核苷酸切除修复基因多态性与肝癌遗传易感性及临床表型的关联研究
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摘要
[研究背景和目的]
乙型肝炎病毒(HBV)感染和黄曲霉毒素B_1(AFB_1)摄入是我国原发性肝细胞癌(HCC)发病的两大主要危险因素。此外,吸烟、饮酒和遗传背景等因素也可能影响疾病的发生。
国内的流行病学调查和研究结果显示,与HCC低发区比较,在高发区,粮食受AFB_1污染的程度更严重,同时当地居民血清AFB_1-白蛋白加合物的水平也显著增高。AFB_1在体内经Ⅰ相代谢酶活化为AFB_1-8,9-环氧化物,该致癌物与DNA上鸟嘌呤(dG)的N~7特异性结合,经过一系列反应后,形成AFB_1-N~7-dG和AFB_1-FAPY两种大加合物,导致DNA发生G＞T碱基颠换等多种突变和损伤,并强烈抑制DNA的复制。这种损伤是基因突变和细胞癌变的分子基础。而核昔酸切除修复(NER)通路被认为是机体内修复AFB_1所致DNA损伤的最主要途径。
NER通路主要基因有:①DNA损伤的识别:涉及XPA、XPC等基因;②损伤部位DNA链的打开:由TFIIH的亚基ERCC3/XPB和ERCC2/XPD解旋酶打开DNA双链;③寡核苷酸链的切断:XPG和XPF-ERCC1复合体分别在损伤部位的3'端和5'端切断DNA单链;④PCNA、RPA等基因完成缺损部位单链片段的再合成和修补缺口。其中,损伤的识别/切除是通路中的限速或调节环节。在通路中,各个基因发挥着相应的独特作用,并按照严格的顺序加入和置换出NER过程,其中任何一个基因的功能异常即可导致整个通路对损伤修复的效率。
研究显示,基因的单核苷酸多态性(single nucleotide polymorphism,SNP)是导致不同个体对复杂疾病的易感性和对药治疗的敏感性等出现差异的最基本分子基础和最主要表现形式。SNP不但与肿瘤的易感性有显著性关联,用于判断疾病的患病风险,还与肿瘤的疗效及患者预后显著相关,能作为反映疾病进展以及预后的重要指标。同样,NER基因的单核苷酸多态性是个体DNA损伤修复能力差异的分子遗传学基础,可影响个体的肿瘤易感性和肿瘤患者的预后。目前,已在肺癌等肿瘤的病例—对照研究中,发现了XPD等NER基因的多态性与肿瘤的易感性以及患者化疗后的预后等显著相关。但在NER基因多态性与肝癌易感性的关联研究方面,目前在国内外仅检索到2篇文献报道,这两项研究检测的位点均是XPD Lys751Gln,均未发现此位点的多态性与HCC的易感性存在显著性关联。NER通路其他基因的多态性与肝癌的易感性、临床表型及预后的关系均未见报道。
在前期的研究中,本研究小组建立了SNP发掘、分型和遗传分析的技术平台,对100多个与HCC等疾病相关的候选基因进行了大规模重测序。在此基础上我们初步确定了NER通路中6个基因共11个位点(ERCC1 C8092A、ERCC1 Asn118Asn、XPA 3'UTR、XPA G-4A、XPC G-5A、XPC Intron 9 PAT、XPD Asp312Asn、XPD Lys751Gln、XPF T26523C、XPG His46His和XPGHis1104Asp(最小等位频率均超过5%),分析这些位点的多态性与中国广西人群HCC发病风险的关系,并初步探讨这些多态性与HCC临床表型及疾病进展(如TNM分期、远处转移、门静脉侵犯等)的关联性,以期阐明NER基因多态性在HCC发生和疾病进展中所起的作用。
[材料与方法]
1.样本和资料收集应用病例-对照研究方法在中国广西的人群中,收集了434例HCC患者和480例健康对照的外周静脉血(4ml/例)及流行病学资料。其中,在广西医科大学附属肿瘤医院收治的HCC 191例,还同时收集详细的临床资料(如疾病TNM分期等)。HCC的诊断依据包括病理组织学/细胞学诊断、血清AFP定量检测、肝脏影像学检查,如B超和(或)CT、肝动脉血管造影等。血清AFP的水平采用放射免疫法分析。HCC疾病分期采用AJCC标准。样本收集过程按照国家人类基因组研究伦理学准则进行,获得了入选者的知情同意。
