Association Studies On Susceptive Genes Of Atrial Fibrillation In The Chinese Han Population And Drug Screening For Prion-Related Disease In Vitro By Protein Misfolding Cyclic Amplification

Atrial fibrillation (AF) is the most common arrhythmia. In mainland China, approximately 10 million people are affected with AF, threatening the public health. The researches on the pathogenesis of AF benifit the correct and effective prevention and treatment of it, being the focus in the rearch fields of arrhythmia. Up to date, three crucial arrhythmia mechanisms have been held to be involved in AF:1) focal ectopic activity,2) multiple-circuit reentry and 3) single-circuit reentry with fibrillatory conduction. But the researches on AF are still gonging on, especially the finding of some un-ionic channels gene with gene mutation related with AF indicated that much unknown factors beside ionic channels participate in the development of AF. These researches will not only rich the knowledge of the pathogenesis of AF, but also afford the theoretic instruction for the individal treatment in the future. It has been certain that heredity is the important correlated factor of AF. In the past years, great achievement has been obtained in the fundamental Genetics researches in AF, most of which denpened on the researches in the family AF, but account for only a small proportion of all AF cases. Most of AF are common AF which are multigenic disease, caused by gene-gene interaction or gene-enviroment interaction. Thus, identification of common AF susceptive genes becomes the most favoured direct methods to understand the mechanisms.It is interested that although the association of single nucleotide polymorphism (SNP) rs2200733 on 4q25 with AF has been replicated in many study cohorts, including our previously reported in Chinese GeneID population, there's no gene in the linkage disequilibrium block containing this SNP. In the AF GWA reports, PITX2 was taken as a candidate gene in this 4q25 locus because it is the nearest gene to rs2200733 and lies 150 kb upstream. But so far, there's no evidence to support the direct association between PITX and atrial fibrillation, whatever through genomic association or expression level association. This leaves us a question on the role of PITX2 gene into the development of AF.In this study, we selected three high minor allele frequency SNPs in PITX2 gene locus and test the association between AF and PITX2 in the GeneID Chinese Han population by the"case-controll" association study, anticipating in providing direct evidence in the relation of PITX2 with AF in heredity level.Aim:To study the association between single nucleotide polymorphism PITX2 gene polymorphism including rs976568, rs994978 and rs2595104 with Atrial Fibrillation in the Chinese Han Population.Method:Genomic DNA of 371 AF patients and 620 non-AF controls were collected randomly from the GeneID Chinese mainland Han population.3 SNPs in PITX2 including rs976568, rs994978 and rs2595104 were genotyped by High Resolution Melt (HRM) methods. The 3 SNPs were tested for Hardy Weinberg equilibrium among controls using PLINK v1.05. Haplotypes construction and frequencies were estimated by softwawre Haploview v4.2. Allelic and genotypic associations of SNPs with AF were assessed using Pearson's 2×2 and 2×3 contingency tableχ2 test (PLINK v1.05). Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by theχ2 test (PLINK vl.05). Multivariate analysis was performed by incorporating age, sex, hypertension (HT), and CAD as covariates by using multivariate logistic regression (PLINK v1.05). Empirical P values were determined using the PLINK v1.05 program with 100,000 Monte-Carlo simulations. linkage disequilibrium (LD) pattern constructions were done by Haploview v4.2.Result:Power analysis suggested that our sample size provides sufficient power to identify the association between these three SNPs and AF in this study.There was no deviation from Hardy-Weinberg Equilibrium of the 3 SNPs genotype distributions in the control group. rs2595104 T allele gained most significant association with AF, observed P value was 4.88×10-4 and odds ratio was 1.39 (95% confidence interval 1.15 to 1.66). rs976568 and rs994978 were in nearly significant with AF,χ2 test P values were 0.054 and 0.078 respectively. In lone AF patients, only rs2595104 reached statistical significant association with the P value of 0.011 and OR was 1.48 (95% CI 1.09-1.99). rs976568 and rs994978 were not statistical significant any more (P value were 0.158 and 0.082 respectively).For genotypic association, all 3 SNPs showed significant association with AF in Chinese GeneID population. The most significant P values were acquired under recessive model for these 3 variants at the same time,0.003 for rs976568,0.004 for rs994978 and 0.002 for rs2595104.There were in total five detectable haplotypes, accounted for 50.0%(in rs976568-rs994978-rs2595104 order G-T-T),41.1%(T-C-G),3.5%(G-C-G),2.8%(G-T-G) and 1.2%(T-C-T) of all haplotype alleles. Among these, only G-T-T and G-T-G haplotypes were significantly associated with AF. T-C-G, G-C-G and T-C-T were negative significant, with P values of 0.063,0.221 and 0.791 respectively.Conclusion:PITX2 is the susceptive gene of Atrial Fibrillation Aim To establish protein misfolding cyclic amplification (PMCA) technology and screen the drug which can inhibit the conversion of PrPc to PrPsc in vitro. Method The brain homogenate of hamster infected with sheep scrapie factor H22# was incubated with normal hamster brain homogenate. After different cycles consisted by sonication followed by incubation, amplified PK-resistant PrPsc was detected by western blotting, to determine the optimal cycle times. Optimal PMCA system was used for drug screen in vitro. By antibodies binding with different domains of PrPc, the effect of Mechlorethamine (MCT) with Dithiothreitol (DTT) on the conformation of PrPc was investigated. Result Established PMCA system has high specificity. Optimal amplification time was 42h, ensuring the best amplification efficiency. By drug screen system, MCT with DTT was found to inhibit the conversion of PrPc to PrPsc in vitro and showed significant dose-effect relationship. Epitope of antibody 6H4 was concealed after treatment by MCT with DTT, which might play a role in the mechanism of inhibition. Conclusion: PMCA technology was successfully established and can be used for the drug screening for TSE. MCT with DTT can inhibit the conversion of PrPc to PrPsc in vitro. Further research on the mechanism will benefit the development for new drug for TSE.