Association Between The Nucleotide Excision Repair (NER) Genes Polymorphisms And Genetic Susceptibility And Clinical Phenotype Of Hepatocellular Carcinoma

Background and ObjectivesAflatoxin B₁(AFB₁)exposure and hepatitis B virus(HBV)infection are two main risk factors in hepatocellular carcinoma(HCC)in China.The other factors, such as smoking and alcohol abuse,and so on,may affect the susceptibility to HCC.Study had showed that people who had higher external aflatoxin exposure in food usually had higher level of serum aflatoxin-albumin adducts.In vivo,AFB₁ is activated by cytochrome P450 enzymes forming the AFB₁-8,9-epoxide,which reacts with DNA preferentially at the N⁷ position of guanine(dG).A number of new products are formed,the initial major adduct is 8,9-dihydro-8-(N⁷-guanyl)-9-hydroxyaflatoxin B₁(AFB₁-N⁷-dG).The resulting cationic adduct,AFB₁-N⁷-Gua, is labile and can suffer opening of its imidazole ring,giving rise to the chemically and biologically stable form amidopyrimidine adduct,AFB₁-formamidopyrimidine (AFB₁-FAPY).Both cationic AFB₁-N⁷-dG and AFB₁-FAPY adducts can react with DNA and cause the same types of mutations(mostly G to T transversions)at the site of the lesion and caused a strong blocking effect on DNA replication.Most of these alterations,if not repaired,can result in genetic instability,mutagenesis and cell death.Nucleotide excision repair(NER)pathway plays an important role in maintaining DNA integrity and removing bulky adducts upon AFB₁ exposure.DNA lesions caused by AFB₁ are preferentially repaired by the NER pathway.The mechanism of NER is quietly complexity,involves in driving the process from damage recognition and removal to DNA resynthesis.Mutations in NER genes can originate many kinds of disorders.In the NER pathways,XPC,RPA and XPA are involved in the recognition step,two subunits of TFIIH,XPB and XPD, exhibit helicase activity and unwind the DNA around the lesion.After binding of XPF-ERCC1,dual incision occurs by XPG and XPF-ERCC1,which cut 3'and 5' to the damage,respectively.In this way,the damage is released in a 24-32 nucleotide long oligonucleotide.Repair is completed by DNA synthesis and ligation.Single nucleotide polymorphisms(SNP)are the most frequent type of variation in the human genome,and they provide powerful tools for a variety of medical genetic studies.They have been hailed as the most common polymorphism found in the human genome and are believed to be responsible for 90%of all inter-individual variation.Not only are they useful markers of susceptibility to complex diseases,SNPs can be utilised as markers of pharmacogenomics and progress and outcomes of diseases.Polymorphisms in the NER genes may contribute to variations in the DNA repair capacity in the general population and may affect genetic susceptibility to cancer.Even a slight reduction in DNA repair capacity could result in a significantly increased risk of cancer.A large number of SNPs in different NER genes have been identified,and some of them have been studied for human cancer susceptibility.For example,there have been many studies found an significantly association between the NER genes XPD polymorphisms and risk of cancers of various kinds.These include lung cancer,head and neck carcer,and so on. Whereas,there are only two studies involved in NER genes polymorphisms and susceptibility to HCC.In these studies,there were only XPD Lys751Gln polymorphisms were tested,and the results both showed that the XPD Lys751Gln polymorphisms was lack association with HCC risk.Overall,association between NER gene polymorphisms and susceptibility to HCC are still not clear.In the previous study,we have established technical platforms for large-scale SNP discovery,genotyping and genetic analysis,respectively.By using these platforms,we screened SNP systematically in more than one hundere functionally important genes in Chinese individuals,and then examined the relationship between SNP markers in these genes and susceptibility to persistent HBV infection, HCC,Nasopharyngeal carcinoma(NPC)and Severe Acute Respiratory Syndrome (SARS)in Chinese population,through case-control and/or transmission/ disequilibrium test(TDT).So,In the present study,by using a candidate gene approach base on the results in the previous study,we selected 11 common(≥5%minor allele frequency)polymorphisms in six NER genes(ERCC1,XPA,XPC,XPD,XPF and XPG),and evaluated the associations between the respective genotypes and risk of HCC.We additionally investigated whether polymorphisms are associated with clinical phenotype of HCC.Materials and MethodsStudy SubjectsA total of 434 HCC and 480 controls were