2.实验方法按照酚-氯仿方法抽提得到的DNA,经过定量并稀释后放于-20℃冰箱保存备用。XPC Intron 9 PAT的PCR产物直接用于琼脂糖凝胶电泳分型,其他位点采用PCR-RFLP方法分析基因型。SNP的等位的频率通过记数得到。
3.统计学分析各基因型在病例组和对照组中的频率,均使用Arlequin软件进行哈-温平衡(Hardy-Weinberg equilibrium,HW-E)检验。P＞0.05被认为各基因型的分布达到Hardy-Weinberg平衡。
以x~2检验比较各基因型在病例与对照组之间分布的差异,以及在不同临床表型组之间的差异。以比值比(Oddsratios,OR)及其95%可信区限(Confidence Intervals,CI)表示基因型与HCC易感性及临床表型的关联强度,以非条件Logistic回归模型计算OR和95%CI,并以性别、年龄、吸烟、吸烟量(Pack-years level)和饮酒等5个潜在混淆因素进行校正。
趋势检验和基因-环境因素的交互作用采用Logistic回归进行分析。
所有的统计检验均为双侧概率检验,P＜0.05被认为差异具有显著性。采用SPSS软件(version 10.0,SPSS Inc.,Chicago,Illinois)进行分析。
使用Bonferroni Correction方法控制假阳性率,计算公式:CorrectedP-value=P-valuexn(number of genes in test),如果纠正P值仍小于错误率(设定为0.05),则认为该基因型的频率在两个组之间的分布差异有显著性。
[主要结果]
本研究是首先对NER整个通路的主要基因的多态性与HCC的遗传易感性及临床表型的关系进行系统分析的研究,得到了以下主要结果:
1.NER基因多态性与HCC患病风险的关联分析
(1)在总体样本中,经过多因素校正和Bonferroni Correction校正后,仅有XPD Lys751Gln的TG基因型(即Lys/Gln基因型)的频率在HCC组和对照组间的差异在有显著性。但经过HBV感染状况调整后显示,不管是在HBsAg(+)人群中,还是在HBsAg(-)人群中,XPD Lys751Gln各基因型的频率在HCC组和对照组间的差异经过Bonferroni Correction校正后均无显著性。
结果表明,在调整了HBV感染状态的情况下,本研究中NER基因的11个多态性位点作为单因素时,其与HCC的遗传易感性均无显著性关联。
分层前后关联结果发生改变,其可能的原因之一是经过分层后,样本例数减少,导致统计效能下降而造成的。同时也说明这11个位点的多态性作为单因素时与个体的HCC遗传易感性的关联性较弱,影响力有限。
(2)本研究中,在合并有HBV感染情况下,共有8个基因的15个基因型与吸烟等环境因素有显著交互作用;而在没有合并HBV感染情况下,只有一个基因的基因型与年龄有显著交互作用。说明HBV感染状况对NER基因多态性与HCC易感性的关系有明显影响,能显著提高NER基因多态性与HCC易感性的关联性。
(3)在合并有HBV感染状态下,以下基因型与环境因素有显著交互作用:
①XPD Lys751Gln TG基因型与饮酒、民族及直系亲属HCC家族史均存在显著的交互作用,与TT基因型比较,携带TG(Lys/Gln)基因型能显著降低非饮酒个体、HCC家族史阴性个体,以及汉族或非汉族个体的HCC易感性。
②XPD Asp312Asn的CT基因型与家族史有显著交互作用,能降低家族史阴性个的患病风险(与CC基因型比较)。
③与CC基因型比较,ERCC1 C8092A的CA+AA基因型与吸烟状况和HCC家族史有显著交互作用,此基因型能显著增加吸烟者或家族史阴性个体的HCC患病风险。
④XPA G-4A的AG基因型与HCC家族史有显著交互作用。进一步分析显示,在家族史阳性的人群中,与携带GG基因型的个体比较,携带AG基因型的个体发生HCC的风险最高(与GG基因型比较)。说明在存在HBV感染以及直系亲属HCC家族史阳性因素联合作用下,XPA G-4A的AG基因型是HCC的风险因素。
⑤XPA 3'UTR的GC基因型与性别及HCC家族史分别存在显著交互作用。其中,与CC基因型比较,携带此基因型的女性和家族史阳性的个体,罹患HCC的风险显著增高。
