| Background:Hepatocellular carcinoma?HCC?is one of common malignancies worldwide and is the second leading cause of tumor-related death.Primary liver cancer,which is the fifth and ninth most frequently occurring cancer in men and women,respectively,has different geographical distribution and changing incidence rates for gender.The incidence of HCC for men is more frequent in East and South-east Asian population worldwide,but for women is in East Africa and West Africa areas.China is the largest area with a higher incidence rate of HCC in East Asian.50 percent of global HCC cases?more than five hundred thousand cases?occured in China every year,furthermore,China contributes more than 50 percent of HCC-related death per year.For now,pathogenic mechanisms in HCC remain mysterious.Previous studies suggest that HCC is a complex disease based on multiple stage with interplay between the host,disease and environmental factors.Infection with chronic hepatitis B virus?HBV?or hepatitis C virus?HCV?is currently the dominant risk factor worldwide,but Infection with chronic hepatitis B virus dominantly contrbute HCC in China.Genetic background play a key role in the risk of HCC development,because HCC is well known to cluster among families.In recent,Genome-wide association studies?GWAS?have identified several susceptibility loci associated with HBV-related HCC,which correlate with the risk of HCC development via multiple signaling pathways,including transforming growth factor??TGF??signaling pathway,insulin/phosphoinositide 3 kinase?insulin/PI3K?signaling pathway,epidermal growth factor receptor?EGFR?signaling pathway,and Wnt/?-catenin signaling pathway.In 2010,our team and Beijing Proteome Research Center together conducted a Genome-wide association study in Chinese population to explore the pathogenic mechanism in HBV-related HCC.The results showed that 1p36.22 was a new susceptibility locus for hepatocellular carcinoma in chronic hepatitis B virus carriers.However,pathogenic mechanisms in HBV-related HCC do not be explained clearly,because many genes with weak effect,which do not be identified,contribute HCC development.Apparently,it is a challenge to identify thses susceptibiliy genes.Huge number of samples collected from different teams can solve the scarcity of statistical power,but genotyping data from multiple genotyping platforms with different human genome reference datasets do not be intergrated for running association study.Following imputation development with a same human genome reference dataset,the gap among the different genotyping platforms began disappearing.Imputation can make allowances for ungenotyped loci among the different genotyping platforms using linkage disequilibrium?LD?.Otherwise,the case-control design based on individuals have population stratification,which can cause false associated signals even they are replicated in several independent populations.Family memebers have a same ancestry and carry similar variants on genome,which means that they have no population stratification and the credible associated signals can be obseved.Recently,researchers have have identified some susceptibility loci associated with diferent complex diseases by some GWASs based on a combination of families and case-control population.However,there is no similiar report about HBV-related HCC.Purpose:To make allowances for the missing heritability,we conducted a GWAS to identify novel loci for HBV-related HCC without false associated signals using a combination of family trios-based and case-control-based designs in the discovery stage.Methods:In this study,we performed a three-stage GWAS among populations of Chinese ancestry,totally consisting of 205 HBV-related HCC trios,4,151 chronic HBV carriers with HCC?cases?and 2,904 chronic HBV carriers without HCC.Specifically,the discovery stage included 205 trios?Guangxi population?and 355 cases and 360 controls?Guangxi population?.To extend the coverage of genomic region in the discovery stage,we performed imputation on the GWAS genotyping data using the IMPUTE2 software?version 2.3.1?.The1,000 Genomes Project data?Version 3,March 2012?was used as the reference dataset.Familytrios-and case-control based association analyses were performed using PLINK?v1.07;ref.14?.In the discovery stage,family trios-based association analyses were conducted using transmission/disequilibrium test?TDT?.Case–control association analyses were conducted using logistic regression under an additive model with adjustment for age,gender,smoking and drinking status,pack-years of smoking and family history of HCC.Then,we combined these two GWAS datasets and used the Fisher method to perform joint association analyses.Totally,we identified candidate SNPs to be significantly associated with HBV-related HCC in the discovery stage(combined P values<5×10-5).We genotyped these SNPs in five independent case-control population recruited from Shanxi,Guangdong,Beijing,Jiangsu,and Guangxi.We performed expression quantitative trait locus?eQTL?analyses to assess the associations between the genotypes of candidate SNPs and mRNA levels in liver tissues in a sample set consisting of 88 HCC cases from the Jiangsu population.The eQTL results were replicated in another independent dataset of HCC tissues,which consists of 82 HCC patients of Asian ancestry from The Cancer Genome Atlas.We used two case-only sample sets?192 cases?to test this priori hoc hypothesis that genotype at candidate SNP was directly associated with the survival of patients with HBV-related HCC.Finally,we explored the regulatory mechanisms for susceptibility genes in HBV-related HCC using HaploReg,3C,and STRING dataset.Results:We identified 14 candidate SNPs to be significantly associated with HBV-related HCC in the discovery stage(combined P values<5×10-5).Then,we genotyped these14 SNPs in five independent case–control population?3796 cases and 2544 controls?.Only the rs10272859 at 7q21.13 survived stage of replication,a meta-analysis combining all the family trios and case–control studies of all stages gave a joint P value of 9.46×10-10?joint OR=1.28?.Furthermore,we observed the at-risk rs10272859[G]allele was significantly associated with higher CDK14 mRNA levels in both sample sets.At the approximately 110 Kb region of7q21.13,there were 75 SNPs in strong LD with rs10272859 based on Asian population in 1000 Genomes Project?all r2>0.7?.22 of them are in enhancer elements of multiple types of cells.We performed 3C assays and Sanger sequencing in HepG2 cells,and successfully confirmed the physical interaction between the DNA fragment F2 containing 3 of the 22 SNPs and the CDK14promoter.Log-rank test showed that the at-risk rs10272859[G]allele was significantly associated with shorter overall survival of HCC patients under an additive model or a dominant model.There is no interaction between the new susceptibility locus and all other susceptibility loci reported early.Conclusions:In the present study,we conducted a new GWAS on HBV-related HCC,which combined family trios-based and case-control-based designs in the discovery stage and identified a novel association signal at 7q21.13?index rs10272859?consisting of a susceptibility gene?CDK14?.These findings might expand our understanding of the genetic susceptibility to HBV-related HCC and would help to improve risk stratification and early therapeutic decision making for this malignancy. |
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