Association Of Expression Regulation Sequence And Related SNPs Of ADH7 Locus With Schizophrenia And Its Forensic Significance Research

Objective: Schizophrenia is a more serious mental illness,and the identification of schizophrenia is often involved in certain criminal cases.Due to the lack of objective indicators for clinical diagnosis,it has caused confusion to the corresponding judicial appraisal.The etiology of the disease is not yet clear,but genetic factors are an important etiology that has become a consensus.Such as dopaminergic,serotonergic theory and so on.Genes that have an effect on the central nervous system may be involved in its occurrence and are considered to be polygenic diseases.The ADH7 gene encodes class IV alcohol dehydrogenase,which oxidizes retinol to retinal,and then participates in the production of retinoic acid,and affects the metabolic pathways and processes of retinoic acid.The changes in the retinoic acid signaling pathway may be related to the pathogenesis of severe mental disorders,such as schizophrenia,depression,and Alzheimer’s disease.There is no report about whether the ADH7 gene and related SNPs are related to schizophrenia.This study will take the Han population in northern China as samples to explore the distribution of SNPs and different haplotypes in specific DNA fragments at the 5’and 3’regions of the ADH7 gene and whether they are related to schizophrenia.And discuss the influence of various haplotypes and different length truncations of specific DNA fragments on gene expression,to analyze the regulatory sequence of gene expression,and provide a working basis for further research on the function of ADH7 gene.Methods: The peripheral blood samples of 313 unrelated individuals of Han nationality in northern China and 275 cases of schizophrenia were collected.Genomic DNA was extracted by phenol-chloroform method and PCR technology was used to amplify three DNA fragments of the ADH7 gene 5’region-693 bp ～+110 bp,3’ region-153 bp ～+1029 bp and +765 bp ～+1162bp.Sanger sequence analysis method is used for the 5’ region product,and Sanger sequence analysis method and RFLP detection technology are used for the 3’ region product to type the SNPs in the sample.Hardy-Weinberg balance test and linkage disequilibrium analysis were performed on the obtained SNPs data using Haploview4.2 software,and forensic parameters such as personal identification ability,heterozygosity,polymorphism information content,and non-paternal exclusion probability were calculated using Power Stats V1.2.The chi-square test and correlation analysis were performed on the genotype,allele and haplotype difference between the healthy group and the schizophrenia patients.Using gene cloning technology,each haplotype and the different truncated bodies of the 5’region and 3’ region products were connected to the expression vector,and the dual luciferase reporter detection technology was used to determine the haplotypes and different truncated bodies in HEK-293,U87,and U87.The relative fluorescence intensity in three kinds of cells including SH-SY5 Y.Use JASPAR,Animal TFDB3.0 software and Target Scan Human7.2 database to predict the binding sites of 5’ region transcription factors and 3’region mi RNAs respectively.Results: This study detected 10 SNPs,including rs2654847,rs2851028,rs17537595,rs4147554,rs284786,rs3805331,rs894369,rs3805329,rs284787 and rs729147,and obtained their polymorphic population data.The frequency distribution of 10 SNPs genotypes accorded with HardyWeinberg equilibrium(P> 0.05).Among them,5 SNPs,rs2851028,rs284786,rs894369,rs284787,and rs729147,had good polymorphism distribution,and their DP values were all greater than 0.5.Analysis of alleles,genotypes and haplotypes of all SNPs found no statistically significant differences between healthy and diseased populations.At the 5’ region of the ADH7 gene,the relative fluorescence intensity of FH1 containing recombinants in the three cell lines HEK-293,U87 and SHSY5 Y were significantly higher than FH2,FH3 and FH4.The relative fluorescence intensity of truncated recombinants containing-1369 bp ～-1625 bp regions was significantly enhanced,while the relative fluorescence intensity of truncated recombinants containing-768 bp ～-987 bp and-1203 bp ～-1369 bp regions was significantly reduced.At the 3’ region of the ADH7 gene,the relative fluorescence intensity expressed by the recombinants containing H2 was the highest in U87 and SH-SY5 Y cell lines,which was significantly different from H1 and H3.In the U87 cell line,the relative fluorescence intensity of H2 and H4 are also significantly different.In the HEK-293 cell line,H1 has the highest relative fluorescence intensity,which is significantly different from H2.The relative fluorescence intensity of truncated recombinants containing the region +468bp ～+564 bp was reduced in the three types of cells.The relative fluorescence intensity of truncated recombinants containing the +258 bp～+380 bp region was also reduced in HEK-293 and SH-SY5 Y cells.Conclusions:1.In the Han population of northern China,the five SNPs of the ADH7 gene,rs2851028,rs284786,rs894369,rs284787 and rs7291475,have good genetic polymorphisms and can be used for forensic personal identification and paternity testing practices.The distri bution of 10 SNPs and haplotypes of ADH7 gene was not found to be associated with schizophrenia.2.Rs2654847 and rs2851028 of ADH7 may play an important role in th e regulation of gene expression.The-1369 bp ～-1625 bp,-768 bp ～-987 bp and-1203 bp ～-1369 bp regions at the 5’ region may contain functional sequences that can be regulated the ADH7 gene expression.3.The +258 bp ～+380 bp and +468 bp ～+564 bp regions at the ADH7 3’region may contain functional sequences that can be regulated the ADH7 gene expression.