⑥XPC Intron 9 PAT的PAT(+/-)基因型与饮酒状况有显著交互作用。亚层分析显示,与PAT(-)等位基因型比较,携带此基因型是饮酒者的HCC患病风险因素。
⑦XPF T26523C的TC基因型与吸烟和家族史有显著交互作用,与TT基因型比较,携带TC基因型能显著降低非吸烟者和家族史阴性者的患病风险。
⑧XPG His46His的突变C等位(TC+CC)基因型与吸烟因素存在显著交互作用。与TT基因型比较,携带TC+CC基因型能显著增加吸烟者,尤其是轻度吸烟个体的HCC患病风险。
(4)在HBsAg(-)状态下,仅有ERCC1 C8092A的CA+AA基因型与年龄存在显著交互作用,CA+AA基因型能显著增加年龄＞49岁个体的HCC患病风险(与CC基因型比较)。
(5)ERCC1 Asn118Asp、XPC G-5A和XPG His1104Asp的各基因型与性别、年龄、吸烟、饮酒和家族史等因素均无交互作用,提示这些多态性位点与HCC的遗传易感性无显著性关联。
(6)趋势分析结果显示,不管是在HBV(+)人群中,还是在在HBV(-)人群中,随着个体携带风险性基因型的个数的增加(达到4个或以上),其罹患HCC的风险显著增加(P-value for trend分别为0.046和0.047)。
(7)XPD Lys751Gln的G等位(Gln)和XPD Asp312Asn的T等位的频率在我国大陆人群中低于台湾人群和白色人种中的频率,有显著的种族特异性。
2.NER基因多态性与HCC临床表型的关联研究
(1)NER基因多态性与原发灶进展的关联分析
(ⅰ)与GG基因型比较,ERCC1 Asn118Asn的AA基因型的频率在T_1组中高于T_(24)组(16.9%vs 7.6%),差异有显著性。提示AA基因型是反映癌灶处于较局限状态的有效指标。
而XPG His1104Asp的CC基因型(与GG基因型比较)在T_2～T_4组中的频率(29.5%)显著高于T_1组中的频率(12.5%)。提示CC基因型是反映癌灶处于较局限状态的有效指标。
(ⅱ)仅有XPC Intron 9 PAT的PAT(+)基因型与癌灶大小显著性关联,其在小肝癌(≤5cm)组的频率显著高于大肝癌组(14.9%vs 4.4%,校正后P=0.009,OR=2.382,95%CI:1.246-4.554)。
(ⅲ)本研究所分析的多态性位点的各基因型与HCC的癌灶数目多少之间无显著性关联。
(ⅳ)上述结果表明,ERCC1 Asn118Asn的AA(突变)基因型和XPC Intron9 PAT的PAT(+)基因型都能导致细胞的DNA修复功能显著下降,而临床上都与病灶局限显著关联;而XPG His1104Asp的CC基因型比突变基因型GG具有更高的DNA修复能力。结果说明,DNA修复能力强的癌细胞,容易向周围发生侵润扩散。
(2)NER基因多态性与门静脉癌栓状况的关联分析
ERCC1 C8092A的CA+AA基因型(与CC基因型比较)、XPG His46His的TC+CC基因型(与TT基因型比较)和XPG His1104Asp的GC+CC基因型(与GG基因型比较)在有门静脉侵犯组中的频率均显著高于无侵犯组中的频率。说明这3个基因型是反映癌细胞侵犯门静脉系统导致癌栓形成的重要指标,并提示携带这3个基因型的HCC患者,容易有门静脉癌栓形成。
其他位点的各基因型与门静脉癌栓形成均无显著性关联。
(3)NER基因多态性与HCC远处转移状况的关联分析
XPG His1104Asp的GC+CC基因型在无远处转移组中的频率显著高于有转移组中的频率(68.7%vs 47.4%,校正后P=0.006)。说明这个基因型与HCC较低的远处转移率显著关联,并提示携带这个基因型的HCC患者出现远处转移的风险最低。
其他位点的各基因型与HCC的远处转移均无显著性关联。
(4)NER基因多态性与血清AFP水平的关联分析
XPC G-5A的AA基因型在AFP阳性组(≥25ng/ml)的频率显著高于阴性组(＜25ng/ml),即AA基因型与AFP水平异常升高有显著性关联。提示联合检测此基因型和血清AFP水平,可能有助于HCC的早发现、早诊断。此外,AFP阳性患者的中位生存期明显短于AFP阴性患者,由于XPC G-5A的AA基因型与AFP水平异常升高有显著性关联,因此,携带此基因型的个体,生存期可能较短,预后较差。