consecutively recruited from July, 2002 to December,2005 in Guangxi Province.The diagnosis of HCC was made by either positive histologic findings or an elevated serum alfa-fetoprotein(AFP)level, combined with at least one positive image on angiography,sonography,and/or high-resolution contrast computed tomography(CT).AFP level was detected by radioimmunity(RI).The controls were randomly selected from a community cancer screening program for early detection of cancer conducted in the same regions during the same period that the HCC cases were enrolled.The selection criteria for the controls included no individual history of cancer and frequency matching to the cases on sex and age(±5 years).At recruitment,informed consent was obtained from each subject,and personal information on demographic factors,medical and reproductive history, tobacco and alcohol use,and family history of cancer were collected via structured questionnaire.The clinical characteristics of 191 HCC cases those were assembled in Guangxi Tumor Hospital were collected.The clinical imformation included primary tumor(T)stage,size of tumor mass,number of tumor mass,portal vein invasion,distant metastasis,serum AFP levels and HBsAg status.Genotyping of PolymorphismsGenomic DNA was extracted from peripheral blood leukocytes of 4 ml whole blood of all participants by means of standard procedures.Polymorphism of XPC Intron 9 PAT were genotyped by PCR/agarose gel electrophoresis analysis only,and the genotypes of other polymorphism points were genotyped bypolymerase chain reaction(PCR)-based RFLP analysis.Statistical AnalysisGenotype and allele frequencies for each polymorphism were determined by direct gene counting,and the fitness to Hardy-Weinberg equilibrium was tested using the randompermutation procedure implemented in the Arlequin package.Pearson's x² test was used to examine differences in demographic and risk factor variables,and,furthermore,to examine differences in polymorphismsand clinical phenotype of HCC.Association between polymorphisms and susceptibility to HCC were estimated by use of unconditional logistic regression using SPSS software(version 10.0;SPSS Inc,Chicago,IL).Logistic regression analysis was used to compute odds ratios(OR)and associated 95%confidence intervals(95%CI)relating each of the SNPs to the risk of HCC,as well as association between SNP and clinical phenotype of HCC.The statistical significance of trends of allele-dosage relationships and interaction between gene polymorphisms with risk factors for HCC were calculated from consistent Logistic regression models,respectively.The odds ratios(ORs)were adjusted for gender,age,smoking status and pack-year,and alcohol use status.Logistic regression was analyzed by using SPSS software(version 10.0;SPSS Inc,Chicago,IL).Bonferroni Correction was used to control the false positive rate of statistical significance examination.Calculation formula was:Corrected P-value= P-valuexn(number of genes in test).P＜0.05 was regarded as significant difference.RESULTSNER polymorphisms and HCC riskThe genotypes of all genes distribution in the control group was in HardyWeinberg equilibrium. Stratified analysis by HBsAg status,no association was observed between each genotype of the ERCC1,XPA,XPC,XPD,XPF and XPG,and risk of HCC, after adjusting for gender,age,tobacco use and pack-year,and alcohol use,and corrected by Bonferroni Correction.We further assessed the interaction of NER polymorphisms and environmental factors stratified by the HBsAg statue because studies have suggested HBsAg statue would make significant effect on HCC risk.In the HBsAg(+)statue,the gene-environmental factor interaction were statistically significant in 15 genotypes.The results were as follow:Stratified analyses revealed that the gene-alcohol use,gene-family-degree HCC history,and gene-nationality interaction,respectively,was statistically significant for XPD Lys751Gln TG polymorphism.And that the association was the strongest in the non-alcohol use,family-degree HCC history negtive,and the Han nationality or the Non-Han nationality,with the multivariate-adjusted OR(TG versus TT)of 0.335(95%CI,0.176-0.640),0.473(95%CI,0.234-0.957)and 0.343 (95%CI,0.183-0.644),respectively.The same direction for the gene-family-degree HCC history interaction was found for the XPD Asp312Asn CT polymorphism,and the interaction was statistically significant.And that the association was the strongest in the family-degree HCC history negtive people with the multivariate-adjusted OR(CT versus CC)of 0.435(95%CI,0.190-0.995).The results revealed that both XPD 751 TGand Asp312Asn CT polymorphism were protect