Functional analysis of haplotypes and promoter activity at the 5' region of the DRD2 gene and associations with schizophrenia.

Background: In previous studies, we researched the association of the DRD2 gene promoter region SNP loci rs7116768, rs1047479195, rs1799732, rs1799978 and schizophrenia using Sanger sequencing. rs7116768 and rs1799978 were found to be slightly associated with schizophrenia. This study investigated the effects of haplotypes consisted of the four SNPs on protein expression level in vitro and identified the functional sequence in the 5’ regulatory region of DRD2 gene which has a potential link with schizophrenia. Methods: Recombinant plasmids with haplotypes, SNPs and 13 recombinant vectors containing deletion fragments from the DRD2 gene 5' regulatory region were transfected into HEK293 and SK-N-SH cell lines. Relative luciferase activity of the haplotypes, SNPs and different sequences was compared using a dual luciferase reporter assay system. Results: Haplotype H4(G-C-InsC-G) could significantly increase the gene expression in SK-N-SH cell lines. Allele C of rs7116768, allele A of rs1047479195 and allele del C of rs1799732 could up-regulate the gene expression. There were 5~7 functional regions in the promoter region of DRD2 gene that could affect the level of gene expression. Conclusion: We cannot rule out the possibility that different haplotypes may influence DRD2 gene expression in vivo. We observed that allele C of rs7116768, allele A of rs1047479195 and allele del C of rs1799732 could up-regulate gene expression. The truncation results confirmed the existence of functional regions in the promoter region of DRD2 gene that could affect the level of gene expression.


Introduction
Schizophrenia is a serious mental illness. More than 21 million people worldwide suffer from schizophrenia, with a lifetime prevalence of more than 1 percent [1,2] . Schizophrenia is a complex mental illness with unknown etiology. It is believed that environmental and genetic factors lead to this disease together. Many hypotheses have been proposed to explain the causes of schizophrenia.
These hypotheses involve many neurotransmitters, such as dopamine, serotonin, and gammaaminobutyric acid. Dopamine is an important neurotransmitter produced in the brain. The dopamine receptors acting together with dopamine belong to the G-protein-coupled receptor family and are involved in many nervous system activities, such as memory, learning, cognition, reward and neuroendocrine signal regulation [3] . Dopaminergic neurotransmission disorder is believed to be one of the causes of schizophrenia [4,5] . Thus, genes associated with dopamine receptors are candidate genes of schizophrenia. The genome-wide association study (GWAS) demonstrated that DRD2 gene is one of the important candidate genes for schizophrenia [6] However, studies on the association between DRD2 gene polymorphisms and schizophrenia have not reached consistent conclusions.
Taking the functional polymorphism site rs1799732 of the DRD2 gene promoter region as an example, Arinami et al. first discovered the polymorphism site rs1799732 in 1997, and proposed that the C del allele could reduce the expression of DRD2 gene [7] But then consistent experimental conclusions can not be gotten in different countries and regions.
In previous studies, we researched the association of the DRD2 gene promoter region SNP loci rs7116768, rs1047479195, rs1799732, rs1799978 and schizophrenia using Sanger sequencing.
Rs7116768 and rs1799978 were found to be slightly associated with schizophrenia [8] . The relative positions of the four SNPS are shown in Fig. 1, and the haplotype composed of the four SNPs is shown in Table 1.
In order to further determine the function of the DRD2 gene promoter region on gene expression, we used the dual-luciferase reporter assay to explore the effects of different haplotypes and different promoter region fragments on DRD2 gene expression.

Sample
Based on our previous analysis of haploid samples from different samples, we selected DNA samples with four haplotypes for follow-up study.

Construction of PGL-3 vector
The functional regions of the 5'-terminal regulatory region of DRD2 gene were predicted by Match TM (http://www.gene-regulation.com/pub/programs.html). The predicted result is shown in Figure   2. Based on the predicted results, the influence of each SNP on gene expression was analyzed, and the truncation basis was provided for truncation experiments to avoid damaging the original functional areas. Primer 5.0 software and PubMed Blast were used to design sequence-specific primers. According to the existing polyclonal sites on the PGL-3 vector, we connected the recognition sequence of the selected restriction enzyme at the 5'-terminal of the primer(Table2). The PCR reaction used PrimeSTAR ® kit (Takara, Dalian, China). We used pGM-T Ligation®Kit (TIANGEN, Beijing,China) to link the target gene fragment with the pGM-T vector. The recombinant vector is transformed into JM109 competent cells by thermal excitation [9] . We used SanPrep®Col-umn

Cell culture and transfection
HEK293 cell lines and SK-N-SH cells cell lines were used for double luciferase reporter gene assay.
HEK293 cells were cultured in HyClone®DMEM/high glucose medium with 10% fetal bovine serum. SK-N-SH cells were cultured in KeyGEN BioTECH®DMEM high glucose medium with 0.011 g/L sodium pyruvate containing 15% fetal bovine serum. When the cell density was above 90%, the cells were inoculated on the 24-well plates(1 × 10 5 cells per well). We transfected the recombinant PGL-3 plasmid(1ug) and PRL-TK (Promega) plasmid (100ng) into cells using Lipofectamine ® 3000 reagent (Invitrogen, California, USA). The cells were harvested after 29 hours. Finally, we standardized firefly luciferase activity (LUC value) using renin luciferase activity (TK value). Standardized activity intensity was used for comparison between different haplotypes or between different fragments. Each experiment was repeated independently in triplicate three times.