[结论]
1.本研究是首个对整个NER通路中涉及的主要基因的多态性与HCC的遗传易感性和临床表型进行关联分析的研究。其中在易感性关联研究方面,除了XPD Lys751Gln外,其他位点的多态性与HCC易感性的关系尚未见文献报道;而NER基因多态性与HCC临床表型的关联研究亦未见报道。
2.在调整了HBV感染状态,以及经过多因素校正和Bonferroni Correction校正后,本研究中11个多态性位点各基因型的频率在HCC组和对照组间的差异均无显著性。说明各基因型作为单因素时,其与HCC的遗传易感性均无显著性关联。
3.HBV感染状况对NER基因多态性与HCC易感性的关系有明显影响。其中,HBV感染能显著增强NER基因各多态性与环境因素的交互作用,共有8个基因的15个基因型与环境因素有显著交互作用,影响个体的HCC患病风险。
4.不管是否合并有HBV感染,随着携带的NER基因风险性基因型数目的增多,个体罹患HCC的风险均显著增高。
5.XPD Lys751Gln和Asp312Asn的突变基因型的频率在我国大陆人群中低于台湾人群和白色人种的频率,有显著的种族特异性。
6.NER基因多态性还与HCC临床表型及疾病进展有显著性关联,可能成为早期诊断HCC和判断疾病进展的重要指标。
Background and Objectives
Aflatoxin B_1(AFB_1)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,AFB_1 is activated by cytochrome P450 enzymes forming the AFB_1-8,9-epoxide,which reacts with DNA preferentially at the N~7 position of guanine(dG).A number of new products are formed,the initial major adduct is 8,9-dihydro-8-(N~7-guanyl)-9-hydroxyaflatoxin B_1(AFB_1-N~7-dG).The resulting cationic adduct,AFB_1-N~7-Gua, is labile and can suffer opening of its imidazole ring,giving rise to the chemically and biologically stable form amidopyrimidine adduct,AFB_1-formamidopyrimidine (AFB_1-FAPY).Both cationic AFB_1-N~7-dG and AFB_1-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 AFB_1 exposure.DNA lesions caused by AFB_1 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 Methods
Study Subjects
A 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 Polymorphisms
Genomic 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 Analysis
Genotype 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 x~2 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.
RESULTS
NER polymorphisms and HCC risk
The 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 phenotypes
We 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).
