Association Of Genetic SNPs Of GABRB3 Gene 5’ Region With Schizophrenia And Its Effect On Gene Expression

Objective: Schizophrenia is a common mental disorder that involves the influence of multiple genetic and environmental factors.Results of numerous epidemiologic studies have indicated that the heritability of schizophrenia is up to 80%,but the precise pathology of this disease remains unknown.The etiology of schizophrenia includes neurotransmitters,such as dopamine,glutamic acid,serotonin,and γ-aminobutyric acid(GABA).GABA is the main inhibitory neurotransmitter in the brain and regulates multiple processes during brain development.Approximately 20% of all central nervous system neurons are GABAergic,and abnormal GABA signaling appears to be correlated with the occurrence of schizophrenia.The key element determining normal functioning of GABA in the nervous system is the GABA receptor.GABRB3 is located in 15q11.2-q13,a region that is prone to chromosomal rearrangements,deletions,and duplications.Microdeletions at 15q11.2 and 15q13.3 are considered susceptibility factors for schizophrenia.But the study of functional fragments of the GABRB3 gene regulatory region and the association between this gene and schizophrenia are not specific enough.The results of functional regions and polymorphisms are still controversial.Therefore,the aim of this study was to explore whether schizophrenia occurrence is associated with polymorphisms in the 5′ regulatory region of GABRB3 and the effect of different haplotypes of SNPs located in the 5′ regulatory region of GABRB3 gene on gene expression at the protein level,to find the functional sequence of the 5′ regulatory region of GABRB3 gene,and explore the effect of this sequence on GABRB3 gene expression.Methods: 1.The study included 324 patients with schizophrenia and 327 unaffected participants;all individuals were northern Han Chinese.Genotype and haplotype frequency distributions were compared for the 2 groups by means of PCR amplification and direct sequencing of the promoter region of GABRB3.2.The recombinant plasmids of different haplotypes and the 10 recombinant vectors containing the deletion fragments of GABRB3 gene 5′ regulatory region were transfected with HEK-293,SK-N-SH and SH-SY5 Y cells.The relative fluorescence intensity of the haplotypes and different length sequences was compared using a dual luciferase reporter assay system.Results: 1.Two common single-nucleotide polymorphism(SNPs)sites were detected in the 5′ promoter region of GABRB3: rs4906902 and rs8179184.Rs8179184 and rs4906902 differed significantly in frequency between controls and cases(p< 0.05);this difference remained significant when only women in each group were compared.The 2 SNPs sites showed linkage disequilibrium,resulting in 2 haplotypes: T-G and C-A.The frequency of C-A was significantly higher among patients with schizophrenia than among controls.2.Using luciferase reporter gene experiments,we further demonstrated that the relative fluorescence intensity of haplotype C-A was significantly lower than that of T-G,which further demonstrated that haplotype C-A might inhibition the expression of GABRB3 gene.We shortened the core promoter sequence of GABRB3 gene 5′ regulatory region from-177 bp to-18 bp(ATG+1).We found that an expression suppression region is from-1735 bp to-1638 bp and an enhanced regulatory region is from-1638 bp to-1335 bp(ATG+1).There are also multiple inhibitory functional elements in the region from-680 bp to-177 bp(ATG+1).Conclusion: Our findings suggest that rs4906902 and rs8179184 in the 5′ promoter region of GABRB3 are associated with schizophrenia.The C-A haplotype may entail an increased risk of schizophrenia,and the onset of schizophrenia may be gender-specific.It also provides possible auxiliary reference indicators for the identification of judicial mental illness.