The SNPs Of Dopamine D5 Receptor (DRD5) Gene 5' Region And The Effect On Gene Expression

Objective: Schizophrenia is a complex disorder of brain dysfunction,which is realted to both heredity and environment.At present,in the forensic psychiatric appraisal,there is still no effective objective indicator to evaluate whether the appraised person suffers from mental illness.The etiology of schizophrenia involves several neurotransmitters and hypotheses.Among them,the hypothesis of dopamine dysfunction was supported by many physiological and pathological studies.Mutations in the dopamine receptor 5(DRD5)gene can alter DRD5 expression level and its ability to bind dopamine(DA),resulting in a number of psychiatric disorders.So far,the mechanism of action of the DRD5 gene in the pathogenesis of schizophrenia remains unclear,and there have been few studies about the polymorphisms of DRD5 gene on the transcriptional level.In the previous study,we investigated the association between four SNPs in DRD5 gene and paranoid schizophrenia and found that the allele A of rs77434921 and the allele G of rs2076907 may be risk factors of paranoid schizophrenia.To further explore the role of DRD5 gene in the pathogenesis of paranoid schizophrenia,we picked two SNPs(rs77434921 & rs2076907)based on the information provided by the previous research,and analyzed the role of 5` regulatory regions and two SNPs in the the expression of DRD5 receptor,in order to provide evidence for the association between the DRD5 gene with schizophrenia in the futher study.Methods: 1.Primers with restriction sites were designed to amplify a 2183 bp fragment from-1599 to +564 of the 5` region of DRD5 gene,containing two SNPs.2.The PCR product was purified and added fragment A,the linear PGM-T vector and PCR product were ligated by T4 ligase to form a circular recombinant vector.3.Vetors were transformed into JM109 cells,select positive monoclonal colony for further culture,and then DNA was extract for identification.4.The recombinant PGM-T vector and PGL3 vector were digested by enzyme at the same time.After agarose gel electrophoresis,the target fragment was purified,and then the target fragment and PGL3 vector were ligated with T4 ligase to construct luciferase reporter gene recombinant vector.5.Vetors were transfected into HEK293 cell and SK-N-SH cell,and the dual luciferase reporter assay was performed.6.Primers for two SNPs were designed and the recombinant vector was constructed separately and transfected into HEK293 cell and SK-N-SH cell for luciferase reporter assay.7.Primers were designed to clone the 5` region of DRD5 gene.The recombinant vectors were transfected into HEK293 cells and SK-N-SH cells for luciferase reporter assay.8.After transfection of the recombinant vectors,the total cellular RNA was extracted and reversed into cDNA and analyzed by the real-time PCR.Results: 1.In dual-luciferase assay,the four haplotypes consisting of two SNPs(rs77434921 and rs2076907)were analyzed.The P1(G-G)vector as wild type,the relative luciferase activity of other three haplotypes were not significant different from P1 in both HEK 293 and SK-N-SH cell lines.2.In the further study,the two SNPs were analysis separately,we still did not found significant differences between different haplotypes,excluding the assumption that these two SNPs were interfere with each other.3.We found that the fragment of +82~+564 on the DRD5 gene was able to down regulate the transcriptional level while the fragment of-1599~-934 played an important role in maintaining the transcriptional level.4.In the truncated study of 5` region of DRD5 gene,we found that the region of-1599~-1486 could promote the transcription in both cell lines by dual-luciferase reporter assay.The region of-1076~-934,could down-regulate transcriptional level in the HEK293 cell line,but promote transcription in the SK-N-SH cell.In addition,the region of-1364~-1233 could promote transcription in the SK-N-SH cell line,while the regions of-1486~-1364 and-1233~-1076 could reduce the transcriptional level.5.In addition,in real-time PCR experiment,we found that the deletion of 5` UTR region could lead to a significant increase in mRNA levels,confirming that the region of +82~+564 has an inhibitory effect on transcription of DRD5 gene.Conclusion: In this study we did not proved the SNPs rs77434921 and rs2076907 had any effect on the expression of DRD5 gene.However,in the study of the 5` region of DRD5 gene,we found some truncated regions could regulate the expression of DRD5 gene.