Conclusions
In 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.
乙型肝炎病毒(HBV)感染和黄曲霉毒素B_1(AFB_1)摄入是我国原发性肝细胞癌(HCC)发病的两大主要危险因素。此外,吸烟、饮酒和遗传背景等因素也可能影响疾病的发生。
国内的流行病学调查和研究结果显示,与HCC低发区比较,在高发区,粮食受AFB_1污染的程度更严重,同时当地居民血清AFB_1-白蛋白加合物的水平也显著增高。AFB_1在体内经Ⅰ相代谢酶活化为AFB_1-8,9-环氧化物,该致癌物与DNA上鸟嘌呤(dG)的N~7特异性结合,经过一系列反应后,形成AFB_1-N~7-dG和AFB_1-FAPY两种大加合物,导致DNA发生G＞T碱基颠换等多种突变和损伤,并强烈抑制DNA的复制。这种损伤是基因突变和细胞癌变的分子基础。而核昔酸切除修复(NER)通路被认为是机体内修复AFB_1所致DNA损伤的最主要途径。
NER通路主要基因有:①DNA损伤的识别:涉及XPA、XPC等基因;②损伤部位DNA链的打开:由TFIIH的亚基ERCC3/XPB和ERCC2/XPD解旋酶打开DNA双链;③寡核苷酸链的切断:XPG和XPF-ERCC1复合体分别在损伤部位的3'端和5'端切断DNA单链;④PCNA、RPA等基因完成缺损部位单链片段的再合成和修补缺口。其中,损伤的识别/切除是通路中的限速或调节环节。在通路中,各个基因发挥着相应的独特作用,并按照严格的顺序加入和置换出NER过程,其中任何一个基因的功能异常即可导致整个通路对损伤修复的效率。
研究显示,基因的单核苷酸多态性(single nucleotide polymorphism,SNP)是导致不同个体对复杂疾病的易感性和对药治疗的敏感性等出现差异的最基本分子基础和最主要表现形式。SNP不但与肿瘤的易感性有显著性关联,用于判断疾病的患病风险,还与肿瘤的疗效及患者预后显著相关,能作为反映疾病进展以及预后的重要指标。同样,NER基因的单核苷酸多态性是个体DNA损伤修复能力差异的分子遗传学基础,可影响个体的肿瘤易感性和肿瘤患者的预后。目前,已在肺癌等肿瘤的病例—对照研究中,发现了XPD等NER基因的多态性与肿瘤的易感性以及患者化疗后的预后等显著相关。但在NER基因多态性与肝癌易感性的关联研究方面,目前在国内外仅检索到2篇文献报道,这两项研究检测的位点均是XPD Lys751Gln,均未发现此位点的多态性与HCC的易感性存在显著性关联。NER通路其他基因的多态性与肝癌的易感性、临床表型及预后的关系均未见报道。
在前期的研究中,本研究小组建立了SNP发掘、分型和遗传分析的技术平台,对100多个与HCC等疾病相关的候选基因进行了大规模重测序。在此基础上我们初步确定了NER通路中6个基因共11个位点(ERCC1 C8092A、ERCC1 Asn118Asn、XPA 3'UTR、XPA G-4A、XPC G-5A、XPC Intron 9 PAT、XPD Asp312Asn、XPD Lys751Gln、XPF T26523C、XPG His46His和XPGHis1104Asp(最小等位频率均超过5%),分析这些位点的多态性与中国广西人群HCC发病风险的关系,并初步探讨这些多态性与HCC临床表型及疾病进展(如TNM分期、远处转移、门静脉侵犯等)的关联性,以期阐明NER基因多态性在HCC发生和疾病进展中所起的作用。
[材料与方法]
1.样本和资料收集应用病例-对照研究方法在中国广西的人群中,收集了434例HCC患者和480例健康对照的外周静脉血(4ml/例)及流行病学资料。其中,在广西医科大学附属肿瘤医院收治的HCC 191例,还同时收集详细的临床资料(如疾病TNM分期等)。HCC的诊断依据包括病理组织学/细胞学诊断、血清AFP定量检测、肝脏影像学检查,如B超和(或)CT、肝动脉血管造影等。血清AFP的水平采用放射免疫法分析。HCC疾病分期采用AJCC标准。样本收集过程按照国家人类基因组研究伦理学准则进行,获得了入选者的知情同意。
2.实验方法按照酚-氯仿方法抽提得到的DNA,经过定量并稀释后放于-20℃冰箱保存备用。XPC Intron 9 PAT的PCR产物直接用于琼脂糖凝胶电泳分型,其他位点采用PCR-RFLP方法分析基因型。SNP的等位的频率通过记数得到。
