【作者】 杨桓；

【导师】 曹佳；

【作者基本信息】 第三军医大学 ， 卫生毒理学， 2009， 博士

【摘要】 结直肠癌(Colorectal Cancer, CRC)是当前人类最常见的恶性肿瘤之一。全球目前每年有超过100万的结直肠癌新发病例,而死亡人数约50万,在肿瘤新发病例数和因肿瘤死亡人数中均处于第三位。中国结直肠癌发病率也正处于上升之中,至2008年,中国结直肠癌标化发病率已达24/10万。随着中国经济的进一步发展,若不采取有效干预措施,结直肠癌的发病率将会继续大幅上升,成为我国的主要“癌症负担”之一。新近制订的《中国癌症预防与控制规划纲要(2004～2010年)》将结直肠癌与肺癌、乳腺癌、肝癌、食管癌、胃癌、宫颈癌和鼻咽癌列为我国现阶段重点防治的8大癌症,这8类癌症死亡约占癌症死因的80 %以上。对结直肠癌的病因研究发现,结直肠癌是在环境因素与遗传因素交互作用下,多步骤、多阶段发展演变而形成的。目前研究认为,环境因素中,膳食结构、体力活动等与结直肠癌发病显著相关;而对于遗传因素与结直肠癌易感性的关系,目前研究较多的是叶酸相关代谢酶编码基因、Ⅱ相代谢酶基因等。全球的结直肠癌流行病学调查发现,饮酒与亚洲人群结直肠癌的发病较欧美人群更为显著。研究人员推测,造成人群间结直肠癌易感性的差异,遗传因素比生活因素起到了更为重要的作用。针对主要乙醇代谢酶编码基因功能多态性位点的研究也确实发现,这些基因多态性在亚洲人群和欧美人群间存在显著差异。就目前亚洲人群的资料来说,已报道的中国人群资料很少,仅我国东部地区一份资料对ADH1B、ALDH2、CYP2E1中的功能性多态位点进行了研究。从文献检索的结果来看,目前尚无应用标签单核苷酸多态性(tag single nucleotide polymorphism, tagSNP)进行主要乙醇代谢酶基因多态性与结直肠癌发病的关联研究,因此,我们在中、日、韩三国协作课题的基础上,利用该课题所建立的结直肠癌病例-对照标本库为基础,加上后续收集的东北结直肠癌病例-对照标本库,进行主要乙醇代谢酶(ADH1B、ALDH2、CYP2E1)编码基因tagSNPs及CYP2E1和环境应答基因JWA中功能多态性位点和与结直肠癌易感性的关联研究。目的:1.筛选中国北京汉族人群(CHB)乙醇代谢酶编码基因ADH1B、ALDH2、CYP2E1中tagSNPs,并在中国西南地区人群中进行tagSNPs与结直肠癌易感性的关联研究;2.研究西南地区人群和东北地区人群CYP2E1功能多态位点与结直肠癌易感性的关系,并对两地间的结果进行比较;3.研究西南地区人群JWA基因功能多态位点与结直肠癌易感性的关系。方法:1.以CHB信息为蓝本,从HapMap(http://www.hapmap.org)获取主要乙醇代谢酶(ADH1B、ALDH2、CYP2E1)编码基因全长及上游1200bp序列内的SNP信息,应用Haploview 4.0软件,按稀有等位频率(minor allele frequency, MAF)大于0.03进行多态性位点的纳入,并构建LD区域,在每个区域选择一个与该区域内其他位点关联最强的SNP,作为tagSNP纳入候选位点,以西南地区人群为研究对象,采用SNPlex技术对tagSNPs进行基因型分型;2.以西南地区和东北地区人群为研究对象,采用PCR-RFLP对CYP2E1中研究较多的功能多态位点RsaⅠ、DraⅠ、TaqⅠ(rs2031920、rs6413432、rs2070676)进行基因型分型;以西南地区人群为研究对象,采用PCR-RFLP对JWA中两个功能多态位点-76 G>C和723 T>G进行基因型分型;3.采用Statistical Analysis System (version 9.0)软件,应用非条件Logistic回归模型,计算各基因型对结直肠癌易感性影响的独立主效应,以比值比(odds ratio, OR)衡量其相对风险度;采用Haploview 4.0软件,构建单倍型;采用unphased 3.0.13软件计算单倍型OR;以MDR 1.0.0软件评估各位点间的交互作用;以分层分析计算吸烟、饮酒与多态性位点的交互作用。结果:1. ADH1B, ALDH2, CYP2E1三个基因中共筛选出16个候选位点,进入分型15个,成功分型14个;ALDH2 rs671 AA基因型、CYP2E1 rs1329149 CC基因型使西南地区人群结直肠癌易感性显著升高(OR值分别为1.86和4.01,95% CI分别为1.12-3.08和2.4-6.66),且两位点均在直肠癌发生中表现出更高的风险(rs671 AA与结肠癌遗传易感性无关联,在直肠癌中OR=2.20, 95% CI=1.25-3.86;rs1329149 CC基因型在结肠癌中OR=3.05,95% CI=1.54-6.06,在直肠癌中OR=4.72,95% CI=2.73-8.16);ALDH2和CYP2E1间存在交互作用,当个体同时携带rs671和rs1329149风险基因型(分别为AA和CC)时,个体患结直肠癌风险升高4.97倍;两位点间与饮酒也存在交互作用,在风险基因型合并作下,饮酒个体患结直肠癌的风险由非饮酒者的2.55升高到7.39,P均小于0.05。2.西南地区人群发病年龄低于东北人群,饮酒人群少于东北人群,两地人群间CYP2E1 rs6413432和rs2070676的基因型频率分布存在显著差异。关联分析发现,西南地区人群中,相对于GG基因型,rs2070676 GC基因型具有微弱升高直肠癌易感性的作用(OR=1.38,95% CI=1.04-1.84);与饮酒间的交互作用分析发现在西南地区人群中,相对携带非风险基因型且不饮酒的个体,当合并饮酒时,携带rs6413432 T等位和rs2070676 C等位个体对结直肠癌易感性升高(OR分别为2.26和3.54,95% CI分别为1.05-4.88和1.03-12.13)。3.西南地区人群中,JWA -76G>C和723T>G这两个位点与结直肠癌的发病不具有显著关联,与吸烟、饮酒也不具有交互作用。结论:1.主要乙醇代谢酶编码基因多态性中,ALDH2 rs671 AA基因型,CYP2E1 rs1329149 CC基因型,rs2070676 GC基因型是西南地区人群结直肠癌的遗传易感因素,CYP2E1和ALDH2间存在基因-基因交互作用,两基因的多态性位点与饮酒间存在环境-基因交互作用,表现出危险因素叠加时,增强西南地区人群结直肠癌易感性。2.西南地区人群和东北地区人群间,CYP2E1存在显著遗传差异,且饮酒习惯、结直肠癌发病年龄也存在显著差异。3.环境应答基因JWA基因多态性可能不是西南人群结直肠癌的遗传易感因素。综上所述,中国西南地区和东北地区人群在所研究的基因中具有遗传差异,提示多人群的对比研究在遗传易感性研究中具有重要意义。西南地区人群中,主要乙醇代谢酶基因多态性与结直肠癌易感性相关,同时与饮酒具有交互作用,提示个体是否罹患结直肠癌,除个体的生活习惯外,与遗传因素的作用也有很大关系,对制定个性化干预措施以降低结直肠癌发病风险具有指导意义。更多还原

