The Study On The Relationship Between The Regulatory Sequence Of GRIN1 Gene Expression And Related SNPs And Schizophrenia And Its Forensic Significance

Objective: Schizophrenia is a chronic and disabling mental illness characterized by emotional impairment,cognitive deficits and social dysfunction.Schizophrenia has affected more than 20 million people all over the world,and has brought a heavy burden to families and society.In addition,schizophrenia is also an important part of the identification of forensic psychiatry,but its identification currently lacks objective indicators.Therefore,the research on the etiology and pathogenesis of schizophrenia has received more and more attention.Schizophrenia is affected by both genetic and environmental factors,and its etiology is complex.Studies have shown that genetic factors play a very important role in the susceptibility and course of schizophrenia.The etiology and pathogenesis of schizophrenia involve the hypothesis that multiple neurotransmitter systems are involved,including glutamate,dopamine,serotonin and gamma-aminobutyric acid.Among them,glutamate is an important excitatory neurotransmitter in the human central nervous system,which plays a biological role by binding to receptors.Glutamate receptors are divided into 4 categories and 18 subtypes.Among them,NR1 receptor has received the most extensive attention.The NR1 receptor is encoded by the GRIN1 gene and has been reported to be associated with a variety of neuropsychiatric disorders,including depression,ADHD,Alzheimer's disease and Parkinson's disease.However,the current research results on the association between the GRIN1 gene and schizophrenia are not unified.There is a lack of data on the Han population in northern China,and the research on the ways in which GRIN1 gene expression is regulated is relatively insufficient.Therefore,this study will investigate the distribution of part of the SNP of the GRIN1 gene in the Han population in northern China,and explore whether it is associated with schizophrenia and its application value to forensic evidence.At the same time,the regulatory sequences that affect gene expression at the 5'and 3'ends of the GRIN1 gene will be verified to lay the foundation for further functional research..Methods: 316 unrelated healthy volunteers and 309 schizophrenic patients of the Han nationality in northern China were selected as the survey subjects.Sanger DNA sequencing analysis was performed on the amplified GRIN1 gene 5'end-2334 bp ~+86bp fragment.The Haploview 4.2 software was used to perform Hardy-Weinberg equilibrium test on the distribution of SNPs,and the Powerstats v1.2 software was used to calculate forensic parameters.A case-control study was conducted to explore whether the distribution of SNPs and its haplotypes are related to schizophrenia.Gene cloning and gene recombination techniques are used to construct expression vectors with different haplotypes at the 5'end and truncated DNA sequences with different lengths at the 5'and 3'ends as recombinants.After transfection of different cell lines,the relative fluorescence intensity of the dual luciferase reporter gene technology was used to study the functional sequences that have a regulatory effect on the expression of GRIN1 gene.Results: In this study,7 SNP loci were detected,including rs112421622,rs138961287,rs117783907,rs181682830,rs7032504,rs144123109 and rs11146020.The genotype distribution of all SNP loci accorded with Hardy-Weinberg equilibrium.The polymorphisms of the three SNPs rs138961287,rs117783907 and rs11146020 were relatively well distributed,and the DP value was all about 0.5.The results of linkage analysis showed that rs138961287 and rs117783907 were in a state of linkage disequilibrium.The results of the case-control study showed that the frequency distribution of the Ins T of rs138961287,the T allele of rs117783907 and the C allele of rs11146020 were significantly different between the schizophrenia group(15.2%,15.2%,and 14.7%)and the control group(70.9% and 16.8%).Among the 6 haplotypes formed by 7 SNPs,there are significant differences in the frequency distribution of haplotypes H1 and H2 between the case group and the control group(p values were 0.005 and 0.046,respectively).Studies have shown that the relative fluorescence intensities of recombinant haplotype vectors H1 and H2 in HEK-293,SK-N-SH and U87 cells are not statistically different(p values were 0.178,0.956,and 0.501,respectively).The relative fluorescence intensities of the truncated recombinant vectors F10 and F11 in the three cell lines are all significantly different(p values were 0.036,4.091E-06,and 1.606E-06,respectively).In addition,in SK-N-SH and U87 cells,the relative fluorescence intensities of recombinant expression vectors F10 and F12 were significantly higher than those of F9 and F11(p values were 0.003 and 3.086E-06,respectively).In the 3'UTR region of the GRIN1 gene,the relative fluorescence intensity of the complete recombinant expression vector F1 in the HEK-293,SK-N-SH and U87 cell lines was significantly lower than that of the pmir GLO-Basic vector(p values were 0.002,6.321E-26,and 3.390E-14,respectively),the relative fluorescence intensity of recombinant expression vector F6 was significantly higher than that of F5(p values were 0.004,3.294E-06,and 3.294E-06,respectively).In addition,in SK-N-SH cells,the relative fluorescence intensities of F4 and F7 were significantly lower than those of F3 and F6(p values were 4.711E-05 and 8.413E-05,respectively).Bioinformatics analysis suggests that has-mi R-212-5p,has-mi R-324-3 and has-mi R-326 may recognize the functional sequence between F6 and F7,and has-mir-491-5p may recognize functional sequence between between F3 and F4.F1 was co-transfected with has-mi R-212-5p mimic,has-mi R-324-3 mimic,has-mi R-326 mimic,has-mir-491-5p mimic,and the control mi RNA NC mimic,then F1 was co-transfected with has-mi R-212-5p inhibitor,has-mi R-324-3 inhibitor,has-mi R-326 inhibitor,has-mir-491-5p inhibitor,and control mi RNA NC inhibitor.The results showed that in HEK-293,SK-N-SH,and U87 cells,the relative fluorescence intensity of F1+has-mi R-491-5p mimic was significantly lower than that of F1+mi RNA mimic NC(p values were 0.021,0.000267,and 0.029,respectively).Compared with the relative fluorescence intensity between F1+mi RNA inhibitor NC and F1+has-mi R-491-5p inhibitor,the statistical difference of relative luciferase expression in the three cell lines were all disappeared.Conclusion: 1.Rs117783907,rs138961287 and rs11146020 of GRIN1 gene are well distributed in the Han population in northern China,and have forensic application value.2.In the Han population in northern China,the T allele of rs117783907,the Ins T of rs138961287 and the C genotype of rs11146020 are protective factors for the occurrence of schizophrenia in the Han population in northern China.3.In the Han population in northern China,the haplotype H1 and H2 of the 5' end of GRIN1 gene might be related to the susceptibility to schizophrenia.4.The two haplotypes composed of 7 SNPs at the 5'end of GRIN1 gene have no significant effect on gene expression.5.The different length sequences of the 5'regulatory region of the GRIN1 gene have different effects on gene expression in the three cell lines of HEK-293,SK-N-SH and U87,indicating that the regulatory mechanism of gene expression is different depending on the cell type,which might provide reference and help for further functional research.6.In nerve cells,-337 bp ~-159 bp and-704 bp ~-556 bp at the 5'end of GRIN1 gene contain transcription repression regions,while-556 bp ~-337 bp contain enhanced transcription enhancement regions,which can provide a reference for the study of gene regulation mechanisms.7.mi R-491-5p can down-regulate the expression of GRIN1 gene in HEK-293,SK-N-SH and U87 cell lines.