| Objective:Ataxin-3（AT3）is a deubiquitination enzyme gene with the characteristics of polymorphic distribution among different individuals,however,it is not clear whether this kind of polymorphisms affect the risk of liver cirrhosis（LC）associated with Aflatoxin B1（AFB1）.This study aimed at exploring the role and corresponding mechanisms of single nucleotide polymorphisms（SNPs）in AT3 gene in the pathogenesis of AFB1 associated LC.Methods:（1）This study is a hospital-based case-control study consisting of 384 LC patients（case group）and 851 controls（control group）without any liver disease.For analyses,we designed a two-stage study model containing a screening stage with small sample（including 100 pairs of cases and controls）and a validation stage with large sample（including284 patients and 751 controls）,to assess the association between polymorphisms in AT3 and AFB1-related LC risk.（2）SNPs in AT3 gene were obtained through the SNPs database,and the most minimum probability of the allele gene distribution for SNPs in the Asian population than than or equal to 0.1 was used as the inclusion criteria.According to these standards,264SNPs were included in the first screening stage in this study.（3）The 264 SNPs genotypes in the first phase were detected with the small sequencing method SnaPshot;and the genotypes of three SNPs from the second phase were detected with the TaqMan probe PCR techniques.（4）The level of AFB1 exosure for individuals was evaluated using the amount of AFB1adducts in their blood samples and adducts were calculated using competitive enzyme-linked immunosorbent assay（ELISA）.（5）The expression of AT3 protein in tissue samples with LC was detected by immunohistochemistry assay.（6）The level of AT3 mRNA expression in tissue samples with LC was detected by TaqMan-PCR.（7）Logistic regression model was used to analyze the effects of AT3 gene SNPs on the risk of LC,and the odd ratio（OR）and the corresponding 95%confidence interval（CI）were used to evaluate the specific risk value.The effects of AT3 gene SNPs on AT3 gene expression and AFB1-DNA adducts were tested by variance analysis and Spearman’s rank correlation method.Results:（1）There were no statistically significant differences in the distribution of clinical data such as age,gender,ethnicity,smoking and alcohol abuse history,hepatitis B virus and hepatitis C virus infection between case and control groups（P<0.05）.（2）Compared with the control group,LC patients had higher serum AFB1-albumin adducts（1.48±0.32 ln fmol/mg and 2.40±0.91 ln fmol/mg,respectively）.（3）Compared with individuals with low AFB1 exposure,these individuals with increasing AFB1 exposure will have a higher risk of LC incidence,and the risk values corresponding to medium and high exposure are 3.59（2.65-4.84）and 7.74（7.74-10.87）.（4）Through the screening analysis in the first stage,we found three AT3 SNPs with different distributions,specifically rs8021276,rs7158733 and rs10146249.（5）Through the confirmation analysis in the second stage,we found that only rs8021276 was related to the incidence risk of LC,and the fusion analysis of the data in the two stages had similar findings.Compared with homozygous rs8021276 A allele（rs8021276-AA）,genotypes containing rs8021276 G allele（heterozygous rs8021276-AG and mutant homozygous rs8021276-GG）significantly increased the risk of LC,with corresponding risk values of 2.48（1.84-3.33）and 6.98（4.35-11.22）,respectively.（6）In the combined analysis of AFB1 exposure and AT3 rs8021276 genotypes,we found that there was a multiplicative interaction between AT3 rs8021276 risk genotypes（OR>1）and AFB1exposure at different levels.（7）In LC tissue samples,AT3 rs8021276 polymorphism was associated with lower AT3 mRNA and protein levels,and the correlation analysis showed a negative correlation(r=-0.25,P=7.30 10-7).（8）AT3 rs8021276 polymorphism affected the level of AFB1-DNA adducts in LC tissues.The DNA adduct concentrations for rs8021276-AA,rs8021276-AG and rs8021276-GG genotypes were 1.51±1.23μmol/mol mol/mol DNA,2.09±1.18 mol/mol DNA,and 3.92±1.81mol/mol DNA,respectively,P=5.61×10-4.Conclusion:The current results show that AT3 rs8021276 polymorphism increases the risk of LC,which is more obvious under the condition of high AFB1 exposure.The possible mechanisms may be that AT3 rs8021276 polymorphism affects the expression and function of AT3.These results suggest that AT3 polymorphism can be regarded as markers to determine the risk of LC in different individuals under AFB1 exposure,in which rs8021276 is a potential optional marker. |
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