factors against HCC risk in HBsAg(+)statue.The frequencies of XPD Lys751Gln G allele and Asp312Asn T allele in our study population were lower than those in Taiwan' population and white people, respectively.For the ERCC1 C8092A CA+AA polymorphism,the gene-smoking status and gene-family-degree HCC history interaction were statistically significant, especially in the smoker and family-degree HCC history negtive people,CA+AA polymorphism would obviously increase the HCC risk(CA+AA versus CC).The same direction for the gene-family-degree HCC history interaction was found for the XPA G-4A AG polymorphism,and gene-gender,gene-family-degree HCC history interaction for XPA 3'UTR GC polymorphism,respectively,were significant.And that the association was the strongest in the family-degree HCC history negtive people for G-4A AG polymorphism(AG versus GG),and male and family-degree HCC history positive people for XPA 3'UTR GC polymorphism (GC versus CC)would significantly increase the HCC risk.For the XPC Intron 9 PAT(+/-)polymorphism,gene-alcohol use status interaction was significant.And PAT(+/-)polymorphism(PAT(+/-)versus PAT(-)) significantly increase the HCC risk in the alcohol use people.XPF T26523C TC polymorphism had a significant interaction with smoke status and family-degree HCC history,respectively,and significantly decreased the HCC risk in non-smoking and family-degree HCC history negtive people. For the XPG His46His TC+CC polymorphism,a significant gene-smoke status interaction was found,and that the TC+CC polymorphism significantly increased the HCC risk in smoker(TC+CC versus TT).Meanwhile,in the HBsAg(-)statue,only the gene-age interaction was statistically significant for the ERCC1 C8092A CA+AA polymorphism(CA+AA versus CC),and that the association was the strongest in the older more than 49 years people.CA+AA polymorphism was a risk factor of HCC.In the presence of HBsAg(+)or HBsAg(-),a dose-dependent association between increasing number of the NER risk genotype and HCC risk was observed (P-value for trend=0.046,and 0.047,respectively).HBsAg carriers or non-HBsAg carriers with 4 or more high-risk genotypes of NER gene polymorphisms had 1.7-fold to 1.9-fold higher risk of HCC than those with only one or none of the high-risk genotypes.The results as above revealed that HBV infected can statistically modify the associations between NER polymorphisms and HCC risk.NER polymorphisms and HCC clinical phenotypesWe analyzed the distribution of NER genotypes among difference clinical characteristics and assessed the relationship between these NER gene polymorphism and clinical characteristics in 191 HCC patients. We found a significant associated between the ERCC1 Asn118Asn polymorphism and primary tumor T stages,and XPG His1104Asp polymorphism and primary tumor T stages,respectively.The significant associated between XPC Intron 9 PAT polymorphism and size of primary tumor mass was found.ERCC1 C8092A CA+AA polymorphism,XPG His46His TC+CC and XPG His1104Asp polymorphism had a significant assocoation with portal vein invasion status,with the multivariate-adjusted OR of 0.492(CA+AA versus CC,95%CI, 0.247～0.983),and 0.724(TC+CC vs TT,95%CI,0.527～0.996),and 0.431(GC+CC vs GG,95%CI,0.202～0.920),respectively.The significant associated between XPG His1104Asp polymorphism and distant metastasis status was foud.XPC G-5A polymorphism had a significant relationship with serum AFP levels increased,with the multivariate-adjusted OR(AA versus GG)of 5.102 (95%CI,1.024-25.641).ConclusionsIn overall,no association was observed between each genotype of the ERCC1, XPA,XPC,XPD,XPF and XPG,and risk of HCC,after stratified by HBsAg status and multivariate-adjusting for gender,age,tobacco use and pack-year,and alcohol use,respectively,and as well as the minimum P value of all genes were corrected by Bonferroni Correction.HBV infected made significantly effect on the associations between NER polymorphisms and HCC risk.In the HBsAg(+)statue,the significant interaction of NER gene polymorphisms and environmental factors were observed in 15 genotypes.Meanwhile,in the HBsAg(-)statue,only the gene-age interaction was statistically significant.In the present of environmental factors(e.g,smoking),the NER gene polymorphisms modified individual susceptibility to HCC.In the presence of HBsAg(+)or HBsAg(-),a dose-dependent association between increasing number of the NER gene risk genotypes and HCC risk was observed. The association between NER genotypes polymorphism and clinical characteristics were statistically significant.NER gene polymorphism may be useful predictor of clinical progress in HCC patients.