3.统计学分析各基因型在病例组和对照组中的频率,均使用Arlequin软件进行哈-温平衡(Hardy-Weinberg equilibrium,HW-E)检验。P＞0.05被认为各基因型的分布达到Hardy-Weinberg平衡。
以x~2检验比较各基因型在病例与对照组之间分布的差异,以及在不同临床表型组之间的差异。以比值比(Oddsratios,OR)及其95%可信区限(Confidence Intervals,CI)表示基因型与HCC易感性及临床表型的关联强度,以非条件Logistic回归模型计算OR和95%CI,并以性别、年龄、吸烟、吸烟量(Pack-years level)和饮酒等5个潜在混淆因素进行校正。
趋势检验和基因-环境因素的交互作用采用Logistic回归进行分析。
所有的统计检验均为双侧概率检验,P＜0.05被认为差异具有显著性。采用SPSS软件(version 10.0,SPSS Inc.,Chicago,Illinois)进行分析。
使用Bonferroni Correction方法控制假阳性率,计算公式:CorrectedP-value=P-valuexn(number of genes in test),如果纠正P值仍小于错误率(设定为0.05),则认为该基因型的频率在两个组之间的分布差异有显著性。
[主要结果]
本研究是首先对NER整个通路的主要基因的多态性与HCC的遗传易感性及临床表型的关系进行系统分析的研究,得到了以下主要结果:
1.NER基因多态性与HCC患病风险的关联分析
(1)在总体样本中,经过多因素校正和Bonferroni Correction校正后,仅有XPD Lys751Gln的TG基因型(即Lys/Gln基因型)的频率在HCC组和对照组间的差异在有显著性。但经过HBV感染状况调整后显示,不管是在HBsAg(+)人群中,还是在HBsAg(-)人群中,XPD Lys751Gln各基因型的频率在HCC组和对照组间的差异经过Bonferroni Correction校正后均无显著性。
结果表明,在调整了HBV感染状态的情况下,本研究中NER基因的11个多态性位点作为单因素时,其与HCC的遗传易感性均无显著性关联。
分层前后关联结果发生改变,其可能的原因之一是经过分层后,样本例数减少,导致统计效能下降而造成的。同时也说明这11个位点的多态性作为单因素时与个体的HCC遗传易感性的关联性较弱,影响力有限。
(2)本研究中,在合并有HBV感染情况下,共有8个基因的15个基因型与吸烟等环境因素有显著交互作用;而在没有合并HBV感染情况下,只有一个基因的基因型与年龄有显著交互作用。说明HBV感染状况对NER基因多态性与HCC易感性的关系有明显影响,能显著提高NER基因多态性与HCC易感性的关联性。
(3)在合并有HBV感染状态下,以下基因型与环境因素有显著交互作用:
①XPD Lys751Gln TG基因型与饮酒、民族及直系亲属HCC家族史均存在显著的交互作用,与TT基因型比较,携带TG(Lys/Gln)基因型能显著降低非饮酒个体、HCC家族史阴性个体,以及汉族或非汉族个体的HCC易感性。
②XPD Asp312Asn的CT基因型与家族史有显著交互作用,能降低家族史阴性个的患病风险(与CC基因型比较)。
③与CC基因型比较,ERCC1 C8092A的CA+AA基因型与吸烟状况和HCC家族史有显著交互作用,此基因型能显著增加吸烟者或家族史阴性个体的HCC患病风险。
④XPA G-4A的AG基因型与HCC家族史有显著交互作用。进一步分析显示,在家族史阳性的人群中,与携带GG基因型的个体比较,携带AG基因型的个体发生HCC的风险最高(与GG基因型比较)。说明在存在HBV感染以及直系亲属HCC家族史阳性因素联合作用下,XPA G-4A的AG基因型是HCC的风险因素。
⑤XPA 3'UTR的GC基因型与性别及HCC家族史分别存在显著交互作用。其中,与CC基因型比较,携带此基因型的女性和家族史阳性的个体,罹患HCC的风险显著增高。
⑥XPC Intron 9 PAT的PAT(+/-)基因型与饮酒状况有显著交互作用。亚层分析显示,与PAT(-)等位基因型比较,携带此基因型是饮酒者的HCC患病风险因素。
