, and G1287A) and schizophrenia. Also, we investigated the relationships of those polymorphisms with clinical severity and characteristics of schizophrenia.
Participants were 220 schizophrenia patients in the acute phase and 167 healthy controls. The genotype, allele frequency, and haplotype of each group were analyzed for T182C, A3081T, and G1287A polymorphisms. Of the 220 schizophrenia patients, 163 patients were evaluated with the Positive and Negative Syndrome Scale (PANSS) and the Korean version of the Calgary depression scale for schizophrenia (K-CDSS) at baseline.
We found no significant differences between the schizophrenia patient group and the control group in genotype distribution or allele frequency of the three tested polymorphisms. Likewise, we could not find any significant differences in genotype or allele frequency by analyzing according to gender. In the haplotype study, no significant association emerged between specific haplotype combinations and schizophrenia. We also found no association between clinical scales (PANSS and K-CDSS) and the studied polymorphisms.
Our results suggest that the investigated polymorphisms of the NET gene are not associated with susceptibility to schizophrenia or its clinical features in a Korean population. However, this study remains significant because it is the first haplotype study to investigate associations between NET gene (SLC6A2) single nucleotide polymorphisms and schizophrenia in a Korean population. Future research with a larger sample size and more genetic markers is needed to replicate our results.
Norepinephrine is one of the neurotransmitters that has been implicated in the pathogenesis of schizophrenia.
Human NET is a NaCl-dependent substrate-specific transporter,
Several studies have tried to show that functional deficits of the NET gene can cause psychiatric diseases including mood disorder, anxiety disorder, substance disorder, and ADHD. It is thought that noradrenergic dysregulation plays an important role in the pathophysiology of those disorders, and agents related to noradrenergic function can improve the symptoms.
Therefore, we focused on the possibility that the NET gene plays a role in schizophrenia. We investigated three NET single nucleotide polymorphisms (SNPs), T182C (rs2242446), A3081T (rs28386840), and G1287A, to determine whether they play a role in schizophrenia. The T182C and A3081T polymorphisms, located in the 5'flanking promoter region of the NET gene, could lead to altered transcriptional activity by changing the DNA structure.
We recruited 220 patients with schizophrenia who met the criteria of the Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV)
The normal controls consisted of 167 healthy individuals recruited through advertisement. These controls were screened with the SCID-I, non-patient edition.
To assess the severity of a patient's psychiatric symptoms, patients were interviewed by trained psychiatrists using the Positive and Negative Syndrome Scale (PANSS)
Written informed consent was obtained from all subjects (or caretakers of the patients), and the study protocol was approved by the Ethics Committee of Korea University Ansan Hospital.
Genotypes of the NET gene were analyzed in 220 patients and 167 controls. Genomic DNA was extracted from leukocytes using a commercial DNA extract kit, the Wizard Genomic DNA purification kit (Promega, Madison, WI, USA). Samples were amplified using a thermocycler (GeneAmp PCR system 2700, Applied Biosystems, Foster City, CA, USA).
The NET T182C SNP was genotyped by the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) methods according to the protocol originally described by Inoue et al.
The NET A3081T SNP was genotyped by PCR, according to the protocol originally described by Suzuki et al.
The NET G1287A SNP was genotyped by PCR according to the protocol originally described by Kazuyuki Inoue et al. The amplification mixture conditions were the same as with T182C. After an initial 5 min at 95℃, each cycle consisted of 30 s at 95℃, 30 s at 55℃, and 30 s at 72℃ for 36 cycles. After a final 10 min at 72℃, the reaction was terminated at 4℃. The amplified DNA was digested with the restriction enzyme Sau96I (New England Biolabs), which cuts at the 1287G site, and the product was electrophoresed in 3.7% agarose gel and stained with ethidium bromide. Homozygous genotypes were identified by the presence of 113, 79, 28, and 21 bp bands (G/G) or bands of 113, 100, and 28 bp (A/A). The heterozygous genotype (G/A) displayed all 5 band sizes (the 28 and 21bp fragments were undetectable because of their small size).
The presence of Hardy-Weinberg equilibrium was tested using a χ2 test for goodness of fit. Allele and genotype frequencies in patients with schizophrenia and healthy controls were also evaluated using the χ2 test. The pattern of linkage disequilibrium (LD) was investigated for the patient and control groups. Haplotype analysis was performed for the evaluation of haplotype association.
We also examined group differences in the PANSS and K-CDSS scores of acute-phase patients related to the possession of specific alleles of the three polymorphisms using an independent t-test.
These analyses were performed using the Statistical Package for the Social Sciences (SPSS) for Windows (version 21.0; SPSS Inc., Chicago, IL, USA) and SNP analyzer (Istech, Seoul, Republic of Korea). The level of statistical significance was set at p-value<0.05.
