基于靶标染色体区域的拷贝数变异对结直肠发病影响的研究

Genetic susceptibility is playing an important role in development of colorectal cancer (CRC). However, bunches of results derived from whole genome wide studies (GWAS), which is a representative of newly developed study strategies with single nucleotide polymorphism (SNP) used as major genetic biomarker, is still not able to explain large part of susceptibility to CRC. Such so called "missing heredity" implies that, as a genetic biomarker that is structurally and functionally distinguished from SNP, how and to what extent DNA copy number variations (CNV) contribute to CRC risk is warranted. So far, there are only a few studies with regarding to CNV associated CRC risk worldwide and almost no relative study in Chinese population is published. Start our former projects in which we had collected CRC cases and controls with a large sample size and been keeping perform CRC related molecular epidemiological studies, in current project, based on mapping of the discovery from total 14 CRC related GWAS and results from CRC related CNV associated studies published heretofore, and on focusing our target on the 39 selected "candidate chromosome regions", we plan to evaluated the effect of polymorphisms of all CNVs localized in the 39 "candidate chromosome regions" on risk of CRC. Briefly, in discovery stage, we plan to perform a custom-designed CNV chip based scan in the 39 "candidate chromosome regions" to test the association between locus CNVs and CRC risk; then the association between CRC risk and statistically associated CNVs will be validated, respectively, in to independent samples (one would be 800 cases and 1600 controls, another one would be 800 cases and 800 controls). As part of functional validation and by taking results of pooling analysis and bioinformatics analysis into fully consideration, we plan to further test and try to uncover the potentially underlying mechanism of whether and how the CNVs regulate the gene expression. We believe that the currently proposing project can help for interpreting undiscovered susceptibility to CRC and providing a theoretical basement for screening "high risk" subpopulation more efficiently.

遗传易感性在结直肠癌（CRC）发病中起着重要作用。以单核苷酸多态（SNP）为遗传标志物、以全基因组关联研究（GWAS）为代表性策略获得的大量结果仍无法解释大部分CRC易感性。这种遗传性丢失提示，作为结构和功能都有别于SNP的遗传标志物，DNA拷贝数变异（CNV）对CRC发病的影响亟需研究。目前仅少数研究涉及CNV与CRC发病的关系，尚未见中国人群的相关研究报道。我们在前期已建立大规模CRC病例-对照样本并开展了系列研究基础上，本项目拟对基于全部14项CRC相关GWAS研究和CNV关联研究筛选到的39个潜在关联"靶标染色体区域"的CNV，第一步先在520病例-对照中全面分析"靶标染色体区域"CNV与CRC风险的关联；结果分别在800病-1600对、800病-对的两个样本中进行验证。并结合生物信息学分析，就关联CNV对基因表达的影响进行功能验证。为解释CRC易感性机制、筛选高危人群提供理论基础

本项目的主要研究目标是研究DNA拷贝数变异（CNV）对我国汉族人群散发性结直肠癌遗传易感性的影响。按照项目研究计划，首先，采用了两阶段病例-对照研究策略，我们首先在第一阶段重庆人群521例病例和532例对照中以CNVPlex多重检测技术、RealTime-PCR等方法对分布在16条染色体上的31个潜在靶标染色体区域内的CNV进行了分析。在全部399个CNV中频率>=0.05的 CNV为130个。其次，对经生物信息学分析后确定的16个优先分析CNV与结直肠癌风险的关联进行了分析，结果提示1q21的NBPF9基因19-22外显子区域约3.5Kb序列的3-8拷贝数增加、位于3p21的SMARCC1基因3’-端约30.6Kb缺失、位于9P21的FOCAD基因11-20外显子区域一个64.7kb的缺失、和位于18q21的PHLPP1基因3’-端约58kb的缺失等4个CNV与重庆人群结直肠癌发病关联显著。但在经进一步第二阶段独立人群样本验证后， FOCAD基因11-20外显子区域的一个64.7kb缺失和PHLPP1基因3’-端约58kb缺失等2个CNV仍与结直肠癌有显著关联。经分析，PHLPP1基因3’-端58kb缺失部分包含了miR-224的结合序列，可能使miR-224对PHLPP1基因表达的阻遏因该缺失而丧失。为此，我们首先对20例正常对照外周血基因组以real-Time PCR分析了该缺失对转录水平的影响，继而在PHLPP1基因3’-端正常的2种结直肠癌细胞模型中以miR-224处理后验证其对该基因表达水平的影响，均未发现差异，结果提示该PHLPP1基因3’-端58kb的缺失可能对该基因表达无直接影响。结直肠癌，靶标区域，拷贝数变异，风险，功能检测