⑦XPF T26523C的TC基因型与吸烟和家族史有显著交互作用,与TT基因型比较,携带TC基因型能显著降低非吸烟者和家族史阴性者的患病风险。
⑧XPG His46His的突变C等位(TC+CC)基因型与吸烟因素存在显著交互作用。与TT基因型比较,携带TC+CC基因型能显著增加吸烟者,尤其是轻度吸烟个体的HCC患病风险。
(4)在HBsAg(-)状态下,仅有ERCC1 C8092A的CA+AA基因型与年龄存在显著交互作用,CA+AA基因型能显著增加年龄＞49岁个体的HCC患病风险(与CC基因型比较)。
(5)ERCC1 Asn118Asp、XPC G-5A和XPG His1104Asp的各基因型与性别、年龄、吸烟、饮酒和家族史等因素均无交互作用,提示这些多态性位点与HCC的遗传易感性无显著性关联。
(6)趋势分析结果显示,不管是在HBV(+)人群中,还是在在HBV(-)人群中,随着个体携带风险性基因型的个数的增加(达到4个或以上),其罹患HCC的风险显著增加(P-value for trend分别为0.046和0.047)。
(7)XPD Lys751Gln的G等位(Gln)和XPD Asp312Asn的T等位的频率在我国大陆人群中低于台湾人群和白色人种中的频率,有显著的种族特异性。
2.NER基因多态性与HCC临床表型的关联研究
(1)NER基因多态性与原发灶进展的关联分析
(ⅰ)与GG基因型比较,ERCC1 Asn118Asn的AA基因型的频率在T_1组中高于T_(24)组(16.9%vs 7.6%),差异有显著性。提示AA基因型是反映癌灶处于较局限状态的有效指标。
而XPG His1104Asp的CC基因型(与GG基因型比较)在T_2～T_4组中的频率(29.5%)显著高于T_1组中的频率(12.5%)。提示CC基因型是反映癌灶处于较局限状态的有效指标。
(ⅱ)仅有XPC Intron 9 PAT的PAT(+)基因型与癌灶大小显著性关联,其在小肝癌(≤5cm)组的频率显著高于大肝癌组(14.9%vs 4.4%,校正后P=0.009,OR=2.382,95%CI:1.246-4.554)。
(ⅲ)本研究所分析的多态性位点的各基因型与HCC的癌灶数目多少之间无显著性关联。
(ⅳ)上述结果表明,ERCC1 Asn118Asn的AA(突变)基因型和XPC Intron9 PAT的PAT(+)基因型都能导致细胞的DNA修复功能显著下降,而临床上都与病灶局限显著关联;而XPG His1104Asp的CC基因型比突变基因型GG具有更高的DNA修复能力。结果说明,DNA修复能力强的癌细胞,容易向周围发生侵润扩散。
(2)NER基因多态性与门静脉癌栓状况的关联分析
ERCC1 C8092A的CA+AA基因型(与CC基因型比较)、XPG His46His的TC+CC基因型(与TT基因型比较)和XPG His1104Asp的GC+CC基因型(与GG基因型比较)在有门静脉侵犯组中的频率均显著高于无侵犯组中的频率。说明这3个基因型是反映癌细胞侵犯门静脉系统导致癌栓形成的重要指标,并提示携带这3个基因型的HCC患者,容易有门静脉癌栓形成。
其他位点的各基因型与门静脉癌栓形成均无显著性关联。
(3)NER基因多态性与HCC远处转移状况的关联分析
XPG His1104Asp的GC+CC基因型在无远处转移组中的频率显著高于有转移组中的频率(68.7%vs 47.4%,校正后P=0.006)。说明这个基因型与HCC较低的远处转移率显著关联,并提示携带这个基因型的HCC患者出现远处转移的风险最低。
其他位点的各基因型与HCC的远处转移均无显著性关联。
(4)NER基因多态性与血清AFP水平的关联分析
XPC G-5A的AA基因型在AFP阳性组(≥25ng/ml)的频率显著高于阴性组(＜25ng/ml),即AA基因型与AFP水平异常升高有显著性关联。提示联合检测此基因型和血清AFP水平,可能有助于HCC的早发现、早诊断。此外,AFP阳性患者的中位生存期明显短于AFP阴性患者,由于XPC G-5A的AA基因型与AFP水平异常升高有显著性关联,因此,携带此基因型的个体,生存期可能较短,预后较差。
[结论]
1.本研究是首个对整个NER通路中涉及的主要基因的多态性与HCC的遗传易感性和临床表型进行关联分析的研究。其中在易感性关联研究方面,除了XPD Lys751Gln外,其他位点的多态性与HCC易感性的关系尚未见文献报道;而NER基因多态性与HCC临床表型的关联研究亦未见报道。
