GENETIC INVESTIGATION OF NPC AND HCC IN A CHINESE POPULATION

鼻咽癌合作研究的主要目标是发现与中国人群鼻咽癌发展相关的基因和变异等位基因的特征。鼻咽癌是中国广东人癌症死亡的主要原因。病例对照研究表明，环境因素发挥了重要作用，包括食品防腐剂（致癌亚硝胺）、盐腌鱼和草药和植物中的酚酯，这些都是鼻咽癌发病率最高的少数民族人群经常食用的食物。在中国和其他国家已经观察到鼻咽癌的家族聚集性和关联证据。由于EBV的慢性复制，如检测EBV病毒衣壳免疫球蛋白A （IgA）抗体所证明的，以及暴露于饮食因素可预测鼻咽癌，但不能解释所有疾病，我们假设也可能存在遗传因素促进鼻咽癌的发展。因此，我们开展了一项病例对照研究，以调查中国南方高度受影响地区鼻咽癌的遗传相关性。HCC是世界上最常见的癌症之一，也是许多国家的主要死亡原因，特别是在东亚和撒哈拉以南非洲。肝癌在发达国家也在增加。在美国，HCC的发病率从1976年至1980年的1.4 / 10万人增加到1991年至1995年的2.4 / 10万人。黄曲霉毒素B1 （AFB1）污染和HBV感染都是公认的HCC危险因素。AFB1是人类接触到的最强致癌物之一。AFB1在人类和动物食品中分布广泛，浓度很高，特别是在东南沿海的HCC流行地区。其中广西是HCC的高危地区，AFB1污染和HBV感染都很常见。在东南亚和非洲某些地区，黄曲霉毒素消费和HBV感染是导致这些地区HCC发病率异常高（24.235.5/10万）的重要因素。hbv诱发的慢性活动性肝炎和肝硬化是肝癌发生的主要因素。在HBV患病率最高的亚洲大陆，HBV与所有HCC病例的70-75%相关。许多研究报道了AFB1和HBV在增加HCC风险中的协同作用。我们假设，如果氧化应激对导致HCC的发生有宿主遗传影响，那么在暴露于环境肝癌致癌物的人群中，暴露和剂量足以表现出遗传效应，这种影响将最为明显。动物和体外研究表明，氧化应激可能在病毒或化学诱导的肝癌发生的关键途径中起作用。有证据表明，在HBV感染和暴露于环境致癌物的研究参与者中，环境致癌物和氧化应激之间存在很强的相关性，这为筛选这一独特人群中HCC风险的候选基因型/单倍型提供了机会。我们将使用候选基因方法选择参与氧化修复途径的基因。对于目标基因，将选择位于调控、剪接位点或编码区域（同义和非同义）内的所有snp，并添加htsnp以捕获95%的变异。在这个模型中，将充分分析基因-基因和基因-环境的相互作用。研究结果将进一步与来自中国北方受试者的HBV结局病例对照研究进行比较。完成NPC研究的入组和数据收集：NPC研究I期和II期的入组、数据收集和DNA提取已经完成。纳入家庭三合会进行单倍型推断和对飞行员的质量控制评估。没有孟德尔错误被观察到在任何家族三合一证明保真的样本处理和基因分型。第二阶段是一项横断面病例对照研究，包括一份调查问卷，以捕捉环境因素，包括职业、饮食和烟草暴露。I期和II期参与者（n=4000）的入组和环境暴露数据的添加为发现主要效应基因和测试基因-环境相互作用提供了足够的动力。家族研究中涉及的4号染色体区域的微卫星调查：我们已经完成了4号染色体短臂的微卫星调查，该区域涉及与家族性鼻咽癌相关的全基因组连锁研究，与非家族性鼻咽癌相关。在我们的I期队列中，我们对4号染色体短臂上跨越18mb的34个微卫星标记进行了基因分型，以确定该区域内的特定等位基因：1)与EBV VCA的IgA抗体证明的慢性EBV复制倾向相关；或2)与NPC相关。尽管我们观察到与鼻咽癌或慢性免疫球蛋白A产生（IgA+）相关的14个等位基因显著相关（P=0.001-0.03），但在进行多重比较校正后，这些等位基因并不显著。II期研究的患者人数增加了三倍，包括环境协变量，提供了验证这个和其他影响鼻咽癌发病的基因组区域的潜力。我们现在正在使用II期研究来确认i期研究中发现的相关性。HCC研究的样本收集和生物标志物状态：来自hbv感染者的HCC肝组织和hbv感染对照组的外周白细胞的DNA，以及HIV感染、黄曲霉毒素暴露和氧化应激的血浆和尿液生物标志物已经完成。候选基因和snp已被选择使用Illumina Goldengate平台进行基因分型。这些基因包括参与致癌、生物转化和氧化损伤修复途径的基因。snp的选择是基于其假定的或已知的功能，或者因为它们是单倍型标记。所有样本都来自广西的HCC患者和当地对照，广西是中国HCC流行地区，AFB1污染和HBV感染都是公认的危险因素。建立了所有实验室和临床记录的数据库，包括致癌物（黄曲霉毒素）和氧化应激生物标志物

The NPC collaborative study has as its primary objective the discovery of genes and characterization of variant alleles associated with the development of NPC in a Chinese population. NPC is a leading cause of cancer deaths in the Cantonese population in China. Case-control studies have indicated a strong role for environmental factors, including food preservatives (carcinogenic nitrosamines), salt-preserved fish, and phorbol esters in herbs and plants that are commonly consumed among ethnic populations with the highest NPC rates. Familial aggregation of NPC and evidence of linkage have been observed in China and in other countries. Since chronic EBV replication, as evidenced by detection of immunoglobulin A (IgA) antibody against EBV viral capsid, and exposure to dietary factors are predictive of NPC but do not explain all of the disease, we have hypothesized that there may also be genetic factors contributing to the development of NPC. We have therefore developed a case-control study to investigate the genetic correlates of NPC in a highly impacted region of southern China. HCC is one of the most common cancers worldwide and a leading cause of death in many countries, especially in East Asia and sub-Saharan African. HCC is also increasing in developed countries. In the United States the incidence of HCC increased from 1.4 per 100,000 population for the period from 1976 to 1980 to 2.4 per 100,000 for the period from 1991 to 1995. Both aflatoxin B1 (AFB1) contamination and HBV infection are well-recognized risk factors for HCC. AFB1 is one of the most potent carcinogens to which humans are exposed. AFB1 has a wide distribution and high concentration in human and animal foods particularly in HCC endemic regions along the southeast seacoast. Among them Guangxi is a HCC high-risk region where both AFB1 contamination and HBV infection are common. In Southeast Asia and certain regions of Africa, aflatoxin consumption and infection by HBV are important factors giving rise to the extraordinarily high incidence rates (24.235.5/100 000) of HCC in these areas. HBV-induced chronic active hepatitis and cirrhosis constitute major factors in liver carcinogenesis. HBV is associated with 70-75% of all HCC cases