Statistic analysis
We used single-factor LSD-t test for anova to compare the relative luciferase activity between different haplotypes and the relative luciferase activity between different lengths of target gene fragments.

Confirmation of constructed haplotype vectors
The haplotype clone fragment region containing 4 SNPs was +447~-1623bp upstream of the 5'terminal of DRD2 gene. It was linked to the PGL-3 vector by cloning and transfected into cells. The luciferase assay showed that the four haplotypes of PGL-3 recombinant vectors were all transcriptional active. The haplotype H1(G-C-InsC-A) with the highest frequency in previous studies was used as the reference haplotype.

Confirmation of the constructed truncated fragment vectors
We used cloning technology to connect segments with different lengths of the 5'-terminal regulatory region of DRD2 gene to the PGL-3 vector, respectively(Figure3). After transfection, luciferase reporter gene analysis showed that all truncated fragments had transcriptional activity.

Luciferase activity detection
There was no significant difference in the expression of 4 haplotypes in HEK293 cell lines (Figure 4a Figure 5h). In HEK293 cell lines, compared with P13, the relative luciferase activity of P12 was enhanced, p<0.001; compared with P11, the relative luciferase activity of P10 decreased, p<0.001; compared with P10, the relative luciferase activity of P9 decreased, p<0.001 (Figure 6a) compared with P5, the relative luciferase activity of P4 decreased, p<0.001; compared with P4, the relative luciferase activity of P3 enhanced, p<0.001; compared with P3, the relative luciferase activity of P2 decreased, p<0.001; compared with P2, the relative luciferase activity of P1 enhanced, p<0.001 (Figure 6c). In the SK-N-SH cell lines, Compared with P13, the relative luciferase activity of P12 decreased, p<0.001; the relative luciferase activity of P9 was lower than that of P10, p<0.001( Figure 7a); compared with P7, the relative luciferase intensity of P6 was enhanced, P=0.001( Figure 7b); compared with P5, the relative luciferase activity of P4 decreased, p<0.001; compared with P3, the relative luciferase activity of P2 enhanced, p<0.001 (Figure 7c).

Discussion
According to the analysis of the results obtained in this experiment, haplotype H4 significantly enhanced relative luciferase activity in SK-N-SH cell lines, but this phenomenon was not observed in SK-N-SH cell lines belong to nerve cells and were used to study the functional regions of the DRD2 gene 3 ' terminal [11]. This indicates that the cell lines selected in this study are reasonable.
Differences in results between different cell lines are likely due to heterogeneity between cell lines. Different transcription factors expressed in different cell lines may lead to inconsistencies in gene expression levels. Although we did not find a significant association between SNPs in the DRD2 gene promoter region and haplotypes and schizophrenia in case-control studies, haplotype H4 was observed to enhance relative luciferase activity in SK-N-SH cell lines. This suggests that we cannot rule out the possibility that different haplotypes may influence DRD2 gene expression in vivo. We observed that allele C of rs7116768, allele A of rs1047479195 and allele del C of rs1799732 could upregulate gene expression. On the other hand, the truncation results confirmed the existence of functional regions in the promoter region of DRD2 gene that could affect the level of gene expression.
This study provides a reference for the association between DRD2 gene and schizophrenia and other psychiatric disorders related to dopamine system disorders. In order to further clarify the causal relationship between them, more experiments are needed to flesh out our conclusions.

Conclusion
This suggests that we cannot rule out the possibility that different haplotypes may influence DRD2 gene expression in vivo. We observed that allele C of rs7116768, allele A of rs1047479195 and allele del C of rs1799732 could up-regulate gene expression. The truncation results, on the other hand, confirmed the existence of functional regions in the promoter region of DRD2 gene that could affect the level of gene expression. This study provides a reference for the association between DRD2 gene and schizophrenia and other psychiatric disorders related to dopamine system disorders. In order to further clarify the causal relationship between them, more experiments are needed to flesh out our conclusions.

Compliance with ethical standards
All participants gave their informed consent in writing after the study aims and procedures were carefully explained to them in their own language. The study was approved by the ethical review board of the China Medical University, Shenyang Liaoning Province, People's Republic of China and in accordance with the standards of the Declaration of Helsinki. rs7116768 rs1047479195 rs1799732 rs1799978 Figure 1 The relative positions of rs7116768, rs1047479195, rs1799732 and rs1799978 in DRD2

Figures
gene.

Figure 2
Prediction of the functional area of DRD2 regulatory area by MatchTM