2.在调整了HBV感染状态,以及经过多因素校正和Bonferroni Correction校正后,本研究中11个多态性位点各基因型的频率在HCC组和对照组间的差异均无显著性。说明各基因型作为单因素时,其与HCC的遗传易感性均无显著性关联。
3.HBV感染状况对NER基因多态性与HCC易感性的关系有明显影响。其中,HBV感染能显著增强NER基因各多态性与环境因素的交互作用,共有8个基因的15个基因型与环境因素有显著交互作用,影响个体的HCC患病风险。
4.不管是否合并有HBV感染,随着携带的NER基因风险性基因型数目的增多,个体罹患HCC的风险均显著增高。
5.XPD Lys751Gln和Asp312Asn的突变基因型的频率在我国大陆人群中低于台湾人群和白色人种的频率,有显著的种族特异性。
6.NER基因多态性还与HCC临床表型及疾病进展有显著性关联,可能成为早期诊断HCC和判断疾病进展的重要指标。
Background and Objectives
Aflatoxin B_1(AFB_1)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,AFB_1 is activated by cytochrome P450 enzymes forming the AFB_1-8,9-epoxide,which reacts with DNA preferentially at the N~7 position of guanine(dG).A number of new products are formed,the initial major adduct is 8,9-dihydro-8-(N~7-guanyl)-9-hydroxyaflatoxin B_1(AFB_1-N~7-dG).The resulting cationic adduct,AFB_1-N~7-Gua, is labile and can suffer opening of its imidazole ring,giving rise to the chemically and biologically stable form amidopyrimidine adduct,AFB_1-formamidopyrimidine (AFB_1-FAPY).Both cationic AFB_1-N~7-dG and AFB_1-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 AFB_1 exposure.DNA lesions caused by AFB_1 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 Methods
Study Subjects
A 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 Polymorphisms
Genomic 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 Analysis
Genotype 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 x~2 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.
RESULTS
NER polymorphisms and HCC risk
The 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 phenotypes
We 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).
Conclusions
In 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.
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