Demographic data on patients and controls are summarized in
The distributions of the T182C, A3081T, and G1287A polymorphisms in the patients and control groups were in agreement with the Hardy-Weinberg equilibrium and were as follows: T182C (patients, χ2=0.284, df=1, p-value=0.594; controls, χ2=0.094, df=1, p=0.759; total, χ2=0.041, df=1, p-value=0.839); A3081T (patients, χ2=0.326, df=1, p-value=0.568; controls, χ2=0.055, df=1, p-value=0.815; total, χ2=0.322, df=1, p-value=0.570); and G1287A (patients, χ2=0.902, df=1, p-value=0.342; controls, χ2=0.068, df=1, p=0.795; total, χ2=0.377, df=1, p-value=0.539).
Comparing the group of schizophrenia patients with the control group, we found no differences in the genotype or allele distribution. When the male and female subjects were analyzed separately, we also found no significant association of these three polymorphisms with schizophrenia (
The LD test revealed that LD was moderately significant between T182C and A3081T (|D'|=0.702, r2=0.275, p-value=<10-5) but not between A3081T and G1287A or between T182C and G1287A (
When male and female subjects were analyzed separately, the LD test revealed that LD was also moderately significant between T-182C and A-3081T in both sexes (
The symptomatic characteristics of acute-phase schizophrenia were determined using PANSS and K-CDSS. Of 220 schizophrenia patients, 163 patients were assessed by PANSS and K-CDSS. We examined the effects of the T182C, A3081T, and G1287A SNPs on these clinical scales using an independent t-test.
No significant differences emerged in most of the analysis (
Specific genes and relevant DNA sequence variations involved in the pathogenesis of schizophrenia have not yet been identified. Molecular genetic studies on schizophrenia have suggested that some genes related to the dopaminergic or noradrenergic system could be etiologic factors of schizophrenia,
If NET is related to the pathogenesis of schizophrenia, we can consider some possibilities according to the symptom profile. First, NET dysregulation could be related to dopaminergic overflow causing psychotic symptoms, mainly in the temporal lobe. Also, NET dysregulation could play a role in negative deficit symptoms, even though the mechanism of deficit symptoms in schizophrenia is not fully understood. Siuta et al.
Several studies have investigated the association between NET gene polymorphisms and various psychiatric disorders, including major depression, bipolar disorder, and substance dependence. However, most of them demonstrated no association with the disease. For schizophrenia, only a few association studies have been performed. Leszczyńska-Rodziewicz et al.
In the present study, we found no significant differences in the genotype or allele frequency between patients and controls for any of the three NET SNPs. The absence of associations was also observed when male and female subjects were analyzed separately. Our results are in accordance with the Polish study. We also found no significant association between haplotype combinations and schizophrenia in a three-locus haplotype analysis. In relation to clinical assessment with G1287A, A/A carriers showed higher K-CDSS score than patients without the A/A genotype (p-value=0.024). Although this suggests that schizophrenia patients with A/A have more depressive symptoms in the acute phase, the interpretation should be done carefully due to our small sample size and multiple comparison problem.
Currently, no evidence suggests that drugs related to norepinephrine reuptake improve the core symptoms of schizophrenia. If NET is involved in the pathogenesis of schizophrenia, drugs related to NET should improve the clinical symptoms. Antidepressant add-on in patients with schizophrenia is restrictively attempted to reduce depressive symptoms, and antidepressants involving norepinephrine reuptake are not preferred because of the potential risk of aggravating psychotic symptoms. Poyurovsky et al.
This study has several limitations. First, the sample size was not large enough. Second, the subjects of our study were all Korean people. Third, antipsychotic agents were not under experimental control. Even though this study has many weak points, it could serve as an important basis for future study.
Our results suggest that polymorphisms of the NET gene we investigate are not associated with susceptibility to schizophrenia or its clinical features in a Korean population. However, it is significant because it is the first haplotype study to investigate associations between NET gene (SLC6A2) single nucleotide polymorphisms and schizophrenia in a Korean population. Future research with a larger sample size and more genetic markers is needed to replicate our results.
*χ2 test, †independent t-test, ‡not significant, §of 220, 163 patients were evaluated by PANSS and CDSS. PANSS: Positive and Negative Syndrome Scale, CDSS: Calgary depression scale for schizophrenia
*p-value after Bonferroni correction
*p-value after Bonferroni correction
*p-value after Bonferroni correction
NET: norepinephrine transporter, LD: linkage disequilibrium, SNP: single necleotide polymorphism
*p-value after Bonferroni correction
PANSS: Positive and Negative Syndrome Scale, K-CDSS: Korean version of Calgary depression scale for schizophrenia