| [Content] There were three parts included in this study: 1 a clinical epidemiological investigation was designed to determine the distributions of serum alpha fetoprotein（AFP）levels in hepatitis B virus（HBV）related Hepatocellular carcinoma（HCC）patients from the first affiliated hospital of Guangxi medical university.2 we take a exome-wide association study for observing the Single Nucleotide Polymorphism（SNP）associated with serum AFP levels.3 Base on the result of candidate SNPs,we tried to verificate in hepatic cell lines.Objective: In this part,we tried to explore the clinical factors associated with serum AFP levels from the HBV related HCC patients;we also try to investigate the clinical value of serum AFP levels in predicting prognosis in the participants.Materials and methods: A total of 223 HBV related HCC patients was collected from the first affiliated hospital of Guangxi medical university.Different distributions of serum AFP levels（400ng/ml）associated with the clinical factors were assessed using logistic regression.Kaplan-Meier methods and Cox regression were used for evaluating the association between clinical factors and the prognosis of patients.All the statistical analysis were conducted by SPSS 16.0.Result: The serum AFP levels ranged 2.17-60,500ng/ml.The result of Logistic regression shows age[Odd Ratio（OR）=0.42,95%confidence interval（CI）=0.45-0.73,P=0.002] and tumor size（OR=0.43,95%CI=0.24-0.76,P=0.004）were significant associated with the different distributions of serum AFP levels（400ng/ml）.Moreover,significant association was observed between the AFP levels and disease-free survival（DFS）[Hazard Ratio（HR）=0.71,95%CI=0.52-0.95,P=0.016].After Adjusted the risk factors such as TACE status,radical resection,antiviral therapies,age and BCLC stage,the different distributions of serum AFP levels still showed significant association with DFS（Adjusted P=0.01）.In the stratification analysis（by age and tumor size）,the patients who got elevated AFP levels（>400ng/ml）got poor prognosis in the sub-groups（age>46 year: HR=0.66,95%CI=0.43-0.99;tumor size>8cm:HR=0.54,95%CI=0.31-0.95）.Furthermore,the different distributions of serum AFP levels was significant association with the 1-year DFS and 2-year DFS（1-year:P=0.0007;2-year:P=0.004）.Conclusion: In this study,age and tumor size were clinical factors significant affecting the distributions of serum AFP levels.Moreover,the different distributions of serum AFP levels may be the independent risk factor for the postoperative HCC recurrence.In the sub-groups（age>46year,tumor size>8cm）,patients with elevated AFP levels may got poorer prognosis.Objective: We performed a exome-wide association study in 223 HBV-related HCC patients and tried to identify the loci associated with serum AFP levels.Materials and Methods: Genomic DNA kit-DP304（Tiangen Inc.Beijing,China）was used to extract DNA from samples.Illumina Human Exome Bead Chip-12-1A（Illumina Inc,USA）was used for genotyping,which including over 240,000 SNPs.The software package Genome Studio 2011.1（Illumina Inc.CA,USA）was used for evaluating the call rate of the SNPs.In total,we genotyped 242,901 SNPs among 223 HCC patients.The quality control was performed by Plink version 1.07,R 3.0.1（http://cran.r-project.org/）and EIGENSOFT package 21.Only SNPs with Hardy–Weinberg equilibrium（HWE）>1×10-6,minor allele frequency（MAF）>0.05 and SNP call rate>95% were kept for further analysis.The samples with call rates<95%,outliers in principal components analysis（PCA）and had genome-wide identity-by-descent（IBD）>0.1875 were removed.At last,a total of 223 samples with 21,353 SNPs were passed the QC.The match group（n=152）was genotyped via Sanger sequencing using ABI Prism3100（Applied Biosystems,Shanghai Sangon Biological Engineering Technology & Services Co.,Ltd.,Shanghai,China）.All the statistical analysis was conducted by SPSS 16.0.Result After the QC procedure,108 cases（AFP>400ng/ml）and 115 controls（AFP<400ng/ml）with 21,353 SNPs were further analyzed.Total genotyping rate in remaining individuals is 99.81%.The result of PCA and small genomic control inflation factor（λ=1.0178）showed no population stratification for this study.Due to the limiting specimen,we focused on the loci of MAF>0.05.Randomly selected 10% of the samples for validation,and the results were 100% concordant.No SNPs satisfied GWAS significant(P<2×10-6,Calculated according to the 21,353 SNPs,α< 0.05)in Single Variant Test.An excerpt of 10 genes of analysis is reported.After analysis the association between the candidate SNPs and match group,the combine calculate result shows three SNPs include rs2125739,rs1644634 and rs379707 strongly associated with AFP levels.The T allele carriers of rs2125739,the A allele carriers of rs1644634 and the G allele carriers of rs379707 got significant higher serum AFP levels(rs2125739: P=1.32×10-4,rs1644634: P=2.33×10-5,rs379707: P=3.47×10-5).Furthermore,the polymorphisms of rs1644634 were significant association with disease-free survival(HR=0.56,95%CI=0.41-0.78,P=5.12×10-4).Conclusion: We found three new SNPs rs2125739,rs1644634,rs379707 strongly associated with serum AFP levels in HBV-related HCC patients.The T allele of rs2125739,the A allele of rs1644634 and the G allele of rs379707 might be the risk factors for elevated serum AFP levels.Moreover,polymorphisms of rs1644634 were significant association with disease-free survival in HBV-related HCC patients.Objective: To analysis the association between the polymorphisms of candidate SNPs and the AFP levels（AFP protein expression）in hepatic cell lines.Materials and Methods: We selected 5 Chinese crowd-sourced hepatic cell lines including BEL-7402,BEL-7404,SMMC7721,MHCC97-H and HL7702 for observation.These hepatic cell lines were co-cultured in the same system.We extracted DNA of the hepatic cell lines,and got the genotypes of the candidate SNPs by directly sequenced.Supernate of hepatic cell lines was collected,and the AFP levels of the hepatic cell lines was tested by clinical laboratory of Guangxi medical university.The AFP protein expression of different hepatic cell line were tested by western-blot.T-test and Mann-whitney U test were used to evaluate the polymorphisms of candidate SNPs and the AFP levels（AFP protein expression）in hepatic cell lines.The SPSS 16.0 program was used for analysis and P value<0.05 was considered statistically significant.Result: In the 5 hepatic cell lines,polymorphisms were presented in 2 candidate SNPs: rs1644634 and rs379707.The candidate SNP rs2125739 polymorphisms all distributed the genotype of TT in the hepatic cell lines.The MHCC97-H take the risk allele（AG）associated with higher AFP level in rs1644634,meanwhile the BEL-7404 and BEL-7402 carry the risk allele（GT）in rs379707.After comparison,the AFP level in supernate of MHCC97-H was significant higher than other hepatic cell lines（P=0.003）.However,significant difference between the AFP level in supernate of BEL-7402/BEL-7404 and the other cell lines was not observed（P=0.49）.The AFP protein expression was compared by different genotypes of candidate SNPs,the risk allele carrier of MHCC97-H in rs1644630 showed higher AFP protein expression than other hepatic cell lines（P=0.01）.Difference genotypes of rs379707 distributed in the hepatic cell lines had negative correlated to the AFP protein expression.Conclusions The polymorphisms of candidate SNP rs1644634 were significant associated with AFP levels and AFP protein expression.The A allele may be the risk allele associated with higher AFP levels.Moreover,the correlation between the polymorphisms of rs379707 and AFP levels/AFP protein expression were not observed. |
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