The Study On Molecular Genetic Epidemiology Of Phospholipase A2Family Genes In Schizophrenia

Schizophrenia is a common and serious mental disorder, whose pathogenesisremains undefined. The studies of schizophrenia on genome scanning and candidategene have acquired many positive results. However, these results with poor repeatshave caused much disputation. So far, the major and specific susceptibility genesleading to schizophrenia remains unidentified. Schizophrenia associatedenvironmental exposures, particularly at key developmental stages, may result inlong-lasting epigenetic alterations that impact on the neurobiological processesinvolved in pathology. Several studies conducted by our laboratory demonstrated thatthere was increased serum PLA2activity but no SNP locus in the PLA2family geneassociation with schizophrenia in case-control samples. Based on the work we havedone before, here we investigated the status of DNA methylation in a typical CpGsisland in the PLA2family genes’ promoter by bisulphite sequencing in schizophreniaand control group, aiming to determine the possible involvement of the epigeneticmechanisms in the schizophrenia associated altered pattern of the PLA2family geneexpression.Objective:The purpose of this study is to explore the association between PLA2G21A genepolymorphisms and schizophrenia and confirm the possible involvement of theepigenetic mechanisms in the schizophrenia. We discuss the relationship betweenschizophrenia and DNA methylation in promoters of PLA2family genes. Also thisresearch is likely to reveal that epigenetic mechanisms play an important role in thepathogenesis of schizophrenia.Final results of this study combined with the earlystudies in our groups, integrated PLA2gene polymorphisms, DNA methylationactivity and multi-angle multi-level studies to clarify the relationship between PLA2family gene and schizophrenia.Methods:Genomic DNA samples were extracted from whole solidification blood using theColumn Blood Clot DNA out (Andybio) and the MALDI-TOF-MS technology was employed to detect genotypes. We apply analysis software SPSS16.0and Haploview4.2to analyze the date. In our study, we also investigated the status of DNAmethylation in promoters of PLA2family genes by bisulphite sequencing withschizophrenic and healthy person. Firstly, we scan the DNA methylation status of thepromoters of PLA2family genes with a few samples and mark the methylation siteswhich distributed in the promoters of PLA2family genes. Then we selected theregions which show significant difference of methylation status between cases andcontrols. Our study detects them using more samples. At last we perform data analysisto find out the differences of methylation status between cases and controls.Result:(l) The distribution of genotypic frequency in PLA2G12AWe selected4tag SNPs in PLA2G12A gene and analyze the genotype frequencydistributions of each tagSNPs in patient group and control group with the χ2test. Theresults showed that the genotype frequency distributions of each tagSNPs in controlgroup were not deviated from the H-W equilibrium (P>0.05) while rs11728699andrs2285714in patient group were deviated form the H-W equilibrium (P＜0.05). Thisresult indicated that each SNP could use as genetic markers to test the relationshipbetween PLA2G12A genes and schizophrenia. Correlation analysis found that thedistribution of genotypic and allelic frequencies at rs7694620site, rs11728699,rs2285714site showed no statiscit significance between the case group and thecontrol group(P>0.05); There was statistical significance for the frequencies of wildgenotype and mutant genotype of rs3087494site between the case group and thecontrol group (P＜0.001), The mutant allele G carrier on rs3087494was a protectfactor to develop schizophrenia (OR=0.773,95%CI:0.665~0.882).(2) Linkage disequilibrium test of four tag SNPs in PLA2G12A and haplotype testWe tested the degree of linkage disequilibrium among four tag SNPs inPLA2G12A using Haploview software. The results showed that there is completelylinkage in rs7694620-rs3087494and rs3087494-rs2285714(D’=1). The haplotyperesult indicates that these haplotype system were not assoeiated withschizophlenia.The haplotype system of rs7694620-rs3087494and rs3087494-rs2285714frequeney distribution in the PLA2G12A gene were statistically signifieantdifference in case and control group (P <0.001). Also the two haplotype system in the PLA2G12A gene rs7694620-rs3087494-rs2285714and rs3087494-rs2285714-rs11728699frequeney distribution in the PLA2G12A gene were statisticallysignifieant difference in case and control group (P <0.001). At the time, the frequeneydistribution of six haplotype in the one haplotype system in the PLA2G12A gene(rs7694620-rs3087494-rs2285714-rs11728699) in the PLA2G12A gene werestatistically signifieant difference in case and control group (P <0.001).(3) Number of methylated sited in the promoter of PLA2familyWe checked DNA methylation of promoter region in PLA2family genes withsmall samples and our results showed that there are17CpGs which are methylated inthe promoter regions of PLA2G4B. At the same time there are7CpGs which aremethylated in the promoter regions of PLA2G4C. The corresponding number ofmethylated CpG sites in the promoter of PLA2G4D, PLA2G4E, PLA2G4F,PLA2R1and PLA2G12A genes is19,15,8,21, and29.(4) The relationships PLA2gene promoter region DNA methylation status andschizophreniaThere are not methylation sites which show significant difference between casesand controls in the promoters of PLA2G4B, PLA2G4D, PLA2G4E, PLA2G4F andPLA2R1genes. However, we find two cytosines (-13site&4site) of whichmethylation status shows significant difference (P <0.05) between schizophrenics andcontrols in PLA2G4C and PLA2G12A respectively. The supplementary study alsofound that there is association between SNP (rs3087494) and schizophrenia whichlocates in PLA2G12A gene.Conclusions:According to the above-mentioned analytic results, we can reach the followingconclusions:(1) There is significant association between the polymorphism ofrs3087494and SCH;(2) Three SNP haplotypes includes rs7694620-rs3087494、rs3087494-rs2285714、rs7694620-rs3087494-rs2285714、 rs3087494-rs2285714-rs11728699and rs7694620-rs3087494-rs2285714-rs11728699have significantassociation with SCH;(3) There are methylation sites in the promoter regions ofPLA2G4B、PLA2G4D、PLA2G4E、PLA2G4F and PLA2R1which are included inthe PLA2gene families;(4) There are7methylation sites in the promoter regions ofPLA2G4C and there is association between the methylation level of the-13CpG sites and schizophrenia.(5) There are29methylation sites in the promoter regions ofPLA2G12A and there is association between the methylation level of the4CpG sitesand schizophrenia.