in Asia, the continent with the highest prevalence of HBV. A synergistic effect of AFB1 and HBV in increasing risk for HCC has been reported in many studies. We hypothesize that if there is a host genetic effect on oxidative stress leading to the development of HCC, it would be most evident in a population exposed to environmental hepatocarcinogens where exposure and dosage is strong enough to manifest the genetic effects. Both animal and in vitro studies indicate that oxidative stress might contribute to the key pathways in either virus or chemical induced hepatocarcinogenesis. There is evidence for strong correlations between environmental carcinogens and oxidative stress in study participants with both HBV infection and exposures to environmental carcinogens, providing an opportunity to screen candidate genotypes/haplotypes for HCC risk in this unique population. We will be using a candidate gene approach selecting genes involved in oxidative repair pathways. For targeted genes, all SNPs within regulatory, splice sites or coding regions (both synonymous and nonsynonymous) will be selected and htSNPs added to capture >95% of the variation. Gene-gene and gene-environment interactions will be fully analyzed in this model. The findings will be further compared with a HBV outcome case-control study of subjects from northern China. Accomplishments: Completion of enrollment and data collection for the NPC study: Enrollment, data collection, and DNA extractions for the NPC study Phase I and Phase II of the NPC study have been completed. Family triads were enrolled for haplotype inference and for quality control assessment of the pilot. No Mendelian errors were observed within any of the family triads attesting to the fidelity of sample handling and genotyping. Phase II is a cross-sectional case-control study and included a questionnaire to capture environmental factors, including occupational, dietary and tobacco exposures. The completion of enrollment of Phase I and II participants (n=4000) and the addition of environmental exposure data provides adequate power to discover main effect genes and to test gene-environment interactions. Microsatellite survey of Chromosome 4 region implicated in family study: We have completed a microsatellite survey of the short arm of chromosome 4, a region implicated in a genome-wide linkage study to be associated with familial NPC, for association with non-familial NPC. Thirty-four microsatellite markers spanning 18 Mb on the short arm of chromosome 4 were genotyped in our Phase I cohort, to determine if specific alleles within this region: 1) were associated with a propensity to develop chronic EBV replication as evidenced by IgA antibodies against EBV VCA; or 2) were associated with NPC. Although we observed significant association for 14 alleles associated with either NPC or chronic immunoglobulin A production (IgA+) (P=0.001-0.03), these were not significant after applying a correction for multiple comparisons. The Phase II study triples patient enrollment and includes environmental covariates offering the potential to validate this and other genomic regions that influence onset of NPC. We are now using the Phase II study to confirm associations found in Phase I. Sample collection and biomarker status for the HCC study: DNA from HCC liver tissues from HBV-infected persons and peripheral leukocytes of HBV-infected controls, together with plasma and urinary biomarkers of HIV infection, aflatoxin exposure and oxidative stress have been completed. Candidate genes and SNPs have been selected for genotyping using the Illumina Goldengate platform. These include genes involved in carcinogenesis, biotransformation and oxidative damage repair pathways. SNPs were selected based on their putative or known function or because they were haplotype tagging All samples are from HCC patients and local controls from Guangxi Province, a HCC endemic area in China where both AFB1 contamination and HBV infection are well-recognized risk factors. A database of all laboratory and clinical records, including carcinogen (aflatoxin) and oxidative stress biomarkers, has been established