【Abstract】 Backgroud:Colorectal cancer (CRC) is one of the most common malignant cancers in modern society. More than 1 million people develop CRC all over the world every year, and about 500 thousand people die of it, which made CRC ranking the 3rd of both morbidity and mortality in all cancers. In China, the morbidity of CRC is increasing sharply. It counted about 24 incidences in 100 thousand people in year 2008 in China. In a newly enacted document“Essentials in the plan of cancer prevention and control in China”, CRC was listed as one of the eight“emergency cancers”which are responsible for 80% of the mortality caused by cancers.Like most cancers, etiology study suggested that both genetic and environmental factors take effects in developing CRC. Studies revealed that diet and phisycal activities are related environmental factors while genes coding folic acid metabolizing enzymes and some phaseⅡmetabolizing enzymes play important roles in the genetic susceptibily to CRC.Most epidemiology studies found alcohol consumption to be a risk factor of developing CRC, especially in Asians. The different effects of alcohol consumption among populations indicate that genetic factors may play important roles in the association between alcohol consumption and CRC development. Studies on the fanctional polymorphisms in alcohol metaolizing enzymes found significant genetic differences between western people and Asians. However, there are only a few reported researches focused on Chinese population, and no tagSNPs based research into alcohol metabolizing enzymes was reported. On account of this, we conducted a case-control study based on the Japan, Korea and China Colorectal Cancer collaboration study, adding the newly recruited samples from northeastern China, to study the genetic association between polymorphisms of genes coding major alcohol metabolizing enzymes, JWA gene and CRC.Objectives:1. To screen tagSNPs in major alcohol metabolizing genes ADH1B, ALDH2, CYP2E1 and to study the association between the tag SNPs and CRC susceptibility in southwestern Chinese.2. To study the association between CYP2E1 functional polymorphisms and CRC susceptibility in both southwestern and northeastern Chinese, and the difference between the two district.3. To study the association between JWA functional polymorphisms and CRC susceptibility in southwestern Chinese.Method:1. Obtaining CHB population SNPs data of whole length plus 1200bp upper strand of ADH1B, ALDH2, CYP2E1 from HapMap (http://www.hapmap.org), accepting the loci that the minor allele frequency (MAF) is above 0.03, constructing LD blocks with software Haploview v4.0, select the locus which ranks the highest r2 with other loci in the very block as the tagSNP, using SNPlex to genotype these tagSNPs in southwestern Chinese.2. Using PCR-RFLP to genotype CYP2E1 functional polymorphisms RsaⅠ, DraⅠ, TaqⅠ(rs2031920, rs6413432, rs2070676) in southwestern and northeastern Chinese; using PCR-RFLP to genotype JWA functional polymorphisms -76 G>C and 723 T>G in southwestern Chinese.3. Using software Statistical Analysis System version 9.0 to apply unconditional logistic regression model to evaluate the risk by odds ratio (OR); using software Haploview v4.0 for reconstructing haplotypes; using software unphased v3.0.13 for evaluating the ORs of haplotypes; using Multifactor dimensionality reduction (MDR) v1.0.0 to assess the interactions between loci; dividing samples into subgroups by drinking or smoking habits to assess interactions between genetic polymorphisms and smoking and alcohol consumption. Results:1. In the three candidate genes coding major alcohol metabolizing enzymes (ADH1B, ALDH2, CYP2E1), sixteen candidate loci were screened and fourteen of them were genotyped successfully; ALDH2 rs671 AA and CYP2E1 rs1329149 CC genotypes were found to be associated with increased risk of CRC in southwestern Chinese (OR=1.86 and 4.01, 95% CI=1.12-3.08 and 2.40-6.66, respectively), both the loci showed higher effect in rectum cancer (rs671 AA genotype was not associated with colon cancer but showed 2.20-fold of increased risk in rectum cancer, 95% CI=1.25-3.86; rs1329149 CC genotype showed 3.05-fold and 4.72-fold of increased risk in colon cancer and rectum cancer, respectively); Interaction between ALDH2 and CYP2E1 was found, individuals with risk genotypes of rs671 AA and rs1329149 CC, showed an increased risk of 4.97-fold; Interactions existed between the two loci and alcohol consumption, the risk to CRC susceptibility of people with rs671 AA and rs1329149 CC increased from 2.55-fold in non-drinkers to 7.39-fold in drinkers.2. The age of diagnosing CRC was significantly lower in southwestern Chinese than that in northeastern Chinese. Drinking people was fewer in southwestern Chinese than that in northeastern Chinese. Genetic differences were found in CYP2E1 between southwestern and northwestern Chinese. In southwestern Chinese, rs2070676 CC genotype was associated with slightly increased risk of CRC (OR=1.38, 95% CI=1.04-1.84), combined with alcohol consumption, rs6413423 T allele and rs2070676 C allele were associated with increased risk of CRC (OR=2.26 and 3.54, respectively), which were not found in northeastern Chinese.3. JWA -76G>C and 723 T>G were not associated with CRC in southwestern Chinese. No interaction was found between the polymorphisms and smoking or alcohol consumption.Conclusion:1. Among the polymorphisms in major alcohol metabolizing genes, ALDH2 rs671 AA, CYP2E1 rs1329149 CC and rs2070676 GC genotypes are genetic susceptibility factors of CRC in southwestern Chinese. Gene-gene interaction exists between CYP2E1 and ALDH2, and gene-environment interaction also exists between CYP2E1, ALDH2 polymorphisms and alcohol consumption, the result showed increased risk with more risk factors in southwestern Chinese.2. Significant genetic differences exist between southwestern and northeastern Chinese, drinking habit, the age of developing CRC are significantly different between the two populations.3. JWA gene polymorphisms may be not associated with CRC risk in southwestern Chinese.To be summary, the genetic background differs in southwestern and northeastern Chinese, indicating the importance of multi population research. Polymorphisms in major alcohol metabolizing enzymes are associated with CRC susceptibility, and they interact with alcohol consumption, indicating that, beyond genetic factors which are hardly to be modified, life style is an important factor in developing CRC, which we can be easily taken into control. This may lead to a new direction of individual intervention in CRC prevention.更多还原